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GENOTYPIC AND PHENOTYPIC
CHARACTERISTICS OF p53, HER/neu AND Bel-2 IN CHINESE
PRIMARY BREAST CANCER
by

XiaoTan Qiao

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pathology

1997

ABSTRACT

GENOTYPIC AND PHENOTYPIC CHARACTERISTICS OF p53, HER/neu
AND Bel-2 IN CHINESE PRIMARY BREAST CANCER

By
XiaoTan Qiao

There is a need to compare the various descriptional parameters currently used to define
breast cancer with the underlying genetic alterations. The work in this dissertation addresses this
need by examining paraffin embedded samples of breast cancer collected previously for routine
diagnosis. Two hundred and five specimens from 178 cases of primary breast cancer in patients
from China, those specimens were evaluated for clinical and histopathology features of cancer. One
hundred thirty-four primary invasive breast carcinomas from this group were examined for p53,
HER/neu (c-erbB-2) and Bel-2 protein expression by immunohistochemical methods; 14.18%,
23.13% and 66.42%, respectively stained positively. p53+/HER+/Bcl-2+, p53+/HER-/Bcl-2+,
p53+/HER+ and p53+/Bcl-2+ were associated with poor breast cancer prognosis. HER/neu (c-
erbB-Z) genes were amplified and detected by differential PCR and band intensity methods;
29.10% was positive. Sixty-four of them were evaluated for p53 gene point mutations by
polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP), followed
by gene sequence analysis; a positivity of 6.15% was found and included 2 missense, l intron and
l silent, mutations. The detected characteristics of p53 and HER/neu gene mutations, the proteins
expressed, together with Bel-2 protein accumulation, were used to define differences between two
groups: the Western population (high breast cancer risk) and Chinese people (low breast cancer
risk) these had similar genotypes and phenotypes except for the Chinese breast cancer patients
tending to be younger, to have a higher frequency of infiltrating ductal cancer and have a longer
disease free survival time. Aspects of these findings that might provide information as to the

mechanisms involved in carcinogenesis are discussed.

ACKNOWLEDGMENTS

My special appreciation and gratitude go to my mentor, Dr. Charles D. Mackenzie, for his
constant guidance, support and encouragement throughout this study, for his invaluable assistance
and in the preparation of the manuscript. Special thanks to Dr. Karen Friderici for her broadening
of my perspectives on molecular pathology, and serving as a committee member. My sincere
appreciation also extended to my other committee members, Dr. Thomas Bell and Dr. James
Potchen for serving on the guidance committee and for advice on pathology and radiology.

This dissertation could not have been completed without the technical assistance, advice,
support, and encouragement of many people both in the United State of America and China. These
include: Dr. Jeffrey Williams, Dr. Norbert Kaminski, Dr.Chia-Cheng Chang, Dr. Shi Zheng, Dr.
Gong Chengben, Dr. Wang Lin, Dr. Zhao Gongha. The Histopathology Laboratory team, Dr.
Kaminski’s, the Tissue Typing (Dr. Bull’s lab) Laboratories and our Environmental Pathobiology
Laboratory (Especially: Kathy Campbell, Josselyn Miller, Amy Porter, Jose Santiago, James, Bob,
Peggy and Dr. James Gerlack) .

Acknowledgment is made to Dr. Raymond H. Murray who first introduced me to the
medical science in the United State of America and provided me with extensive educational
opportunities.

I am grateful to my family, especially my mother Dr. Li, Yan, and my father Mr. Qiao,
Gouyu, he has passed away two years ago, who have always supported and encouraged me

through all my endeavors, and my dear daughter Tina who has supportively accompanied me and

brightening up the study days.

iii

TABLE OF CONTENTS

I. IMODUCTION ......OOOOOOOOOOOO0.........00..........
1.0.0 Historical Aspects of Breast Cancer .. ........... ..
2.0.0 General Aspects of Breast Cancer ........... ..... ..

OEpidemiology ........................ ........... .

1
2
3
4
n
r
1

2
3
4
5
C

1
2
3
a
i
1
2
3
4

Geographical Influence ...... ..... ............

Environmental Influence .. .................. ..
Racial Factors ........... ............. .......
Age
Gender Distribution ..........................
Trends ......................................
sk Factors of Breast Cancer ..................
Genetic Factors (Family History) ............
Hormonal Influence ..........................
Nutritional Influence .......................
other Factors ...............................
ical Diagnostic Approaches of Breast Cancer ..
ognostic Indicators ..........................
Tumor Staging ............ . .......... ........
Tumor Size ...... ..... .......................
Tumor Type ..................................
Morphologic Parameters ......................
Lymph node Metastases ..... . ........ . ........
reening ......................................
Physical Examination ........................
Breast Imaging ............. ..... ............
Breast Biopsy ...... ................... ......
st Cancer Diagnosis of Pathology ...... ..... ..
stopathology .................................
Histochemical Presentation ..................
Anatomical Features of Histopathology .......
Carcinoma Differentiation (Grading) .........
Carcinoma Classifications ...................

Cytopathology .................................

1
2

Cytochemistry of Fine Needle Biopsy Sample ..
Morphological Characteristics of Cytologic
Samples ........................... ...... ....

4.2.3 Grading .....................................
4.2.4 Other Techniques ............................

4.3.0 Immunohistochemical Pathology ..... ......... ....
4.3.1 Features of Immunohistochemistry

of the Mammary Tissues ......................

4.3.2 The Immunomarkers used on Breast Cancer

Tissues ......OOOOOOOOOOOOOOOO.......OOOOOOIO

4.3.3 The Immunomarkers of Specific Interest(p53,

c-erbB-Z and Bel-2) ......OOOOOO......OOOOOOO

4.4.0 MOlecular PathOIOgy 0............OOOOOOOOOOOOOOO
4 4.1 in situ Hybridization .......................

fichh

4.
4
4.

2

3
4

in situ Hybridization on Malignant Diseases .
in situ Hybridization on Cytology Samples ...
Flow Cytometry on Breast Cancer Cells .......

W

00¢“ OtOtml-‘H

10
10
11
12
12
13
14
15
15
16
16
17
17
17
18
19
20
22
22
22
23
23
24
26
26

27
28
29
3O

30

31

32
32
33
34
35

4.4.5 Polymerase Chain Reaction (PCR)

in Cancer Studies ...........................
4.4.6 PCR in situ hybridization ...................
4.4.7 Other Molecular Techniques Used in

Breast Cancer Studies ....... ................

5.0.0 Involvement of Genes in Oncogenesis ...... ....... .

5

5

5.1.0 p53 Tumor Suppressor Gene and Product .........
5 1.1 Background ..................................
5 1.2 Structure and Function ......................
5 1. 3 Oncogenesis Feature .........................
5 1. 4 Clinical Approaches .........................
5.2 0 HER/neu Proto-oncogene and Product ............

5 2 1 Background ..................................

5 2 2 Structure and Function ......................

5 2 3 Oncogenesis Feature .........................

5 2 4 Clinical Approaches .........................
.3 c1-2 Cell Death Regulating Gene and Product ...
1 Background ..................................
2 Structure and Function ......................
3 Oncogenesis Feature .........................
4 Clinical Approaches .........................

.0 B
5.3.
5.3.
5.3.
5.3.
.4.0 p53,Bcl-2 and HER/neu(c-erbB-2),Apoptosis and
Oncogenesis ..... ............. . .................
.1 Apoptosis ...................................
.2 Apoptosis and Oncogenesis ...................
.3 p53,Bc1-2 and HER/neu (c-erbB-2)involvement

of Apoptosis and Oncogenesis ................

5.4
5.4
5.4

6.0.0 Immunohistochemical and Molecular Studies of

I
1
2
3
4

I

O
0
0
0

Breast Cancer in China ............... ............

PATIENTS, MATERIAL AND METHODS .....................
.0 Patient Clinical Information .....................
.0 Histopathology .. ....... ..........................
.0 Immunohistochemistry .. ............. . ......... ....
0 Molecular Analysis ...............................
. Cell Lines and Culture Conditions .............
. DNA Extraction Method for Formalin Fixed
Paraffin Embedded Tissue ......................
DNA Extraction Method for Cell Cultures .......
HER/neu Gene Amplification Mutation Analysis
by Differential PCR ...........................
4.5.0 p53 Gene Point Mutation Analysis ..... .........

4
4

NH

00 CO

.3.
.4.

5.0.0 Statistical Analysis .............................

III. RESULTS ...........................................
1.0.0 Patient Clinical Information ........ ...... .......
2.0.0 Histopathology ...................................
3.0.0 Immunohistochemistry .............................

4.0.0 Molecular Analysis . ............................. .
Iv. DISCUSSION .00..00.0000000000000000000.......0......
1.0.0 Comparison of Breast Cancer Characteristics on

Low Risk (Chinese) with High Risk (Western)

36
36

38
39
40
40
4O
42
43
45
45
46
47
48
49
49
50
52
53

54
54
55

56

59

61
61
61
62
67
67

68
68

69
72
75

77
79
80
83
88

92

Breast cancer Populations ......COOOOOOOOOOOOOOOOO 92

1.1.0 General Aspects of Breast Cancer ............... 92
1.1.1 Age Difference ............................ 92
1.1.2 Size of Tumor ............................. 94
1.1.3 Survival Rate ............................. 95
1.2.0 Histopathology of Breast Cancer ................ 96
1.2.1 Types of Breast Cancer .................... 96
1.2.2 Breast Cancer Histological Grading ........ 98
1.3.0 Immunohistochmical Studies ..................... 99
1.3.1 General Aspect ............................ 99
1.3.2 Combined Three Immunohistochemical Studies.101
1.3.2.1 The Effects Combining Three
immmunostaining Results ........... 102
1.3.2.2 The Effects Combining Three
immmunostaining Results ........... 103
1.4.0 Gene Mutation Studies ......................... 104
1.4.1 HER/neu Gene Amplification Mutation ...... 104
1.4.2 p53 Gene Point Mutation .................. 106
2.0.0 Characteristics of p53, c-erbB—z and Bcl-2
and Their Relationship to Oncogenesis .......... 108
2.1.0 General Concept of Oncogenesis ................ 108
2.2.0 Our Findings in Oncogenesis ................... 110
2.2.1 p53 ................................... 110
HER/neu ............................... 111
Correlation of p53, HER/neu and Bel-2 . 112
The Feature of p53 Gene Point Mutation
in Chinese Primary Breast Cancer ...... 113
3 0 0 Clinical Approach .............................. 115
4.0.0 Technical Approaches in The Study .............. 116
5 0 0 Conclusion ..................................... 117

NNN

.20
.2.
.2.

uwa

v. Rnrnmczs ..........OOOOOOOO......OOOOOOOOOOO..0... 119

Table
Table
Table
Table
Table
Table
Table
Table

Table
Table

Table
Table
Table
Table
Table
Table
Table
Table
Table

Table

Table
Table
Table

Table

Table

Table

I-1:
I-2:
I-3:
I-4:
I-S:
I-6:

I-7:
I-8:

LIST OF TABLES

Factors influencing the risk of developing
breast cancer
The influence of various indicators on the
prognosis of breast carcinoma .... ............
Randomized clinical trials of breast cancer
screening ....................................
World Health Organization (WHO) histologic
classification of breast tumors
World Health Organization classification of
carcinomas ...................................
Breast cancer classification in China
Common cellular features of breast cancer ...
Some immunocytochemical markers used to
define breast cancer .........................

II-l: Histologic grading of invasive breast cancer
II-2: The methods used for immuno-staining

evaluation ......OOOOOOOOOOOO0.0.0.0000.......

II-3: The amplimer sequences of p53 gene

III-
III-
III-
III-
III-
III-
III-
III-

III-

III-

III-

III-

III-

III-

III-

exon 5 to 9
1: The age and percentage distribution of the
breast cancer patients .......................
2: The correlation of age with lymph node
metastasis
3: Survival information and the % results of
Life Table Analysis ..........................
4: Histopathologic types and frequency of
Chinese breast cancer
5: Histology grading of invasive breast cancer
6: Immunohistochemical studies
7-1: Immunostaining positivity by pathologic
tumor types ..................................
7-2: Immunostaining negativity by pathologic
tumor types ..................................
8: protein expression for p53, cerbBZ and
Bcl-2 and their relationship to clinical
indicators
9: The results of Chi-square test from
the table III-8
10-1: Relationships between clinical
indicators and immunostain results
10-2: Telationships between clinical
indicators and immunostain results
11-1: The associations between three
immunostaining and clinic-pathological
features
11-2: The associations between three
immunostaining and clinic-pathological
features ..............
11-3: The associations between two
immunostaining and clinic-pathological
features

Wi

14
15
18
24
25
25
28

31
62

67
72
77
77
79
79
80
81
82

82

83

83

84

84

85

Table

Table

Table

Table
Table
Table
Table
Table
Table
Table
Table
Table

Table

III-11-4: The associations between two

immunostaining and clinic-pathological

features ........................... .......... 86
III-11-5: The associations between two

immunostaining and clinic-pathological

features ..................................... 87
III-11-6: The associations between two

immunostaining and clinic-pathological

features ..................................... 87
III-12-1: Amplification of HER/neu gene mutation

related to clinical information .............. 88
III-12-2: Amplification of HER/neu gene mutation

related to clinical information .............. 89
III-12-3: Amplification of HER/neu gene mutation

related to clinical information .............. 89
III-13: p53 gene point mutation related to other

information .................................. 90
III-14-1: p53 gene and HER/neu gene

mutation related to clinicla information ..... 90
III-14-2: p53 gene and HER/neu gene

mutation related to clinicla information ..... 91
III-14-3: Sum of the results of the gene mutation

and clinical information ..................... 91
IV-l: The frequency of breast cancer types

the USA . ............. .......... ....... ..... ..97
IV-2: The frequency and comparison of breast

cancer grade ................................. 98
IV-3: Comparison of different groups ............. 99

\riii

LIST 0? FIGURES

Figure 1: H & E stain section of a primary

breast cancer ................................ 65
Figure 2: Immunohistochemical stain positive of p53 .... 65
Figure 3: Immunohistochemical stain positive of

c-erbB-2 .. ..... ........................ ..... . 66
Figure 4: Immunohistochemical stain positive of Bcl-2 .. 66
Figure 5: The HER/neu dPCR products are in

a 2% agarose gel without detection box ....... 71
Figure 6: The HER/neu dPCR products are in a 2% agarose

gel with detection box ....................... 71
Figure 7: PCR products of p53 gene at exon 5 in a 2.7%

agarose gel .................................. 73
Figure 8: p53 SSCP analysis. Controls on a 6%

nondenaturing polyacrylamide gel ............. 74
Figure 9: p53 SSCP analysis. Sample No.43 on a 6%

nondenaturing polyacrylamide gel ............. 75
Figure 10: The size tistribution of the breast cancer .. 78
Figure 11: Contribution of genotype and phenotype to

risk determination .......................... 109
Figure 12: Schematic representation of events of the

transformation of cytosine to thymine ....... 114

k

I. INTRODUCTION

Breast cancer is the most common malignant disease of women. It is a disease that
in all probability has more than one cause, and indeed may be the reflection of many
interacting mechanisms. Breast cancer understandably has been the subject of extensive
studies. This current project is designed to compare the characteristics of p53, HER/neu
(c-erbB-Z) gene mutation, protein expression and Bel-2 oncoprotein accumulation in a
low risk breast cancer population (China) with that in a high risk population (Western
countries). Furthermore, the correlative features of gene point mutations of p53, the gene
amplification mutations of HER/neu (c-erbB-Z), their protein products and Bel-2
oncoprotein in these primary invasive breast cancers are analyzed and evaluated in the

hope that this information can help to better understand breast cancer.

1.0.0 Historical Aspects of Breast Cancer

Breast cancer has had the attention of physicians for several centuries and has a
place in medical history because its uncertain causes, the confusion of treatment and the
ofien unhappy endings.

The oldest of medical records comes from ancient Egypt about 1600 BC through
engravings or painted hieroglyphics on stone where were recorded the 45 case earliest
known cases of breast cancer. The examiner was told that “a breast with bulging tumors,
very cool to the touch, is an ailment for which there is no treatment”; Edwin Smith (1822-
1906) acquired this old information about breast cancer at Thebes in 1862 (Henry, 1984).

Records existed in ancient Greece by 460-136 BCs Hippocrates (460-370), father

of medicine and the originator of the theory that the four cardinal humors of the body
(blood, phlegm, yellow bile, and black bile) with the four universal elements (earth, air,
water, and fire) balance maintained the health, put forward the notion that the origin of
cancer was natural causes. He described a woman with carcinoma of the breast with
bloody discharge from the nipple, and clearly stated that in cases of deep-seated cancer it
was better to give no treatment, because treatment hastened death (Boyd, 1987).

During the Greco-Roman Period (150 BC-SOO AD), Leonides, a Greek physician
in the first century AD who practice at Rome, is credited with the first recorded operative
treatment for breast cancer. In the same century, Aurelius Cornelius Celsus, an
encyclopedist and not a physician, published the first clinical description of breast
pathology: “a fixed irregular swelling with hardness or softness, dilated tortuous veins
and with or without ulceration.” He mentioned four clinical stages: namely malignancy
(apparently simple or early), carcinoma without ulcer, ulcerated cancer, and ulcerated
cancer with flower-like excrescences that bleed easily. He opposed treatment of the last
three stages by any method (Wagner, 1991).

Galen (131-201 AD), who was born on the Mediterranean coast of Asia Minor,
studied in Alexandria and practiced in Rome, described mammary cancer as a swelling
with distended veins resembling the shape of a crab’s legs. He is know for his humoral
theory (i.e. “ to prevent accumulation of black bile the patient should be purged and
bled”), but he also claimed to have treated breast cancer by surgery in its early stage when
the tumor was on the surface of the body and all the roots could be extirpated (Wagner,

1991).

There was no significant progress in this field during Medieval Period until the
Renaissance. Andreas Vesalius (1514-1564), a Flemish surgeon comparative anatomist,
advised wide excision and the use of ligatures instead of cautery in breast cancer
operations. Ambrose Pare (1510-1590) studied medicine in Paris where he made the
important observation that breast cancer often caused swelling of the axillary lymph
glands. Henri Francois le Dran (1685-1770) noted that lymphatic spread worsened the
prognosis of breast cancer (Robinson, 1986).

Significant changes in pathological and physiological concepts were slow to
develop in the eighteenth century. Pieter Camper (1722-1789) first described and
illustrated the internal mammary lymph node, and Paolo Mascagni (17 52-1815) later
reported the same for the pectoral lymph drainage. The textbooks described a variety of
diseases: breast cancer was considered as a benign growth that under adverse
circumstances could undergo malignant degeneration. Death caused by metastasis from
cancer was not yet be understood at that time. Mastectomies were performed in larger
numbers. Bernardine Ramazzini, an Italian physician, noted the high incidence of breast
cancer in nuns and hypothesized that this was in some way related to their celibate life
style (Yu 1981; Wagner, 1991).

The nineteenth century brought several important contributions to the field. In
1822, James Elliott first examined a tumor under the microscope: an axillary node
removed during breast surgery and diagnosed as sarcomatous. Improvements in surgical
technology occurred to assist breast cancer surgery. Bresler, William and Morton first

introduced anesthesia in the United States in 1846 and Joseph Lister in Great Britain in

1867 established the principle of antisepsis. The modern radical mastectomy technique
and specific investigations into the epidemiology statistics both having a great effect on
the study of breast cancer were established. Methods for removing axillary nodes,
cutting off the blood supply of tumor, and for taking out the entire breast no matter how
small the primary tumor, were developed for use in surgical treatment (Elliott, 1822;
Wagner, 1991). In 1856, Sir James Paget reported that scirrhous carcinoma patients
who were not operated on lived longer than those with surgery (Paget, 1856). By the
end of the century, the Halsted radical mastectomy had been established as the ideal
method for surgical treatment of operable breast cancer; this method, where “the
suspected tissues should be removed in one piece”, was provided by Mlliam Steward
Halsted (1852-1922). It is still used in today’s surgical treatment. William Welch, a
pathologist at Hopkins was the first to employ frozen sections in the diagnosis of breast
lesions, in 1891 (Cullen, 1895). As medical education increased towards the end of the
century research into breast cancer increased accordingly.

For most of the Twentieth century, the causes of breast cancer remain poorly
understood, but it was known that a strong family history led to a high risk for breast
cancer and it was believed that the solution was bilateral mastectomy. Cushman
Dhaagensen classified breast cancer according to the size, clinical findings and nodal
status and first proposed self-examination of the breast; he suggested that lobular
neoplasm (in situ) was not actually a carcinoma (Haagensen, 1986). The modified
radical technique for mastectomy in surgical treatment for carcinoma of the breast was

developed in 1948 by Patey and Dyson in London. In the same year, McWhirter

introduced a simple mastectomy technique and included radiotherapy treatment, however
the survival rates remained Similar to tradition surgical methods (McWhirter, 1948; Patey,
1948)

In 1973 estrogen receptors and their properties were demonstrated in human
breast tumors by McGuire (McGuire,1975), and in 1975, Horowitz identified
progesterone receptors in hormone-dependent breast cancer (Horowitz, 1975). The
presence of circulating malignant cells in the blood stream, reported in 1955 by Engell,
stimulated interest for therapy through destruction of these cells by systemic
administration of chemicals (Engell, 1955).

In 1895, Emile Cnibbe (1875-1960), a second-year medical student in Chicago,
first irradiated a breast cancer patient by X-ray, with such radiation therapy becoming the
common form of enhanced standard treatment in many clinical centers until 1920 (de
Moulin, 1983); the five-year survival of Stage 11 breast cancer patients was improved
through this approach, although Stage 1 cases remained unchanged. Keynes (1932) in
London, used radium as a source of radiation, and obtained a five-year survival of 77 .1
percent in those cases with negative axillary nodes, and of 36.3 percent in those with
axillary-involved breast cancer. In 1948, Robert McWhirter proposed the approach of
simple mastectomy followed by radiotherapy (McWhirter, 1948). Higher X-ray voltage
up to 6000 to7000 rads, including the implantation of Ir'”, were also used during 1960s.
Even to the present day there is no certainty as to whether the more intensive surgical
procedures or the addition of radiation was most benefit breast cancer patients.

During this present century, improved management of pain and infection through

the advent of anesthesia and aseptic technique have helped breast cancer control.
Mammographic screening in 1978 (Nation Institutes of Health, 1978), hormonal therapy
in 1939, chemotherapy in 1963, all are important additional treatments introduced for
breast cancer patients; the results seen include the observation that five-year survival rates
have increased particularly in premenopausal women with metastases (Bonadonna, 1981).
Irnmunotherapy and genetherapy are not yet accepted as practical clinical treatments yet,

but are under detailed research (Wagner, 1991).

2.0.0 General Aspects of Breast Cancer
2.1.0 Epidemiology

Although, globally breast cancer is the most frequent malignancy in women,
international statistics reveal widely varying rates of breast cancer in different geographical
areas. This may reflect different effects of the various environments or perhaps ethnic
variations on gene expression and mutation in the development in this disease.
2.1.] Geographical Influence

Each year, over 512,100 new cases of breast cancer are detected, and some
250,000 people in the world die of malignant breast cancer (WHO, 1982). However, the
morbidity and mortality appear to be different between the Eastern and the Western areas
of the world. For the period 1986-1988, the highest death rate in malignant diseases was
from breast carcinoma and was recorded in England and Wales (293/ 100,000), followed
by Denmark (28.3/100,000) and Scotland (27.5/100,000). The rate in the United States
was 22.4/100,000 - 16th in a list of 50 nations. The countries at the bottom of the list

were Thailand with 1.0/ 100,000, Korean Republic 6.0/ 100,000 and China with 4.7 deaths

per 100,000 population (Tavassoli, 1992). Thus there is almost a thirty fold difference
between Thailand and England, the East and the West.

In relative terms as the incidence of breast cancer in the United States and Canada
is Six times that in Asia and Afiica. In Western Europe it is four times that in Japan.
South America, the Caribbean region, and Eastern Europe have rates that lie between
these (Waterhouse, 1976). Of pertinence to the current study is the fact the mortality
from breast cancer among Chinese women is reported to be only about one-fifth that of
white females the USA (Fisher, 1978; Hoare, 1996)

2.1.2 Environmental Influences

Approximately 80 to 90% of all cancers are thought to be a result of exposure to
environmental carcinogens. The importance of environmental factors can be deduced
from the considerable geographical variation reported, and from the changes in rates
amongst migrants. An interesting example of this occurs in Japanese who have emigrated
to the continental United States and have a higher mortality rate than those living in Japan;
correspondingly the rates are higher for Japanese born in the United States than for
immigrants who were born in Japan (Dunn, 1977). Since changes in risk usually occur
slowly, and over several generations (as illustrated by the migrants from Japan and China
in the USA), it is assumed that some of the determinants of the risk must act early in life
(American Cancer Society, 1988; Jigginson, 1992).

Chemical induced carcinogenesis has been noted for many areas of cancer, often
related to such carcinogens present in the environment; chemicals are also purported to be

causative in breast cancer. Exposure to more than one hundred chemicals have been

implicated in studies of carcinogenicity in humans (Higginson, 1992a), with 25 - 30
substances proven to cause cancer in human beings. Some pesticides are regarded as
causal in breast carcinogenesis (Miller, 1977).

Environmental microorganisms are also considered as important influences in
oncogenesis, and it has been suggested that viruses may act as triggers but are not
sufficient alone for oncogenesis (Dmochowski, 1969, 1972; Rapp and Reed, 1977).
Mammary cancer in animals can been produced by infectious viruses, with the first
discovery of a B-type RNA tumor virus in mice made over 50 years ago (Higginson,
1992b). Most of the naturally occurring viral induced tumors of animals are associated
with the RNA viruses, and these cause mammary cancer, leukemia, as well as sarcomas in
mouse, cat and chicken (Klein and Smith, 1977). There is no convincing evidence yet of
a viral agent linked to human breast cancer, despite many epidemiological and laboratory
Studies, and most experts favor an etiology involving nonviral carcinogens for breast
cancer (Miller, 1977).

2.1.3 Racial Factors

In the United States, breast cancer is not only the leading cause of cancer mortality
among Caucasian women, but also that of Afiican American and Hispanic groups. The
poorer prognoses and higher mortality rates among African-American and Hispanic
women developing breast cancer are believed to be attributable to a lack of awareness
about the disease and to poor-quality health care (Bair and Cayleff, 1993). The
demographic features of Black and Caucasian women with breast carcinoma have been

compared in several studies (Kovi, 1989; Natarajan, 1985; Polednak, 1986; Vernon,

1985). Incidence rates for female breast cancer (per 100,000) in the different racial
groups in Hawaii were as follows: Caucasian, 80.3; Hawaiian, 66.2; Chinese, 54.2;
Japanese, 44.2; and Filipino, 21.5 (Waterhous,1976; Mant and Vessey, 1991). In contrast
to the outcome, the incidence of breast cancer is lower in black women compared to
Caucasian women: for the period 1978- 1981, the age-adjusted incidence rate was
71 .9/100,000 for Black women and 85.6/100,000 for Caucasian women (Tavassoli, 1992).

Aside from economic social reasons, there are reports of more fundamental racial
difl'erences in that breast cancer in Afiican-American women has a poor progression
(Jacob, 1993).
2.1.4 Age

It is well known that the incidence of breast cancer increases progressively with
age throughout the lifetime. The annual risk of breast cancer at age 75 is more than three
times higher than the risk at age 35. It is rare for breast cancer to occur before age 15,
and only 2% of breast cancers are found in women aged 30 or younger; 96% of breast
cancer patients die at age 35 or older. Most breast cancer patients are aged 40 to 60,
usually in the menopausal period. More than 70 percent of women over the age of 50
who present with breast cancer do not have any other identifiable risk factors. Thus
women older than 50 years of age are a high risk group in Western countries (Hayes and
Schnitt, 1993; Vogel and Love, 1991).
2.1.5 Gender Distribution
Breast cancer is rare in men in any country, usually averaging 100:1 female : male

in population where the female to male ratio ranges from 70 to 130. In males, the

10

incidence rates are below 1.0/ 100,000 for most countries, and they, when compared with
females, are usually not epiderniologically significant (Jigginson, 1992).
2.1.6 Trends

Death rates fi'om breast cancer in females have generally remained stable, but the
morbidities in China, in Japan, and in the USA, are all increasing. In the world as a
whole, it was estimated that more than 500,000 new cases were diagnosed in 1975, but
that by the year 2,000 the global total may well exceed 1,000,000 year. Clearly this form
of cancer is becoming an increasingly important disease in all parts of the world (Mant and
Vessey, 1991).
2.2.0 Risk Factors of Breast Cancer

The factors identified as increasing a woman's risk of breast cancer are recorded in
several ways. There is the "absolute" risk and the "cumulative" risk. The absolute risk is
based on the population, and is expressed as cases per 100,000. More commonly used is
the cumulative, or “lifetime” risk, which assumes that all patients have the same risk and
that the entire population will live to be a certain age. For breast cancer, this means that
one in 10 women will develop cancer if the population lives to be 80 years of age. If one
assumes that the population will live longer, for example, to age 85 or 100, the risks go up
to 1 in 9 or 1 in 8, respectively, because the entire population is living longer and has more
time to develop cancer.

Factors other than increasing age which Significantly increase the risk of breast
cancer include prior breast cancer, a strong family history, and the presence of atypical

hyperplasia in a biopsy.

11

2.2.1 Genetic Factors (Family History)

The tendency for breast carcinoma to develop in certain families has been known
for many years and it is now recognized that a positive family history of breast carcinoma
constitutes a major risk factor. The risk is especially significant when the diagnosis of
breast cancer is made in a first degree relative (i.e. mother, sister, daughter) (Anderson,
1977; Anderson and Badzioch, 1986).

Li-Fraumeni Syndrome, a specific gene defect, is associated with a higher
frequency of breast carcinoma. Patients with this autosomal dominant syndrome are
characterized by a defect in the tumor suppressor gene, p53; multiple cancers (breast,
brain, sarcomas, and adrenal cortical tumors) are observed in the close relatives (Birch,
1990; Li, 1988; Malkin, 1990).

Recently a breast cancer susceptibility gene, BRCAl, was identified on
chromosome 17 and appears to be involved in approximately 5% of all breast cancers.
BRCAl has been found to be associated with most inherited breast and ovarian cancer
combinations. However, only 45% of the familial breast cancer appears to be linked to
this gene. BRCAI is located in same area as other genes that are known to be involved in
breast cancer such as c-erbB-2 (HER/neu)(Howell, 1995). c-erbB-Z (HER/neu) gene
correlates with a shorter time to relapse and to disease-free survival when this gene's
product is overexpressed (King, 1990, 1992; Sattin, 1985). Recently a second breast
cancer susceptibility gene BRCA2 has been reported (Wooster, 1995). Based on the
inheritance of chromosomal markers in families with hereditary cancers that are not linked

to BRCAI, BRCA2 genes have been are mapped to chromosome 13q12-13. This is a

12

region known to exhibit loss of heterozygosity in 20-40% of sporadic breast cancer. The
new gene should provide the basis for tests to screen for cancer causing mutations in
women fi'om families with a high incidence of mammary cancers, even though no one
knows yet what normal function these susceptibility genes have or how their mutation or
loss many lead to cancer (Marx, 1996; Collins, 1995).
2.2.2 Hormonal Influence

A woman's own hormonal environment is likely to be a very important factor,
perhaps more important than others such as diet, in considering her risk for developing
breast cancer. Three major hormone-related events in the life of a women influence the
risk: these are - age at onset of menstruation (menarche); secondly the age at the
menopause; and lastly the age at which she has her first fiill-tenn pregnancy. Breast
cancer risk is increased by an early menarche, a late menopause, or a late first firll-term
pregnancy (Iqbal and Taylor, 1989). Reproductive steroid hormones act mainly as
promoters rather than as initiators of carcinogenesis, and therefore are most likely exert an
influence on carcinogenesis by altering the kinetics of proliferation, differentiation or
atrophy of the stem cell, resulting in an increase in the number of susceptible cells
(Thomas, 1984).
2.2.3 Nutritional Influence

There is suggestive evidence generally relating diet to cancer (American Cancer
Society, 1975), and nutrition, diet, and obesity have also been linked to an increased risk
of breast cancer. This link was suggested by findings of large differences in national

breast cancer incidence rates between different countries. As already mentioned, the age-

13

adjusted incidence rates of breast cancer are five times higher in the United States and
other Western industrialized nations than in Asia; this many have a dietary component.
Changes in dietary habits may also be partly responsible for the changes observed in the
incidence and mortality of breast carcinoma in migrant populations (Lodon, 1992;
Tammenbaum, 1942; Tavassoli, 1992).

A significant correlation between breast cancer mortality and consumption of fat
and animal protein has been reported (Vorherr, 1980). A number of population studies
have correlated the incidence and mortality of this disease with total dietary fat (Ening,
1978). For example, in the USA most individuals derive 30 and 45 % of their calories
from fat, compared with 15 per cent in China (Whittemore, 1990). Serum cholesterol
levels have also been linked to breast cancer (Hiatt, 1982). Nowadays not only are Single
elements in food receiving attention, but also the patterns of the food consumption are
being considered. In addition, investigators have found that a high total caloric intake in
human beings (e. g., obesity) is associated with a modestly increased risk of developing
many diseases, particularly breast cancer; alcohol and coffee intake are also linked with
breast cancer risk (Willett, 1987; Harvey, 1987; Willet, 1985).

A 13 year study funded by the National Institutes of Health and the Women's
Health Initiative began in 1993 to focus on whether or not a low fat diet can reduce the
incidence of breast cancer; this study should present some answers to the many
outstanding questions (O'Grady and Rippon, 1994).

2.2.4. Other Factors

A previous history of benign breast disease, oral hormone exposure, low

14

socioeconomic status, abnormal marnmographic pattern, and radiation exposure, are all of
concern as factors related to the development of breast cancer. Considering all the data
available is not implausible to suggest that the major determinant of breast cancer risk is
the total cumulative exposure of breast tissue to bioavailable estrogen, and that this
exposure is influenced by nutritional factors and by reproductive history. Energy-rich
diets during adolescence may enhance the occurrence of precancerous lesions, with
pregnancy having a strong protective effect. Table 1-1 shows the factors influencing risk

of developing breast cancer.

 

 

 

 

 

 

 

 

 

 

Table I-1 . Factors influencin I risk of developing breast cancer
Factors High Risk Group Low Risk Group

SEX AND REPRODUCTION

Sex Female Male

Menarche Early age Late age

Menopause Late age Early age

Ovarian Function Artificial menopause, Normal, praous

nulliparous
m (first) Later age Early gge

HORMONAL EXPROSURE

Oral Contraceptives Use Non-use

Postmenopausal Estrogen Use Non-use

HORMONE PROFILES

Circulating Oeatradiol High Low

RACE AND ENVIRONMENT

Race Weetemized Developing

Socioeconomic Status High Low

Body Statute Obese Lean

Diet High fat calories Low fat calories

Radiation Exposure Childhood None

FAMILY HISTORY

First-degree Relative Breast cancer No breast cancer

Premenopausal Bilateral History No history

Breast Cancer

MEDICAL HISTORY

Age Old Young

Previous Benign Disease, History No history

Particularly Biopsy

Man'mog'aphic Pattern Prominent duct dense No prominent ducts not
dense

 

(Forrest, 1990)

3.0.0 Clinical Diagnostic Approaches of Breast Cancer

A variety of features have been evaluated as prognostic indicators for breast

 

15

carcinoma. The use of some of these potentially can markedly influence the management
of breast carcinoma.
3.1.0 Prognostic Indicators

The prognostic indicators for breast cancer are varied in their features. Some can
be used to divide breast carcinoma into types with good or a poor prognosis.

Table 1-2. The influence of various indicators on the prognosis of breast carcinoma.

 

 

 

 

 

 

 

 

 

 

Good Prognosis Poor Prognosis
Size < 1 cm > 5 cm
Local Extension Absent Present
Grade Low High
Receptor status ER+, PR+ ER-, PR-
Axillary nodes Tumor - Tumor +

 

(Tavassoli, 1992). ER - eostrogen receptor; PR - progesterone receptor.

3.1.1 Tumor Staging

The staging of breast cancers is essential in the evaluation of the tumors, the
determination of prognosis and in guiding the type of management used. This process is
divided into pathologic staging and clinical staging. The two staging systems do not
always correlate; for example, inflammatory changes in the Skin of the breast do not
necessarily reflect dermal lymphatic invasion in inflammatory carcinomas 030mm, 1995;
Tavassoli, 1992).

The most widely used system is based on the TNM guidelines, where TNM stands
for _T_umor, Nodes and Metastases, which was proposed in 1954 by the International
Union Against Cancer. The current classification was agreed upon in 1987 by both the

International Union Against Cancer and the American Joint Committee on Cancer. The

16

TNM classification is presented in table and is derived fiom Sobin (1988) and Harmanek
(1987)

Other classifications are usually only used within individual institutions. For
example, the cTNM system, which is an evaluation of clinical presentation made before
treatment, usually includes physical examination, imaging, and laboratory testing. The
pTNM category is referenced as a pathological evaluation based on the macroscopic and
microscopic examination of specimens, and it adds precision to clinical evaluation
(Tavassoli, 1992; Hammond, 1995).

3.1.2 Tumor Size

The gross size (maximum diameter) of the invasive primary mass is one of the
most important prognostic factors (Carter, 1989; Page, 1991). Tumor masses 2 cm or
smaller in maximum diameter have a significantly better prognosis and survival compared
to those that are larger (Palmer, 1982). Patients with tumors 1.0 cm or smaller had a 20
year survival of 86%; survival dropped to 69 % for those with tumors over 1.0 cm in size
(Rosen, 1989a). The size of tumor also been a direct relationship to the probability of
axillary node metastases: tumors 1.0 cm or less in diameter produced 26% axillary node
metastases, whereas tumor size over 1.0 cm show 78% (Haagensen, 1986).

3.1.3 Tumor Type

Tumor type, independent fi'om the stage, is an important indicator. Certain types
of breast cancer, such as medullary, tubular, mucinous (colloid) and adenoid cystic
carcinoma, are “low grade”, and are associated with a low frequency or the absence of

axillary node metastases and have a relatively good prognosis. In contrast, inflammatory

17

breast carcinoma, which a merely implies widespread dissemination and tends to develop
with pregnancy (Bonnier, 1995), has a high grade, with a low five-year survival (Donegan,
197 7). Moreover, recurrences are comparatively less fiequent in other types such as
tubular, medullary, invasive papillary, and colloidal cancer (Rosen, 1989b; Ciatto, 1990).
3.1.4 Morphologic Parameters

The morphologic parameters of breast cancer have been well defined. The
morphological detection of stromal invasion is one of the most significant prognostic
indicators; in situ or noninvasive carcinomas are almost invariably cured by mastectomy
(Boring, 1992) or less radical removal of the tumor tissues. Also, intraductal proliferative
lesions have been studied for an associated relative risk for subsequent development of
cancer: only the development of invasive carcinoma has been usefiil in analyses (Page,
1985; Schintt, 1984, 1987).
3.1.5 Lymph Node Metastases

The status of the regional lymph nodes with regard to invading breast cancer cells
is one of the most important prognostic indicators in breast cancer; determination that a
tumor has become invasive largely establishes the survival rate (Cotran, 1994). The
larger the number of cancer positive lymph nodes identified, the more serious a particular
neoplasm is (Fisher, 1970), the higher the failure rate in subsequent treatment (Fisher,
1975), and the lower the survival rate (Neville, 1991).
3.2.0 Screening

Increased emphasis has been placed on breast cancer screening in the last several

years, with the mortality being significantly reduced by mammography and physical

18

examination.

Table I-3. Randomized clinical trials of breast cancer screening (5 to 13 year follow 11 )

 

 

 

. Breast Cancer Mortality T f Scree -
Trial Reduction in Those Screened ype o nrng
Health Insurance Plan ( New 29% Annual two-view
York) mammograrn+BPE
Swedish Two County 31% Single view mammogram

every 2-3 years

 

Malmo 0 Initial two-view
mammogram, then single-
or two-view mammogram
every 18-24 months

 

 

 

 

 

Canadian NBSS 0 Annual two-view

mammogram + BPE
Combined Swedish 24% Mammogram at variable
Trials intervals

 

 

BPE = breast physical examination; NBSS = National Breast Screening Study.
(Lindfors, 1995)
3.2.1 Physical Examination

Physical examination for palpable tumors is one of the most widely used methods
of breast cancer screening. It is also simple and cheap as no equipment is needed and in
fact, most palpable tumors are found by physical examination. Palpable breast lesions of
various types can be found during physical examination of women with different kinds of
breast symptoms. When a woman has detected a lump in her breast , an experienced
physician must examine and evaluate the lesion. This examination includes assessment of
the size, mobility, skin changes associated with the tumor, together with an assessment of
the size and mobility of regional lymph nodes, and any evidence of distant metastases

(Berndt, 1969; Danforth, 1988; Mackenzie, 1994; Potchen, 1993).

19

3.2.2 Breast Imaging

Various breast imaging procedures are widely used to detect cancer.

X-ray mammography is the only proven method capable of detecting nonpalpable breast
cancer. It is successful in detecting carcinomas in situ or those that are minimally
invasive, as well as those less than 5 mm in diameter; associated with a survival rate is
around a 93 percent over 20 year (Gold, 1987). Thus, X-ray mammography has the
greatest emphasis in the disclosure of early, nonpalpable, and often curable cancer. Since
this is the technique that has proven most successfirl on detecting early breast cancer, it is
the standard used to screen for breast cancer and the one to which all other imaging
alternatives are compared (Lindfors, 1995; Potchen, 1993, 1991).

Ultrasound is increasingly used as a possible alternative to X-rays, but has not yet
proven to be superior. Diagnostic ultrasonography nevertheless stresses differentiation of
benign cysts fiom diagnostically indetemiinate solid masses, with the latter requiring
biopsy (Lindfors, 1995). Ultrasound is useful when used with fine needle aspiration
biopsy (FNA) technique.

Recent interest has been focused on Magnetic Resonance Imaging (MRI) as a
possible alternative to mammography. Preliminary data indicates that MRI (with
contrast) can reveal lesions in dense breasts that are often missed by conventional
mammograms. The cost of MRI and the time required to perform it will likely prohibit its
use for general screening, but it may prove to be a useful ancillary technique to
mammography (Lindfors, 1995).

Trgtsilluminatign light-scanning is based on the concept that breast cancer

20

increases blood supply and absorbs more near-infrared and red electromagnetic radiation.
This procedure is rapid, noninvasive, risk-free, and relatively inexpensive, and is eliciting
increasing interest. However, it is also less sensitive than x-ray mammography in the
detection of nonpalpable cancer (Geslien, 1985; Sickles, 1984).

Thermography is an older procedure that seems to be only usefirl for advanced
cancer. The cancer detection rate using thennography is only 42 percent, compared with
57 % for physical examination, and 91% for mammography; thennography gives a high
number of false positive examination, and thus is clinically unacceptable (Baker, 1982;
Gold, 1986).

3.2.3 Breast Biopsy

The majority of palpable breast lumps are not malignant, but it is often difficult to
definitively make that determination by palpation or by mammography or both.
Therefore, tissue sampling is an important adjunct for diagnosis.

Surgical excision is the classical standard because it gives a direct examination of
the entire lesion through histological examination. The non-palpable lesion can be
successfiilly examined also with marnmographic localization using indicator needles.
However, excisional biopsy carries the typical surgical risks of potential wound infection ,
hemorrhage and development of tissue scars postoperatively. Moreover, the cost, the
morbidity, and the frequency of multifocal lesions requiring multiple surgical procedures,
make it not always practical, nor desirable, to do such surgical biopsies.

Fine needle aspiration biopsy (FNA) is a very usefiil technique. It can be

performed not only on palpable lesions but also on non-palpable lesions using

lalsl

21

mammographic or ultrasound guidance. FN A is a simple, safe procedure with only little
discomfort. By using X-ray guided (Hann, 1989: Lofgren, 1988; Masood, 1990) or
sterotactic (Azavedo 1989; Dowlatshahi, 1987; Lofgren, 1990) procedures to ensure
placement of the aspiration needle in the lesion. The sensitivity and specificity are
reported to be from 70 to 100% and the Specificity is 100%. Multiple lesions can be
easily aspirated during the same consultation (Rosen, 1996b). The ease and accuracy of
this biopsy method allows suspicious lesions to be sampled immediately, thus reducing the
patient’s anxiety usually associated with long drawn out clinical follow-ups. However,
FNA cannot distinguish in situ from invasive carcinomas, and not all pathologists are
equally proficient or comfortable at interpreting cytopathologic breast samples. False-
negative and false-positive results may be generated by the less experienced
cytopathologists.

Cutting needle biopsy or large core biopsy techniques have been regularly
performed with 12 to 16 gauge cutting needles; both methods obtain a solid core of tissue
for histologic diagnosis. Pathologists are generally more proficient at examining this type
of specimen as it is a true tissue sample. The procedure however, unlike FN A, is very
painfirl, produces much local hemorrhage, and may lead to tumor cell seeding along with
the needle track.

Rotex needle biopsy (MB) is performed by a needle with the inner screw cutting
component covered by a 22 gauge cannula. The procedure maintains the advantage of
FNA and has proved more adept at obtaining specimens for pathologic diagnoses. The

false-positive and false-negative results have been reduced remarkably (Qiao, 1992).

22

Needle biopsy techniques can therefore be used in breast cancer diagnosis to
distinguish between cysts and solid tumors, and to provide material not only for cytologic
pathology evaluation, but also for immunohistochemical analysis, flow cytometric studies,

morphometric analysis and molecular research (Potchen, 1993, 1991; Schnitt, 1993).

4.0.0 Breast Cancer Diagnosis of Pathology

Many delays in the diagnosis of breast cancer occur because breast masses are
followed clinically without any microscopic evaluation. The best clinical laboratory
support for a patient with a breast mass is based on tissue diagnostic techniques.
4.1.0 Histopathology

Histopathology is the well established method for breast cancer diagnosis. Even
though there are many new theories and new methods in the today's world for
understanding, detecting, and diagnosing breast cancer, histopathology remains the
essential component in practical diagnosis.
4.1.1 Histochemical Pathology

Classical histopathology is a direct microscopic observation of a tissue section
treated with tinctorial stains that assist in the visualization of the cellular and tissue
structure. This permits observation of defining morphological characteristics of the breast
lesion morphology. Histochemical stains can also be used to visualize the nucleic acids,
carbohydrates, proteins, lipids and enzymes in the constituent cells. The information
obtained from the histopathology forms the basics of the information to guide the breast

cancer patient’s prognosis and treatment.

23

As the Cancer Committee of the College of American Pathologists practice
protocols suggests, a pathology report concerning breast carcinoma should include the
anatomic extent of tissue removed, the anatomic extent of the carcinoma in the specimen,
the histologic type and any other information that may be usefirl to the referring physician
in the selection of treatment, such as the type of adjuvant, the evaluation of new types of
therapy, prognosis, and analyses of outcome (Hammond, 1995).

4.1.2 Anatomical Features of Histopathology

Breast carcinomas are almost all adenocarcinomas and are usually derived from the
glandular epithelium of the terminal duct lobular unit. Various subtypes derive their
names fi'om a combination of their histologic patterns and their cytologic characteristics,
rather than their site of origin (Bartow, 1988).

4.1.3 Carcinoma Differentiation (Grading)

Histological grading of breast cancer is potentially of great clinical value in
determining prognosis. Several different grading systems are used; however, almost all
grading criteria in the different systems are based on an assessment of cytologic factors
that includes tubule formation, nuclear size, nuclear pleomorphism and mitotic count
(Robbins, 1995; Elston, 1987). The overall grade of a tumor is classified by a three-point
scale based on from cytologic features, with the final grade depending on the specific
features. Grade I is a well differentiated tumor; Grade II is moderately differentiated; and
Grade 111 is poorly differentiated. Importantly, studies of the relationship between grade
and prognosis show a strong correlation, even when data are collected from many

pathologists, use different grading systems, and are without standardized guidelines

24

(Robbins, 1995; Henson, 1991).

Recently, thymidine labeling index (TLI), and Flow Cytometry have also been used
to define tumor grades.
4.1.4 Carcinoma Classifications

The histological classification of breast tumors is descriptive, and probably the

most widely used classification is that proposed by the World Health Organization

(WHO).

Table I-4. World Health Organization (WHO) histolqgic classification of breast tumors
1. Epithelial Tumors
A. Benign
1. Intraductal papilloma
2. Adenoma of the nipple
3. Adenoma
a. Tubular
b. Lactating

 

B. Malignant
1. Noninvasive
a. Intraductal carcinoma
b. Lobular carcinoma in situ
2. Invasive (infiltrating)
a. Invasive ductal carcinoma
b. Invasive ductal carcinoma with Paget's disease
c. Invasive lobular carcinoma
d. Mucinous carcinoma (colloid carcinoma)
e. Medullary carcinoma
1‘. Secretory (juvenile) carcinoma
3. Tubular carcinoma
h. Adenoid cystic carcinoma
i. Apocrine carcinoma
j. Invasive papillary carcinoma
1:. Carcinoma with metaplasia
i. Squamous type
ii. Spindle cell type
iii. Cartilaginous and osseous type
iv. Mixed type
1. Others
3. Paget's disease of the nipple
11. Mixed Connective Tissue And Epithelial Tumors
Fibroadenoma
Phyllode Tumor (cystosarcoma phyllodes)
Carcinosarcoma
Miscellaneous Tumors
Soft Tissue Tumors
Skin Tumors

assoc?

25

C. Tumors of Hematopoietic and Lymphoid Tissues
IV. Unclassified Tumors
V. Mammary Dysplasia/Fibrocystic Disease
VI. Tumor-like Lesions
A. Duct Ectasia
B. Inflammatory Pseudotumors
C. Hamartoma
D. Gynecomastia
E. Others
(Azzopardi, 1982; WHO, 1982).

Variations on this basic system appear in other texts, such as the following which
is from Bartow and Fenoglio-Preiser (1988):

T_able 1-5. World Health Organization classification of carcinomas
Non-invasive

Intraductal

Lobular carcinoma in situ

Intraductal papillary carcinoma
Invasive

Invasive ductal carcinoma

Paget's disease

Invasive lobular carcinoma

Medullary carcinoma

Mucinous carcinoma

Tubular (well-differentiated) carcinoma

Invasive papillary carcinoma

Adenoid cystic carcinoma

Secretory carcinoma

Apocrine carcinoma

Carcinoma with metaplasia

Mixed time

The classification of breast cancer used mainly in China today is that proposed in
1987 at the Third National Breast Cancer Meeting in China. This system is shown in the
following Table 1-6.

Table 1-6. Breast carcinoma classification in China

1. Non-infiltrative carcinoma
Ductal carcinoma in situ
Lobular carcinoma in situ

2. Early infiltrative carcinoma
Ductal carcinoma with early invasion
Lobular carcinoma with early invasion

3. Infiltrative specific carcinoma
Papillary carcinoma
Medullary carcinoma with massive lymphocytic infiltration

26

Tubular carcinoma (well-difi‘erentiated—adenoearcinoma)
Adenoid cystic carcinoma
Mucinous adenocarcinoma
Apocrine carcinoma
Squamous cell carcinoma
Paget’s disease
4. Infiltrative not specific carcinoma
Infiltrating lobular carcinoma
Infiltrating ductal carcinoma
Scirrhous carcinoma
Medullary carcinoma
Simple Carcinoma
Adenocarcinoma
5. Other rare carcinomas
Signet-ring cell carcinoma
Smell cell undifferentiated carcinoma
Secretory carcinoma
Carcinosarcoma
Pseudosarcoma
Anaplastic carcinoma
Papillary carcinoma
(Goung, 1993; He, 1990)

 

 

Other classifications exist in other countries but are not widely used.
4.2.0 Cytopathology

There are three different types of materials for cytologic examination of the breast:
nipple secretions, aspirations from cysts, and various types of needle samples from solid
tumors. The more needle biopsies performed, the more important cytopathology
diagnosis on breast specimens becomes to disease management. Accuracy of the
cytologic diagnosis plays a major role in the breast cancer patient's life today. Clear
differential diagnosis between benign and malignant lesions is possible for most breast
cytologic specimens when there is careful application of the cytologic criteria for
malignancy (Carter, 1990).
4.2.1 Cytochemistry on the Fine Needle Biopsy Sample

Histochemistry on cytology samples is essentially similar to that used with tissue

 

 

27

samples, where different chemicals stain specific cellular structures. For breast tumors,
the commonly used histochemical stains are Papanicolaou, modified Wright-Giemsa, H &
E, "Diff-Quik”, and the modified May-Grunwald-Giemsa stains.

The ”Diff-Quik" stain offers rapid results with good cellular detail on air-dried
smears and usually provides a good presentation of the cytoplasm. The rapid
Papanicolaou stain provides a fast staining method usefiil for immediate FNA diagnosis.
Mth the Papanicolaou stain, cell nuclear and nucleolar details provide a very clean image
for pathologists to used in diagnosis (Bedrossian, 19913; F eldman, 1985).

4.2.2 Morphological Characteristics of Cytologic Samples

Features of the breast cancer cytology present in certain patterns; for example, the
solid cell ball, psammoma bodies, small semi-cohesive cell clusters, and "indian files" are
all seen.

The solid cell ball configuration is a typical presentation of breast carcinoma. The
cell balls immediately stand out, even under low-power examination, not only because of
their size, but also because of their distinct configuration. Psammoma bodies are curious
structures that represent degeneration and calcification of cells commonly arranged in
papillary clusters in the tumor. The calcific concretions are usually larger and more
irregular than calcospherites, and commonly fragment along straight fracture lines.
However, a diagnosis of malignancy should not be made unless well-preserved neoplastic
cells are clearly identified. The small partially cohesive tumor cell clusters often do not
have overt differentiating features in their cytoplasm. There is usually only a small

amount of nonvacuolated cytoplasm exhibited by these cells and they lack a tight-ball,

28

acinar, or papillary configuration. The cell clusters may break up into smaller groups that
have no consistent pattern of size and shape. Tandem formations of tumor cells are
known as ”Indian files", a phenomenon that occurs commonly in invasive breast cancer.
The arrangement into rows is understandable, with the tumor cells appearing to fall into
line because of the pressures from the stroma surrounding of the neoplasm. Electron
microscopy can identify the intercellular connections between the various members of the
rows of cells in this linear formation (Bedrossian, 1991a,b; Spriggs, 1984; Salhadin, 1976;
Ashton, 1975).

The features of malignant cells in cytology include cellular and nuclear variation in
size and shape, hyperchromatism, irregular chromatin clumping, abnormal chromatin
distribution, nuclear molding, prominent or macro nucleoli, abnormal mitotic figures,
increased nuclearzcytoplasmic (N :C) ratio, and overlapping and crowding of nuclei. Such
features must be carefully observed for diagnosis (Feldman, 1985). The malignant
features in breast cytology are summarized in table I-7.

Table I-7. Common cellular features of breast cancer
High cellularity

Lack of cohesion of cellular groups

Presence of isolated intact single cells

Cellular monomorphism

Variation in nuclear size from cell to cell

(anisonucleosis)

Nuclear membrane infolding

Chromatin Clamping and radial dispersion of chromatin
Prominent nucleoli or macronucleoli

(Wilkinson, 1991).
4.2.3 Grading
In recent years, cytological grading of breast carcinoma specimens has been well

described. Early on, a modified histological scoring grade was used in cytological

29

grading, but it failed to become an independent prognostic indicator after a median follow
up study of 8 years (de Graaf, 1994). The grading which may be satisfactorily substituted
for the histological grade, therefore, is a combination of fine needle aspiration sarnpleing
(FNA) and mammography; it can provide information on tumor type, grade, and size
before surgery (Robinson, 1994). Furthermore, the proliferating cell nuclear antigen
(PCNA), a proliferation marker, detected by the PC 10 monoclonal antibody (MAb), can
also be used as an indicator for cytological grading in determining prognosis (Betta,
1993)
4.2.4 Other Techniques

Recently, newer techniques have been reported with fine needle aspiration samples
of breast cancer. Transmission electron microscopy as well as scanning electron
microscopy, has been used. Immunocytochemical staining for keratin, estrogen receptors
(ER), progesterone receptor(PR), carcinoembryonic antigen (CEA), c-erbB2, human nrilk-
fat globules (HMF G) have been successfully applied in stereotictic fine needle aspiration
sampling (SFNA) of breast cancer (Redard, 1989; Corkill, 1994; Bedrossian, 1991b).
Immunofluorescence and immunoperoxidase techniques are also often used to detect
steroid hormone receptors (Wilkinson, 1991). In addition, flow cytometric DNA
analysis, and determination of thymidine labeling indices and morphometric analysis are
also now common (Barbieri, 1994; Andoh, 1991). Most recently, oncogene
investigations on FNA samples have been carried out, using c-erbBZ oncogene

amplification (Barbieri, 1994).

30

4.3.0 Immunohistochemical Pathology

Immunocytochemical pathology is the application of immunological principles and
techniques to the study of the microscopic structure of diseased tissue and cells (Smith,
1993). The principle of immunohistochemical reactions is the specific recognition by an
antibody of an antigen that is present, and is chemically fixed, in tissue sections or a cell
suspension, followed by the visualization of the antibody (DeLellis, 1979).

Classical histopathology, using chemical stains for the visualization of the cellular
and tissue structure usually cannot differentiate among specific antigenic components of
breast cancer. Immunocytochemical histopathology on the other hard led to a great
expansion in the range of substances identified as being relevant to diseases, and provides
a higher level of specificity. For example, with immunocytochernistry it is often possible
to delineate the internal architecture of the cell "ball" described above found in cytology
samples.

4.3.1 Features of Immunohistochemistry of the Mammary Tissue

Immunocytochemical features of the normal breast unit have been described. The
epithelial cells display a strongly positive reaction for various cytokeratins. Positivity for
alpha-lactalbunrin is evident in the epithelial cells during secretory activity, while this trait
is much less strong and uniform in resting epithelial cells. Myoepithelial cells show a
strongly positive reaction for aim and a variable degree of positivity for S—100 protein
and cytokeratins. A positive reaction for am indicates a myoepithelial cell type in the
mammary duct system. The basal lamina shows positive reactions for law and gm

IV collagen (Berg, 1984; Owings, 1990).

 

31

4.3.2 The lmmunomarkers Used on Breast Cancer Tissues
Monoclonal antibodies have been generated against (1) human milk-fat globule

membranes, (2) human mammary tumor cell lines, and (3) extracts of human breast
tumors. Recently, immunocytochemical serological and in situ stains for hormone

receptors and tumor markers have been the most frequently used probes in either routine

 

diagnostic or laboratory investigations of breast carcinoma (Kufe, 1988).
Estrogen (ER) and progesterone receptor (PR) immunochemical staining are used

as indicators for classification, prognosis, and to provide information for the treatment of

breast cancer. A breast cancer with a positive stain for ER and PR responds better for
endocrine therapy, prognosis and survival rate than does a negative tumor (Roulston,
1993a). There are very many immunocytochemical markers that have been used on

breast cancer for histological and cytological evaluations. Most of them are lactation-

related antigens. They are listed in the table 1-8.

Table 1-8. Some immunohistochemical markers used to define breast cancer
Carcinoembryonic antigen (CEA)

Ferritin
Human chorionic gonadotropin (IICG)
Polyamines (putrescine, Sperrnidine, spermine)

Hydroxyproline
Monoclonal antibodies

Tissue polypeptide antigens
Calcitonin

Casein

Alpha-Lactalbumin

Sialyl transferase, galactosyl transferase

Ceruloplasmin
Alkaline phosphatase
r-Glutamyl transpeptidase
Erythrocyte sedimentation rate
Pregnancy-associated alpha-glycoprotein (PAM)
Oncogene product
Tumor associated gene brodpct

(Veach 1987; Klavins 1985)

 

 

32

4.3.3 The Immunomarkers of Specific Interest

The specific immune tumor markers of interest to this present study are p53 tumor
suppressor protein, c—erbBZ oncoprotein, and Bic-2 oncoprotein, and they have been
selected for our immunocytochemical investigation. Comparison of these markers by
immunohistochemical stain has not yet been reported in breast cancer, including for the
disease in Asian countries. Neither have p53 gene point mutations and c-erbBZ gene
amplification of breast cancer patients ever been sought in any study on Chinese patients.
4.4.0 Molecular Pathology

Molecular pathology is the study at a molecular level of the causative agents and
mechanisms underlying disease by molecular methods (McGee, 1992). It focuses on the
DNA, RNA, and expressed proteins present in the tissues and cells, and has been used to
analyze the causative agents and mechanisms in intact cells by relating phenotypes to
genotypes. Most techniques used for this approach employ immunocytochernistry on
both light and electron microscopic preparations. Some techniques use RNA or DNA
probes for in situ hybridization, estimation of the DNA of index, karyotyping, and in situ
hybridization to detect chromosomal aberrations by cytogenetics. Flow cytometry to
measure DNA content is now incorporated into many experimental studies and practical
diagnostic pathology. Primed in situ labeling, or PRIN S, is used for determine the
distribution of high copy number repeats within human chromosomes, and for the
localization of target DNA and RNA within cells (Mackenzie, 1994).
4.4.1 In situ Hybridization

In situ hybridization has become a valuable tool in the world of molecular studies

33

on nucleic acid (DNA and RNA). The principle behind in situ hybridization is the specific
annealing of a labeled RNA or DNA probe to complementary sequences in genes of
chromosomes, in cell preparations or in fixed tissues, followed by visualization of the
specific location of the probe (Lipshaw, 1995; Wilkinson, 1992). With the in situ
hybridization technique, the nucleic acids are held within the perfectly preserved structure
of the sample. This technique can be used to locate DNA sequences in chromosomes, or
to detect RNA or DNA in tissues (Lux, 1990; Van den Berg, 1989). Pathologists can
visualize these targets at the molecular level, and this technique has opened up a new
approach for studying many diseases and their pathogenesis.

In situ hybridization can be used with light microscopy and also at the electron
microsc0pic level (Binder, 1986). F urtherrnore, it and immunochemistry can be viewed
as complementary techniques with the interpretation of immunochemistry findings can
often be aided by in situ hybridization (Pringle, 1993, 1990; Ruprai, 1991).

4.4.2 In situ Hybridization in Malignant Diseases

The in situ hybridization technique has been used to study malignant tumors,
permitting detection of uncontrolled cellular proliferation, terminal differentiation and
excessive gene transcription. Oncogenesis is a complex process which is reflected in the
fact that most malignancies probably arise from a combination of abnormalities in both
proto-oncogenes and tumor-suppressor genes via a multi-step process. Extraction
methods for DNA/RNA analysis and in situ analysis of malignancy should be viewed as
being complementary to immunohistochemistry (Bishop, 1991; Fleming, 1994).

In situ hybridization has been used to investigate human proto-oncogene

34

expression in both non-malignant tissue and malignant tumor tissues; the products of
oncogenes can be detected and localized in tissues. Examples include: c-sr's (PDGF-2)
mRNA which is overexpressed in the lung epithelial cells (Antoniades, 1990); oncogene
WT] detected in Wilms' tumor (Pritchard-J ones, 1991); N-myc in neuroblastoma, c-myc in
non-Hodgkin's lymphoma, and ms family genes (Ha-ms, Ki-ras, N-ras) in colon, lung,
pancreas and thyroid tumors (Basset-Seguin, 1991; Bishop,1991; Fleming, 1994).

C-erbBZ mRN A has been detected by 3’ S-labelled probe in a poorly differentiated
infiltrating ductal breast carcinoma. c-myc gene and its product have been compared with
an immunolabelling and in situ hybridization in breast cancer (Walker, 1989) and found to
have gene ampilification mutation, limited to invasive duct carcinoma type, and positivity
correlated with shorter disease -free survial time; but not related to age at diagnosis,
tumor grade or estrogen receptor status of the tumor (Rosen, 1995b). Similar data were
also obtained for mRNA of fins, a growth factor and its receptor related gene (Le Roy,
1991; Fleming, 1994).
4.4.3 in situ Hybridization on Cytology Samples

Theoretically, any cytological specimen can be analyzed by in situ hybridization.
In practice, the technique has not been widely applied because of difficulties in the
methodology; most experience has been obtained from its use in viral detection. As yet in
situ hybridization on cytology samples from breast cancer cases has not yet been reported,
although, there are techniques for cultured cells which could be adapted for use on such

cytologic specimens (Gan, 1994; Lewis, 1992; Yap, 1992).

35

4.4.4 Flow Cytometry on Breast Cancer Cells

Flow cytometry, especially DNA flow cytometry, is often performed for routine
clinical application and provides additional information for breast cancer patient
management.

The basic principle of flow cytometry is the use of particular wavelengths to excite
fluorescent dyes attached to phenotype markers which are detected by a focused light
beam (generally a laser), resulting in separation of the various specifically identified
components of a single cell suspension. A wide variety of cellular constituents can be
measured by flow cytometry, such as DNA, RNA total protein content, and the presence
of specific proteins (by using monoclonal antibodies). Furthermore, intracellular pH,
calcium transport, and enzyme kinetics can be measured by the flow cytometry technique
(Melamed, 1990). Many diagnostic and research flow cytometric studies have been
performed on breast cancer. Using DNA histograms, flow cytometry can show clearly
defined G, S and Gz/M phases of the cell cycle: these are now routinely used on breast
cancer samples. DNA aneuploid cells with a high S-phase fi'action can be found in
malignant breast tumors: DNA aneuploidy has been shown to be more common in high-
grade tumors in virtually all published studies. Correlation of ploidy with other
clinicopathological variables such as tumor size, nodal status and steroid receptor presence
has yet be established (Merkel, 1990; Camplejohn, 1992; Camplejohn, 1993 a,b).

This technique can be performed on fresh cell biopsy material, fresh solid tumors,
and on paraffin-embedded archival material. It is therefore a popular routine method used

with breast cancer needle biopsy studies. The results of DNA flow cytometry are critical

36

to the care of the majority of breast cancer patients, and serve as a very useful adjunct for
pathological studies and basic research (Levack, 1987; Vindelov, 1990; Camplejohn,
1992)

4.4.5 Polymerase Chain Reaction (PCR) on Cancer Studies

PCR has proven to be a valuable tool applicable to a number of research and
diagnostic areas. PCR is the in vitro enzymatic replication of DNA by a repetitive cyclical
process that results in approximately a millinon fold amplification of a specific DNA
sequence (Spadoro, 1993).

PCR applications in cancer research include: 1) Detection of tumor viruses; 2)
Detection of immunoglobulin (1g) or TCR gene rearrangements; 3) Detection of
chromosomal translocation in hematological malignancies; 4) Detection of point
mutations, 5) Detection of tumor-suppressor genes, and 6) Detection of amplified
oncogenes.

A variety of sample types can be used for PCR studies and mutation analysis.
Venous blood, biopsy samples, cultured cells, fixed tissue specimens, dried blood spots
and mouthwash samples are all suitable for PCR analysis.

PCR has been used successfully in breast carcinoma to study c-erbBZ oncogene
amplification and p53 gene point mutations. It is also a well established method in the
study of the genetics of breast cancer.

4.4.6 PCR in situ Hybridization
PCR in situ hybridization combines the techniques of nucleic acid amplification

and in situ hybridization together to increase the copy number of specific sequences for

37

visialization within single cell. The amplified sequence product can be either detected by
detector markers as used with in situ hybridization or by immunohistochemistry on
histological specimens presently (Komminoth, 1995; Staecker, 1994).

In situ gene amplification theoretically can be used with any DNA analysis together
with appropriate sample fixation. By using reverse transcriptase in situ, gene
amplification has successfirlly allowed the amplification of mRNA targets (Chen, 1993).
This will be a most useful when other techniques, such as histochernistry,
immunohistochemistry or in situ hybridization, fail to answer a scientific or diagnostic
question. It allows the direct correlation of molecular analysis with cytopathology and
histopathology.

PCR in situ has been particularly useful for detecting human papilloma virus,
human immunodeficiency virus-1, and cytomegalovirus (CMV). Recently, the technique
has been extended to detect nerve growth factor, T cell cytotoxic marker expression,
CMV, lentivirus, EGF receptor, tumor necrosis factor, hepatitis C cDNA, and
immunoglobulin gene rearrangement. Many different types of specimens, such as blood
cells and formalin fixed/paraffin embedded tissue sections, can be evaluated by this
procedure (Nunovo, 1994; Komminoth, 1995). However, PCR in situ is still essentially a
research technique that is difficult to perform and has many variables. Its relatively low
sensitivity when compared with other methodologies remains a challenge. Tissues fixed
in solutions that contain picric acid or heavy metals such as mercury are generally not
acceptable for either PCR or in situ hybridization, and consequently these fixatives will not

support PCR in situ hybridization (Nunovo, 1994).

38

4.4.7 Other Molecular Techniques Used with Breast Cancer

Thm'dine labeling index (TLI), is also utilized for breast cancer diagnosis and
study. This technique determines the percent of cells within the tumor that are in S-
phase; the median TLI for breast cancer is approximately 5%; the results directly relating
to the histological grade. In general, low grade carcinoma such as mucinous and adenoid
cystic carcinomas, have a low TLI. Carcinomas with high TLIs have the characteristics
of growing rapidly, and recur relatively quickly (Tubiana, 1981; Tavassoli, 1992).

Ka_ryotypic analysis of interphase and metaphase chromosomes from human cancer
has been able to reveal interstitial deletions in several chromosomal arms. Specific
information on chromosome number and structural abnormalities can be produced with all
chromosomes being viewed simultaneously. However, this analysis cannot detect the
large majority of the small gene deletions, and is also dependent on the culturing of the
cells; it may also select for unrepresentative subpopulations (Waters, 1994; Fleming,
1994). In breast cancer, loss of heterozygosity on chromosome 17p (the location of p53
gene) has been reported (Fleming, 1994).

Primed in situ labeling (ERIN S ), and Cycling PRINS are rapid alternative methods
to fluorescence in situ hybridization for the localization of DNA and mRN A sequences in
chromosomes. They are used in the detection of unique sequences within short,
synthesized oligonucleotides without the need for genomic cloning. These techniques
have been reported from the human genome mapping project, and are useful in
chromosome and PCR product band mapping, and for research into the location of alpha

satellite DNA and RNA. Theoretically, these types of analytic techniques can be used in

39

many areas of breast cancer molecular evaluation (Miller, 1995).

5.0.0 Involvement of Genes in Oncogenesis

Cancer as a disease is characterized by an autonomous proliferation of neoplastic
cells that are genetically dysfunctional. All cellular functions are controlled by proteins,
and because these proteins are encoded by DNA organized into genes, molecular studies
have shown that cancer is a paradigm of acquired genetic disease. Carcinogenic agents
are known to mutate three types of genes that regulate cell growth: proto-oncogenes,
tumor suppressor genes and programmed cell death regulator genes (Weinberg, 1991).

The existence of mutant proto-oncogenes which code for the grth factor related
proteins has been demonstrated in human tumors (Weinberg, 1994). A change in the
sequence structure of a gene has been pinpointed as being responsible for converting a
proto-oncogene into an active oncogene. In addition, understanding that normal cells
regulate their growth provided a possible explanation for the mechanism of non-
oncogenes action in oncogenesis (Weinberg, 1994). A number of tumor-suppressor genes
have been discovered over the past five years (Weinberg, 1991; Weinberg, 1994), and
their role in tumorgenesis appears to be as important as that of oncogenes.

Roughly 10 percent of breast cancers are due to a genetic predisposition and two
breast cancer genes have been identified, BRCAland BRCA2. They showed up in about
80% of gennline breast cancer, but only in less than 5% of general breast cancer cases

(Miki, 1994; Lerman, 1995; Szabo, 1995; Neuhausen, 1996).

40

5.1.0 p53 tumor Suppressor Gene and Product
5.1.1 Background

The p53 tumor suppressor protein was first identified in 1979 as a cellular protein
that coimmunoprecipitated with the transforming protein of Siman Virus 40(SV40), the
large T antigen in SV40 transformed cells. In the early experiments, p53 was thought to
be a tumor antigen, because the large T antigen is needed to maintain the transformed
phenotype, and p53 coprecipitated with the large T antigen during purification from SV40
(Lane, 1979). Later, it was found in SV40 transformed cells and tumor derived cell lines
fi'om both human and nonhuman tumors that p53 was present in much higher levels in
tumor than the normal cells (Oren, 1981).
5.1.2 Structure and Function

The p53 gene encompasses a 6 to 20 kilo base (Kb) of DNA on the short arm of
human chromosome 17 at position 17p13.1. This gene is composed of 11 exons
(Mcbride 1986, Miller 1986). In a cross-species comparison, the p53 protein is seen to
have five highly (>90%) conserved regions within the amino acid residues 13-19, 117-142,
171-181, 234-258, and 270-286 (Soussi, 1990). These five evolutionarily conserved
domains within the coding regions are regarded as essential to the function of the p53
(Pavletich, 1993).

The product of the p53 gene is a 393-amino acid, 53-Kda nuclear phosphoprotein
(Soussi, 1990), containing an acidic amino terminal (amino acids 1-75) possessing the
transcriptional activation region followed by a central, extended hydrophobic proline rich

domain (amino acids 75-150) and a highly charged basic carboxy terminal domain (amino

41

acids 275-390) containing nuclear localization signals (Shaulsky, 1990; Addison, 1990), as
well as the oligomerization (Milner, 1991) and DNA biding domains (Raycroft, 1991).
Oligomerization refers to multiple binding sites on the protein molecules which results in
formation of protein complexes (Milner, 1991). The central region is the highly
conserved portion of the protein, and the majority of oncogenic mutations are located here
(Soussi, 1990).
Wild type p53 is virtually undetectable in normal cells as it is known to be
expressed at only low levels and has a short half-life (20-30 minutes); it is generally
located in the nucleus (Oren, 1981). In serum stimulated, nontransforrned Balb/c 3T3
cells, the cellular localization of the p53 protein was seen to vary during the cell cycle.
This protein is found in cytoplasm during the GI phase, then enters the nucleus during the
Gl/S transition where it remains until the end of the G2/M phase. After DNA synthesis,
it is again found in the cyt0plasm. In tumour or transformed cells, the location of p53
protein is still generally unclear but in certain cases p53 protein has been found in
cytoplasm (Shaulsky, 1990).
p53 tumor suppressor protein appears to have multiple fiinctions that include
regulation of DNA synthesis (Lane, 1979, Gannon, 1987, Friedman, 1990), repair
(Nelson, 1994) and programmed cell death regulation (Nelson,1994, Lane, 1993, Rand,
1 996), as well as acting as a transactivator of gene expression (Vogelstein, 1992). Loss
of normal p53 fiinction predisposes cells to multiple deleterious effects. Inactivation of
p53 protein can occur through mutation (Levine, 1991; Soussi, 1994; Chang, 1993),

protein complex formation with viral oncoproteins (Mietz, 1992; Yew, 1992), binding to

42

cellular gene products (Harris, 1993; Momand, 1992; Leach 1993), and dislocation of p53
between the cell compartments (Moll, 1992). Allelic deletions (Rand, 1996; Levine,
1991; Soussi, 1994; Chang, 1993), structural rearrangements (Levine, 1991; Soussi, 1994;
Chang, 1993), missionse mutations (Levine, 1991; Soussi, 1994; Chang, 1993) and
gerrnline mutations (Malkin, 1993; Santibanez-Koref, 1991; Kleihues, 1997) have all been
reported for the p53 gene.

5.1.3 Oncogenic Features

There are mutational “hot spots” in p53 at amino acids 175, 248-249, 273, and
282 (Soussi, 1990); mutations at these amino acids account for approximately 30% of the
total missense mutations in human tumors (Finlay, 1993). The codon 248 is also a hot
spot for germline mutations (Birch, 1994). Interestingly, different “hot Spots” are present
in different tissues: for example, mutations at amino acid 175 have been found repeatedly
in colorectal carcinomas (Baker, 1990; Rand, 1996), in lymphoma (Diller, 1990) and
breast cancer (Prosser, 1991), glioblastoma (Nigro, 1989), and an esophageal squamous
cell carcinoma (I-Iollstein, 1991a). The mutation at 175 has not yet been observed in lung
carcinomas (Finlay, 1993).

Recent laboratory data suggest that specific carcinogens can cause point mutations
in the p53 gene and have their specific features; for example, exposure to ultraviolet (UV)
light is correlated with transition mutations at dipyrimidine sites (CC to TT double base
change mutation) (Brash, 1991; Kress, 1992). Mutations resulting from G:C to T:A
transversion that occur commonly in hepatocellular carcinomas, are a result of exposure to

Aflatoxin (Aguilar, 1993; Bressac, 1991). Exposure to cigarette smoke is correlated with

43

G:C to T:A transversions in lung, esophageal, and head/neck carcinomas (Miller, 1992;
Takeshima, 1993). In addition, endogenous mutagenesis can have the same effect; for
example the high frequency of C to T transitions at CpG dinucleotides in colon carcinomas
is consistent with an endogenous mutational mechanism due to deamination of the 5-
methylcytosine residue found at CpG dinucleotides in the mammalian genome (Lehman,
1994). Thus analysis of p53 mutations may provide clues to the etiology and
pathogenesis of human cancers.

The phenotypic alterations in p53 gene mutation patterns are different in different
races. For example, Asians tend to have more frequent G:C to A:T transitions at the
CpG site (Sasa, 1993), Blacks an A:T to G:C transition (Blaszyk, 1994), and Caucasians
have G:C to A:T transversions, and rarely at CpG site (Fukushima, 1995).

5.1.4 Clinical Approaches

The p53 tumor suppressor protein was first associated with human mammary
neoplasia in 1982 and it was found that 90% of patients with breast cancer, particularly
those cases with visceral metastases, had circulating antibodies to human p53 protein
(Crawford, 1982). An hypothesis was developed that “p53 is altered in amount, and type
in breast tumors”. The accumulation of p53 protein in breast carcinoma was found to be
associated with point mutations within highly conserved regions of the p53 gene
(Hollstein, 1991). These altered genes encode stable proteins detectable by standard
immunohistochemical techniques that can detect both wild and mutant form of the p53
protein. In addition, a significant association between high levels of p53 in late stages of

the disease with metastatic tumor spread was found (Humphrey, 1994; Isola, 1992).

44

Results of these and other studies, clearly indicate that immunohistologic expression of
p53 is associated with a clinically advanced and highly aggressive forms of breast cancers
(Elledge, 1993; Mazars, 1992; Iwaya, 1991).

Li-Fraumeni syndrome is a rare autosomal dominant syndrome. The disorder is
characterized by diverse neoplasms at multiple sites, including breast carcinoma,
adrencocortical carcinoma, soft tissue sarcoma, brain tumors, osteosarcoma, leukemia,
and possibly other tumors. Tumors develop in the family members at unusually early
ages, and multiple primary tumors are fiequent. Members of these families carry one
mutant p53 allele and one wild type allele (Malkin, 1993; Santibanez-Koref, 1991). The
wild-type allele present in the normal tissues of these individuals is generally deleted in the
tumor, while the mutant allele is retained. It is therefore thought that Li-Fraumeni
syndrome patients are predisposed to cancer because one p53 allele is inactivated in the
gerrnline and only the remaining allele is altered by somatic mutation (Malkin, 1993;
Santibanez-Koref, 1991).

p53 gene mutations and the mutant protein are frequently found on human cancers
including breast cancer. It has been shown that the p53 gene is a tumor suppressor gene
and that is a fi'equent target for inactivation in many types of tumors (Finlay, 1989;
Raycroft, 1990; Ginsberg, 1991; Kern, 1992). These p53 gene mutations occur on the
short arm of chromosome 17 and the point mutations frequently occur in association with
neoplastic transformation in human tissues (Harris, 1990). It is believed that mutations of
the p53 gene play an important role in the development of many human malignancies

(Nigro, 1989; Hollstein, 1991b; Coles, 1990). Normal p53 protein has a very short half-

45

life and thus the protein level is too low to be identified immunohistochemically.
However, in transformed cells or malignantic cells were found p53 was lOO-fold higher
concentration than that in normal cells, and the half-life was increased to over 6 hours
instead 20-30 min, and thus can be detected by immunohistochemical methods (Finlay,
1988; Runnebaum, 1991).

Only limited data relating p53 expression to the clinical features of mammary
carcinoma exists in the literature. Several investigators have failed to detect a significant
correlation with age at diagnosis or menstrual status (Isola, 1992; Rosen, 1995a;
Thompson, 1992; Barbareschi, 1992; Silvestrini, 1993). Clinically, mutated p53 protein
appears in breast cancer tissues when it is of a poor differentiated carcinoma type. p53
has been used as a biologic gene therapy (Roth, 1996) and could be used to generate new
clinically active agents (W einstein, 1997).

5.2.0 HER/neu (c-erbB-Z) Proto-oncogene and Product
5.2.1 Background

HER (c-erbB-Z or neu) is an proto-oncogene. The name neu was defined in
1974, when female BDIX rats were injected with the chemical carcinogen N-ethyl-N-
nitrosourea at 4 mm months after birth: 93% of these animals developed central nervous
system tumors such as neuroblastomas or glioblastomas (Schubert, 1974). The tumor
cells were cloned and the DNA extracted from these cell lines was able to induce
transformation of mouse embryo fibroblast NII-I3T3 cells (Shih, 1981); they induced
tumors (fibrosarcomes) when injected into nude mice (Padhy, 1982). The DNA acted as

a potential transforming oncogene, and was responsible for the original induced rat

46

neuroblastomas during 1974 to 1982 (Schubert, 1974; Shih, 1981; Padhy, 1982). Then,
another group researchers found the neu gene to be homologous to the avian
erythroblastosis virus (AEV) transforming gene v-erbB (Schechter, 1984). Later, the
human homologue of the rat neu gene was also cloned independently and named c-erbB-Z
or HER-2, or MAC117, or NGL by several laboratories (Coussens, 1985; Yamamoto,
1986; King, 1985; Semba, 1985).

5.2.2 Structure and Function

Human HER-Z/neu gene was mapped to chromosome 17q21 (Coussens, 1985;

F ukushige, 1986). It contains an open reading flame of 3765 nucleotides and has major
transcript of 4.8 kb (Coussens, 1985; Fukushige, 1986; Semba, 1985), which translates
into a 1255 amino acid polypeptide (Coussens, 1985; Yamamoto, 1986; King, 1985).

The firnctional gene product of the HER-Z/neu gene was termed p185 in accordance with
its molecular weight (185,00 Daltons), while the primary translation product has a relative
molecular mass (Mr) of 137,895 daltons (Padhy, 1982; Drebin, 1984). This difference in
apparent molecular masses is due in part to N-linked glycosylation and probably O-linked
oligosaccharides as well (Stern, 1988a). Phosphorylation has also been found to be
involved in the modification of I-IER-2/neu-encoded protein.

The HER/neu gene sequence and its protein product are closely related to those
associated with epidermal growth factor receptor (EGF-R) with show an overall
homology of 50% (Schechter, 1985; Yamamoto, 1986; Bargmann, 1986). Similar to
EGF-R, neu encoded pl 85 is a transmembrane glycoprotein having intrinsic tyrosine

kinase activity (Coussens, 1985; Stern, 1986; Akiyama, 1986), and can be grouped as a

47

member of the growth factor receptor tyrosine kinase (RTK) gene family (Yarden, 1988).

The p185 consists of the following domains: 1) 16 of the first 19 amino acids following
the first ATG codon are hydrophobic residues, thus representing a cleaved Signal peptide
sequence; 2) 640 residues constitute the extracellular ligand-binding domain, containing
two cysteine-rich regions that may be important for ligand binding; 3) a second
hydrophobic stretch from residues 650 to 680 suggests a transmembrane domain; and 4)
the remaining carboxy-terminal 580 amino acids constitute the intracellular/cytoplasmic
domain (Coussens, 1985; Yamamoto, 1986; Bargmann, 1986).

The extracellular domain of pl 85 shows 44% homology with the ligand-binding
domain of the EGF-R. Within the ligand-binding domain are two cysteine-rich regions
that are completely conserved between these two erbB related proteins. The cytoplasmic
component of the protein encompasses residues 727-986 and contains the tyrosine kinase
domain with an ATP binding site. Some important structures in this region that are
identical between the two proteins include threonine (Thr) 954, which is the site of
phosphorylation mediated by protein kinase C (PKC), and several tyrosine residues
(Y1023, Y1248, Y1139,Y1222) for autophosphorylation (Hazan, 1990). All of the
autophosphorylation sites of neu reside within the carboxy-tenninal tail (Margolis, 1989).
5.2.3 Oncogenic Features

In the rat, c-neu proto-oncogene has a single point mutation of T to A at
nucleotide position 2012 that results in the proto-oncogene being activated to the neu
oncogene (Bargmann, 1988). This point mutation results in a change at amino acid

residue 664 fi'om valine (Val) to glutaminc acid (Glu) in the transmembrane domain of the

48

protein product and was shown to activate the intrinsic tyrosine kinase activity
(Bargmann, 1986; Stern, 1988 a ; Barmann, 1988 a, b). In humans, amino acid 664
mutation is a “hot spot” for activation of increasing tyrosine kinase activity in vitro
(Stern,1988b; Weiner, 1989).

The human HER/neu gene was originally isolated as an amplified v-erbB related
sequence in human mammary carcinoma, salivary gland adenocarcinoma, and gastric
cancer cell lines (Yamamoto, 1986; King, 1985). This gene is amplified or overexpressed
in many different human primary tumors (Tal, 1988). Abnormality of the HER/neu
proto-oncogene has been studied most frequently in human breast cancer with the
HER/neu gene found to be amplified or rearranged in many primary breast cancers and in
breast cancer cell lines (Kraus, 1987; Slamon, 1989; van de Vijver, 1987). One of the
early reports described a 2 to 20 fold amplification of the HER-Z/neu gene in about 30%
of primary human breast cancer in a 189 cases study. In addition there is a significant
correlation between the level of HER/neu gene amplification fold and its protein
overexpression (Slamon, 1987).

5.2.4 Clinical Approaches

HER/neu gene has been reported to be amplified or overexpressed many different
cancers including breast cancer, ovarian cancer (Slamon, 1989; Zhang, 1989; Berchuck,
1990), gastric tumors, (Yokota, 1988; Park, 1989), colon cancer (D’Emilia, 1989), lung
cancer (Schneider, 1989; Weiner, 1990), oral cancer (Hou, 1992) and cervical cancer
(Mitra, 1994). In contrast to gene amplification mutation, a very low frequency of

rearrangement mutation of the HER/neu gene has been observed in breast cancer and

49

gastric carcinoma (Yokota, 1988; Park, 1989).

C-erbBZ oncoprotein is already an established marker for poor prognosis in breast

cancer, and may also be predictive of response to treatment (Soomro, 1993; Allan, 1993).

The HER/neu gene was found to correlate with the number of lymph node metastases in
reoccurring breast cancers, and therefore is considered to play an important role in tumor
metastasis. The correlations found from clinical data have however been insuficient to
prove that HER/neu gene expression is responsible for aggressive breast cancer (van de
Vijver, 1987; Varley, 1987; Venter, 1987; Adnane, 1989; Garcia, 1987; King, 1989).
Nevertheless, HER/neu gene amplification or overexpression in human primary breast
cancers was shown to be a powerful predictor of the risk of recurrence (N agai, 1993).

It was reported that breast cancers showing HER-2 overexpression are less
responsive to chemotherapy than those with a normal amount of the gene product
(Gusterson, 1992). However, the p185” on the cell surface of HER-2 overexpressing
tumor cells may be a good target for receptor-directed immunotherapies. Using the anti-
human p185 monoclonal antibody 4D5 together with chemotherapy may increase the
chemosensitivity of the p185 overexpressing breast cancer cells to Taxol and Taxotere
(Cole, 1992; Moscow, 1988). Gene therapy has been studied by different groups, but
more study is needed before this therapy becomes beneficial to the cancer patients (Hung,
1995; Wels, 1995; Dhingra, 1995).

5.3.0 Bel-2 cell death regulate gene (proto-oncogene) and product
5.3.1 Background

In 1984, while studying the t (14: 18) chromosome translocation that occur

50

frequently in B cell leukemia and non-Hodgkin’s follicular lymphoma, a gene, Bel-2, was
isolated from the breakpoint of the translocation between chromosomes 14 and 18; it was
found to be present in a high proportion of the most common human lymphomas, the
follicular B cell lymphomas (Yunis, 1982; Tsujimoto, 1984). In 1988, it was shown that
introduction of the gene into interleukin-3 -dependent myeloid and lymphoid cell lines
promoted survival of these cells after withdrawal of interleukin-3, but did not stimulate
their proliferation (Vaux, 1988). Subsequently, the gene was shown to specifically inhibit
apoptosis (Hockenbery, 1990). Thus, Bel-2 emerged as a new type of proto-oncogene,
one that suppresses cell death rather than stimulating proliferation. However, it did not
inhibit the apoptosis occurring in all circumstances as it fails to block apoptosis induced by
cytotoxic T-lymphocytes (Vaux, 1992).

5.3.2 Structure and Function

The Bel-2 gene was cloned as a novel gene located at chromosome 18q21 and it
has a three exon structure with an untranslated first exon, a facultative 220 bp intron 1, but
with an enormous 370 kb intron II (Seto, 1988).

Bel-2 is 717 nucleotides long and codes for a Bcl-2 alpha molecule of 239 amino
acids with a molecular mass of 26 Kda (Tsujimoto, 1984). It is a putative membrane-
associated protein containing a hydrophobic carboxyl terminus located in the inner
mitochondria membrane; its lipophilic character suggests that perhaps if is a membrane-
spanning protein (Cleary, 1986; Tsujimoto, 1984). Its location suggests that it is involved
in some way with the metabolic functions of the inner mitochondria membrane, i.e.

oxidative phosphorylation and electron transport (Hockenbery, 1990). It has been noted

51

that decreasing cytosolic Ca2+ and increasing mitochondrial Ca” are correlated with Bcl-2
overexpression suggesting that Bcl-2 may be involved in the regulation of intracellular
Ca2+ distribution (Bafy, 1993). However, Bel-2 protects against apoptosis even in cells
without mitochondria (Jacobson, 1993). Bel-2 has also been localized to the nuclear
membrane and to the mitotic nuclei in epithelial cell lines suggesting that it may protect
DNA fiom damage caused by nuclease activation (Lu, 1994). It is known that Bel-2
expression can occur at any stage of the cell cycle (Fanidi, 1992; Bissonnette, 1992), but
the mechanism by which Bel-2 inhibits apoptosis is still unclear.

The function of Bel-2 as a cell death suppressor provided the first clue that altered
gene expression could enhance cell survival without affecting cell proliferation. In the
Bel-2 gene family, a group of genes with a sequence homology to Bel-2 produce protein
that share two highly conserved domains, Bel-2 homologue l and 2 (BH1,BH2). They
are divided functionally into two groups: 1) cell death suppressors such as Bel-2 and Bcl-
XI, and MCL-l, and 2) cell death promoters such as bax, bcl-xs, Bak and Bad cell death
promotors. BHI and BH2 domains regulate heterodimerization and regulation of cell
death by members of these gene family may be achieved through competing dimerization
(Cleary, 1986; Lin, 1993; Farrow, 1995; Yang, 1995).

Normal Bel-2 expression is associated with proliferating cells and cells which have
a need for longevity. Bel-2 is often demonstrated in glandular epithelial cells where
regulation of hyperplasis and involution is controlled by hormones and growth factors
(breast), in complex differentiating epithelium with long-lived stem cells (skin, intestine,

memory B cells ) and in fully differentiated long-lived stem cells and non-cycling cells

52

(neurons) (Nunez, 1990).

The protein product of the Bcl-2 gene that acts to inhibit apoptosis is maximally
expressed in the normal fetal breast (Nathan, 1994a) where there is immunohistochemical
localization in the basal epithelium of the developing breast bud and in the surrounding
stroma; similar patterns of staining have been reported in male and female tissues. Bc1-2
reactivity is lost soon after birth and it is not present in the epithelium of the normal adult
breast (Nathan, 1994b). The observations suggest that up-regulation of Bcl-2 contributes
to morphogenesis of the fetal breast by its inhibitory effect on apoptosis.

Bcl-2 proto-oncogene actively blocks apoptosis and is triggered by several factors
and events such as withdrawal growth-factor (Nunez, 1990), wild-type p53 protein
(Chiou, 1994), and X-radiation (Sentman, 1991). Roles for Bel-2 in epithelial
differentiation, morphogenesis, its response to hormone regulation, and in tumorgenesis
have been reported from different groups and it seems to be a important factor in
regulating complex pathways.

5.3.3 Oncogenic Features

The most common translocation of Bcl-2 was found in human lymphomas, at
t(14;18) (q32;21). This translocation point is located within exon III of the Bel-2 gene at
chromosome 18q21 and at the joining regions of the gene to immunoglobulin heavy chain
(IgH) loci at chromosome 14q32; this creates a Bcl-2/IgH fusion gene resulting in high
levels of Bcl-2 (Seto, 1988; Hanada, 1993). Expression of Bel-2 was thought initially to
be restricted to those B cell malignancies with the translocation, but subsequent studies

have reported its expression in a wide range of lympho-proliferative diseases that lack this

53

chromosomal abnormality as well as in normal lymphoid cell.

The mechanism by which Bel-2 promotes tumorgenesis is unique and the gene
expression confers survival advantage to a variety of cell types by inhibiting apoptosis, an
important feature in many normal biological processes.

5.3.4 Clinical Approaches

The detection of positivity for the Bel-2 gene and its product protein in neoplastic
cells has revealed a wide range of manifestations and heterogeneous staining pattern
throughout the tumors. Bel-2 oncoprotein is expressed in 79% of invasive breast
carcinomas, 97% of normal breast epithelium specimens, and 91% of in situ cancer
samples. Loss of Bel-2 expression is generally regarded as a relatively late event in the
progression of the disease. In breast cancer, its expression is associated with favorable
clinicopathological features. In addition, loss of Bcl-2 gene expression tends to be linked
to poor prognosis (Russel, 1994). It is an unique oncoprotein and is proving to be an
independent marker for prognosis in neoplastic breast disease (Leek, 1994;
Vanhaesebroeck, 1993; Joensuu, 1994; Carson, 1993; Bissonnette, 1992).

One study suggests that Bel-2 cooperates with c-myc in vitro to promote
proliferation and in some cases inducing tumors in nude mice (Strasser, 1990). Because
Bel-2 expression prolongs the life span of cells, it increases the risk for those cells to
acquire other changes, such as chromosomal abnormality and viral infection, which may
result in malignant transformation or overt tumor progression (McDonnell, 1989, 1991;
Nunez, 1990).

The association of Bcl-2 expression with the sensitivity of epithelial cells to drug,

54

radiation and hormone therapies very much depends on the type of malignancy (Lu, 1996).

Its effect on therapeutic responses and hence on prognosis is not clear cut and additional
studies are needed.
5.4.0 p53, Bel-2 and HER/neu (c-erbB-Z), Apoptosis and Oncogenesis
5.4.1 Apoptosis

Apoptosis is a form of cell death where single cells are deleted in the midst of

living tissue. The term drives from a Greek word to describe the dropping off of leaves
from trees. It is characterized by structural changes that appear with a great fidelity in
cells of widely different lineage and presumably represent a pleiotropic effector response
(Kerr, 1972a; Wyllie, 1980; Arends, 1991). Most or all of the programmed death is
responsible for tissue modeling in cell development, for the cell loss that accompanies
atrophy of adult tissues following endocrine and other stimuli, and in some tissues for the
physiological death of cells that occurs in the course of normal tissue turnover. The
morphological changes in apoptosis have been extensively reviewed (Kerr, 1972a; Wyllie,
1980; Arends, 1991).

Morphological evaluation shows that apoptosis characteristically affects scattered
single cells, not groups of adjoining cells, such as the case of necrosis. Early apoptosis is
characterized by compaction and margination of nuclear chromatin, condensation of
cytoplasm, and convolution of nuclear and cell outlines; at a later stage, the nucleus
fragments, and protuberances that form on the cell surface separate to produce apoptotic
bodies, which are phagocytosed by nearby cells and degraded within lysosomes. In

contrast, the development of necrosis is associated with irregular clumping of chromatin,

55

marked swolling of organelles and focal disruption of membranes. Membranes
subsequently disintegrate, but the cell usually retains its overall shape until removed by
mononuclear phagocytes (Wyllie, 1980; Kerr, 1987; Arends, 1991). Apoptosis can be
distinguished biochemically and morphologically from necrosis by the following summary
criteria: 1) chromatin condensation; 2) membrane blebbing; 3) appearance of apoptotic
bodies; and 4) fragmentation of the genomic DNA (Ellis, 1991).

Apoptotic cells do not induce an inflammatory reaction even when they present in
large numbers, but they are targets of immediate phagocytosis, either by macrophages
already present nearby, or by other adjacent viable cells. The compacted organelles and
condensed chromatin of the apoptotic cells may be visible for a few hours but eventually
reduce to large nondescript lysosomal residual bodies (Kerr, 1972b; Wyllie, 1980; Arends,
1991)

5.4.2 Apoptosis and Oncogenesis
An increase in cell number is one of the most prominent characteristics of a tumor.
It may be caused by either an increase in cell proliferation or a decrease in cell death, or
both. Previous studies of oncogenesis have focused predominantly on abnormalities of
cell proliferation but recently it has been recognized that abnormalities in the control of
cell death and survival are of equal significance (Huebner, 1969; Bishop, 1983; Oren,
1985; Lee, 1991).

The fact that apoptosis occurs in tumors is not new. It can be found in virtually

all untreated malignant tumors (Searle, 1973; Wyllie, 1985; Harmon, 1987). More than

20 years ago it was suggested that apoptosis may account for much of the spontaneous

56

cell loss known from kinetic studies to occur in many tumors (Kerr, 1972 a, b). It has
been clear for some time that abnormal apoptosis is enhanced in tumors by common
treatments such as, irradiation (Kerr, 1980; Stephens, 1991; Macklis, 1992), cytotoxic
chemotherapy (Searle, 1975; Dive, 1991; Gunji, 1991; Cotter, 1992; Johnston, 1992), heat
(Dyson, 1986; Barry, 1990; Harmon, 1990, 1991), and hormone ablation (Szende, 1990;
Kyprianou, 1991; Redding, 1992). The genetic analysis of programmed cell death during
the development of the nematode Caenorhabditis elegans has proved very useful in
identifying important events in the cell death program (Hale, 1996). Advances in
understanding of the control of apoptosis at the molecular level have potential oncologic
significance far for beyond the mere provision of a mechanistic explanation for tumor cell
deletion (Marx, 1993; Wyllie, 1987).
5.4.3 p53, Bel-2 and HER/neu (c-erbB-Z) Involvement in Apoptosis and
Oncogenesis

It has been suggested that exposure to environmental DNA damaging agents
contributes to the development of the vast majority of human tumors (Doll, 1981).
Therefore, an understanding of the molecular events involved in the cellular responses to
such exposures should provide insights into mechanisms of human carcinogenesis. There
are many interrelating effector events in apoptosis and the malignant phenotype, p53,
HER/neu (c-erbB-Z) and Bel-2 mutations in breast cancer have attracted the great
attention (Cope, 1991) to those studying this phenomenon.

The p53 tumour suppressor gene is involved on genetic regulation of apoptosis.

It acts as a direct “apoptogene”, a gene that causes apoptosis (Carson, 1993), and it has

57

been suggested that the product of the p53 gene acts as a “molecular policeman”
monitoring the integrity of the genome. If a cell’s DNA is damaged, p53 product
accumulates through a post-translational stabilization mechanism and arrests the cell cycle
at G1 to allow extra time for repair. If repair fails, p53 may trigger deletion of the cell by
apoptosis (Lane, 1992). To what extent p53 is involved in regulating apoptosis under
normal conditions is unknown (Donehower, 1992). A major mechanism whereby
abnormalities of p53 contribute to the development and progression of tumors may be the
abrogation of the normal pathway that leads to the self-destruction of mutant cells (Lane,
1993). Mutation in p53 confer resistance to apoptosis (Yonisch-Rouach, 1991; Shaw,
1992)

Bel-2 is unique among oncogenes because it exerts its oncogenic effect via the
inhibition of apoptosis and not via enhanced cell cycle progression. Bel-2 is normally
expressed in tissues characterized by extended viability or self-renewal that involves the
process of apoptosis (Hockenbery, 1991). In many malignancies, Bel-2 overexpression
confers an inhibition of programmed cell death upon malignant cells, even in the absence
of gene arrangements (Pezzella, 1990). Bel-2 has been shown to inhibit apoptosis
triggered by wild-type p53, and an inverse correlation between Bcl-2 expression and p53
mutation has been observed in breast cancer and other malignant diseases (Tomita, 1996).

The loss of function of p53 in cells may effect the function of Bel-2. Tumor cell
numbers would increase as a result of both unregulated proliferation and resistance to cell
death. The study has suggested Bel-2 oncoprotein expression is frequently associated

with an overexpression of p53 (Siziopikou, 1996). Loss of the p53 tumour suppressor

58

gene or overexpression of the apoptosis inhibitor protein Bel-2, has been reported in
oncogenically transformed cells that have lost their apoptotic potential (Graeber, 1996).
p53 was also found to down regulate Bel-2 and to regulate the Bcl-2/Bax balance. Bax is
one of member in Bel-2 family which acts as a apoptosis promotor (Oltvai, 1993;
Krajewski, 1994); wild-type p53 function may be required for the optimal expression of
Bcl-2 since p53 mutation in some cell types results in a marked reduction of the Bax and
an increase of Bcl-2 expression (Haldar, 1994; Lohmann, 1993).

HER/neu (c-erbB-Z) is known as an EGFR proto-oncogene, playing an important
role in the prognosis, and in transformation of neoplasms (Lupu, 1996); its involvement in
apoptosis is thought to be limited. One report, however, demonstrated that erbB-2
overexpression in ovarian tumor cell lines transfected with an endoplasmic reticulum form
of an anti-erB-Z single-chain antibody undergo specific cytotoxicity through the induction
of apoptosis (Grim, 1996). Another study using anti-HER2 monoclonal antibodies
induced tyrosine phosphorylation of HER proteins, causing cell morphology changes and
apoptosis (Kita, 1996).

Most of the studies on the mechanisms of apoptosis have been in vitro with only
limited in vivo studies. Overexpression of those entities in breast cancer has been
demonstrated with the finding that Bel-2 expression is negatively associated with
overexpression c-erbB-Z and p53 protein expressions in breast by immunohistochemical
methods (Binder, 1996; Ceccarelli, 1995; Nathan 1994a,b); however, it is strongly
associated with steroid hormone receptors. Bel-2 negative is associated with estrogen

growth factor receptor (EGFR) positivity (Gee, 1994; Leek, 1994). p53, c-erbB-2 and

59

Bel-2 are rarely co-expressed at any stage of breast cancer (Cohen, 1995; Reson, 1996a).

Combined studies of Bel-2, p53, and c-erbB-Z are hard to find.

6.0.0 Breast Cancer Immunohistochemical and Molecular Studies in China

Breast cancer in China is not a major malignant disease. However Chinese
researchers have made a number of basic and clinical investigations. The epidemiology,
histopathology, immunocytochemistry and molecular biology of breast cancer in China
have been discussed separately (Li, 1994). ER and PR are determined routinely in the big
hospitals, and the diagnostic value of various tumor markers in breast cancer has been
reported (Shi, 1988; Yiao, 1991; Song, 1993; Gong, 1993).

There are also studies from mainland China on oncogenes, tumor-suppressor genes
and their products, but the most them were on the other kinds of cancer. For example,
oncogene product overexpression, metastasis-suppressor gene and metastatic cell markers
were all studied by immunocytochemical methods and in situ hybridizations with myc, ras
family, jun, fos, int2, NM23, EGF, JCZ30, DCC, POR, TF, CEA, CA19-l, c-erbBZ, and
p53 (Fu, 1993; Chen, 1994; Ni, 1993; He, 1990; Liu, 1993; Lu, 1993), and the majority
of the reports were published in Chinese language only. A report of using flow cytometry
on breast cancer research also exists (Li, 1994; Guo, 1994). There is a single report of
the detection of point mutation of p53 gene by PCR-SSCP only without DNA sequencing
in paraffin-embedded breast cancer tissue: Fourteen of sixty (23.33%)cases were found
SSCP abnormal shifting bands. Another group showed 13 of60 (21.67%) cases where
p53 overexpression was detected by an immunohistochemical method. However, no

information on p53 gene point mutation sequence was presented, nor correlations with

60

other indicators, in these studies (Chen, 1994).

II. PATIENTS, MATERIAL AND METHODS

1.0.0 Patients Clinical Information

Two hundred and five formalin fixed, paraffin embedded specimens, which were
originally obtained for diagnostic purposes, were examined from 178 cases of primary
breast cancer patients at teaching hospitals in mainland China with the full
acknowledgment of the patients as part of the clinical function of these teaching hospitals.
Adjacent normal skin was also studied in 54 of these cases of primary breast cancer. All
specimens were obtained from surgical operations medically directed as part of treatment
with the consent of the patients. The age, sex, tumor location, Size of tumor, status of
lymph node metastasis and certain patient’s disease free follow up for various times were

recorded.

2.0.0 Histopathology

Tumors were diagnosed for type according to the World Health Organization
(WHO) classification (Hartrnanek, 1987) with all histological grading made by certified
pathologists (in most cases more than three observers). The Specimens were grouped into
three groups depending on duct or tubule malformation, the nuclear morphology and the
mitotic rate. The method used in the Nottingham/Tenovus breast study (shown in the

table II-l) was used for our specimen evaluation (Elston, 1987; Powell, 1994).

61

62

Table II-l. Histological grading of invasive breast cancer

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Feature Score
1. Tubule formation
Tubules with visible lumina as major component of tumor mass 1
Tubules with visible lumina as moderate component of tumor mass
Tubules with visible lumina as minor component of tumor mass 3
11. Nuclear morphology
Nuclei Show little variation in size and shape 1
Nuclei Show moderate variation in size and shape 2
Nuclei Show marker variation in Size and shape 3
III. Mitotic rate
<10 mitoses per 10 high-power fields (mitoses per 1000 cells at 1
standardization of high-power field size).
10 - 19 mitoses per 10 high-power fields 2
20 or more mitoses per 10 high-power fields 3
Grade 1 (well differentiated) 3-5
Grade 2 (moderately differentiated) 6-7
Grade 3 (poorly differentiated) 8-9

 

 

 

(Elston, 1987; Powell, 1994)

3.0.0 Immunohistochemistry

For the immunohistochemistry studies, five 4pm sections were cut, deparaffinized
in xylene, transferred to 100 %, 95%, 80%, 70%, 50% ethanol, distilled water and to Tris
Buffered Saline (TBS, Commercial). These sections were digested with 0.03% Pronase E
in 0.2N HCL, at 37°C for 10 min, and rinsed well for 5 min, and stained using an
automated HISTOSTAIN ER lg immuno-stainer (Leica Instruments Gmb H, Germany).

p53 immuno-staining: The deparaffmized sections for p53 immuno-staining were
treated with a 1:30 dilution of normal horse serum (Vector Co.) for 30 min; then with
anti-p53, clone BP53-12 (BioGenex), 1:1000 dilution for 720 min; followed by

biotinylated anti-mouse IgG (H+L) made in the horse, 1:150 (Vector Co.) dilution for 40

63

min; then streptavidin alkaline phosphatase (Kirkegaard & Perry Labs) for 60 min, and
finally the substrate 1 (Vector Red) for 8 min. BP53-12 is an IgGZ, monoclonal antibody
which recognizes a fixation-resistant epitope of both mutant and wild-type p53 proteins
located on the amino terminus between residues 1 and 45 (out of range of most identified
mutational ‘hot spots’). This clone yields highly consistent results even in poorly fixed
and overfixed tissue.

c-erbB-2 (HER/neu) immuno-staining: Deparaffinized sections were treated with
a 1:30 dilution of normal horse serum for 30 min (Vector Co.); then treated with c-
erbB-2 antibody (NOVA CASTRA Co.) 1:40 dilution for 90 min; biotinylated anti-
mouse 1:150 dilution for 40 min; streptavidin alkaline phosphatase for 60 min; and
finally substrate 1 (Vector Red) for 8 min. The c-erbB-Z clone CBll is a mouse IgGl
monoclonal antibody which recognizes a site on the internal domain of the c-erbB—Z
oncoprotein.

Bel-2 immuno-staining : the deparaffinized tissue sections at the room
temperature were immersed into a preheated Antigen Retrieval Solution (95-100°C) in
the waterbath, and incubated for 40 min; the slides in the incubation container were
stood at room temperature for 20 min for clone 124.3, and at 37°C for clone 100; then
the sections were rinsed two to three times with distilled water. These antigen pretreated
sections were then exposed to 1:30 dilution of normal horse serum for 30 min (Vector
Co.), treated with Bel-2 antibody (BIOGENEX Co.) 1:50 dilution for 720 min for clone
124.3; a 1:20 dilution of clone 100 for 120 min followed by the biotinylated horse anti-

mouse IgG (H+L) (VECTOR Co.) at a 1:150 dilution for 40 min; and finally

64

streptavidin biotin alkaline phosphatase was applied for 60 min and substrate 1 (Vector
Red) for 20 min. Both Bel-2 clone 124.3 and 100 can positively stain the 380 cell line
which carries a 14:18 chromosomal translocation mutation.

The slides were counterstained with Lerner 2 Hematoxylin for 15-30 seconds,
differentiated in 1% glacial acetic acid, blued in running tap water for 3 min and
dehydrated through graduated percentages of ethyl alcohol (95%, 100% several changes
of each), cleared in several changes of xylene, and coverslipped immediately with
synthetic mounting media.

The Cflokeratin (AE3/AE1) and Proliferating cell nuclear my; (PCNA)
antibodies were used to evaluate the quality of antigens in the breast cancer tissues as
“house control” markers. AE3/AE1 monoclonal antibodies have been used in many
studies to characterize an epithelial source for neoplasms and to study the distribution of
keratin-containing cells in epithelia during normal development. A_E_3 was directed at
basic keratins, and ;A_F:1_ at low molecular weight cytokeratin antigens. LCM
monoclonal antibody is used to identify a proliferating cell nuclear antigen which is
usually present in any normal tissue components that contain proliferating cells.

A cell was regarded as staining positively for p53 if there was red nuclear
staining; for c-erbB-Z, if there was red membrane staining; and for Bel-2, if there was red
cytoplasmic staining. Known positive and negative control specimens were stained

within each run. See the figure 1 - 4.

65

 

Figure 2. Immunohistochemical stain positive of p53 mutated protein
overexpression in a primary breast cancer.

 

Figure 3. Immunohistochemical stain positive of HER/neu (c—erbB-Z)
oncoprotein overexpression in a primary breast cancer.

I ‘r'.
o v)

 

... a...» .../«v . - .: ...?

Figure 4. Immunohistochemical stain positive of Bel-2 oncoprotein

overexpression in a primary breast cancer.

67

The immuno-stains were graded by using two semiquantitative methods (Berg
1993; Joensu 1994). The number of positive stain cells (semiquantity grade) or color
intensity of such positive stain cells (color intensity grade) were evaluated and grade
methods showed on the table II-2:

Table II-2. The methods used for immuno-staining

 

 

 

 

 

 

 

 

 

 

Grade Semiquantity Grade Color Intensity Grade
0 No stain No stain
l < 1 per cent positive cells Weak staining
2 2-9 per cent positive cells Intermediate positive staining
3 10 - 24 per cent positive cells Intense staining
4 25 - 49 per cent positive cells -
5 > 50 per cent positive cells -

 

 

4.0.0 Molecular Analysis
4.1.0 Cell Lines and Culture Conditions

The human breast cancer cell line BT474 (ATCC), known to have 4 to 8 fold
amplifications of HER/neu gene, was used to calibrate the relationship for the differential
PCR ratio. This cell line was also known to have a p53 point mutation GAG to AAG at
codon 285 and was used as a positive control for SSCP at exon 8. The BT474 breast cell
line was grown in RPMI 1640 medium with bovine insulin 10 ug/ml, L-glutamine
medium 300 mg/ml and 90 % fetal bovine serum, 10% and maintained in a 5 % CO2
humidified atmosphere for 10 days (Soto and Sukumar, 1992). Another breast cancer cell
line T47D (provided by Dr. Cheng) known to have a p53 point mutation CTT to TTT at

codon 194 and used as a positive control for SSCP at exon 6 (Soto and Sukumar, 1992).

68

4.2.0 DNA Extraction Method for Formalin Fixed Paraffin Embedded Breast
Tissues

Five of Sam tissue sections from the each primary breast cancer patient were put
in a microcentrifuge tube, 1 ml of xylene added to each tube, mixed at room temperature
for about 30 min or mixed 15 min at 56°C, pelleted the tissue by centrifugation 5 min at
full speed and carefiilly aspirated the xylene; this was repeated with a second change of
xylene and then 1 ml absolute ethanol was added; the solution mixed by inverting,
centrifuging, and aspirated the ethanol; this was repeated with a second change of
ethanol; finally then dried tissue pellet in a speed vacuum about 15 min.

One hundred pl (200 pl if the tissue pellet was large) digestion buffer (1M Tris-
HCL, pH8.5, 0.5 M EDTA, Tween-20) containing 200 ug/ml of proteinase K was
vortexed briefly with the pellet, and the solution incubated for 3 hours at 55°C or 37°C
overnight. The tubes were then spun briefly, heated and inactivated proteinase K at 95°C
for 8 to 10 min, centrifiiged for 5 min and samples frozen at -20° C. Aliquots of the
supernatant were used for amplification (typically 1 to 10 pl) (the method was provided
by Dr. K Friderici).

4.3.0 DNA Extraction Method for Cell Cultures

The cell culture medium was removed and centrifuged for 10 min at 1500g; 10
ml Trypsin 0.25% with 0.03 % EDTA was added to the culture well for 10 min, or until
the cells detached from the well walls and then the cells removed into a centrifuge tube;
the medium was centrifuged for 10 min at 1500 g. Phosphate-buffered saline (PBS) 10
ml used to wash the cells twice with centrifugation after which the PBS was removed

until about 50 ul cell solution was left. Fifty mM NaOH 100 pl was added to the pelleted

69

cells at 95°C for 10 min, and this placed on ice for 5 min.; 1 M Tris, pH 8, 10p] was
added, and the solution was then ready to use on PCR analysis.
4.4.0 HER-Z/nue Gene Amplification Analysis by Differential PCR

Differential PCR (dPCR) is a method used to detect quantitative alterations in
genes within small samples of cells or paraffin embedded specimens. This procedure
involves coamplification of a single copy reference gene, interferon-y (IF N-y) and the
target gene. The level of sample gene amplification is reflected in the ratio between the
results of PCR product bands of the target and reference gene (Frye, 1989; Evers,
1992)

CLONTECH’S HER-Z/neu ONCO-LYZER Kit was used for the HER/neu (c-
erbBZ) gene amplification analysis. The amplimer sequences were: HER/neu gene 5’
primer: 5’ CCT CTG ACG TCC ATC ATC TC3’; 3’ primer: CGG ATC TTC TGC
TGC CGT CG3’. IFN-y gene 5’ primer: S’TCT TTT C’I'I’ TCC CGA TAG GT3’; 3’
primer: 3’ CTG GGA TGC TCT TCG ACC TC5’ (Yamamoto 1986; Gray 1982).

A differential PCR amplification mixture for 50 pl final reaction volume of the
following composition was used. The reagent included: distilled water 31.75 pl; 10X
PCR reaction buffer 5.0 pl (PERKIN ELMER); dNTP mix (10 mM each) 1.0 pl ; 5’
HER/neu amplimer (20 pM) 2.5 pl ; 3’ HER/neu amplimer (20 pM) 2.5 pl; 5’ IFN-y
amplimer (20 pM) 2.5 pl ; 3’IFN-y amplimer (20 pM) 2.5 pl (CLONTECH); genomic
DNA (0.25pg/pl) 2.0 pl; AmpliTaq DNA polymerase (5 U/pl) 0.25 pl (PERKIN
ELMER). The temperature cycling program was as follows: segment 1: 94°C, 1 min;

segment 2: 55°C,] min; segment 3: 72°C, 1 min for 40 cycles; then 72°C, 7 min; and

70

finally held at 4°C (Gene Amp PCR System 9600, PERKIN ELMER).

For agarose gel electrophoresis, 8 pl PCR product was mixed with 2 p1 of gel-
loading solution (0.25% xylene cyanol, 0.25% bromophenl blue and 30% glycerol, in
water), loaded in the wells of 2% agarose gel containing 0.5 pg/ml ethidium bromide in
0.5 X TBE (45 mM Tris—Borate-EDTA) buffer. ¢Xl74/Haee III (Gibco BRL) were used
as the DNA size markers each time the gel was run. Electrophoresis was run in the same
TBE buffer for 1-1.5 hours at 60-70 V.

BIORAD’s DOClOOO gel detection and image analysis system was used for the
sample DNA band intensity analyses. The gel detector was set at optimal range of the
sample intensity with the analysis box covering the whole density of each band; the size
of the analysis area was kept constant for all the samples. Normal placenta tissue and
normal skin were used as normal limitation ratio controls. The breast cancer cell line
BT474 was chosen to assign positivity. The HER/neu gene band’s intensity of sample
was compared with IFN-y band’s intensity each time and the ratios between neu and IFN-
7 were calculated for each sample. The sample ratio higher than normal placenta tissue
and normal skin was considered as positive, HER/neu gene amplified >1 fold. A ratio
higher than BT474 cell line was considered HEWneu gene amplified >4-8 fold. A
HER/neu gene band with intensity brighter than the IFN-y that sample was considered as
positive gene amplification. Figure 5 and 6 show the (1 PCR products on 2% agarose gel

contains 0.5 pg/ml ethidium bromide in 0.5 X TBE.

7l

 

    

'3 3 4 5 ".7 I 910lllZl3umlGu||uzo

 

 

Figure 5. The HER/neu dPCR products are in a 2% agarose gel contains 0.5 pg/ml ethidium
bromide in 0.5 X TBE. The DNA bands on the top are lFN-y band’s intensity. The DNA bands
below are neu gene PCR products. Lane I to 20 is from lefi to right. Lane 1,2: normal placenta
tissue (dPCR positive control); lane 3: dPCR negative control (water); lane 4: BT474 cell line,
as a known positive control; lane 5: normal skin; lane 6-l9: specimens; lane 20: ¢Xl74/Haee
[II were used as the DNA size markers. From the top ofthe gel picture, first band is lFN-y and
the next band below that is neu .

 

I z a a s "i7 c omniznumlawuwzo

 

!?
Figure 6. The HER/neu dPCR products are in a 2% agarose gel contains 0.5 pg/ml ethidium
bromide in 0.5 X TBE. The sample intensity was defined with the analysis box covering the
whole density of each band with the intensity detection box. Lane 1 to 20 is from left to right.
Lane I, 2: normal placenta tissue (dPCR positive control); lane 3: dPCR negative control
(water); lane 4: BT474 cell line, as a known positive control; lane 5: normal skin; lane 6-19:
specimens; lane 20: 4>X174/Haee III were used as the DNA size markers. From the top ofthe gel
picture, first band is lFN-y and the next band below that is neu .

 

 

 

72

4.5.0 p53 Gene Point Mutation Analysis
CLONTECH’s Human p53 Amplimer Panels kit was used for p53 gene point

mutation analysis. The exons 5, 6, 7, 8, and 9 were analyzed.

Table II-3. The amplimer sequences for p53 gene exon 5 to 9

 

Exon Sequence of Amplimers

 

Exon 5 PU5 5’- CTC TTC CTG CAG TAC TCC CCT GC-3’
PD5 5’- GCC CCA GCT GCT CAC CAT CGC TA-3’

 

Exon 6 PU6 5’- GAT TGC TCT TAG GTC TGG CCC CTC-3’
PD6 5’- GTG TTG TCT CCT AGG TTG GCT CTG-3’

 

Exon 7 PU7 5’- GTG TTG TCT CCT AGG TTG GCT CTG-3’
PD7 5’- CAA GTG GCT CCT GAC CTG GAG TC-3’

 

Exon 8 PU8 5’- ACC TGA TTT CCT TAC TGC CTC TGG C-3’
PD8 5’- GTC CTG GTT GCT TAC CTC GCT TAG T-3’

 

 

Exon 9 PU9 5’- GCC TCT TTC CTA GCA CTG CCC AAC-3’
PD9 5’- CCC AAG ACT TAG TAC CTG AAG GGT G-3’

 

 

 

 

A PCR mixture for 50 pl final reaction volume was used containing the
following: distilled water 40.06 pl; 10X PCR reaction buffer (PERKIN ELMER) 5.0 pl;
dNTP mix (10 mM each) 1.0 pl; 5’ exon X amplimer (20 pM) 1.0 pl ; 3’ exon X
amplimer (20 pM) 1.0 pl; genomic DNA (0.25pg/pl) 1.0 pl; AmpliTaq® DNA
polymerase (5 U/ pl) 0.4pl. The temperature cycling program was: segment 1: 95°C, 30
sec; segment 2: 66°C, 45 sec; segment 3: 72°C, 1.5 min for 35 cycles; then 72°C, 7
min; soak in 4°C (Gene Amp PCR System 9600, PERKIN ELMER).

To determine whether the PCR amplification yielded product, an agarose gel
containing 2.7% agarose and 0.5 pg/ml ethidium bromide in 0.5X TBE buffer was used
for the agarose gel electrophoresis. Ten pl of PCR product mixed with 2 pl of gel-
loading buffer was loaded into the well and ¢X174/Haee III digest used in place of the
sample as size markers. The electrophoresis was run in the same 0.5X TBE buffer at 80

V for 1-1.5 hr. and the DNA bands were visualized with a UV transilluminator.

73

Any sample with enough PCR product was subjected to a Single-Strand
Conformation Polymorphism (SSCP) study; the PCR amplification was repeated up to
three times on a sample before this sample was abandoned for p53 gene point mutation

analysis. Figure 7 shows p53 PCR products run on 2.7% agarose gel.

 

Figure 7. PCR products of p53 gene at exon 5 in 2.7% agarose gel. Lane 1: PCR positive
control; lane 2: PCR negative control; lane 3-19: breast cancer specimens; lane 20:
¢Xl74/Hae lll digest used in place ofthe sample as size markers. Lane 3-6, 9, 12,13, 15, 16, 18,
and 19 showed optimal PCR products for SSCP and DNA sequencing. Lane 7, 8, 10, I l, 14, and
17 showed less than optimal PCR products and PCR were repeated for these samples.

For SSCP, formamide loading dye (95% forrnamide, 10 mM NaOH, 0.25%
bromophenol blue and 0.25% xylene cyanol) of 6% polyacrylamide with and without
5%g1ycerol gel (Biochem Co.) was used for each sample denaturation. One pl of each
PCR product was mixed with 10 pl fonnamide loading dye in a microcentrifuge tube,

then heated at 95° C for 5 min and cooled quickly on ice for more than 5 min. A 6%-

nondenaturing polyacrylamide gel containing no or 5 % (v/v) glycerol in 0.5X TBE

74

buffer was used for SSCP. Three pl of each denatured DNA sample was loaded on each
lane, and same volume of denatured control DNA loaded in an adjacent lane.
Electrophorese the gel at 0.5 W/cm for 1.5 to 2 hr at room temperature, and visualize the
bands by Silver Stain (BIORAD Co.) the gel. The appearance of a band shift in a tumor
sample when compared with the normal band pattern of a particular exon was
characteristic of a change in the secondary structure of the mutant single strand.

The breast cancer cell line T47D (which has a known gene point mutation at
codon 194 CTT to TTT) was used as a positive control for exon 6. BT474 (known to
have a gene point mutation at codon 285 GAG to AAG) was used as a positive control for
exon 8. (Soto and Sukumar 1992). The polymorphism bands were compared with

normal controls to ascertain difference.

1 2 3 4 5 6

da-
7
5

EH 2

. 1' u a» ' i i '
Figure 8. SSCP analysis of exon 6, T47, (lanes 1 - 4) and exon 8, BT474, (lanes 5 and 6) of the
p53 gene in breast cancer cell lines in a 6% nondenaturing polyacrylamide gel in 0.5X TBE
buffer. Lane 1 and 2 are nondenatured normal placenta and T47D samples. The appearances of
the band mobility shift in the breast cancer cell lines compared with the normal placenta tissue

bands (lanes 3 and 5) are characteristiced, because of two different conformations of the
sequence.

75

Figure 9. SSCP analysis of exon 5 of p53 gene in breast cancer sample No. 43 in a 6%
nondenaturing polyacrylamide gel in 0.5x TBE buffer. Lane 1 is denatured breast cancer tissue.
The appearances of the band mobility shift are characteristiced, because of two different
conformations of the sequence. Lane 2 is denatured normal skin tissue.

After detection of mutations by PCR-SSCP, DNA sequencing was used for
confirmation purposes and for pinpointing the exact base change. The PCR products and
primers were diluted to 1:10, then 6pl sample and 3 pl primer were sent to the Tissue
Typing Laboratory for sequencing. ABI PRISM Cycle Sequencing Day Terminator with

ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (PERKIN ELMER)

are used for DNA sequencing. Each sample was sequenced from both directions.

5.0.0 Statistical Analysis
Statistical analysis for characteristics of Chinese breast cancer were performed by
using Jandel Scientific program, SigmaStat, for GATEWAYZOOO, IBM Personal

Computer. Student t test was used for age distribution between two variables. Mann-

76

Whitney Rank Sum test was used to compare the difference of treatments. Chi-square
test, Fisher Exact test, Pearson Product Moment Correlation and Spearman Rank Order
Correlation tests used for analyzing the degree of association between two or more
variables Linear Regression test and Best Subsets Regression tests were applied for
analyzing the significance of functional relationship between two or more variables. The
Life Table test used for general five-year survival analysis.

All statistics studies were to examine the confidence interval allows a 95%
confidence interval overall at a standard power (0.80) with two-tail for each test.

(Arrnitage 1994).

III. RESULTS

The results of this study are divided to four parts: patient clinical information,
histopathology, immunohistopathology and molecular pathology.
1.0.0 Patient Clinical Information

Sex: All tumors were defined as breast cancer. One hundred seventy - eight of
the samples were from females and one from a male (0.57%).

Age: The age range of patients was from 22 - 79 years, with a mean of 49 years
and a median of 48 years. The age distribution data summarized in the Table 111-1.

Table IH-l. (n = 176)

  
  

33.5
24.4
14.2
6.25
0
The relationship between age and lymph node metastasis was analyzed by
Fisher’s Exact tests: the data and the results show in the table III-2.

Table III-2. The (n=85)

The Chi-square test was used to analyze the effect of age on survival rate. The age
range groups used were 20 to 39, 40 to 59, and 60 to 79. The Chi-square test, probability

value greater than 0.05 (P>0.05). Indicating that there was no significant difference

77

78

amongst the three age range groups. The effecting of young or old of age on breast
cancer patient tendency were also tested by Chi-square test. The ages were grouped as
mentioned before. The result of P>0.05 indicated that there was no significant difference
between those groups.

Tumor Location: The location of tumors were recorded in fifty-eight cases and
these involved the left side breast in 55.17% (32/58), and the right side in 44.83% (26/58).

Tumor Size: The sizes of the breast cancers are shown in the Figure 10. These
ranged from 1.3 cm to 12 cm in diameter, with a mean of 4.5 cm. The most frequent size

for palpable breast cancer detection was 3 - 4 cm.

 

 

 

 

 

/
/
/
/

No. of Cases

 

 

 

IO '1 I2 .3 I4 .5 I6 .7 I8 39 I10 I>11

Figure 10. The size distribution of the breast cancer.

Metastasis: Lymph node metastases were assessed and found to be as follows:
Positivity of 60.7%, 51/84 cases had positive lymph nodes. The number of lymph nodes
evaluated in each patient was from 1 to 29 (average 17). The years of survival following

the primary breast cancer were also analyzed with regard to whether or not the lymph

79

nodes were positive for metastases by the Chi-square test. A statistically significant

difference with primary breast cancer patient’s five-year survival between those with and

those without lymph node metastasis was seen with a probability value of below 0.05

 

 

 

 

 

 

 

 

 

(P=0.006).
Table III-3. Survival information and the % results of Life Table Analysis
Survival Breast Cancer Breast Cancer with Lymph Breast Cancer without
Years Node Metastasis Lymph Node Metastasis
No. of Dead or Survial No. of Dead or Survial No. of Dead or Survial
Missed/Total % Missed/1’ otal % Missed/'1’ otal %
1 3/41 95.12 2/26 92.3] 1/15 93.33
2 8/41 83.52 6/26 71.00 2/15 80.89
3 11/41 63.15 8/26 49.16 3/15 64.71
4 6/41 55.44 5/26 39.70 1/15 60.40
5 7/41 47.33 5/26 32.07 7/15 32.2]
>5 6/41 41.55 0/26 0.00 1/15 30.06

 

 

 

 

 

 

 

2.0.0 Histopathology

2.1.0 Types

Ten histopathological diagnostic types of breast cancer were identified in these

specimens and shown in the table III-4.

Table 111-4. Histopathological types and their frequency of in the Chinese

breast cancer samples (n=178)

 

 

 

 

 

 

 

 

 

 

 

 

 

Pathologic Cancer Type No. of Cases %
Adenocarcinoma 2 1. 12
Apocrine carcinoma 1 0.56
Carcinoma 1 0.56
Intraductal carcinoma 3 1.69
Infiltrating ductal carcinoma 153 86.0
Infiltrating lobular carcinoma 7 3.93
Infiltrating papillary carcinoma 1 0.56
in situ lobular carcinoma 2 1.12
Medullary carcinoma 3 1.69
Mucinous adenocarcinoma 5 2.80

 

 

 

 

2.2.0 Grade

80

The histopathological grading was recorded and the specimens scored into

categories of well, moderate and poor differentiation depending on duct and tubule

malformation, nuclear morphology and mitotic rate. The results are shown in the table HI-

5. Two in situ breast cancer cases were excluded in this table, because the grade system of

in situ tumor is different with infiltrating tumor grading system.

Table 111-5. Histology grading of invasive breast cancer (n=178)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pathologic grading Well Moderately Poorly
differentiated differentiated differentiated
No. % No. % No. %
Adenocarcinoma l 50 0 0 l 50
Apocrine carcinoma 0 0 l 100 0 0
Carcinoma 0 0 0 0 1 100
Intraductal carcinoma 3 100 0 0 0 0
Infiltratig ductal carcinoma 51 33.33 56 36.60 46 30.07
Infiltrating lobular carcinoma 2 28.57 2 28.57 3 42.86
Infiltratinipapillary carcinoma 1 100 0 0 0 0
Medullary carcinoma 0 0 2 66.67 1 33.33
Mucinous adenocarcinoma 2 40.00 3 60.00 0 0
Total number of cases 60 64 52
% 33.71 35.96 29.21

 

3.0.0 Immunohistochemistry

3.1.0 Control Used

The cytokeratin (AE3/AE1) and proliferating cell nuclear antigen (PCNA)

antibodies were used as “house keeping control” markers to assess the quality of antigens

in the breast cancer tissues. All the Chinese breast cancer specimens stained positively for
the positive controls and all negative controls were negatively (100%).

3.2.0 Immunohistochemical Study

The overall results for the immunohistochemical study were 14.18% (19/134)

 

81

positive for p53, 23.13% (31/134) positive for c-erbB—Z (HER/neu) and 66.42% (89/134)
positive for Bel-2, when assess using a semiquantification grading method. The color
intensity grade was assessed and used for technique comparison only. The results are in
the table III-6.

Table III-6. Immunohistochemical studies (n = 134)

 

 

 

 

 

Immuno-stain Quanti {Grade‘ Color Intensity Grade"
Grade - 1+ 2+ 3+ 4+ 5+ - 1+ 2+ 3+
p53 99 13 3 3 5 l l 99 13 14 8
c—erbB-Z 95 5 3 5 7 19 95 3 10 26
Bel-2 37 2 6 12 19 58 35 10 34 55

 

 

 

 

 

 

 

 

 

 

 

 

 

* data are based on number of positively stained cell, ** data are based on intensity of stain

The semiquantity grade and the color intensity grade were analyzed by Spearman
Rank Order Correlation statistical technique. There were no significant relationship
between the two evaluation methods (P>0.05). In this study, the results of the
semiquantity grade were used for the rest of the studies and analyses as is reflect cell
positively the best.

Mann-Whitney Rank Sum Test was used to analyze for differences between
semiquantitying grade and the color intensity grade methods. For p53 and HER/neu
immuno-stains, there were no statistically significant differences (P = 0.704 and P =
0.513). There was a statistically significant difference (P<0.0001) for the Bcl-2 immuno-
stain.

3.3.0 Correlation Analysis of Immunohistochemical and Other Studies
3.3.1 Correlation Immunostain with Breast Cancer Types

The correlation of immuno-stain positive and with pathological cancer type were

recorded and shown in the tables III-7.

A comparison of immuno-stain positive for p53, HER/neu and Bcl-2 with the

82

different type of breast cancers were analyzed by Pearson Product Moment Correlation

test. Within each pattern, there was no significant relationship between the variables

 

 

 

 

 

 

 

 

 

 

 

 

 

(P>0.05).
Table III-7-1. Immunostaining positivity by pathological tumor type
Pathologic diagnosis Frequency p53 (+) HER/neu (+) Bel-2 (+)
(n=l34) (19/134, 14.18%) (31/134, 23.13%) (89/134, 66.42%)
No. % No. % No. % No. %
Adenocarcinoma 0 0 0 0 0 0 0 O
Apocrine carcinoma 0 0 0 0 0 0 0
Carcinoma 1 0.74 0 0 0 0 l 100
Intraductal carcinoma 3 2.24 0 0 0 0 2 66.67
Infiltrating ductal cancer 116 86.57 18 15.52 31 26.72 74 63.79
Infiltrating lobular cancer 7 5.24 0 0 0 0 5 71.43
Infiltrating papillary cancer 1 0.74 0 0 0 0 1 100
in situ lobular carcinoma 2 1.49 0 0 0 0 2 100
Medullary carcinoma 1 0.74 1 100 0 0 l 100
Mucinous adenocarcinoma 3 2.24 0 0 0 0 3 100

 

 

 

 

 

 

 

 

 

 

Table HI-7-2. Immunostaining negativity by pathological tumor type

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pathological Diagnosis Frequency p53 (-) HER/neu (-) Bel-2 (-)
(n = 134) (115/134, 85.82%) (103/134, 76.87 %) (45/134, 33.58%)
No. % No. % No. % No. %
Adenocarcinoma 0 0 0 0 0 0 0 0
Apocrine carcinoma 0 0 0 0 0 0 0 0
Carcinoma l 0.74 l 100 1 100 0 0
Intraductal carcinoma 3 2.24 3 100 3 100 l 33.33
Infiltratingductal cancer 1 16 86.57 98 84.48 85 73.28 42 36.21
Infiltrating lobular cancer 7 5.24 7 100 7 100 2 28.57
Infiltratingpyillary cancer 1 0.74 1 100 l 100 0 0
in situ lobular cancer 2 1.49 2 100 2 100 0 0
Medullary carcinoma 1 0.74 1 100 l 100 0 0
Mucinous adenocarcinoma 3 2.24 3 100 3 100 0 0

 

 

 

 

 

3.3.2 Correlation Immunostain Studies with Each Other

One Way ANOVA (Kruskal-Wallis) test was used to compare the p53, HER/neu
(c-erbB-Z) and Bel-2 positive stains, and showed those immunohistochemical stains that
were significantly different (P<0.0001). The Spearman Rank Order Correlation test was

used for the correlation analysis. No significant relationships between any pair of variables

83

were detected (P>0.05) i.e. for immuno-stain positive results of p53, HER/neu, and Bcl-2.

3.3.3 Correlation Immunostain Studies with Clinical Indicators

clinical indicators, such as age, lymph node metastasis, cancer type, cancer cell

The immunohistological results with p53, HER/neu and Bel-2 were compared with

differentiation and cell mitotic rate. (Table IH-8).

Table 111-8. Protein expression for p53, c-erbBZ and Bel-2 and their relationship to
clinical indicators

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Study p53 (+) p53 (-) irrnnuno- c-erbB-2(+) c-erbB-Z (-) Bel-2 (+) Bel-2 (-)
(19/ 134) (1 15/134) (31/134) (103/134) (89/ 134) (45/ 134)
No. % No. % No. % No. % No. % No. %
Grade 1 4/19 21.05 34/113 30.09 381 9.68 35/ 101 34.65 2087 29.89 12/45 26.67
Grade 2 4/19 21.05 42/113 37.17 1461 45.16 32/101 31.68 28/87 32.18 18/45 40.00
Grade 3 11/19 57.89 37/113 32.74 14/31 45.16 34/101 33.66 33/87 37.93 15/45 33.00
M l 3/ 19 15.79 33/1 13 29.20 7/31 22.58 29/ 101 28.71 24/87 27.59 12/45 26.67
M 2 6/19 31.58 44/113 31.86 11/31 35.48 31/101 30.69 23/87 26.44 19/45 42.22
M 3 10/19 52.63 43/113 38.94 13/31 41.94 41/101 40.59 40/87 45.98 14/45 31.11
L+ 9/ 13 69.23 43/72 59.72 18/22 81.82 33/63 52.38 20/55 36.36 20/32 62.50
L - 4/13 17.00 29/72 40.28 4/22 18.18 30/63 47.19 35/55 63.64 12/32 37.50
<50 7/19 36.84 63/115 54.78 1631 51.61 45/103 52.43 46/89 51.69 24/45 53.53
>50 12/19 63.16 52/1 15 45.22 1581 48.39 49/103 47.57 43/89 48.31 21/45 46.67
IDC 18/19 94.74 98/115 85.22 3131 100.00 86/103 83.50 74/89 83.15 43/45 95.56
1113 0/19 0.00 7/115 6.09 0.00 0.00 6/103 5.82 5/89 5.62 2/45 4.44
MA 0/19 0.00 3/115 2.61 0.00 0.00 3/103 2.91 3/89 3.37 0/45 0.00
in situ 0/19 0.00 2/115 1.74 0.00 0.00 0/103 0.00 2/89 2.25 0/45 0.00
MC 1/ 19 5.26 1/115 0.87 0.00 0.00 0/103 0.00 1/89 1.20 0/45 0.00
IC 0/19 0.00 3/1 15 2.61 0.00 0.00 0/103 0.00 3/89 3.37 0/45 0.00

 

 

 

 

 

 

 

 

 

 

 

 

 

Grade 1 = cell well differentiation, Grade 2 = cell moderate differentiation, Grade 3 = cell poor differentiation; M 1 =
cell with low mitotic rate, M2 = cell with moderate mitotic rate, M3 = cell with high mitotic rate; L + = lymph node
metastasis positive, 1.- = lymph node metastasis negative; IDC = infiltrating ductal cancer, ILC = infiltrating lobular
cancer, Ca = cancer; MA = mucinous adenocarcinoma; in situ = in situ lobular carcinoma; MC = medullary cancer,
IC = intraductal cancer.

The table III-9 shows these results analyzed statistically.

Table IH-9. The results of Chi-square test from the table III-8 (n=134)

 

 

 

 

 

 

 

 

 

 

p53 irnmuno (+) HER/neu Bcl-2
& (-) immuno- (+) & H (+) & (-)
stain rmm’ uno-starn' 1mm' uno-stam'
x2 P x2 P x2 P

mammal 4.50e+000 0.105 7.23e+000 0.671 5.43e+000 0.066
Mitotic rate 2.20e+000 0.333 5.09e-001 0.140 1.45e+000 0.484
Lymph Node + 1.146001 0.735 4.72e+000 0.033 3.69e+000 0.055
Age < > 50 1.45e+000 0.229 2.386002 0.998 6.566001 0.418

 

 

 

 

 

The clinical indicators correlated with immunostain results were also recorded and

 

84

analyzed statistically. The results show in the tables III-10.

Table III-10-1. Relationships between clinical indicators and immunostain results Q1=134)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Age Ca in Breast Lymph Node
<50 >50 Lefi Ri t + -
N0 % No % No % No % No % No %
r6011 70 52.23 64 47.76 32 55.17 26 44.82 52 61.18 33 38.82
1353... 7 10.00 12 18.75 4 12.50 3 11.54 9 17.31 4 12.12
p53- 63 90.00 52 81.25 28 87.50 23 88.46 43 82.69 29 87.88
P value of p53 0.215 1.000 0.758
HER+ 16 22.86 15 23.44 4 12.50 3 1 1.54 19 36.54 3 9.09
HER- 54 77. 14 49 76.56 28 87.50 23 88.46 33 63.46 30 90.91
P value of HER 1.000 1.000 0.005
Bel-2+ 46 55,71 43 67.19 22 68.75 21 80.77 32 61.54 21 63.64
Bel-2 - 24 34.29 21 32.81 10 31.25 5 19.23 20 38.46 12 36.36
P value of Bcl-2 1.000 0.374 1.000

 

 

 

 

Fisher’s Exact tests are used for all P value calculations. Ca = carcinoma

Table III-10-2. Relationships between clinical indicators and immunostain results (n=134)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cell Differentiation Cell Mitotic Rate
Well Moderate Poor Low Moderate High

No % No % No % No % No % No %
Total 38 28.36 46 34.85 48 36.36 36 27.27 42 31.82 54 40.91
53+ 4 10.53 4 8.69 11 22.92 3 8.33 6 31.82 54 40.91
p53- 34 89.47 42 91.30 37 77.08 43 91.67 36 85.71 44 81.48
P value ofp53/1 1.000‘ 0.090” 0.161 ‘" 0.300‘ 0.783“ 0.133‘“
#11 0105”" 0209""
HER+ 3 7.89 14 30.43 14 29.17 7 19.44 11 26.19 13 24.07
HER- 35 92.11 32 69.57 34 70.83 29 80.56 31 73.81 41 75.93
P value ofHER # 0.013‘ 1.000" 0.016‘” 0.593‘ 0.817" 0.796‘”
## 0027"" 0_776aua
Bcl-2+ 26 68.42 28 60.87 33 68.75 24 66.67 23 54.76 40 74.07
Bcl-2- 12 31.58 18 39.13 15 31.25 12 33.33 19 45.24 14 25.93
P value ochl-2# 0.502’ 0.518" 1.000‘” 0.355‘ 0.055“l 0.483‘“
Mi 0247"” 0.67ltttl

 

# Fisher’s Exact tests are used for all P value calculations. ## Chi-square tests are used for all P value
calculations. ’ = the result of compared with Well (Low) and Moderate groups; " = the result of compared with
Well (Low) and Poor (High) groups; ‘" = the result of compared with Moderate and Poor (High) groups; "" = the
result of compared with all groups.

3.4.0 Relationship Between Immunostain Results

Associations between immunohistochemical findings and other pathological

parameters are shown in the tables III-11.

 

 

85

Table IH-l 1-1. The associations between three immunostaining and clinic-pathological

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

features
p53+lHER+chl-2+ p53+/l-1ER+/Bcl-2- p53+/HER-/Bcl-2- 53+/HER-IBcl-2+
No. % No. % No. % No. %

Total ease 5/134 3.73 2/134 1.49 7/134 5.22 5/134 3.73

Mitotic rate 1 0/5 0 1/2 50.00 2/7 28.57 0/5 0.00
Mitotic rate 2 2/5 40.00 1/2 50.00 2/7 28.57 1/5 20.00
Mitotic rate 3 3/5 60.00 0/2 0 3/7 42.86 4/5 80.00
Differentiated 1 0/5 0 [/2 50.00 3/7 42.86 1/5 20.00

Difl’erentiated 2 1/5 20.00 1/2 50.00 2/7 28.57 0/5 0.00
Differentiated 3 4/5 80.00 0/2 0 2/7 28.57 4/5 80.00
Lymph node + 3/5 60.00 2/2 100.00 3/5 60.00 1/3 33.33
Lymph node - 2/5 40.00 0/2 0 2/5 40.00 2/3 66.67
<50 years 1/5 20.00 212 100.00 2/7 28.57 2/5 40.00
>50 4/5 80.00 0/2 0 5/7 71.43 3/5 60.00

IDC 5/5 100 2/2 100.0 7/7 100.00 4/5 80.00
Medullary Ca 0 0 0 0 0 0 1/5 20.00

 

 

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer; Ca = cancer.

Table III-11-2. The associations between three immunostaining and clinic-pathological

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

features
p53-MER-chl-2- p53-MER-IBcl-2+ p53-/HER+/Bc1-2+ p53-lHER+/Bcl-2-
No. % No. % No. % No. %

Total case 28/134 20.90 63/ 134 47.01 16/134 11.94 8/ 134 5.97
Mitotic rate 1 8/28 28.57 19/61 31.15 5/ 16 31.25 1/8 12.50
Mitotic rate 2 11/28 39.24 17/61 27.87 3/16 50.00 5/8 62.50
Mitotic rate 3 9/28 32.14 25/61 40.98 8/16 12.5 2/8 25.00

Differentiated 1 9/28 32.14 23/61 37.70 2/16 43.75 0/8 0.00
Differentiated 2 10/28 35.71 20/61 32.79 7/16 43.75 5/8 62.50
Differentiated 3 9/28 32.14 18/61 29.51 7/16 25.00 3/8 37.50
Lymph node + 10/20 50.00 19/35 54.29 3/ 12 25.00 5/5 100.00

Lymph node - 10/20 50.00 16/63 45.71 9/ 12 75.00 0/5 0.00
<50 years old 17/28 60.71 38/63 60.32 8/16 50.00 5/8 62.50
>50 11/28 39.29 25/63 39.68 8/16 50.00 3/8 37.50
IDC 26/28 92.86 50/63 79.37 0/ 16 0.00 8/8 100.00

ILC 2/28 7.14 2/63 3.17 0/16 0.00 0/8 0.00

MA 0/28 0.00 3/63 4.76 0/ 16 0.00 0/8 0.00

Intraductal Ca 0/28 0.00 3/63 4.76 0/16 0.00 0/8 0.00

in situ lobular 0/28 0.00 2/63 2/63 0/16 0.00 0/8 0.00

IPC 0/28 0.00 1/63 1.59 0/16 0.00 0/8 0.00

 

 

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer; ILC = infiltrating lobular cancer; Ca = eancer; MA = mucinous

adenocarcinoma; Intraductal Ca = intraductal carcinoma; in situ lobular = in situ lobular carcinoma; IPC
= infiltrating papillary carcinoma.

 

 

86

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table IH-11-3. The associations between two immuno-stains and clinic-pathological
features
p53+/HER+ p53+/Bcl-2+ p53-IHER-
No. % No. % No. %
Total case 7/134 5.22 10/134 7.46 91.134 67.91
Mitotic rate 1 1/7 14.29 0/134 0.00 27/89 30.34
Mitotic rate 2 3/7 42.86 3/ 10 30.00 28/89 31.46
Mitotic rate 3 3/7 42.86 7/10 70.00 34/89 38.20
Difl'erentiated 1 1/7 14.29 1/ 10 10.00 32/89 35.96
Differentiated 2 2/7 28.57 1/ 10 10.00 30/89 37.71
Differentiated 3 4/7 57.14 8/10 80.00 27/89 30.34
Lymflr node + 5/5 100.00 4/6 66.67 29/55 52.73
Lymph node - 0/5 0.00 2/6 33.33 26/55 47.27
<50 years 3/7 42.86 3/10 30.00 41/91 45.05
>50 4/‘7 57.14 7/10 70.00 56/91 61.54
IDC 7/7 100.00 9/10 90.00 75/91 82.42
ILC 0/7 0.00 0/10 0.00 6/91 6.59
MA on 0.00 0/10 0.00 3/91 3.30
Medullary Ca 0/7 0.00 0/10 0.00 0/91 0.00
Intraductal Ca 0/7 0.00 1/10 10.00 3/91 3.30
in situ lobular 0/7 0.00 0/10 0.00 2/91 2.20
IPC 0/7 0.00 0/ 10 0.00 1/91 1.10

 

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer; ILC = infiltrating lobular cancer; Ca = cancer; MA = mucinous
adenocarcinoma; Intraductal Ca = intraductal carcinoma; in situ lobular = in situ lobular carcinoma;
IPC = infiltrating papillary carcinoma.

Table III-11-4. The associations between two immunostaining and clinic-pathological

features

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

p53-[Bcl-2 - HER+IBcl~2+ HER-chl-Z-
No. % No. % No. %
Total case 36/ 134 26.87 l6ll34 11.94 28/134 20.90
Mitotic rate 1 9/36 25.00 5/16 31.25 8/28 28.57
Mitotic rate 2 16/36 44.44 3/16 18.75 11/28 39.29
Mitotic rate 3 11/36 30.56 8/ 16 50.00 9/28 32.14
Difl'erentiated 1 9/36 25.00 2/ 16 12.50 9/28 32. 14
Difl‘erentiated 2 15/36 41.67 7/ 16 43.75 10/28 35.71
Differentiated 3 12/36 33.33 7/ 16 43.75 9/28 32. 14
Lymph node + 15/25 60.00 9/12 75.00 10/20 50.00
Lyrmth node - 10/25 40.00 3/ 12 25.00 10/20 50.00
<50 years 22/36 61.11 8/16 50.00 l7/28 60.71
>50 14/36 38.89 8/ 16 50.00 11/28 39.29
IDC 34/36 94.44 15/ 16 93.75 26/28 92.86
ILC 2/36 5.56 1/16 6.25 2/28 7.14

 

 

 

 

IDC = infiltrating ductal cancer; ILC = infiltrating lobular cancer.

 

Table III-1 1-5. The associations between two immunostaining and clinic-pathological

87

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

features
p53-ll-lER+ p53+chl-2- p53+lHER-
No. % No. % No. %
Total case 24/14 17.91 9/134 6.72 12/134 8.96
Mitotic rate 1 6/24 25.00 3/9 33.33 2/ 12 16.67
Mitotic rate 2 8/24 33.00 3/9 33.33 3/ 12 25.00
Mitotic rate 3 10/24 41.67 3/9 33.34 7/12 58.33
Differentiated 1 2/24 8.33 3/9 33.33 3/ 12 25.00
Differentiated 2 12/24 50.00 3/9 33.33 2/ 12 16.67
Differentiated 3 10/24 41.67 3/9 33.34 7/ 12 58.33
Lymph node + 3/17 17.65 5/7 71.43 4/8 50.00
Lymph node - 14/ 17 82.35 2/7 28.57 4/8 50.00
<50 years 13/24 54.17 4/9 44.44 4/ 12 33.33
>50 1 1/24 45.83 5/9 55.56 8/ 12 66.67
IDC 24/24 100.00 9/9 100.00 11/12 91.67
ILC 0/24 0.00 0/9 0.00 O/ 12 0.00
MA 0/24 0.00 0/9 0.00 0/ 12 0.00
Medullary Ca 0/24 0.00 0/9 0.00 1/12 8.33
Intraductal Ca 0/24 0.00 0/9 0.00 0/ 12 0.00
in situ lobular 0/24 0.00 0/9 0.00 0/ 12 0.00
IPC 0/24 0.00 0/9 0.00 0/ 12 0.00

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer, ILC = infiltrating lobular cancer, Ca = cancer, MA = mucinous adenocarcinoma;
Intraductal Ca = intraductal carcinoma; in situ lobular = in situ lobular carcinoma; IPC = infiltrating papillary

carcinoma.

Table III-11-6. The associations between two immunostaining and clinic-pathological

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

features
p53-/Bcl-2+ HER-l-chl-Z- HER-chl-2+
No. % No. % No. %

Total case 78/134 58.21 10/134 7.46 68/134 50.75
Mitotic rate 1 23/‘76 30.26 2/ 10 20.00 l9/66 28.79
Mitotic rate 2 20/76 26.32 6/ 10 60.00 18/66 27.79
Mitotic rate 3 33/76 43.42 2/10 20.00 29/66 43.94
Differentiated 1 24/76 31.58 l/10 10.00 24/66 36.36
Differentiated 2 26/76 34.21 6/10 60.00 20/66 30.30
Difi’erentiated 3 25/76 32.89 3/10 30.00 22/66 33.33
Lymph node + 24/34 70.59 7/7 100.00 20/38 52.63
Lymph node - 10/34 29.41 0/7 0.00 18/38 47.37
<50 years 40/74 54.05 6/9 66.67 37/68 54.41
>50 34/74 45.83 3/9 33.33 31/68 45.59

IDC 61/78 78.21 10/10 100.00 54/68 79.41

ILC 5/78 6.41 0/10 0.00 4/68 5.88

MA 3/78 3.85 0/ 10 0.00 3/68 4.41
Medullary Ca 0/78 0.00 0/10 0.00 1/68 1.47
Intraductal Ca 3/78 3.85 0/10 0.00 3/68 4.41
in situ lobular 2/78 2.56 0/ 10 0.00 2/68 2.94
IPC 1/78 1.28 0/ 10 0.00 1/68 1.47

 

 

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer, ILC = infiltrating lobular cancer, Ca = cancer, MA = mucinous adenocarcinoma;
Intraductal Ca = intraductal carcinoma; in situ lobular = in situ lobular carcinoma; IPC = infiltrating papillary
carcinoma.

4.0.0 Molecular Analysis

88

4.1.0 HER/neu Gene Amplification Mutation

When tissues were examined for HER/neu (c-erbB-2) gene amplification, thirty-

nine specimens (29.10%) had HER/neu gene amplification equal to and/or greater than

two fold as detected by a differential PCR technique (Frye, 1989; Evers, 1992). Two

folds of HER/neu gene amplification was the most frequent gene amplification mutation

21/39 (53.85%) compared with 3 fold 7/39 (17.95%), 4 fold 5/39 (12.82%), and greater

than 4 fold 6/39 (15.38%). Relationships between HER/neu gene amplification rate and

clinical information is shown in the tables III-12.

Table III-12-l. Amplification of HER/neu gene mutation related to clinical information

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Normal rate 51 HER/neu rate 2 HER/neu rate 3 HER/neu rate 4 INF/neu rate >4

No. % No. % No. % No. % No. %

Total case 95/134 70.90 21/134 15.67 7/134 5.22 5/ 134 3.73 6/134 4.48
Grade 1 30/94 32.26 6/20 30.00 1/7 14.29 1/5 20.00 0/6 0.00
Grade 2 27/94 29.03 8/20 40.00 6/7 85.71 3/5 60.00 2/6 33.33
Grade 3 37/94 39.78 6/20 30.00 0/7 0.00 1/5 20.00 4/6 66.67
Mitosis 1 27/94 29.03 5/20 25.00 3/7 42.86 1/5 20.00 0/6 0.00
Mitosis 2 27/94 29.03 7/20 35.00 3/7 42.86 3/5 60.00 2/6 33.33
Mitosis 3 40/94 43.01 8/20 40.00 1/7 14.29 1/5 20.00 4/6 66.67
Lymph Node + 33/60 55.00 7/ 12 58.33 6/6 100 2/2 100.0 4/5 80.00
Lymph Node - 27/60 45.00 5/ 12 41.67 0/6 0.00 0/2 0.00 1/5 20.00
<50 47/95 49.47 13/21 61.90 4/7 57.14 2/3 66.67 1/6 27.78
>50 48/95 50.53 8/21 38.10 3/7 42.86 1/3 33.33 5/6 83.33
IDC 82/95 86.32 17/21 80.95 7/7 100.0 5/5 100.0 6/6 100
ILC 5/95 5.26 2/21 9.52 0/7 0.00 0/5 0.00 0/6 0.00
MA 2/95 2.11 1/21 4.76 0/7 0.00 0/5 0.00 0/6 0.00
M04111“ c. 1/95 1.05 0/21 0.00 0/7 0.00 0/5 0.00 0/6 0.00
mm C‘ 2/95 2.11 0/21 0.00 0/7 0.00 0/5 0.00 0/6 0.00
M 3"" lobular 2/95 2. 1 1 1/21 4.76 0/7 0.00 0/5 0.00 0/6 0.00
IPC 1/95 1.05 0/21 0.00 0/7 0.00 0/5 0.00 0/6 0.00

 

 

 

 

 

 

 

 

 

 

 

IDC = infiltrating ductal cancer; ILC = infiltrating lobular cancer; MA = mucinous adenocarcinoma;
Intraductal Ca = intraductal carcinoma; in situ lobular = in situ lobular carcinoma; IPC = infiltrating
papillary carcinoma.

 

89

Table III-12-2. Amplification of HER/neu gene mutation related to clinical information

 

 

 

 

 

 

 

 

 

 

Age Cancer in Side of Breast Lymph Node Metastasis
<50 >50 Lefi Right + -
No % No % No % N0 % No % No %

TO“! M 70 52.24 64 47.76 32 55.17 26 44.83 52 61. 18 33 38.82
HER gene we“ 26 37.14 46 71.88 13 40.63 11 42.31 30 57.69 28 84.85
HER gene we 1 21 30.00 2 3.13 12 37.50 7 26.92 2 3.85 1 3.03
HER saw me 2 13 18.57 1 1.56 7 21.88 6 23.08 1 1.92 l 3.03
HERsencme3 4 5.71 l 1.56 0 0.00 1 3.85 2 3.85 1 3.03
HER gene we 4 4 5.71 4 6.25 0 0.00 l 3.85 4 7.69 0 0.00
HER gene W4 2 2.86 10 15.63 0 0.00 0 0.00 13 25.00 2 6.06

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P value 0.263 0.550 0.013

 

 

Fisher’s Exact tests are used for all P value calculations. P value is comparison of HER gene rate 51 and
22.

Table III-12-3. Amplification of HER/neu gene mutation related to clinical information

 

 

 

 

Cell Differentiation Cell Mitotic Rate
Well Moderate Poor Low Moderate High
No % No % No % No % No % No %
Totalcase 38 28.79 46 34.86 48 36.36 36 27.27 42 30.95 54 40.91

 

HERWWI 9 23.68 17 36.96 17 35.42 9 25.00 13 31.82 21 38.89

 

HERSWMU 21 55.26 10 21.74 20 41.67 18 50.00 14 33.33 19 35.19

 

HERsemmz 6 15.79 8 17.39 6 12.50 5 13.89 7 16.67 8 14.81

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

HER 86m rate 3 1 2.63 6 13.04 0 0.00 3 8.33 3 7.14 1 1.85
HER 8mm4 1 2.63 3 6.52 1 2.08 1 2.78 3 7.14 1 1.85
HER gene "194 0 0.00 2 4.35 4 8.33 0 0.00 2 4.76 4 7.41
P value # *0.062 “1.000 "*0767 "' 0.336 "1.000 "*0372
P value ## 0.066 — 0.484

 

P value is comparison of HER gene rate $1 and 22. # = Fisher’s Exact tests are used. ## = Chi-square
tests are used. "‘ = the result of compared with Well (Low) and Moderate groups; " = the result of
compared with Well (Low) and Poor (High) groups; ”* = the result of compared with Moderate and
Poor (High) groups; **“ = the result of compared with all groups.

How the amplification results for HER/neu gene mutations relate to clinical
indictors were analyzed by Chi-square test. The characteristics that define in the table
were not significantly related (P>0.05) except lymph node metastasis.

4.2.0 p53 Gene Mutations

A total of 4 cases (6.15%) from 65 samples were found to have p53 tumor

suppressor gene mutations in our study. The features of p53 gene mutation and the

mutations related to clinical and histopathological information are in the table III-13

 

90

Table III-13. p53 gene point mutation related to other information

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Case No. 43 57 73 80
Age 62 52 64 39
Sex female female female female
No. of lymph node metastasis 2 18 5 19
Type of breast cancer IDC IDC IDC IDC
Cell differentiation poor poor poor well
Cell mitotic rate high high high low
Survive year >5 years >2 years unknown 3 years
p53 immunostain - + - -
c—erbB-Z immunostain + + - +
Bel-2 immunostain + + - +
PIER/nggene amplification mutation + - - -
p53 gene mutation c-tt t-tc t-ta g-ta
p53 gene mutation location (codon) 151 14436 309 248
(exonS) (intron7) (exon8) (exon8)
Characteristic of Mutation transition, transition transversion transition
CpG site non-CpG srte
Amino acid sequence change Pro-*Ser - Cys->Ser Arg
(soc-3 tcc) (tgceagc) (agar
Types of mutations missense intron rrrissense silent

 

 

 

 

 

 

IDC = infiltrating ductal carcinoma

4.3.0 Correlation of Gene Mutation with Clinical Indicators

The relationship of p53 gene and HER/neu gene mutations with breast cancer

clinical indicators were recorded and showed on the tables III-14.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table III-14-1. 353 gene and HER/neu ene mutations related to clinical information

A e Ca in Breast Lymph Node

<50 >50 Left Ri t + -
N0 % No % No % No % No % No %

p53 ggne + 1 25.0 3 75-0 - - - l - 4 100 0 0.00
p53 gene - 37 60.7 24 39.3 23 57.5 17 J 42.5 18 72.0 7 28.0
P value 0.30 - 0.55
HER gene + 23 32.86 16 25.00 7 21.88 3 30.77 19 36.54 6 18.18
HER gene - 47 67.14 43 75.00 25 78.13 13 69.23 33 63.46 27 81.82
P value 0.346 0.550 0.089

 

 

 

 

Fisher’s Exact tests are used for all P value calculations. Ca = carcinoma.

 

91

Table III-14-2. p53 gene and HER/neu gene mutations related to clinical information

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cell Differentiation Cell Mitotic Rate
Well Moderate Poor Low Moderate Hi

No % No % No % No % No % No %
p53 gene + 1 0 3 1 0 3
p53 gene - 20 26 15 20 23 18
P value # 0.446’ 0.318“ 0062'” 0.477‘I 0.606“ 0.100‘”
P value #311 0.074 0.137
HER gene + 8 21.05 19 41.30 11 22.92 9 85.00 15 35.71 14 25.93
HER gene - 30 78.95 27 58.70 37 77.08 27 75.00 27 64.29 40 74.07
P value # 0.062‘ 0.077" 1.000‘" 0.336‘ 0.372” 1.000‘”
P value ## 0066”” 0_484aaaa

 

# Fisher’s Exact tests are used for all P value calculations. ## Chi-square tests are used for all P value
calculations. ‘ = the result of compared with Well (Low) and Moderate groups; " = the result of compared with
Well (Low) and Poor (High) groups; "" = the result of compared with Moderate and Poor (High) groups; ”“ = the
result of compared with all groups.

Table III-l4-3. Sum of the results of the gene mutations and clinical information

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

HER/neu gene Normal p53 gene with p53 gene with

“'1‘" "“P'ifi‘mlion HER/neu gene mutation no mutaion

(39/134) (95/134) (4/65) (61/65)

No. % No. % No. % No. %
Grade 1 8/38 20.5 30/94 31.9 1/4 25.0 20/61 32.8
Grade 2 19/38 50.0 27/94 28.7 0/4 0.00 26/61 42.6
Grade 3 11/38 29.0 37/94 39.4 3/4 75.0 15/61 24.6
Mitosis 1 9/38 23.7 27/94 28.7 1/4 25.0 20/61 32.8
Mitosis 2 15/38 39.5 27/94 28.7 0/4 0.00 23/61 37.7
Mitosis 3 14/38 36.8 40/94 42.6 3/4 75.0 18/61 29.5
Lymph Node + 21/27 77.8 33/60 55.0 4/4 100 18/46 39.1
Lymph Node - 6/27 22.2 27/60 45.0 0/4 0.00 2846 60.9
<50 23/39 59.0 47/95 49.5 1/4 25.0 37/58 63.8
>50 16/39 41.0 48/95 50.5 3/4 75.0 24/58 41.4
IDC 35/39 89.7 81/95 85.3 4/4 100 53/61 86.9
ILC 2/39 5.1 5/95 5.3 0.00 0.00 5/61 8.20
MA 1/39 2.6 2/95 2.1 0.00 0.00 1/61 1.64
in situ 1/39 2.6 1/95 1.1 0.00 0.00 0/61 0.00
Medullary Ca 0/39 0.00 0/95 0.00 0.00 0.00 0 0.00
Intraductal Ca 0/39 0.00 0/95 0.00 0.00 0.00 2/61 3.28

 

 

IDC = infiltrating ductal cancer, ILC = infiltrating lobular cancer; Ca = cancer; MA = mucinous
adenocarcinoma; in situ = in situ lobular carcinoma.

VI. DISCUSSION

Earlier studies reported that Asian countries had a lower morbidity rate of breast
cancer than Western countries. In particular, that patients with breast cancer in Asia have
better prognosis than in the USA (Sakamoto, 1979). However, considerable caution is
required when comparing Chinese and Western data through comparison of literature as
the diagnostic criteria used often are different. Therefore, in this present study, the most

commonly used criteria and methods are employed.

1.0.0 Comparison of Characteristics of High Risk (Western Countries) with Low
Risk (Chinese) Breast Cancer Populations

Although breast cancer is globally the most frequent malignancy in women,
international statistics reveal that the rates of breast cancer vary widely in different
geographical areas.

1.1.0 General Aspects of Breast Cancer
1.1.1 Age Difference

The most frequent age of patients with breast cancer in the USA is 40-50 years
(Potchen, 1993). The American Cancer Society’s Cancer Prevention Study reported:
21% of breast cancer cases occur in the 30-54 age group and 79% of cases in the 55-84
age group (Seidman, 1982). Mettlin’s (1994) study showed that only 4.8% of breast
cancer in the United States occurred in women with the age of 30 —39, 17.8% with in ages
40-49, 23.8% of ages 50-59, 26.3% in ages 60 ~70; and 2.66% in women older than 70;
this report thus indicates that 95% of all breast cancer was found in women in the US who
were older than 40. In the Leis’s report, only 1.8% of breast cancers occur below the age

of 30 and 4% below the age of 35 with 75% of breast cancers appearing after the age of

92

93

40 (Leis, 1970).

From the records of our 178 primary breast cancer patients, the mean and median
ages of Chinese breast cancer patients is younger, than the findings from the Michigan
State University (MSU) data bank (data not shown) and from that reviewed in the
literature. In addition, the Student t test confirmed that there is a statistically significant
difference in age distribution between Chinese primary breast cancer group and MSU
group (personal observation). However, the obvious differences between these groups
make such comparisons difficult, for example the patients from MSU group have non-
palpable lesions and are in early stage of breast cancer; whereas the patients from Chinese
group all have palpable lesions with tumor sizes fiom 1.3 to 12 cm (average size 4.5 cm),
some with lymph node metastases; therefore they are at a later stage than those in MSU
group.

It is clear from our results and observations that primary breast cancer occurs
earlier in Chinese women than in the Western women. Reasons for this are undoubtedly
complicated, and probably include the environment, life style, as well as genetic reasons.
Further study and investigation of a larger sample size are needed to confirm these current
findings.

Some factors may be similar between Western and Chinese women. The peak age
distribution in both Chinese and the USA groups is in the over 40 years olds, which
suggests that breast cancer may occur by 40 years or older that not only in the Western
population, but also in the Chinese. By the age of 40 menopausal influences is a major

involvement, supporting the contention that female hormones may play important roles in

94

both populations with regards oncogenesis.
1.1.2 Size of Tumor

Tumor size is one of the most important indicators for evaluating breast cancer
development. The breast cancer specimens from China were all from breast surgical
operations where the tumors were palpable masses. A 3 cm sized breast tumor is the most
often detected size of tumor in physical examinations and this has been reported by
Robbins (1994), Tavassoli (1990) and Lagios (1982) which similar to our study.
Mammography screening has been widely used in the West for patients over 40 years old,
and this has decreased the detectable size of breast cancer dramatically with many more in
situ breast carcinomas detected than occurs in China.
1.1.3 Survival Rate

Only some of the Chinese breast cancer patients in this current study had followed
up investigations involving more than five years. The Life Table analysis, a method for
reporting clinical trials and presenting data from comprehensive registries, and Chi-square
tests are both used for our data analyses (Mueller, 1994). The general five-year survival
rate in our Chinese group is 47.33%. Others report breast cancer five-year survival rate in
Chinese populations as 58.1% (826 cases) (Liu, 1992), 59% and 47.6% (626 cases)
(Fisher, 1978). In the USA, the survival rates on primary breast cancer are 40.1% (1,335
cases) and 44% (1,700 cases). Rates in Southeast England were 42% (16,516 cases) and
43% (1,045 cases) (Mueller, 1994). The Mann-Whitney Rank Sum test analysis showed
there is a statistically significant difference in survival rate between Chinese and Western

breast cancer patients (P = 0.0286).

95

As previous stated, patients with breast cancer in Asia are known to have a better
prognosis than that of the patients in the USA. In our present study, this figure seems to
indicate that the Chinese have a higher five-year survival rate. However, the method of
analysis, the size of the samples, the method of treatment and the other factors may effect
the final result. The approach taken in our study will hopefully enrich knowledge of the
breast cancer, and will serve as a reference for the breast cancer research and investigation
of the future.

Analysis of our results showed that the patient’s age does not effect her survival
chance from breast cancer. Whether a patient has breast cancer at 20, or 40, or at 60
years of age does not appear to influence the patient's subsequent survival time. Support
for this contention exists in the literature as to the age of breast cancer patients not being a
good clinical prognostic indicator, as has be reported in the West for breast cancer in
women (Potchen, 1993; Leis, 1970; Seidman, 1982; Mettlin, 1994). In summary, the age
of breast cancer patient can not be used as a prognostic indicator in the Western or in the
Chinese populations.

We also analyzed the effect of lymph node metastases from a primary breast cancer
on patient survival rate. As expected, there was a significant difference between the
primary breast cancer patients with and without lymph node metastases. The primary
breast cancer patient with positive lymph node metastasis had a poor prognosis when
compared with lymph node negative breast cancer patients. These results support the
concept that lymph node metastases in primary breast cancer patients can be used as a

general prognostic indicator for different racial groups, and a fact well accepted in the

96

West (Tavassoli, 1992).

In our study, the sample size of the patients with primary breast carcinoma and a
five-year survival rate are too small to be fully analyzed. Only 3 cases of well
differentiated, 2 of moderately differentiated, and 3 cases of poorly differentiated group
were recorded for their five-year survival rate. For same reason the influence of the type
of breast carcinoma on five-year survival rate, with only 17 cases of infiltrating ductal
carcinoma, can not be adequately assessed.

1.2.0 Histopathology of Breast Cancer

In our study, one hundred seventy-eight primary breast cancer cases were
evaluated for the type of breast cancer, the cell mitotic rate and the degree of cell
differentiation and compared with Western breast cancer patients.

1.2.1 Types of Breast Cancer

The type of breast carcinoma is regarded as another important prognostic
indicator. It can divide breast cancers into favorable or unfavorable types in relation to
progression. The classifications of breast cancer in various countries used are not exactly
same, even though WHO has a standard classification for breast tumors. The variety of
breast cancer classifications and sampling methods used by different groups makes
comparing diagnostic data between Chinese and Western breast cancer groups somewhat
difficult. Thus we only used direct comparison for this part of study and those which
followed WHO guide lines.

The frequencies of histological types of invasive breast cancer in three large series

in the Western woman are showed in the table IV-l.

97

Table IV-l. The frequency of breast cancer es in the USA

 

 

 

 

 

 

 

Types of Breast Cancer Fisher Bartow Rosen
(unknown No.
(1,000 cases) of cases) (1024 cases)

Infiltrating ductal cancer 81.0 % 52.6% 66.0 %
Infiltrating lobular cancer 5.00 % 4.90% 6.48%
Medullary carcinoma 6.00 % 6.20% 5.66%
Mucinous carcinoma 2.00% 2.40% -
Tubular carcinoma 1.00 % - -
Others 5.00 % 5.90% 21.86%

 

 

 

 

 

 

(Fisher, 1975; Bartow, 1988; Rosen, 1982).

Invasive, or infiltrating, ductal carcinoma is by far the most common form of breast
cancer in the world when considering all sources of data: for Chinese it is 85.96%, and for
Western women reports range between 81% and 52.6%, with an average of 70%. There
is apparently no racial influence on the type of tumor which is the most common.
Infiltrating lobular carcinoma is the second most common type of breast cancer found by
most observers, with all other types of breast cancer considerably less common. These
studies demonstrated that racial grouping probably dose not influence the frequency of
primary breast cancer types. In addition, when comparing the differences between these
two racial groups, the average percentage of the infiltrating ductal cancers in Chinese and
Western women by Chi-square test, had probability values with a wide range (0.098 to
0.0013). Comparison of the averages from these groups shows a statistically significant
difl’erence between the Chinese and the Western women (P = 0.0454). Furthermore, fiom
our results, it is suggested that Chinese have a higher fi'equency of infiltrating ductal

cancer than do the Western group.

98

1.2.2 Breast Cancer Histological Grading

The grade of a breast cancer is another important prognostic indicator though it is

not usually used as a parameter in staging a tumor. The results from other Chinese

investigators and from USA studies are summarized in table IV-2.

Table IV-2. The frequency of breast cancer grades

 

 

 

 

 

 

 

 

 

Cell MSU (134) Liu (826) Keshgegian (135) Fisher (1000)
Difl‘erentiation (Chinese) (Chinese) (Western) (Western)
Well 33.5% 35.2% 31.0% 2.3%
Moderately 35.8% 49.9% 48.0% 28.1%
Poorly 30.7% 14.9% 21.0% 69.0%

 

(Keshgegian, 1995; Fisher, 1975; Liu, 1992).

The results from different reports are quite variant for both Chinese women as well

as Western women, and statistical analysis shows that those groups are not a significantly

different (all P>0.05). In a recent report, interobserver agreement in histological grading

of breast cancer study has been investigated. Complete agreement in grade in different

studies range from 54 - 80 %. An adequate training as a pathologist, the use of optimally

fixed tissue, and adaptation of precise guidelines are, all useful quality guidelines for the

diagnostic histopathologist to pursue. Fortunately, statistical studies of the relationship

between grade and prognosis show a strong correlation, even with data collected from

many pathologists in the USA, and even when different grading systems are used without

standardized guidelines. However, it is inevitable that some disagreement in breast cancer

histological grading will occur among the individual pathologists. Special training

following proper guidelines for pathologists will be helpful for limiting the vary in the

breast cancer grading (Robbins, 1995; Davis, 1986; Elston, 1991; Henson, 1991).

 

99

1.3.0 Immunohistochemical Studies

Immunohistochemical methods were used to detect p53 mutated protein, HER/neu
(c—erbB-Z) onCOprotein, and Bc1-2 oncoprotein in our investigation. The results were
evaluated and a comparison made between Chinese breast cancer and reports of Western
breast cancer.
1.3.1 General Aspect

The positivity of the immunohistochenrical studies on protein overexpression from

our Chinese breast cancer tissues are in the similar range to that of the Western (table IV-

 

 

 

 

 

3).
Table IV-3. Comparison of difi’erent groups
Study MSU (134) Alsabeh“ Luna-More. Markss Ceccarelli‘
@hinese) (208) (33) (230) (291)
53 14.18% 52.7% 12.1% 24% 30.6%
c-erbB-2 23.13% 18. 9% 36. 3% 17% 31. 6%
Bel-2 66. 42% 79.3% 69. 6% 85. 9%

 

 

 

 

 

 

*The Western group (Alsabeh, 1996; Marks, 1995, Luna-More, 1996; Ceccarelli, 1995)

Overexpression of p53 mutated tumor suppressor protein was very common in

patients age of >50 regardless of the type of cancer. Similarly overexpression of p53 was

common in these with the types of infiltrating ductal and medullary breast cancers, with

the highest cell mitotic rate, with poor differentiation, and in the cancers with lymph node

metastases regardless of age. However the data was not clearly statistically significant

different and thus we report trend rather than a clear statistical differences.

HER/neu (c-erbB-Z) oncoprotein overexpression was present in around half of

both <50 and >50 year ie. apparent different with age. Overexpression was usual in those

tumors of the well differentiated cell group, with poorly and moderated differentiated cell

 

100

groups being sirrrilar (45%). Overexpression appear and increase with mitotic rate. The
most significant correlations were with stage of differentiation (P=0.027) and lymph node
metastases (P=0.005). Lymph node positivity was strongly related to the presence of
erbB2 positivity

Bel-2 oncoprotein expression was present in similar frequency in the breast cancer
patients age <50 and >50, with regard to breast cancer type the most commonly present in
infiltrating ductal cancer, and with other forms presenting positive much less common in
infiltrating lobular cancer, mucinous adenocarcinoma, intraductal carcinoma, in situ
lobular carcinoma, infiltrating papillary cancer, and medullary carcinoma. Bel-2 positivity
was with common in those with the moderate cell mitotic rate, poorly differentiation, and
the those cancers without lymph node metastasis (63.64%) compare with Bel-2 negativity
common with lymph node metastasis (62.50%). These results are not statistically
significant different between Bcl-2 oncoprotein expression positive and negative, however,
it gives us the useful information in breast cancer prognosis.

Results from different groups indicate that p53 protein overexpression occurs in
13% to 53% cases (Rosen, 1996a; Elledge, 1993). Several investigators failed to detect
any significant correlation with age (Isola, 1992; Rosen, 1995a,b; Silvestrini, 1993), tumor
size or stage (Isola, 1992; Rosen, 199Sa,b). p53 expression was more frequent in ER-
negative tumors (Isola, 1992; Rosen, 199Sa,b; Iwaya, 1991).

HER/neu protein overexpression is an established marker for aggressive breast
cancer in Western women (Van de Vijver, 1987; King, 1989; Adnane 1989). This is also

true for our Chinese breast cancer group as it was associated with poorly differentiated

101

cancer and lymph node metastasis.

Bel-2 protein overexpression was detected by an immunohistochemical method in
our study; the positive results seem to be lower than cases studied in the West (Leek,
1994; Vanhaesebroeck, 1993; Joensu, 1994; Carson, 1993). An explanation may be the
immuno-staining technique, itself, or perhaps racial difi‘erences could be in play. In
addition, our study shows that Bcl-2 immuno-stain positivity appears to correlate with the
poor prognostic indicator, lymph node metastasis, that has been reported in a study in
Spain (Sierra, 1995). In contrast many studies have indicated that Bcl-2 presence relates
to a good prognosis (Luna-More, 1996; Ceccarelli, 1995).

Protein overexpression by p53, c-erbB-Z and Bel-2 may not be of significant
benefit to the pathologist in clinical diagnosis if used individually. However, if used in
combinations they may have more value. They could be usefirl as an alternative method to
confirm those which are not “clean cut” breast cancer cases, especially where the breast
cancer cytological diagnosis (especially the needle biopsy) is becoming more and more
popular.

From our experience, it is clear that standardized criteria for classifying and
evaluating immunohistochemical results is urgently needed to allow comparison of various
studies of the irnmunohistochenrical studies today.

1.3.2 The Combination of Immunostaining Results

In our study, we compared the significance of associations between either all three

or just two of p53, HER/neu and Bel-2 protein overexpressions in each breast cancer case.

Each combination group appears to have its own specific associations.

102

1.3.2.1 The Effects of Combining Three Immunostaining Results

When combining three protein overexpressions. p53-lHER-chl-2+ was found to
the most frequent combination pattern; and p53+/HER+/Bcl-2- was the least frequent
combination pattern in breast cancer tissues (refer to the tables 111-10). The combination
of p53+lHER+/Bcl-2- pattern is the most common in breast cancer patient age of <50; and
p53+lHER+/Bcl-2+ pattern relate to shows with the highest percentage at breast cancer
patient age >50. p53+lHER+chl-2- and p53-/I-IER+/Bcl-2- patterns are with same the
highest percentage in the breast cancer cases with lymph node metastases, when p53-
/HER+/Bcl-2+ pattern is with the highest percentage in the breast cancer cases without
lymph node metastasis.

When all the three combinations are considered, the characteristics in the different
combination pattern are found in the breast cancer patient age, the lymph node metastasis,
and the feature of histopathology. All combination patterns were shown with the highest
percentage in infiltrating ductal carcinoma; the combinations of p53+/I-IER+/Bc1-2+,
p53+/HER+/Bcl-2-, p53+lHER-chl-2-, and p53-/HER+/Bcl-2- patterns are detected in
infiltrating ductal carcinoma type only. p53+lHER-chl-2+ is with the highest percentage
in medullary cancer; p53-lHER-chl-2- is with the highest percentage in infiltrating lobular
cancer; p53-lHER-chl-2+ is found with the highest percentage in mucinous
adenocarcinoma, intraductal carcinoma, in situ lobular carcinoma and infiltrating papillary
carcinoma.

The characteristics of the histopathological evaluations, p53+lHER-chl-2+ is

with the highest percentage in the cell high mitotic rate, while p53+lHER+chl-2- shown

103

with the highest percentage in the low mitotic rate; p53+/I-IER+/Bcl-2+ and p53+/HER-
/Bcl-2+ showing with same the highest percentage in cell poorly differentiated group, and
p53+/I{ER+/Bcl-2- is with the highest percentage in cell well difi‘erentiated group.

The information of the combination of p53, HER/neu, and Bel-2 protein
overexpression correlated with clinical, histopathological features, and lymph node
metastases are not available in literature for the different population comparison.
1.3.2.2 The Effects 01' Combining Two Immunostaining Results

With the combining two of p53, HER/neu and Bcl-2 protein overexpression in the
breast cancer are records. p53-/HER- pattern is the most frequent combination, and
p53+lI-IER— pattern is least frequent combination in our present study.

Every combination of staining was seen, usually as the highest percentage, in
infiltrating ductal carcinomas; indeed the combination patterns of p53+lHER+, p53-
/HER+, p53+chl-2-, and HER+/Bcl-2- were detected in infiltrating ductal carcinoma type
only. The highest percentage of p53+ll-IER- showed in medullary cancer and in situ
lobular carcinoma; HER-chl-2+ was in infiltrating lobular cancer; and p53+lHER+ was
found within intraductal carcinoma, in situ lobular carcinoma and infiltrating papillary
carcinoma; and p53-IHER- was the most common in infiltrating papillary carcinomas.

With regards in the hisotopathological characteristics, p53+chl-2+ was highest
percentage in the group with the high cell mitotic rate, while p53+chl-2- was seen with a
low mitotic rate; p53+lI-IER- was associated with poor cell differentiation; and I-IER-chl-
2+ occurred with a high percentage of well differentiated cells.

The interaction of p53 and HER/neu has been extensively investigated, but

104

combination studies are not very common. Iwaya, et al reported that the majority of
tumors were p53-lHER-, and that the least frequent combination was p53 +/HER+.
Combinations of p53-/HER+ and p53+lHER- occurred with intermediate frequency
(Iwaya, 1991); our data essentially showed the same results as the Iwaya group.
Combined expression of p53 and HER was significantly associated with the histological
characteristics of the carcinomas in the USA (Rosen, 19958,b) and that was true also in
the most cases in our studies. In addition, medullary carcinomas are almost always p53
+/HER- and very rarely p53- /HER+ in the Western group as well as in Chinese;
infiltrating lobular carcinomas tend to be p53-lHER-.

The joint expression of p53 and HER was not found by others to be significantly
related to prognosis in the USA; patients with the favorable stage, T1NOM0 and
T2NOM0, were found to have a immunostain profile of p53+/I-IER+ (Rosen, 1995a,b,
1996a). Our study does not have enough information for such comparison.

1.4.0 Gene Mutation Studies

Genetic change is essential to the role of genotype and phenotype in process of
oncogenesis. p53 tumor suppressor gene point mutations and in HER/neu oncogene
amplification mutations are both investigated in this study.

1.4.1 HER/neu Gene Amplification

Several investigators have suggested that HER/neu (c-erbB-Z) amplification is an
independent variable predictive for shorter disease-free survival, high metastatic spread
and time to give remove all tissue in breast (Dickson, 1987). The results of the molecular

analysis of gene amplification procedures from different researches vary. The fi'equency of

105

gene amplification of HER/neu (c -erbB-2) averages 20-30% (Zhou, 1987; Clark, 1991;
Gasparini, 1992; Guerin, 1988; Rosen, 1995b; Slamon, 1987; van de Vijver, 1987; Varley,
1987; Marks, 1995) with a range of 9% (Digiovanna, 1996) to 52% (Tandon, 1989).

Clinical and pathological studies of the expression of the HER/neu oncogene in
human breast carcinomas have employed molecular and immunohistochemical procedures.
Molecular analysis of gene amplification offers a more quantitative approach, and there
appears to be a close correlation between amplification (reflected in increased copy
number) and HER/neu immunohistochempositivity (Borg, 1990; Tsuda, 1990).
Furthermore, the HER/neu gene amplification occurred in a higher frequency in primary
breast cancers (22.5%), and in those breast cancers with lymph node metastases (50%).
HER/neu gene amplification can thus be used as a molecular prognosis indicator for
cancer (Borresen 1990).

From the results of our study, the Chinese population is seen to be no exception
from other areas having a positive detection percentage in the same range of the USA and
another Asian group, the Japanese (Tsuda, 1990). HER/neu gene amplification mutation
tends to be more frequent in those whose age <50, with moderate differentiation, with
moderate mitotic rate, with positive lymph node metastasis that frequency is higher than
the Western group. However, the results are not strongly statistically significant different.

In addition, our study showed that HER/neu oncogene amplification mutation
cases correlate with immunohistochemical stain positivity. Our semiquantifying evaluation
of the immunohistocherrrical positivity (using HER/neu oncogene amplification >2 fold)

suggests that the higher fold the gene is amplified, the higher level of the protein is

106

expressed. Immunohistochemical studies of HER/neu protein expression can thus be used
to screen for HER/neu gene amplification. By the same token, the HER/neu gene
amplification can provide information on HER/neu oncoprotein expression.

HER/neu gene amplification is thought to be a poor prognosis for breast cancer
(Prost, 1994; Ravdin, 1995). It is our impression that HER/neu gene amplification can be
used as a molecular marker and an independent predictor for breast cancer management
and study. The number of amplifications of HER/neu gene seems to be not as important
as the gene amplification mutation itself. In the present study, there are no statistically
significant differences between different amplification rates (2 to >4 fold) for HER/neu
gene amplification it related to age 50, cell differentiation, cell mitotic rate and lymph node
metastasis.

In our study, there were more cases of HER/neu gene amplification mutation
recorded than HER/neu protein overexpression. There are at least two possible
explanations for this phenomenon. (1) the dPCR method is more sensitive in detection the
gene DNA amplification mutation than the immuno-stain method used to detect the
mutated protein; (2) there are other factors acting in the controlling mechanism pathway
of gene expression, as has suggested from in vitro study (Slamon, 1989).

1.4.2 p53 Gene Point Mutation

Mutations in the human tumor suppressor gene p53 have been found in most
human cancers. In breast cancer, the mutation in p53 have been proposed to occur in 16-
50% and appeared to be a strong indicator of poor prognosis, independent of other risk

factors (Callahan, 1992; Eytjord, 1992; Friedrichs, 1993). Missense mutations are the

107

most common finding and more than 90% of missense mutations are clustered in four
mutation “hot spot” regions (Pavletich, 1993).

In this study, p53 gene mutations have been detected in 4 cases (6%) of 65
Chinese primary breast cancer patients, a percentage which is lower than that in the USA.
Our group detected two missense, 1 intron and 1 silent mutations. Three mutations in
exons were associated with p53 immune-stain negativity, however, the mutation in intron
was associated with p53 immuno-stain positivity. Most breast cancer cases with p53 gene
mutations were found in age >50 (75%), with infiltrating ductal carcinoma (100%), lymph
node metastasis (100%), 7 5% with poorly differentiated cell and with a high cell mitotic
rate. p53 gene point mutation was not statistically associated with its protein
overexpression, lymph node metastasis, cell differentiation, and cell mitotic rate. A
possible explanation is that only a few cases with positivity of p53 gene mutation were

studied, and the sample size is too small to analyses.

In our study, the most common mutation type is the transition mutation (50%).
One case mutation being found in a “hot spot” (25%). One case mutation was found in
CpG site (25%), reportedly a less common mutation for Western women. One mutation
was found at base 14436 in intron 7, with the expected T but a novel C. The primers used
in our study were to detect exon mutation only, but with exon 8 primer also included an
end part of intron 7, fiom 14404 to 14452; SSCP and both direction DNA sequencing
confirmed this mutation. This intron mutation site has not been previously reported.
Intronic point mutation was noted in 1990, one study identifying p53 intronic mutation in

intron 3 where mutation inactivation of p53 tumor suppressor gene during cell

108

transformation in a cancer cell line occurred (Takahashi, 1990). Later, there were reports
of intron 3, 4, and 10 mutations in breast cancer (Sjalander, 1996; Wazer, 1994, Buller,
1995). Our finding indicates that for detection of p53 gene mutation, intronic sequencing

is also as important as exon sequencing.

2.0.0 Characteristics of p53, HER/neu (c-erbB-Z )and Bel-2 and Their Relationship
to Oncogenesis

2.1.0 General Concept of Oncogensis

Foulds (1954) codified and expanded the concept of multistage oncogenesis.
Evidence obtained twenty years after Rous’s work indicating that the first stage, initiation,
is characterized by damage to DNA, and showed that the second stage, promotion, usually
does not involve damage to the DNA, but rather a stimulation of cellular proliferation.
Prior to the promotional stage, the process is reversible and exhibits a distinct dose
response and a measurable threshold. The third stage, progression, leads to
morphological changes and increased grades of malignant behavior, such as invasion,
metastasis, and drug resistance (Robbins, 1994). Carcinogenic agents can mutate two
type of genes that regulate cell growth: proto-oncogenes that code for growth factors
(Hirsh-Ginsberg, 1995), and tumor suppressor genes which code for growth suppression
(Weinberg, 1991). Human carcinogenesis can involve several steps, including activation
of an oncogene, inactivation of a tumor suppressor gene, and abnormal activation of a cell
death suppressor gene.

The illustration below shows the contribution of genotype and phenotype to risk

determination for cancer. It reveals the primary importance of genotype, which influences

109

not only the characteristics of the tumor but also those of the host. It also indicates the
effect of the host on the tumor’s characteristics, the oncogene, tumor suppressor gene and
cell death suppressor gene mutations. The immune and endocrine systems are the most
likely to be important modulating mechanisms. The magnitude of risk could be assessed in
a more refined way either by a sophisticated integration of all known phenotypic
characteristics or, alternatively, by pinpointing the crucial locus in the DNA - either a
single gene of an unidentifiable factor which responsible for the phenotype. Integrated
knowledge and quantification of the phenotypic and genotypic characteristics may provide

the ultimate risk assessment (Ragaz and Ariel, 1989).

GENOTYPE PHENOTYPE

Immuno system
Endocrine system

 

Ploidy I
:GENES / HOST — mféiqsiific
Oncogene Proliferation
Tumor suppressor gene TUMOR Differentiation _ Pathology
Cell death suppressor gene \\ 13:33:38 /

TNM Stage
Kinetics (% S-Phase)
Oncoprotein overexpression

\ RISK//

 

Figure 11. Contribution of geneotype and phenotype to risk determination.
The characteristics of the genotype and the phenotype are major players. In the

understanding of the oncogenesis of breast cancer, behaviors of the p53 tumor suppressor

110

gene and HER/neu oncogenes, both genotype and phenotype, are investigated in this
study. The gene product overexpression of the proto-oncogene, HER/neu, the tumor
suppressor gene, p53; and the cell death suppressor gene, Bel-2, which is also called
prom-oncogene, are all investigated with our primary breast cancers. These elements
have been reported to involve the alteration of breast genotype and phenotype in many
complex ways. HER/neu acts as a transmembrane growth factor receptor pathway, whilst
p53 and Bel-2 regulate though the apoptosis pathway and they are affected and regulated
in many ways.
2.2.0 Our Findings in Oncogensis

Our studies produced some interesting phenomena which may help to understand
oncogenesis of breast cancer.
2.2.1 p53

The p53 mutated protein, the HER/neu (c-erbB-2) oncoprotein overexpression
without p53 tumor suppressor gene point mutation and HER/neu oncogene amplification
revealed in our study have been previously reported in human breast cancer as well as in
established mammary carcinoma cell lines (Guerin, 1988; Kraus, 1987; Elledge, 1993).
Many different mechanisms possibly explain the overexpression of a gene without gene
mutation, and which involve the regulation of gene expression at the transcriptional level.
p53 gene and HER/neu gene overexpression in the absence of gene point mutation or
amplification mutation were found in a few of our cases, suggesting that mechanisms other
than gene amplification or gene point mutation can contribute to gene overexpression. In

vitro studies, involving the use of a novel transcription factor OB2-1, was found to be

111

required for HER/neu overexpression in several breast cancer cell lines (Hollywood,
1993). Also, the gene promoter, the hormone regulator, and transforming grth factor
(TGF) are all involved in protein expression. The expression of human HER/neu gene has
been shown to be inhibited by estrogens (Read, 1990; Dati, 1990; Russell, 1992) and by
ligand-activated thyroid hormone and retinoic acid receptors (Hudson, 1990).

The intronic base mutation of p53 gene attracts attention recently. Our finding
indicates that the p53 gene mutation in the intron may be related to p53 protein
overexpression. It may serve as a marker of risk for malignancy or as a clinical prognosis
indicator is uncertain and needs more complete researches.

2.2.2 HER/neu

Two interesting phenomena were found in our study. (1) The immuno-stain
profile for HER/neu oncoprotein overexpression when compared with the cases that have,
or do not have, HER/neu gene amplification mutation showed that HER/neu gene
amplification was common in well and moderated cell differentiated groups, and when its
oncoprotein overexpression frequent was more in poorly differentiated group. (2) The
behaving of HER/neu gene amplification, by our record, suggests differently with protein
expression during the carcinogenesis. This characteristic, such as, HER/neu gene
amplification, is not correlated with other oncogenesis factors, such as p53 gene mutation,
p53 or Bcl-2 protein overexpression. The question is whether the HER/neu gene
amplification, which has a different mediate potential in HER/neu protein expression and
this specific potential could effect the cancer cell differentiation, or this just shows the

gene mutation present in early oncogenesis stage and the protein change in the later

112

cancergenesis stage. This information has not be reported before. (3) There is significant
correlation between the number of HER/neu gene amplifications and HER/neu
oncoprotein overexpression, indicating that with more copies of amplificated DNA
sequence, there is more oncoprotein expression in the cancer cells. In addition, the
number of HER/neu gene amplifications is not correlated with specific characteristics of
genotype and phenotype. The impression seems to be that overexpression protein level is
a more impotent change that occurs during the processing of breast oncogenesis.
However, the phenotypic HER/neu gene is associated with lymph node metastases and as
strong as the oncoprotein dose. This suggests that HER/neu gene mutation has the
metastatic potential in the breast cancer.
2.2.3 Correlation of p53, HER/neu and Bcl-2

The third interesting finding relates to the basic fact that breast cancer oncogenesis
has multiple elements involved and is a complicated process. Our consideration of joint
protein expressions of p53, HER/neu and Bcl-2 in breast cancer provides novel
information to help understand the mediating mechanisms. We found that p53, HER/neu
and Bel-2 are acting individually, and there are no apparent correlations among them.
Each combination pattern had its specific feature related to clinical, pathological, and
immunohistological evaluations. In the study of the combination of three immuno-stains,
the combinations p53+/I-lER+/Bcl-2+ and p53+lHER-/Bcl-2+ indicated a bad breast
cancer prognosis; with the cancer cells poorly differentiated, highest in mitotic rate, and
likely to metastasize to lymph nodes, as well as tending to be of the infiltrating ductal

cancer type. In the two immuno-stain combination, p53+lHER+ and p53+chl-2+ were

113

associated with the worse characteristics of poor differentiation, high cell mitotic rate, a
likelihood of lymph node metastasis and a tendency to be infiltrating ductal cancer. These
results with multiple immuno-stain studies indicate that abnormal or lack of action in the
regulation of the normal cell apoptosis pathway (p53 and Bel-2) and a transmembrane
growth factor receptor pathway ( HER/neu) have a strong potential for oncogenesis.
Features of multiple difference in breast phenotype from genotype are the worse patterns.
However, many breast cancer cases in our study show that there is no evidence of an
association with p53, HER/neu, or Bcl-2, either gene mutation or protein expression.
This phenomenon may suggest the specific factors which we detected and their pathways
are only involved in a particular period during oncogenesis procession. In addition, we
observed that p53-lHER-chl-2+ combination is the most fi'equent in our combinational
analysis. That seems to demonstrate that Bcl-2 may play an alternative rule during
oncogenesis; Bel-2 is down regulated by p53 during cell apoptosis (Diebold, 1996; Haldar,
1994). Therefore, the t(14)(18) traslocation mutation of the Bcl-2 gene, with its mutated
protein expression, seems to change more frequently during oncogenesis. That event may
indicate that beside cell differentiation, the cell apoptosis regulation is very important for
breast cancer oncogenesis.
2.2.4 The Feature of p53 Gene Point Mutation in Chinese Primary Breast Cancer
Finally, in our study 2 rrrissense, 1 intron, 1 silent mutation were detected. The
missense mutation is the most common mutation as reported in the USA. The intronic
point mutation was detected in intron 7 this phenotypic characteristic has not been

previously reported, even p53 gene mutation in intron 3, 4, 6 and 10in cancer that have

114

been reported in a few studies (Merlo, 1994; Takahasi, 1990; Sjalander, 1996). The
sequence features of the p53 gene point mutation found in case 80 which is an non-CpG
site mutation in the “hot spot”. By the contract, case 43, which is a CpG site, is not a
common “hot spot” of p53 gene mutation location. However, both of the mutation are in
the conserved domain area (Harris, 1996). An endogenous mutational mechanism due to
deamination of the methylcytosine could be the cause of this mutation (see figure 12)

(Antonarakis, 1996; Lehamn, 1994).

 

 

\. “”be wrf...
2° “" .../1° «L

Cytoatne 5- methytcytoalne Thymlne

Figure 12. Schematic representation of structure of cytosine, 5’-methylcytosine
and thymine, and the chemical events for the transformation of mutational mechanism for
cytosine to thymine due to methylation and deamination reactions.

Both cases were not associated with mutated p53 protein overexpression. There
could be three explanations: (1) gene mutation present earlier than protein does; (2)
protein overexpression not associated with gene mutation; (3) the pathway was
dysfunctional between gene mutation and mutated protein overexpression,.

Indeed, our data showing the differences and similarities between Chinese and

Western women in terms of their breast cancer are still early evaluation. This underscores

115

the need for more studies such as those with large sample sizes, more complete clinical
information, with long-term follow-up, in the near fiJture for a better understanding of
breast oncogenesis and the benefit for the breast cancer detection, diagnosis and treatment

of molecular techniques.

3.0.0 Clinical Approach

Since p53 and HER/neu (c-erbB-Z) protein overexpression occurs in less than
24% of our primary breast cancer specimens, and Bel-2 appears in a high percentage of
normal tissues, the immuno-staining can only be used to help pathologists in unclear
diagnostic cases. Positive staining of p53, HER/neu and Bel-2 can provide cancer
prognostic information for the clinical management. Since there is no correlation among
the overexpression of each protein that can be of benefit to pathologist on clinical
diagnosis for use of them individually as a immunohistochemical tumor markers; or to use
them in combinations to increase positive stain case numbers from p53’s 14. 18%,
HER/neu’s 23.13% up to 32.09% (which is 1.27 fold higher than us p53 immuno-stain
alone and 0.72 fold higher than by using HER/neu immuno-stain alone).

HER/neu gene amplification mutations detected by dPCR method provide more
positive finding of primary breast cancer cases than does immunohisto-staining methods.
It can thus be used as a molecular breast cancer marker for detecting and studying this
gene in the breast cancer and the other malignant diseases.

Today, clinical screenings are becoming very important part in breast cancer

management. It follows from 8 increasing number mammograms in breast cancer

116

screening, and the stereo-mammography guiding breast needle biopsy, that more cytology
samples are expected for the pathologists. With the breast cytology samples, the
pathological diagnosis is more difficult when compared with the surgical biopsy samples,
p53 and HER/neu can be used as the immunohistological marker, and HER/neu gene
amplification mutation could be used as the molecular marker to help the pathological
diagnosis.

Immune therapy and the gene therapy are novel treatments for the fight with the
malignant diseases. The immunochemical and the molecular tumor markers are very
important information for those therapies. p53 and HER/neu are the most common
factors studied and are already used on immuno therapy and gene therapy (Hung, 1995;
Roth, 1996). In a clinical or pathologic review, when a immunohistocherrrically positive
case shows, this case most likely also has the HER/neu gene amplification mutation, and
that can be a usefirl reference information for the gene therapy application in the firture.
Following the continuing maturity of those treatments, p53 and HER/neu genes and their

proteins will be very useful clinically.

4.0.0. Technique Approach in The Study

The various types of tissue preparation, the different antibodies, and the lack of
standardized criteria for classifying carcinomas by immunohistocherrristry evaluation,
makes comparisons very difficult.

For a large sample size breast cancer study, most specimens will probably be

paraffin embedded tissues. Preparations needs on these specimens can vary and some

117

specimens may for technical reasons not be able to be worked with. Therefore, for
immunohistochemical methods or molecular analysis, it is important to compare different
methods and select the most suitable one.

By using difl‘erential PCR to analyze HER/neu gene amplification mutations, each
PCR run and gel electrophoresis is compared with the normal controls and to known
positive control sample to provide a confident interpretation.

Interpretation of SSCP is often difficult without experience. From our
experiences, the pattern of denatured DNA bands are a major concerned for positive

results. Whereas, the band intensity is not important in gene mutation finding.

5.0.0 Conclusion

The genotypic and phenotypic characteristics of breast cancer in Western (high
risk) and Chinese (low risk) population are sirrrilar, such as, sex , tumor location of side
breast, size of palpable tumor, survival rate of lymph node metastasis, type of tumor,
grade of tumor, protein expression of p53 and HER/neu and HER/neu gene mutation.
There are however variations between Western and Chinese populations in terms of
phenotypic characteristics of breast cancer on age, survival rate, frequency distribution of
tumor types, Bel-2 protein expression, p53 gene point mutation.

Two missense, 1 intron and 1 silent, mutations of p53 gene were detected. p53
gene point mutation does not correlate with overexpression of its protein or clinical
indicators. p53 protein overexpression was detected in those samples with the highest
percentage of cell poor differentiation and cell mitotic rate, and had showed in infiltrating

ductal and medullary carcinomas. HER/neu gene amplification mutation is correlated with

118

HER/neu oncoprotein overexpression. HER/neu gene mutation tends to occur in
moderately differentiated tumors, and its oncoprotein tends to be over-expressed in the
group with poor cell differentiation. HER/neu gene amplification mutations occurred in
infiltrating ductal, infiltrating lobular carcinoma, mucinous adenocarcinoma as well as in
situ lobular carcinomas. HER/neu protein overexpression was detected in infiltrating
ductal carcinoma only. HER/neu gene amplification mutation and its product
overexpression is strongly correlated with lymph node metastasis. Bel-2 oncoprotein
overexpression is correlated with lymph node metastasis and was seen in infiltrating
ductal, infiltrating lobular, in situ lobular, medullary, intraductal carcinoma and mucinous
adenocarcinomas. p53, HER/neu and Bel-2 protein overexpressions are not correlated to
each other in breast cancer tissues.

p53-lHER-/Bc12+ and p53-IHER- are the most frequent combination patterns;
p53+lHER+/Bcl-2- and p53+/HER- are the least frequent combination patterns.
p53+lHER+/Bcl-2+ , p53+lHER-chl-2-, p53+/HER+ and p53+chl-2+ were associated
with the poor in breast cancer prognosis and considered as a “bad indicator” for a breast

cancer patient

V. REFERENCE

Addison C, Jenkins JR, Sturzbecher HW: 1990. The p53 nuclear localization signal is
structurally linked to a p34cdc2 kinase mortification. Oncogene 52423-426.

Adnane J, Gaudray P, Simon M, et a1: 1989. Proto-oncogene amplification and human
breast tumor phenotype. Oncogene 4: 1389-1395.

Aguilar F, Hussain SP, Cerutti P: 1993. Aflatoxin Bl induces the transversion of G—9T
in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proceedings of
National Academy of Science of the United States of A merica 90:8586-8590.

Akiyama T, Sudo C, Ogawara H, et al: 1986. The product of the human c-erbB-Z gene:
a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 232: 1644-1646.

Ali IU, Campbell G, Libereau R: 1988. Lack of evidence for the prognostic significance
of c—erbB-2 amplification in human breast carcinoma. Oncogene Research 3: 139-146.

Allan S, Dean D, Fernando I, et al: 1993. Radioimmunolocalisation in breast cancer
using the gene product of c-erbBZ as the target antigen. British Journal of Cancer
67 :706-712.

Almoguera C, Shibata D, Forrester K, et a1: 1988. Most human carcinomas of the
exocrine pancreas contain mutant c-K-ras genes. Cell 53:549-554.

Alsabeh R, Wilson CS, Ahn CW, et al: 1996. Expression of bcl-2 by breast cancer: a
possible diagnostic application. Modern Pathology 9:439-444.

American Cancer Society: 1975. A symposium sponsored by the American Cancer
Society and the National Cancer Institute on Nutrition in the Causation of Cancer.
Cancer Research 35 :323 1-3 543.

American Cancer Society, Silverberg E, Lubera A: 1988. Cancer statistics. CA: A
Cancer Journal for Clinicians 3825-22.

Anderson DE, Badzioch MD: 1986. Survival in familial breast cancer patients. Cancer
58:360-365.

Anderson DE: 1977. Breast cancer in families. Cancer 40:1855-1860.

Andoh Y, Kimijima I, Takita K, et a1: 1991. Flow cytometric DNA analysis using fine
needle aspiration sample in breast cancer. Journal of Japan Surgical society 92:228-232.

119

120

Antonarakis SE: 1996. Mutations in human disease: nature and consequences. m
Principles and Practice of Medical Genetics Third Edition. Edited by Rimoin DL,
Connor JM and Pyeritz RE. Churchill Livingstone. pp53-66.

Antoniades HN, Bravo MA, Galanopoulos T, et al: 1990. Platelet-derived growth factor
in idiopathic pulmonaryfibrosis. Journal of Clinic Investigation 86: 1055-1064.

Amritage P, Gerry G: 1994. Probability. I_n Statistical Methods in Medical Research
Edited by Armitage P and Berry G. Third Deition. Blackwell Scientific Publications.

pp41-76.

Arends MJ, Wyllie AH: 1991. Apoptosis: mechanisms an roles in pathology.
International Review of Experimental Pathology 32:223 -254.

Ashton PR, Hollingsworth AS Jr, Johnston WW: 1975. The cytopathology of metastatic
breast cancer. Acta C ytologica 19:1-6.

Azavedo E, Svane G, Auer G: 1989. Stereotactic fine-needle biopsy in 2294
mammographically detected nonpalpable lesions. Lancet 1:1033-1036.

Azzopardi JG, Chepick OF, Hartmann WH et al: 1982. The World Health Organization
histological typing of breast tumors-second edition. American Journal of Clinical
Pathology 78:806-816.

Bafy G, Miyashita T, Williamson JR, et al: 1993. Apoptosis induced by with draw] of
interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with
repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein
production. Journal of Biologic Chemistry 286:6511-6519.

Bair B and Caylefl‘ S: 1993. Part 4 Breast cancer: images of self. I_n Wings of Gauze
Women of Color an the Experience of Health and Illness. Edited by Bair, Barbara and
Cayleff, Susan E. Wayne State University Press. Detroit. pp241-243.

Baker SJ, Preisinger AC, Jessup JM, et al: 1990. P53 gene mutations occur in
combination with 17 p allelic deletions as late events in colorectal tumorgenesis. Cancer
Research 50:7717-7722.

Baker LB: 1982. Breast cancer detection demonstration project: five-year summary
report. Cancer 32:196-225.

Barbareschi M, Leonardi E, Mauri FA, et al: 1992. P53 and erbB-2 protein expression in
breast carcinomas. American Journal of Clinical Pathology 98:408-418.

121

Barbieri F, Parodi G, Massa T, et al: 1994. Feasibility of thymidine labeling index on fine
needle aspirates from breast cancer: comparison with surgical samples. Anticancer
Researches 14(5A): 1973-1977.

Barry MA, Behnke CA, Eastman A: 1990. Activation of programmed cell death
(apoptosis) by cisplatin, other anticancer drugs, toxins and hypertherrnia. Biochemical
Pharmacology 40:23 53-2362.

Bargmann CI, Hung M-C, Weinberg RA: 1986. The neu oncogene encodes an epidermal
grth factor receptor-related protein. Nature 319:226-230.

Bargmann CI, Weinberg RA: 1988a. Increased tyrosine kinase activity associated with
the protein encoded by the activated neu oncogene. Proceedings of National Academy of
Science of the United States of America 85:5394-5398.

Bargmann CI, Weinberg RA: 1988b. Oncogenic activation of the neu-encoded receptor
protein by point mutation and deletion Engineering in Medicine and Biology Journal
7:2043-2052.

Bargmann CI, Hung M-C, Weinberg RA: 1986. The neu oncogene encodes an epidermal
grth factor receptor-related protein. Nature 319:226—230.

Bartow SA and F enoglio-Preiser C: 1988. The breast. I_n Pathology Edited by Rubin E
and Farber JL. J .B Lippincott Company Philadelphia pp990-1013.

Basset-Seguin W, Escot C, Moles JP, et a1: 1991. c-fos and c-jun proto-oncogene
expression is decreased in psoriasis: a in situ quantitative analysis. Journal of
Investigation of Dermatology 97:672-678.

Bedrossian CWM: 1991a. Epithelial Malignancies. I_nMaIignant Eflusions A
Multimodal Approach to C ytologic Diagnosis Edited by Bedrossian, C.W.M. IGAKU-
SHOIN Medical Publishers, Inc. New York. pp120-147.

Bedrossian CWM: 1991b. In search of the primary site. I_n Malignant Effiisions A
Multimodal Approach to Cytologic Diagnosis Edited by Bedrossian, C.W.M. IGAKU-
SHOIN Medical Publishers, Inc. New York. pp190-210.

Berchuck A. Kamel A, Whitaker R, et a1: 1990. Overexpression of HER/neu is
associated with poor survival in advanced epithelial ovarian cancer. Cancer Research
50:4187-4091.

Berg FM, Baas IO, Polak 00, et al: 1993. Detection of p53 overexpression in routinely
paraffin-embedded tissue of human carScinomas using a novel target unmasking fluid.
American Journal of Pathology 142:381-385.

122

Berg JW: 1984. Clinical implications of risk factors for breast cancer. Cancer 53 :589-
591.

Bemdt H, Titze U: 1969. TNM clinical stage classification of breast cancer.
International Journal of Cancer 4:83 7-844.

Betta PG, Bottero G, Pavesi M, et al: 1993. Cell proliferation in breast carcinoma
assessed by a PCNA grading system and its relation to other prognostic variables.
Surgical Oncology 2:59-63.

Binder C, Marx D, Binder L, et al: 1996. Expression of Bzx in relation to Bcl-2 and
other predictive parameters in breast cancer. Annals of Oncology 7: 129-133.

Binder M., Tourrnente S, et al: 1986. in situ hybridization at the electron microscope
level: localization of transcripts on ultrathin sections of Lowicry K4M-embedded tissue
using biotinylated probes and protein A-Gold complexes. Journal of Cell Biology
102:1646-1653.

Birch JM, Horrley AL, Tricker KJ, et a1: 1994. Prevalence and diversity of constitutional
mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Research 5421298-
1304.

Birch JM: 1990. The Li-Fraumeni cancer family syndrome. Journal of Pathology 161:1-
2.

Bishop JM: 1983. Cellular oncogenes and retroviruses. Annals Review of Biochemistry
52:301-3 54.

Bishop JM.: 1991. Molecular themes in oncogenesis. Cell 64:235-248.

Bissonnette RP, Echeveni F, Mahboubi A: 1992. Apoptotic cell death induced by c-myc
is inhibited by Bel-2. Nature 359:552-556.

Blaszyk H, Vaughn CB, Hartmann A, et al: 1994. Novel pattern of p53 gene mutations
in an American black cohort with high mortality fiom breast cancer. Lancet 343: 1 195-
l 197.

Bonadonna G, Valagussa P: 1981. Dose-response effect of adjunct chemotherapy in
breast cancer. New England Journal of Medicine 304: 1-15.

Bonnier P; Charpin C; Lejeune C, et al: 1995. Inflammatory carcinomas of the breast: a
clinical, pathological, or a clinical and pathological definition? International Journal of
Cancer 62:382-385.

123

Borg A, Tandon AK, Sigurdsson H, et al: 1990. HER/neu amplification predicts poor
survival in node-negative breast cancer. Cancer Research 50:4332-4337 .

Boring CC, Squires TS, Tong T: 1992. Cancer statistics. CA: A Cancer Journal for
Clinicians 42:19-38.

Boyd NF: 1987. The epidemiology of cancer: principles and methods. I_n The Basic
Science of Oncology. Edited by Hill RP, Tannock LF, Pergamon Books, Inc. pp1-4.

Borresen AL, Ottestad L, Gaustad A, et al: 1990. Amplification and protein over-
expression of the neu/HER-Z/c-erbB-Z protooncogene in human breast crcinomas:
relationship to loss of gene sequences on chromosome 17, family history ad prognosis.
British Journal of Cancer 622585-90.

Bragmann CI Hung M, Weinberg RA: 1986. Multiple independent activations of the neu
oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649-
657.

Brash DE, Rudolph JA, Simon JA, et a1: 1991. A role for sunlight in skin cancer: UV-
induced p53 mutations in squamous cell carcinoma. Proceedings of National Academy of
Science of the United States of America 88: 10120-10128.

Bressac B, Kew M, Wands J, et al: 1991. Selective G to T mutations of p53 gene in
hepatocellular carcinoma fi'om southern Afiica. Nature 350:429-431.

Callahan R, Cropp CS, Merlo GR, et al: 1992. Somatic mutations an dhuman breast
cancer. Cancer 69: 1582-1588.

Camplejohn RS: 1993a. A role for proliferative measurements in clinical oncology?
Annals of Oncology 4: 184-186.

Camplejohn RS, Brock A, Barnes DM, et al: 1993b. A novel proliferative marker: flow
cytometric assessment of staining in human breast carcinoma cells. British Journal
Cancer 67:657-662.

Camplejohn RS: 1992. Flow cytometry in clinical pathology. I_n Diagnostic Molecular
Pathology A Practical Approach volume 1. Edited by Herrington CS and McGee JO'D
IRL PRESS pp239-258.

Carson DA, Ribeiro JM: 1993. Apoptosis and disease. Lancet 34: 1251-1254.

Carter CL, Allen C, Henson DE: 1989. Relation of tumor size, lymph node status, and
survival in 24,740 breast cancer cases. Cancer 63: 181-187.

124

Carter D: 1990. Biopsy. I_n Interpretation of Breast Biopsy 2nd Edition Biopsy
Interpretation Series Edited by Carte D. Raven Press New York pp9-28.

Ceccarelli C, Santini D, Chieco P, et al: 1995. Multiple expression patterns of
biopathological markers in primary invasive breast cancer: a usefirl tool for elucidating its
biological behavior. Annals of Oncology 6:275-282.

Chang F, Syrjanen, Kurvinen K, et al: 1993. The p53 tumor suppressor gene as a
common cellular target in human carcinogenesis. American Journal of Gastroenterology
88:174-1186.

Chen YH, Dong JP, Zhang SJ: 1994. Detection of point mutation of p53 gene by non-
isotopic PCR-SSCP in parafiin-embedded breast cancer tissue. Chung Hua Ping Li Hsueh
Tsa Chih 23:327-329.

Chen RH, Fuggle SV: 1993. in situ cDNA polymerase chain reaction a novel technique
for detecting mRN A expression. American Journal of Pathology 143:1527-1534.

Chiou SK, Rao L, White B: 1994. Bcl-2 blocks p53-dependent apoptosis. Molecular
and Cellular Biology. 14:2556-2563.

Ciatto S, Biggeri A, Del Turco CR, et al: 1990. Risk of breast subsequent to proven
gross cystic disease. The European Journal of Cancer 26:555-557.

Clark GM, McGuire WL: 1991. Follow-up study of HER/neu amplification in primary
breast cancer. Cancer Research 51:1944-1948.

Cleary MI, Smith SD, Sklar J: 1986. Cloning and structural analysis of cDNAs for Bcl-2
and a hybrid Bcl-2/immunoglobulin transcript resulting from the t(14218) translocation.
Cell. 47:19-28.

Cohen RJ, Cooper K, Haffejee Z, et al: 1995. Immunohistochemical detection of
oncogene proteins and neuroendocrine differentiation in different stages of prostate
cancer. Pathology 27 :229-232.

Cole SPC, Bhardwaj G, Gerlach JH, et al: 1992. Overexpression of a transporter gene in
a multidrug-resistant human lung cancer cell line. Science 258: 1650-1654.

Coles C, Thompson A, Elder P, et al: 1990. Evidence implicating at least two genes on
chromosome 17p in breast carcinogenesis. Lancet 336:761-763.

Collins N, McManus R, Wooster R, et al: 1995. Consistent loss of the wild type allele in
breast cancers from a family linked to the BRCA2 gene on chromosome 13q12-13.
Oncogene 10: 1673-1675.

125

Cope F0, Wille JJ: 1991. Carcinogenesis and apoptosis: paradigms and paradoxes in cell
cycle and differentiation. hi. Apoptosis: The Molecular Basis of Cell Death. Edited by
Tome LD, Cope F0. Cold Spring Harbor Laboratory Press. pp61-86.

Corkill EM, Katz R: 1994. Immunocytochemical staining of c-erbB2 oncogene in fine-
needle aspirates of breast carcinoma: a comparison with tissue sections and other breast
cancer prognostic factors. Diagnosis of Cytopathologl 11:250-254.

Cotran RS, Kumar V, Ribbins SL: 1994. Chapter twenty-four. The breast. In Robbins,
Pathologic Basis of Disease, 5th Edition. Edited by Schoen FJ. W.B. Saunders Company
pp1089-1111.

Cotter TG, Glynn JM, Echeverri F, et al: 1992. The induction of apoptosis by
chemotherapeutic agents occurs in all phases of the cell cycle. Anticancer Research
12:773-780.

Coussens L, Yang-Feng TL, Liao YC, et al: 1985. Tyrosine kinase receptor with
extensive homology to EGF receptor shares chromosomal location with neu oncogene.
Science 230:1132-1139.

Crawford DC, Pim DC, Bulbrook RD: 1982. Detection of antibodies against the cellular
protein p53 in sera from patients with breast cancer. International Journal of Cancer.
30:403-408.

Cullen IS: 1895. A rapid method of making permanent specimens from frozen sections
by the use of formalin. Bull J Hopkins Hospital 6:67-73.

Danforth D, Lichter AS, Lippmann ME: 1988. The diagnosis of breast cancer I_n
Diagnosis and Management of Breast Cancer Edited by Lippmann ME, Lichter AS, and
Danflo D. WB Saunders, Philadelphia. pp50-94.

Dati C, Antoniotti S, Taverna D, et al: 1990. Inhibition of c-erbB-2 oncogene expression
by estrogens in human breast cancer cells. Oncogene 5: 1001-1006.

Davis BW, Gelber RD, Goldhirsch A, et al: 1986. Prognostic significance of tumor grade
in clinical trials of adjutant therapy for breast cancer with axillary lymph node metastasis.
Cancer 58:2662-2670.

de Graaf H, Willemse P, Ladde BE, et al: 1994. Evaluation of a cytological scoring
system for predicting histological grade and disease-free survival in primary breast cancer.
C ytopathology 51294-300.

126

de Moulin D: 1983. A short history of breast cancer. The Hague, Martinus Nijhoff,
pp99.

DeLellis RA, Stemberger LA, Mann RB, et al: 1979. Immunoperoxidase techniques in
diagnostic pathology. American Journal of Clinical Pathology 71 :83.

D’Emilia J, Bulovas K, D’Ercole K, et al: 1989. Expression of the c-erbB-2 gene
product (p185) at different stages of neoplastic progression in the colon. Oncogene
4: 1233-1239.

Dhingra K, Hittelman WN, Nortobagyi GH: 1995. Genetic changes in breast cancer -
consequences for therapy? Gene 159:59-63.

Dickson RB, Aitken S, Lippmen ME, et al: 1987. Part II: Peptide Growth Factors. Assay
of mitogen-induced effects on cellular thymidine incorporation. I_n Methods in
Enzymology Hormone Action. Edited by Barnes D, Sirbasku DA . New York, Academic
Press, pp327-430.

Diebold J, Baretton G, Felchner M, et al: 1996. Bcl-2 expression, p53 accumulation, and
apoptosis in ovarian carcinomas. American Journal of Clinical Pathology 1052341-349.

Digiovanna MP, Carter D, Flynn SD, et al: 1996. Functional assay for HER-2/neu
demonstrates active signalling in a minority of I-IER-2/neu-over expressing invasive human
breast tumours. British Journal of Cancer 74:802-806.

Diller L, Kassel J, Nelson CE, et al: 1990. PS3 function as a cell cycle control protein in
osteosarcomase. Molecular and Cellular Biology 10:5772-5781.

Dive C, Hickman JA: 1991. Drug-target interactions: only the first step in the
commitment to programmed cell death? British Journal of Cancer 64: 192-196.

Dmochowski L, Seman G, Gallager HS: 1969. Viruses as possible etiologic factors in
human breast cancer. Cancer 24: 1241-1249.

Dmochowski L: 1972. Viruses and breast cancer. Hospital Practice 7:73-81.

Doll R, Peto P: 1981. The causes of cancer in the United States today. Journal of the
National Cancer Institute 66: 1 192-1 196.

Donegan EL: 1977. The influence of untreated internal mammary metastases upon the
course of mammary cancer. Cancer 39:533-538.

Donehower LA, Harvey M, Slagle BL, et al: 1992. Mice deficient for p53 are
developmentally normal but susceptible to spontaneous tumors. Nature 356.215-221.

127

Douc-Rasy S, et al: 1994. Association of c-erbBZ-gene amplification with poor
prognosis in non-inflammatory breast carcinomas but not in carcinomas of the
inflammatory type. International Journal of Cancer 58:763-768.

Dowlatshahi K, Jokich PM, Schmidt R, et al: 1987. Cytologic diagnosis of occult breast
lesions using stereotaxic needle aspiration. A preliminary report. Archives of Surgery
122: 1343-1346.

Drebin AJ, Stern DF, Link VC, et a1 : 1984. Monoclonal antibodies identify a cell-surface
antigen associated with an activated cellular oncogene. Nature 321:545-548.

Dunn JE: 1977. Breast cancer among American Japanese in the San Francisco Bay area.
National Cancer Institute Manager 7: 157-166.

Dyson JED, Simmons DM, Daniel J, et al: 1986. Kinetic and physical studies of cell death
induced by chemotherapeutic agents or hyperthennia. Cell and Tissue Research 19:311-
324.

Elledge, RM, Fuqua SA, Clark GM, et al: 1993. The role and prognostic significance of
p53 gene alterations in breast cancer. Breast Cancer Research and Treatment 27:95-102.

Elliott J: 1822. Sarcomatous tumour of the breast successfully removed. London
Medicine Physical Journal 48:305-308.

Ellis RE, Yuan J, Horvitz HR: 1991. Mechanisms and functions of cell death. Annals
Review of Cell Biology, 7:663 -698.

Elston CW, Ellis 10: 1991. Pathological prognostic factors in breast cancer. I. The
value of histological grade in breast cancer: Experience from a large study with long-term
follow-up. Histopathology 19:403-410.

Elston CW: 1987. Grading of invasive carcinoma of the breast. I_n Diagnostic
Histopathology of the Breast. Edited by Page DL and Anderson TJ. Churchill
Livingstone. Edinburgh London Melbourne and New York. pp300-311.

Engell HC: 1955. Cancer cells in the circulating blood. Acta Chirurgiae Scandinavica
207: 1-70.

Ening MG, Munn RJ, Keeney M: 1978. Dietary fats and cancer trends-a critique.
Federation of Proceeds 37:2215-2220.

Evers BM, Rady PL, Tyring SK, et al: 1992. Amplification of the HER/neu
protooncogene in human endocrine tumors. Surgery 1122211-218

128

Eyfiord JE and Thorlacius S: 1992. Genetic changes in breast carcinomas in an Icelandic
population. Pharmacogenetics 22309-316.

Fanidi A, Harrington EA, Evan GI: 1992. Cooperative interaction between c-my and Bcl-
2 proto-oncogenes. Nature 359:554-556.

Farrow SN, White JHM, Martinou I, et al: 1995. Cloning of Bel-2 homologue by
interaction with adenovims ElB 19 K. Nature 374:731-733.

Fearon ER, Vogelstein B: 1990. A genetic model for colorectal tumorigenesis. Cell
61 :759-767.

Feldman PS, Covell JL: 1985. Technique and Interpretation. I_n Fine Needle Aspiration
Cytology and its Clinical Applications: Breast & Lung. American Society of Clinical
Pathologists Press. Chicago. pp25-44.

F eto M, Jaeger U, Hockett RD, et a1 : 1988. Alternative promoters and exons, somatic
mutation and deregulation of the Bcl-2-Ig firsion gene in lymphoma. Engineering in
Medicine and Biology Journal 7: 123-131.

Finlay CA: 1993. Chapter 17. Normal and malignant growth control of p53. In oncogene
and Tumor Suppressor genes in human malignancies. Edtied by Benz CC and Liu ET. By
Kluwer Academic Publisher: Boston. pp327-344.

Finlay C, Hinds P, Levine A: 1989. The p53 proto-oncogene can act as a suppressor of
transformation. Cell 57:1083-1093.

Finlay C, Hinds P, Tan T, et al: 1988. Activating mutations for transformation by p53
produce a gene product that forms an hsc70-p53 complex with an altered half-life.
Molecular and Cellular Biology 82531-539.

Fisher TC, Milner AE, Gregory ED et al: 1993. Bel-2 modulation of apoptosis induced
by anticancer drugs: resistance pathways. Cancer Research 53:3321-3326.

Fisher B: 1978. Cancer of the breast. In Cancer in China. Edited by Kaplan HS and
Tsuchitani PJ. ALAN R.LISS, INC. New York. pp143-147.

Fisher B, Slack N, Katrych D, et al: 1975. Ten year follow-up results of patients with
carcinoma of the breast in a cooperative clinical trial evaluating surgical adjutant
chemotherapy. Surgery, Gynecology and Obstetrics 140: 528-534.

Fisher B, Slack NH: 1970. Number of lymph modes examined and the prognosis of
breast carcinoma. Surgery, Gynecology and Obstetrics 131:79-88.

129

Fleming KA, Morey AL, Yap EPH: 1994. Genetic analysis of malignancy in
histopathological material using filter and in situ hybridization. h Molecular Biology in
Histopathology Edited by Crocker J. JOHN WILEY & SONS. pp15-3 8.

Fobbins CK: 1993. Neoplasia. I_n Pathology Basis of Disease. W.B.Saunders Company.
pp23 9-435.

Forrest SP: 1990. Questions of morbidity and cost. In Breast Cancer: the Decision to
Screen. Edited by Forrest SP. Nufiield Provincial Hospitals Trust London. pp147-178.

Foulds L: 1954. A Review. Cancer Research 14:327-339.

Friedman PN, Kern SE, Vogelstein B, et al: 1990. Wild-type, but not mutant, human p53
proteins inhibit the replication activities of simian virus 40 large tumor antigen.
Proceedings of National Academy of Science of the United States of America 87:9275-
9279.

Friedrichs K, Gluba S, Eidtmann H, et al: 1993. Overexpression of p53 and prognosis in
breast cancer. Cancer 72:3641-3647.

Frye RA, Benz CC, Liu E: 1989. Detection of amplified oncogenes by differential
polymerase chain reaction. Oncogene 421153-1157.

Fukushige SI, Matsubar KI, Yeshiva M, et al: 1986. Localization of novel v-erbB-2
related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer
cell line. Molecular and Cellular Biology 62955-058.

Fukushima T, Onda M, Abe R, et al: 1995. P53 mutations and overexpressions in
Japanese breast cancer. European Journal of Surgical Oncology 21:595-600.

Gan FY, Luk GD, Gesell MS: 1994. Nonradioactive in situ hybridization techniques for
routinely prepared pathology specimens and cultured cells. The Journal of
Histotechnology 17:3 13-3 19.

Gannon JV, Lane DP: 1987. PS3 and DNA polymerase alpha compete for binding to
SV40 T antigen. Nature. 329:456-458.

Garcia G, Dietrich M, Aapro M, et al: 1987. Genetic alterations of c-myc, cerbB-2, and
cHa-ras protooncogenes and clinical associations in human breast carcinomas. Cancer
Research 49:6675-6679.

Gasparini G, Gullick WJ, Bevilacqua P, et al: 1992. Human breast cancer. Prognostic
significance of the c-erbB-2 oncoprotein compared with epidermal growth factor receptor,
DNA ploidy, and conventional pathologic features. Journal of Clinical Oncology
102686-695.

130

Gee JM, Robertson JF, Ellis 10, et al: 1994. Immunocytochemical localization of Bel-2
protein in human breast cancers and its relationship to a series of prognostic markers and
response to endocrine therapy. International Journal of Cancer 592619-628.

Geneva, Wildenhain Y, Pawson T, Blackstein M, et al: 1990. p185”u is phosphorylated
on tyrosine in human primary breast tumors which overexpression neu/erbB-2. Oncogene
52879-883.

Geslien GE, Fisher JR, DeLaney C: 1985. Transillumination in breast cancer detection:
Screening failures and potential. American Journal of Radiology 144:691-622.

Ginsberg D, Mechta F, Yaniv M, et al: 1991. Wild-type p53 can down-modulate the
activity of various promoters. Proceedings of National Academy of Science of the United
States of America 88:9979-9983.

Goelz SE, Hamilton SR, VogelsteinB: 1985. Purification of DNA fi'om formaldehyde
fixed and paraflin embedded human tissue. Biochemistry and Biophysics Research
Communication 1 30: l 1 8-126.

Gold RH, Bassett LW, Kimme-Smith C: 1986. Breast imagining: state of the art.
Investigation of Radiology 21:298-304.

Gold RH, Bassett LW, Kimme-Smith C: 1987. Introduction to breast imaging: state of
the art and fixture directions. I_n Breast Cancer Detection: Mammography and Other
Methods in Breast Imaging. 2nd Edition. Edited by Bassett LW and Gold RH. Grunt &
Stratton, Inc. Harcourt Brace Jovanovich, Publishers Orlando, New York, San Diego,
London, San Francisco, Tokyo, Sydney, Toronto. ppl-S.

Goung XY, Wang BT, et al: 1993. Breast tumor. I_n Atlas of Practical Tumor
Histopathology (Chinese) AnHuei Science and Technology Press. pp288-321.

Graeber TG, Osmanian C, Jacks T, et al: 1996. Hypoxia-mediated selection of cells with
diminished apoptotic potential in solid tumours. Nature 379288-91.

Gray PW, Goeddel DV: 1982. Structure of the human immune interferon gene. Nature
298(5877):859-63.

Grim J, Deshane J, Feng M, et al: 1996. erbB-2 knockout employing an intracellular
single-chain antibody (st) accomplishes specific toxicity in erbB-2-expressing lung
cancer cells. American Journal of Respiration Cell Molecular Biology 15;348-3 S4.

Guerin M, Barrois M, Terrier MJ, et al: 1988. Overexpression of either c-myc or c-erbB-
2/neu photo-oncogenes in human breast carcinomas; correlation with poor prognosis.
Oncogene Research 3 221-3 1 .

131

Gunji I-I, Kharbanda S, Kufe D: 1991. Induction of inemucleosomal DNA fragmentation
in human myeloid leukemia cells by l-B- D-arabinofirranosylcytosine. Cancer Research
51:741-743.

Guo WD, Chow WH, Zheng W: 1994. Diet, serum markers and breast cancer
mortality in China. Japan Journal of Cancer Research 85:572-577.

Gusterson BA, Gelber RD, Goldhirsch A, et al: 1992. Prognostic importance of c-erbB-2
expression in breast cancer. Journal of Clinical Oncology 10: 1049-1056.

Haagensen CD: 1986. Diseases of the Breast, edition 3, Philadelphia, WB Saunders,
pp656.

Haldar S, Negrini M, Monne M, et al: 1994. Down-regulation of Bcl-2 by p53 in breast
cancer cells. Cancer Research 54:2059-2097.

Hale A], Smith CA, Sutherland LC, et al: 1996. Apoptosis: molecular regulation of cell
death. Europeanp Journal of Biochemistry 23621-26.

Hammond EH, Henson DE: 1995. The role of pathologists in cancer patient staging.
American Journal of Clinical Pathology June2679-680.

Hanada M, Delia D, Aiello A, et al 2 1993. Bel-2 gene hypomethylation and high-level
expression in B- cell chronic lymphocytic leukemia. Blood 82(6): 1820-1828.

Hann L, Ducatman BS, Wang HH, et al: 1989. Non-palpable breast lesions: Evaluation
by means of fine-needle aspiration cytology. Radiology 171:373-376.

Harmanek P, Sobin LH: 1987. TM! Classification of Malignant Tumors, 4th edition.
International Union against Cancer, Berlin.

Harmon BV, Takano YS, Winterford CM, et al: 1991 The role of apoptosis in human
and murine tumour cell lines: a study using electron microscopy and DNA gel
electrophoresis. Journal of Pathology 163:329-336.

Harmon BV, Corder AM, Collins RJ, et al: 1990. Cell death induced in a mourine
mastocytoma by 42-47°C heating in vitro: evidence that the form of death changes from
apoptosis to necrosis above a critical heat load. International Journal of Radiation
Biology 58:845-858.

Harmon BV: 1987. An ultrastuctureal study of spontaneous cell death in a mouse
mastocytoma with particular reference to dark cells. Journal of Pathology 153 2345-355.

132

Hanis CC, Hollstein M: 1993. Clinical implications of the p53 tumor-suppressor gene.
New England Medicine 329: 13 18-1327.

Hanis CC: 1996. p53 tumor suppressor gene: from the basic research laboratory to the
clinic - an abriged historical perspective. Carcinogenesis 1721187-1198.

Harris A: 1990. Mutant p53: The commonest genetic abnormality in human cancer?
Journal of Pathology 16225-6.

Harvey EB, Schairer C, Brinton LA, et al: 1987. Alcohol consumption and breast cancer.
The Journal of National Cancer Institute 78:65 7-661.

Hayes DF, Schnitt SJ: 1993. Risk factors, epidemiology, and development of breast
cancer. In Atlas of Breast Cancer. Edited by Hayes DF. Published by Mosby Europe
Limited London, St. Louis, Baltimore, Boston, Chicago, Philadelphia, Sudney, and
Toronto. pp2.2-2.9.

Hazan R, Margolis B, Domblagian M, et al: 1990. Identification of autophosphorylation
sites of HER/neu. Cell Growth and Differentiation 1:3-7.

He GH, et a1: 1990. Breast cancer. In Diagnosis and Treatment of Malignant Tumor
(Chinese) JingXi Science & Technology Press pp124-133.

Henson DE, Ries L, Freedman LS, et al: 1991. Relationship among outcome, stage of
disease and histologic grade for 22,616 cases of breast cancer. The basis for a prognostic
index. Cancer 68:2142-2149.

Henry J 2 1984. In The Edwin Smith Papyrus. The Classics of Medicine Library. p405.

Hiatt RA, Friedman GD, Bawol RD, et al: 1982. Breast cancer and serum cholesterol.
The Journal of the National Cancer Institute 60:955-960.

Hiatt RA, Friedman GD, Bawol RD, et al: 1982. Breast cancer and serum cholesterol.
Journal of National Cancer Institute 68: 885-9.

Higginson J, Muir CS, Munoz N2 1992a. Chemical factors. I_n Human Cancer:
Epidemiology and Environmental Causes. CAMBRIDGE UNIVERSITY PRESS New
York pp75-96.

Higginson J, Muir CS, Munoz N: 1992b. Breast and genitourinary system. In Human
Cancer: Epidemiology and Environmental Causes. CAMBRIDGE UNIVERSITY
PRESS New Yor pp375-397.

133

Hilkers J, Buijs F, Hilger J 2 1984. Monoclonal antibodies against human milk-fat globule
membranes detecting differentiation antigens of the mammary gland and its tumour.
International Journal of Cancer 34:197-206.

Hirsh-Ginsberg CF, Stass SA, and Freireich EJ 2 1995. Principles of Molecular Oncology.
I_n_ Molecular Basis of Oncology. Edited by Freireich EF, Stass SA. Blackwell Science.
pp1-21.

Hoare T: 1996. Breast screening and ethnic minorities. British Journal of Cancer
74 2 3 8-4 1 .

Hockenbery DM, Oltval ZN, Yin XM, et al: 1993. Bel-2 fimctions in an antioxidant
pathway to prevent apoptosis. Cell 75:241-251.

Hockenbery DM, ZutlerM, Hickey, et al: 1991. Bel-2 protein is topographically
restricted in tissues characterized by apoptotic cell death. Proceedings of National
Academy of Science of the United States of America 88:6961-6965.

Hockenbery D, Nunez G, Milliman C: 1990. Bel-2 is an inner mitochondrial membrane
protein that blocks programmed cell death. Nature, 348: 334-336.

Hollstein MC, Peri L, Mandard AM, et a1 2 1991a. Genetic analysis of human esophageal
tumors from two high incidence geographic areas: frequent p53 base substitutions and
absence of ras mutations. Cancer Research 51:4102-4106.

Hollstein M., Sidransky D, Vogelstein B, et al: 1991b. p53 mutations in human cancers.
Science 253249-53. '

Hollywood DP, Hurst HC: 1993. A novel transcription factor, OBZ-l, is required for
overexpression of the proto-oncogene ce-erbB-2 in mammary tumor lines. Engineering in
Medicine and Biology Journal 12:23 69-23 75.

Horowitz KB: 1975. Progesterone receptors and hormone dependent breast cancer.
Doctoral Dissertation. University of Texas Southwestern Medical School, Dallas.

Hou L, Shi D, Tu SM, et al: 1992. Oral cancer progression and c-erbB-Z/neu proto-
oncogene expression. Cancer Letter 652215-220.

Howell LP: 1995. The pathway to cancer. I_n A Practical Approach to Breast Disease
Edited by O'Grady LF, Lindfors KK, Howell LP, and Rippon MB. Little, Brown and
Company. Boston, New York, Toronto, London. pp23-39.

Hudson LG, Santon JB, Glass CK: 1990. Ligand-activated thyroid hormone and retinoic
acid receptors inhibit growth factor receptor promoter expression. Cell 62:1165-1175.

134

Huebner RJ, Todaro G] 2 1969. Oncogenes of RNA tumor viruses as determinants of
cancer. Proceedings of National Academy of Science of the United States of America
64:1087-1094.

Humphrey PA, Franquemont DW, et al: 1994. Applied Immunohistochemistry. 2:22-28.

Hung MC, Matin A, Zhang YJ, et a1: 1995. HER/neu-targeting gene therapy- a review.
Gene 159265-71.

Iqbal M], Taylor W: 1989. Hormonal and reproductive factors - new evidence. In
Women at High Risk to Breast Cancer. Eedited by Stoll BA. KLWER ACADEMIC
PUBLISHERS. Dordrecht, Boston, London. pp41-45.

Isola J, Visakorpi T, Holi K, et al: 1992. Association of overexpression of tumor
suppressor protein p53 with rapid cell proliferation and poor prognosis in node-negative
breast cancer patients. Journal of National Cancer Institute 8421109-1117.

Isola J, Visakorpi T, Holli K, et al: 1992. Association of overexpression of tumor
suppressor protein p53 with rapid cell proliferation and poor prognosis in node-negative
breast cancer patients. Journal of National Cancer Institute 84:1109-1114.

Iwaya K, Tsuda H, Hiraide G, et a1 : 1991. Nuclear p53 immunoreaction associated with
poor prognosis of breast cancer. Japanese Journal of Cancer Research 82:835-840.

Jacob TC, Spieth LE, Penn NE: 1993. Breast cancer, breast self-examination, and
African-American women. In Wings of Gauze Women of Color an the Experience of
Health and Illness. Edited by Bair B and Cayleff S. Wayne State University Press.
Detroit. pp244-265.

Jacobson MI), Bume JF, King MP et al: 1993. Bel-2 blocks apoptosis in cells lacking
mitochondrial DNA. Nature, 3612365-369.

Jigginson J, Muir CS, Munoz,N 1: 1992. Part X Breast and genitourinary system. 40
Breast I_n Human Cancer: Epidemiology and Environmental Causes. CAMBRIDGE
UNIVERSITY PRESS. Great Britain. pp377-387.

J oensu H, Pylkkanen L, Toikkanen S: 1994. Bel-2 protein expression and long-term
survival in breast cancer. American Journal of Pathology 145:1191-1198.

Johnston JB, Lee K, Verburg L, et al: 1992. Induction of apoptosis in CD4+
prolymphocytic leukemia by deoxyadenosine and 2’-deoxycoformycin. Leukemia
Research 162781-788.

Kern S, Pietenpol J, Thiagalingarn S, et al: 1992. Oncogenic forms of p53 inhibit p53-
regulated gene expression. Science 256:827-830.

135

Kerr JFR, Searle J : 1980. Apoptosis: its nature and kinetic role. In Radiation Biology in
Cancer Research. Edited by Meyn RE, Withers HR. Raven Press, New York 19802367-
384.

Kerr JFK, Wyllie AH, Currie AH: 1972a. Apoptosis, a basic biological phenomenon with
wider implications in tissue kinetics. British Journal of Cancer 26:23 9-245.

Kerr JFR, Searle J, Harmon BV, et al: 1987. Apoptosis. m Perspectives on Mammalian
Cell Death. Edited by Potten CS. Oxford: Oxford University Press 1987:93-128.

Kerr JFR, Searle J 2 1972b. A suggested explanation for the paradoxically slow grth
rate of basal-cell carcinomas that contain muerous mitotic figures. Journal of Pathology
107241-44.

Keshgegian AA, Cnaan A: 1995. Proliferation markers in breast carcinoma. American
Journal of Clinical Pathology 104242-49.

Keynes GL2 1932. The radium treatment of carcinoma of the breast. British Journal of
Surgery 19:415-480.

King CR, Kraus MH, Aaronson SA: 1985. Amplification of a novel v-erbB-related gene
in a human mammary carcinoma. Science 229:974-976.

King CR, Rwain SM, Porter L, et a1: 1989. Heterogeneous expression of erbB-2
messenger in human breast cancer. Cancer Research 4924185-4191.

King MC: 1990. Linkage of early-onset familial breast cancer to chromosome 17q21.
Science 25021684.

King MC: 1992. Breast cancer genes: How many, where, and who are they? Nature
Genetics 2: 125.

Kinzler KW, Vogelstein B: 1996. What’s mice got to do with it. Nature 382;670-671.

Kita Y, Tseng J, Horan T, et al: 1996. ErbB receptor activation , cell morphology
changes, and apoptosis induced by anti-Her2 monoclonal antibodies. Biochemical and
Biophysical Research Communications 226259-69.

Klavins JV 2 1985. Carcinomas of various organs and organ systems. In Tumor Markers
Clinical and Laboratory Studies Edited by Klavins, J .V. ALAN R. LISS INC. New
York. pp29-39.

Kleihues P, Schauble B, zur Hausen A, et al: 1997. Tumors associated with p53 gerrnline
mutations: a synopsis of 91 families. American Journal of Pathology 15021-13.

136

Klein PA, Smith RT: 1977. The role of oncogenic viruses in neoplasia. Annual Review
of Medicine 28:311-327.

Komminoth P2 1995. in situ gene amplification - theory and practice. In situ
Applications Guide. SHANDON LIPASHAW pp53-75.

Kovi J, Mohla S, Norris HJ, et al: 1989. Breast lesions in black women. Annual of
Pathology 24(pt 1): 199-218.

Krajewski S, Krajewska M, Shabaik A, et al: 1994. Immunohistochemical determination
of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. American Journal of
Pathology 14521323-1336.

Kraus MH, Popescu NC, Amsbaugh SC, et al: 1987. Overexpression of the EGF
receptor-relate proto-oncogene erbB-2 in human mammary tumor cell lines by different
molecular mechanisms. Engeming in Medicine and Biology Journal 62605-610.

Kress S, Sutter C, Strickland PT, et al: 1992. Carcinogen-specific mutational pattern in
the p53 gene in ultraviolet B radiation-induced squamous cell carcinomas of mouse skin.
Cancer Research 5226400-6403.

Kufe D, Inghirami G, Abe M, et al: 1984. Differential reactivity of a monoclonal
antibody (DF3) wit human malignant versus benign breast tumors. Hybridoma 3 2223-
232.

Kufe DW: 1988. Monoclonal antibody assays for breast cancer. In Cancer Diagnosis In
Vitro Using Monoclonal Antibodies. Edited by Kupchik HZ. MARCEL DEKKER, Inc.
New York and Basel. pp68-70.

Kute T, Shao Z, Keith Sugg N, et al: 1992. Cathepsin D as prognostic indicator for
node-negative breast cancer patients using both immunoassays and enzymatic assays.
Cancer Research 52:5198-5203.

Kyprianou N, Englis HF, Davidson NE, et al: 1991. Programmed cell death during
regression of the MCF-7 human breast cancer following estrogen ablation. Cancer
Research 512162-166.

Lagios MD, Westdahl PR Margolin FR, et al: 1982. Duct carcinoma in situ.
Relationship of extent of noninvasive disease to the frequency of occult invasion,

multicentricity. lymph node metastases and short-term treatment failures. Cancer
50:1309-1314.

Lane DP, Crawford LV: 1979. T antigen is bound to a host protein in SV 40-transformed
cells. Nature 2782261-263.

137

Lane DP: 1992. p53, guardian of the genome. Nature 358215-16.
Lane DP: 1993. A death in the life of p53. Nature 3622786—787.

Le Roy X Escot C, Brouillet JP, et al: 1991. Decrease of c-erbB-2 and c-myc RNA
levels in tamoxifen-treated breast cancer. Oncogene 62431-437.

Leach FX, Tokino T, Meltzer P, et al: 1993. PS3 mutation and MDM2 amplification
inhuman soft tissue sarcomas. Cancer Research. 53:2231-2234.

Lee ET-HP: 1991. Tumor suppressor genes: Anew era for molecular genetic studies of
cancer. Breast Cancer Research and Treatment 1923-13.

Leek RD, Kaklamanis L, Pezzella F, et al: 1994. Bel-2 in normal human breast and
carcinoma, association with oestrogen receptor-positive, epidermal growth factor
receptor-negative tumours and in situ cancer. British Journal of Cancer 692135-139.

Lehman TA, Greenblatt M, Bennett WP, et a1: 1994. Mutational spectrum of the p53
tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Drug
Metabolism Review 262221-23 5.

Leis HP Jr.2 1970. The pill and the breast. NY State Journal of Medicine 70:2911-2918.

Lerman C, Seay J, Balshem A, et al: 1995. Interest in genetic testing among first-degree
relatives of breast cancer patients. Amercan Joural of Medicine Genetics 572385-92

Levack PA, Mullen P, Anderson T], et al: 1987. British Journal of Cancer 562643.

Levine AJ, Momand J, Finlay CA: 1991. p53 tumour suppressor gene. Nature. 3512453-
456.

Lewis FA, Wells M: 1992. Detection of virus in infected human tissue by in situ
hybridization. In in situ Hybridization A Practical Approach. Edited by Wilkinson DG
IRL PRESS Oxford University Press Oxford New York Tokyo pp121-136.

Li FP, F raumeni JF Jr, Mulvihill JJ, et al: 1988. A cancer family syndrome in twenty-four
kindred. Cancer Research 48:5358-5362.

Li SL: 1994. Review of protection and treatment on breast cancer. In Studies of the
Research and Clinic Progresses of Malignant Tumors. Edited by Suan JK (Chinese)
China Medical University and Beijing Medical University Press. pp53-62.

138

Lin EY, Orlofsky A, Bergar M, et al: 1993. Characterization of A1, a novel hemopoietic-
specific early-response gene with sequence similarity to Bel-2. Journal of Immunology
15121979-1988.

Lindfors KK: 1995. Screening for breast cancer. I_n A Practical Approach to Breast
Disease Edited by O'Grady LF, Lindfors KK, Howell LP, and Rippon MB. Little, Brown
and Company. Boston, New York, Toronto, London. pp41-58.

Lipshaw S: 1995. Introduction In in situ Applications Guide. Edited by Lipshaw S.
handon, Inc. pp1-3.

Liu FS, Li X, Change X, et al: 1992. Pathological investigation of the breast cancer. The
monograph of International Meeting pp32-33.

Lodon SJ, Willet WC: 1992. Relationship to diet and body size. I_n Women at High Risk
to Breast Cancer edited by Stoll, B.A. KLUWER ACADEMIC PUBLISHERS.
Dordrecht, Boston, London. pp47-56.

Lofgren M, Andersson I, Bondeson L, et al: 1988. X—ray guided fine-needle aspiration for
the cytologic diagnosis of nonpalpable breast lesions. Cancer 61:1032-1037.

Lofgren M, Andersson I, Lindholm K, et al: 1990. Sterotactic fine-needle aspiration for
cytologic diagnosis of nonpalpable breast lesions. American Journal of Roentgenology
154:1191-1195.

Lohmann DR, F esseler B, Putz B, et al: 1993. Infrequent mutation of the p53 gene in
pulmonary carcinoid tumors. Cancer Research 53:5797-5801.

Lu QL, Abel P, Foster CS, et al: 1996. Bel-2: role in epithelial differentiation and
oncogenesis. Human of Pathology 272102-110.

Lu QL, Hanby AM, Hajibagheri MA, et al 2 1994. Bcl-2 protein localizes to the
chromosomes of mitotic nuclei and is correlated with the cell cycle in cultured epithelial
cell lines. Journal of Cell Sciences 107: 363-371.

Luna-More S, de los Santos F, Breton JJ, et al: 1996. Estrogen and progesterone
receptors, c-erbB-Z, p53, and BcI-2 in thirty-three invasive micrpapillary breast
carcinomas. Pathology, Research and Practice 192227-32.

Lupu R, Cardillo M, Cho C, et al: 1996. The significance of heregulin in breast cancer
tumor progression and drug resistance. Breast Cancer Research and Treatment 38257-
66.

Lux SE, Tse WT, et al: 1990. Hereditary spherocytosis associated with deletion of
human erythrocyte ankyn'n gene on chromosome 8. Nature 345:736-739.

139

Mackenzie CD: 1994. Molecular diagnosis of breast cancer. In Molecular Pathology
Course.

Macklis RM, Lin JY, Beresford B, et al: 1992. Cellular kinetics, dosimetry, and
radiobiology of m-particle radioimmunotherapy: induction of apoptosis. Radiation
Research 1302220-226.

MacMahon CE, Cahill JL: 1840. Practical Surgery. 3rd ed. London Churchill, pp336.

Malkin D: 1993. PS3 and the Li-Fraumeni syndrome. Cancer Genet Cytogenet. 66:83-
92.

Malkin D, Li FP, Strong LC, et al: 1990. Germ line p53 mutations in a familial syndrome
of breast cancer, sarcomas, and other neoplasms. Science 25021233-1238.

Mant D, Vessey MP: 1991. Section V Epidemiology and primary prevention of breast
cancer. I_n The Breast: Comprehensive Management of Benign and Malignant Diseases.
Edited by Bland KI, Copeland EMIII. W.B. Saunders Company. Philadelphia. pp235-
246.

Margolis BL, Lax 1, Kris R, et al: 1989. All autophosphorylation sites of epidermal
growth factor (EGF) receptor and her2/neu are located in their carboxyl-terminal tails.
Identification of a novel site in EGF receptor. Journal of Biology and Chemistry
264:10667-1071.

Marks JR, Humphrey PA, Wu K, et al: 1995. Overexpression of p53 and HER-2/neu
proteins as prognostic markers in early stage breast cancer. Annals Surgery 2212433-
435.

Marshall E: 1996. Rifldn’s latest target: genetic testing. Science 272 1904.

Marx J 2 1993. Cell death studies yield cancer clues. Science 2592760-761.

Marx J 2 1996. A second breast cancer susceptibility gene is found. Science 271230-31.

Mazars R, Spinardi L, BenCheikh M, et al: 1992. p53 mutations occur in aggressive
breast cancer. Cancer Research 52(14):3918-3923.

Masood S, Frykerg BIL McLellan GL, et al: 1990. Prospective evaluation of
radiologically directed fine-needle aspiration biopsy of nonpalpable breast lesions. Cancer
6621480-1487.

Mcbride OW, Merry D, Givol D: 1986. The gene for human p53 cellular tumor antigen is
located on chromosome 17 short arm (17p13). Proceedings of National Academy of
Science of the United States of America 83: 130-134.

140

McDonnell TJ, Deane N, Platt FM, et al: 1989. Bcl-2-immunoglobu1in transgenic mice
demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57:79-88.

McDonnell TJ, Korsmeyer SJ: 1991. Progression from lymphoid hyperplasia to high-
grade malignant lymphoma in mice transgenic for the t(14;18). Nature 349:254-256.

McGee JO'D: 1992. Molecular techniques in laboratory medicine. I_n Diagnostic
Molecular Pathology A Practical Approach Edited by Herrington CS, McGee J O'D.
Oxford University Press. Oxford, New York, Tokyo pp1-6.

McGuire WL, De La Garza M: 197 5. Similarity of estrogen receptors in human and rat
mammary carcinoma. Journal of Clinical Endocrinology and Metabolism 362 548-552.

McWhirter R: 1948. The value of simple mastectomy and radiotherapy in the treatment
of cancer of the breast. British Journal of Radiology 212599-610.

Melarned MR, Lindmo T, Mendelsohn ML, et al (ed): 1990. F low Cytometry and
Sorting 2nd edition. Wiley-Liss Publish. New York.

Merkel DE, McGuire WL: 1990. Ploidy, proliferative activity and prognosis: DNA flow
cytometry of solid tumors. Cancer 65: 1 149-1205.

Merlo GR, Venesio T, Taverna D, et al: 1994. Growth suppression of normal mammary
epithelial cells by wild-type p53. Oncogene 92443-453.

Mettlin C: 1994. The relationship of breast cancer epidemiology to screening
recommendations. Cancer 742228-30.

Mietz JA, Unger T, Huibregtse JM, et al: 1992. The transcriptional transactivation
function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6
oncoprotein. Engeming in Medicine and Biology Journal 1 125013-5020.

MikiY, Swensen J, Shattuck-Eldens D,Futreal PA et al: 1994. A strong candidate for the
breast and ovarian cancer susceptibility gene BRCAl. Science 266266-71.

Miller RW: 1977. Relationship between human teratogens and carcinogens. Journal of
National Cancer Institute 582471-474.

Miller C, Mohands T, Wolf D, et al: 1986. Human p53 gene localized to short arm of
chromosome 17. Nature 3192783-784.

Miller CW, Simon K, Aslo A, et al: 1992. PS3 mutations in human lung tumors. Cancer
Research 5221695-1698.

141

Miller B: 1995. PRINS & Cycling PRINS Theory & Practice In [N SI TU Applications
Guide SHANDON LIPSHAW Pittsburgh pp37-50.

Milner J, Medcalf EA, Cook AC: 1991. Tumor suppressor p53: analysis of wild-type and
mutant p53 complexes. Molecular and Cellular Biology 11212-19.

Milner J, Medcalf EA, Cook AC: 1991. Tumor suppressor p53: analysis of wild-type and
mutant p53 complexes. Molecular and Cellular Biology 11212-9.

Mitra AB, Murty W, Pratap M, et al: 1994. ERBB2 (HERZ/neu) oncogene is frequently
amplified in squamous cell carcinoma fo the uterine cervix. Cancer Research 542637-
639.

Moll UM, Riou G, Levine AJ, et al: 1992. Two distinct mechanisms alter p53 in breast
cancer: mutation and nuclear exclusion. Proceedings of National Academy of Science of
the United States of America 89:7262-7266.

Momand J, Zambetti GP, Olson DC, et a1 1992. The mdm-2 oncogene product forms a
complex with the p53 protein and inhibits p53-mediated transactivation. Cell 6921237-
1245.

Moscow JA, Cowan KH: 1988. Multidrug resistance. Journal of National Cancer
Institute 80214.

Mueller CB: 1994. Breast cancer: reporting results with inflationary arithmetic.
American Journal of Clinic Pathology 17:86-92.

Nagai MA, Marques LA, Torloni H, et al: 1993. Genetic alterations in c-erbB-2
protooncogene as prognostic markers in human primary breast tumors. Oncology
50:412-417.

Natarajan N, Nemoto T, Metllin C, et al: 1985. Race-related difl‘erences in breast cancer
patients. Results of the 1982 national survey of breast cancer by the American College of
Surgeons. Cancer 56:1704-1709.

Nathan B, Anbazhagan R, Clarkson P, et al: 1994a. Expression of BCL-Z in the
developing human fetal and infant breast. Histopathology 24:73 -76.

Nathan B, Gusterson B, Jadayel D, et al: 1994b. Expression of Bcl-2 in primary breast
cancer and its correlation with tumour phenotype. For the International breast cancer
study group. Annals of Oncology 52409-414.

National Institutes of Health: 1978. National Institutes of Health /National Cancer
Institute Consensus Development Meeting on Breast Cancer: Issues and
Recommendations. Journal of National Cancer Institute 6026.

142

Nelson WG, Kastan MB: 1994. DNA strand breaks: the DNA template alterations that
trigger p53-dependent DNA damage response pathways. Molecular and Cellular Biology
14:1815-1823.

Neubauer A, He M, et al: 1992. Analysis of gene amplification in archival tissue by
differential polymerase chain reaction. Oncogene 721019-1025.

Neuhausen S, Gilewski T, Norton L, et al: 1996. Recurrent BRCA2 6174de1T mutations
in Ashkenazi Jewish women affected by breast cancer. National Genetics 132126-128

Neville AM, Price KN, Gleber RD, et al: 1991. Axillary node micrometastases and breast
cancer. Lancet 33721110-117..

Nigro JM, Baker SJ, Preisinger AC, et al: 1989. Mutation in the p53 gene occur in
diverse human tumour types. Nature 342:705-708.

Nunez G, London L, Hockenbery D et al: 1990. Deregulated Bel-2 gene expression
selectively prolongs survival of grth factor-deprived hemopoietic cell lines. Journal of
Immunology 144:3602-3610.

Nunovo G] 2 1994. PCR in situ hybridization. I_n_ PCR in situ Hybridization Protocols
andApplications Second Edition RAVEN PRESS New York pp169-213.

O'Grady LF, Rippon MB: 1994. The high-risk patient. In A Practical Approach to
Breast Disease. Edited by O'Grady, L.F., Lindfors, K.K., Howell, LP, and Rippon, M.B.
Little, Brown and Company. Boston, New York, Toronto, London. pp173-182.

Oltvai ZN, Milliman CL, Korsmeyer SJ 2 1993. Bel-2 heterodimerizes in vivo with a
conserved homology, Bax, that accelerates programmed cell death. Cell 742609-619.

Oren M, Maltzman W, Levine AJ 2 1981. Post-translational regulation of the 54K cellular
tumor antigen in normal and transformed cells. Molecular and Cellular Biology 12101-
110.

Oren M: 1985. The p53 cellular tumor antigen: Gene structure, expression and protein
properties. Biochemistry and Biophysics Acta 823 267-78.

Orita M., Iwahana H, Kanazawa H, et al: 1989. Detection of polymorphisms of human

CNA by gel electrophoresis as single-strand conformation polymorphisms. Proceedings
of National Academy of Science of the United States of America 8622766—2770.

Owings D, Hann L, Schnitt SJ 2 1990. How thoroughly should needle localization breast
biopsies be sampled for microscopic examination? A prospective mammographic-
pathologic correlative study. American Journal of Surgical Pathology 14:578-583.

143

Padhy LC, Shih C, Cowing D, et al: 1982. Identification of a phosphoprotein specifically
induced by the transforming DNA of rat neuroblastomas. Cell 28:865-871.

Page DL, Dupont WD, Rogers, L.W., and et al: 1985. Atypical hyperplastic lesions of
the female breast. Cancer 5521563-1571.

Page DL: 1991. Prognosis and beast cancer. Recognition of lethal and favorable
prognostic types. The American Surgical Pathology 15:334-349.

Paget J 2 1856. On the average duration of life in patients with scirrhous cancer of the
breast. Lancet 1:62-63.

Palmer MK, Lythgoe JP, Smith A: 1982. Prognostic factors in breast cancer. British
Journal of Surgery 692697-698.

Park JB, Rhim J S, Park SG, et a1 1989. Expression of the c-erbB-2 gene product (p185)
at different stages of neoplastic progression in the colon. Oncogene 421233-1239.

Patey DH, Dyson WH: 1948. The prognosis of carcinoma of the breast relation to the
type of operation performed. British Journal Cancer 227- 1 3.

Pavletich NP, Chambers KA, Pabo CO: 1993. The DBA-binding domain of p53 contains
the four conserved regions and the major mutation hot spots. Genes and Development
722556-2564.

Pezzella F, Tse AG, Cordell JL, et al: 1990. Expression of the Bcl-2 oncogene protein is
not specific for the 14;18 chromosomal translocation. American Journal of Pathology
137 2225-232.

Polednak AP: 1986. Breast cancer in black and white women in New York state. Case
distribution and incidence rates by clinical stage at diagnosis. Cancer 572807-815.

Posser J, Thompson AM, Cranston G, et al: 1990. Evidence that p53 behaves as a tumor
suppressor gene in sporadic breast tumors. Oncogene 521573-1579.

Potchen, E] 2 1993. Investigation of breast abnormalities. I_n Screening Mammography
Breast Cancer Diagnosis in Asymptomatic Women Edited by Svane G, Potchen EJ,
Sierra A, and Azavedo E. MOSBY St. Louis, Baltimore, Boston, Chicago, London,
Philadelphia, Sydney, Toronto. pp23 7-254.

Potchen EJ, Sierra A, Mackenzie CD, et al: 1991. Svane localization of non-palpable
breast lesions. Lancet 338(8770)2816.

144

Powell DE, Stelling DB: 1994. Chapter 12 Carcinoma of breast In The Diagnosis and
Detection of Breast Cancer Published by Dedicated to Publishing Excellence. St. Louis,
Baltimore, Boston, Chicago, London, Madrid, Philadelphia, Sydney, Toronto. pp289.

Pringle, J.H., Baker, 1., and et al: 1993. The detection of cell proliferation in normal and
malignant formalin-fixed paraffin-embedded tissue sections using in situ hybridization for
histone mRN A. Journal of pathologic Supplementum 1692155A.

Pringle, J.H., Ruprai, AK, and et al: 1990. in situ hybridization of immunoglobulin light
chain mRNA in paraffin section using biotinylated or hapten-labelled oligonucleotide
probes. Journal of Pathology 1622197-207.

Pritchard-Jones, K. and Fleming, 8.: 1991. Cell types expressing the Wilms' tumor gene
(WT 1) in Wilms' tumors: implications for tumor histogenesis. Oncogene 622211-2220.

Prosser J, Thompson AM, Cranston G, et al: 1991. Evans-H] Evidence that p53 behaves
as a tumour suppressor gene in sporadic breast tumours. Oncogene 52 1573 -9.

Qiao XT: 1992. Chapter one. Tisse sampling: a comparison of the weight and the
cellular nature of the material obtained by fine needle aspiration and Rotex needle biopys
techniques. I_n Techniques in the Detection and Treatment of Breast Cancer. Thesis for
the Degree of MS. MSU. pp7-48.

Ragaz J, Ariel IM: 1989. High-risk breast cancer: definition of the risk. In High-Risk
Breast Cancer Edited by Ragaz J and Ariel IM. Springer-Verlag Beidelberg New York.
Pp3 -23.

Rand A, Glenn KS, Alvares CP, et al: 1996. P53 function loss in a colon cancer cell line
with two missions mutations (218eu and 248trp) on separate alleles. Cancer Letters.
982183-191.

Rapp F, Reed CL: 1977. The viral etiology of cancer. A realistic approach. Cancer
40:419-429.

Ravdin P and Chamness G: 1995. The c-erbB-2 proto-oncogene as a prognostic and
predictive marker in breast cancer: a paradigm for the development of other
macromolecular markers - a review. Gene 159219-27.

Raycroft L, Schmidt JR, Yoas K, et al: 1991. Analysis of p53 mutants for transcriptional
activity. Molecular and Cellular Biology 1 126067-6074.

Raycrofi L, Wu H, Lozano G: 1990. Transcriptional Activation by wild-type but not
transforming mutants of the p53 anti-oncogene. Science 249:1049-1051.

145

Read LD, Keith DJr, Slamon DJ, et al: 1990. Hormonal modulation of HER-Z/neu
proto-oncogene messenger ribonucleic acid and p185 protein expression in human breast
cancer cell lines. Cancer Research 50:3947-3951.

Redard M, Vassikos P, Weintraub JA: 1989. A simple method for estrogen receptor
antigen preservation in cytologic specimens containing breast carcinoma cells. Diagnosis
of C ytologic Pathology 5218-193.

Redding TW, Schally AV, Radulovic S, et al: 1992. Sustained release formulations of
luteinizing hormone-releasing hormone antagonist SB-75 inhibit proliferation and enhance
apoptotic cell death of human prostate carcinoma (PC-82) in male nude mice. Cancer
Research 5222538-2844.

Resnick RM, Comelissen MT, Wright DK, et al: 1990. Detection and typing of human
papilloma virus in archival cervical cancer specimens by DNA amplification with
consensus primers. Journal of National Cancer Institute 8221477-1484.

Robbins P, Pinder BS, Klerk N, et al: 1995. Histological grading of breast carcinomas: a
study of interobserver agreement. Human Pathology 26:873-879.

Robinson IA, McKee G, Nicholson A: 1994. Prognostic value of cytological grading of
fine-needle aspirates from breast carcinomas. Lancet 3432947-949.

Robinson J02 1986. Treatment of breast cancer through the ages. American Journal of
Surgery 151:321-327..

Robbins SL 2 1994. The breast. I_n Pathologic Basis of Disease 5th Edition Edited by
Cotran RS, Kumar V and Robbins SL. W. B. SAUNDERS COMPANY pp1089-1112.

Rogel A, Popliker M, Webb CG, et al: 1985. P53 cellular tumor antigen: analysis of
mRNA levels in normal adult tissues, embryos, and tumors. Molecular and Cellular
Biology. 522851-2855.

Rosen PP: 1996a. Biological markers of prognosis. In Breast Pathology. Edited by
Rosen PP. Lippincott-Raven Publishers. Philadelphia. pp295-320.

Rosen PP: 1996b. Role of cytology and needle biopsy in diagnosis. I_n Breast Pathology.
Edited by Rosen PP. Lippincott-Raven Publishers. Philadelphia. pp817-835.

Rosen PP, Lesser ML, Arroyo CD, et al: 1995a. P53 in node-negative breast carcinoma:
an immunohistochemical study of epidemiologic risk factors, histologic features and
prognosis. Journal of Clinical Oncology 132821-830.

146

Rosen PP, Lesser ML, Arroyo CD, et al: 1995b. Immunohistochemical detection of
HER-2/ neu in node-negative breast carcinoma: a study of epidemiologic risk factors,
histologic features and prognosis. Cancer 75: 1320-1326.

Rosen PP, Goshen S, Saigo PE, et al: 1989a. A long-term follow-up of survival in Stage
I (TlNOMO) and Stage II (TlNlMO) breast carcinoma. Journal of Clinical Oncology
72355-366.

Rosen PP, Goshen S, Saigo PE, et al: 1989b. Pathologic prognostic factors in stage I
(TINOMO) and stage II (TlNlMO) breast carcinoma: a study of 655 patients with median
follow-up of 18 years. Journal of Clinical Oncology 721239-1251.

Rosen PP, Lesser ML, Senie R, et al: 1982. Epidemiology of breast carcinoma IV: age
and histologic tumor type. Journal of Surgical Oncology 19:44-47.

Roth JA, Nguyen D, Lawrence DD, et al: 1996. Retrovirus—mediated wild-type p53 gene
transfer to tumors of patients with lung cancer. Nature Medicine 2(9):985-991.

Roulston JE, Leonard RCF 2 1993a. Breast cancer. In Serological Tumor Markers An
Introduction Edited by Roulston JE and Leonard RCF. Churchill Livingstone
Edinburgh, London , Madrid, Melbourne, New York, and Tokyo. pp39-46.

Roulston JE, Leonard RCF: 1993b. Breast cancer. In Serological Tumor Markers An
Introduction. Edited by Roulston JE and Leonard RCF. Churchill Livingstone
Edinburgh, London, Madrid, Melbourne, New York, and Tokyo. pp75-85.

Runnebaum I, Nagarajan M, Bowman M, et al: 1991. Mutations in p53 as potential
molecular markers for human breast cancer. Proceeding of the National Academy of
Sciences of the United Stated of America 88(23): 10657-10661.

Ruprai AK, Pringle JH, et al: 1991. Localization of immunoglobulin light chain mRNA
expression in Hodgkin's disease by in situ hybridization. Journal of Pathology 164237-
40.

Russel DL, Kaklamanis] L, Pezzella F et a1: 1994. Bel-2 in normal human breast cancer
carcinoma, association with oestrgen receptor-positive , epidermal growth factor receptor-
negative tumours and in situ cancer. British Journal of Cancer 692135-139.

Russell KS, Hung MC: 1992. Transcriptional repression of the neu protooncogene by
estrogen. Cancer Research 52:6624-6629.

Sakamoto G, Sugano H, Kasumi F2 1987. Bilateral breast cancer and familial
aggregations. Preventive Medicine 7(2): 225-229.

147

Salhadin A, Nasiell M, Nasiell K, et a1: 1976. The unique cytologic picture of oat cell
carcinoma in effusions. Acta Cytology 202298.

Santibanez-Koref MF, Birch JM, Hartley AL, et al: 1991. PS3 gremlin mutations in Li-
Fraumeni syndrome. Lancet 338:1490-1491.

Sasa M, Kondo K, Komaki K, et al: 1993. Frequency of spontaneous p53 mutations
(CpG site) in breast cancer in Japan. Breast Cancer Research and Treatment 272247-
251.

Sattin RW, et a1: 1985. Family history and the risk of breast cancer. Journal of the
American Medical Association 253:1908-1913.

Schechter AL, Hung M-C, Vaidyanathan L, et al: 1985. The neu gene: an erbB-2
homologous gene distinct from and unlinked to the encoding the EGF receptor. Science
229:976—978.

Schechter AL, Stern DF, Vaidyanathan L, et al: 1984. The neu oncogene: an erb-B-2
related gene encoding a 185,000-Mr tumour antigen. Nature 3122513-516.

Schneider PM, Hung MC, Chiocca SM, et al: 1989. Differential expression of the c-
erbB-2 gene in human small cell and non-small cell lung cancer. Cancer Research
49:4968-4971 .

Schnitt SJ 2 1993. Processing of breast biopsies. I_n Atlas of Breast Cancer. Edited by
Hayes DF. Mosby Europe Limited London, St. Louis, Baltimore, Boston, Chicago,
Philadelphia, Sudney, and Toronto. pp6.2-6.6.

Schnitt SJ, Connoly JL, Khettry U: 1987. Pathologic findings on re-excision of the
primary site in breast cancer patients considered for treatment by primary radiation
therapy. Cancer 592675-681.

Schnitt SJ, Connoly JL, Harris JR, et al: 1984. Pathologic predictors of early local
recurrence in stage I and H breast cancer treated by primary radiation therapy. Cancer
5321094-1057.

Schubert D, Heinemann 8, Carlisle W, et a1. 1974. Clonal cell lines the rat central
nervous system. Nature 249:224-227.

Schubert, EL, and et al: 1993. A method to isolate DNA from small archival tissue
samples for p53 gene analysis. Human Mutation 22123-126.

Searle I, Lawson TA, Abbott P], et al: 1975. An electron-microscope study of the mode
of cell death induced a cancer-chemotherapeutic agents in populations of proliferating
normal and neoplasic cells. Journal of Pathology 1162 129-138.

148

Searle J, Collins DJ, Harmon B, et al: 1973. The spontaneous occurrence of apoptosis in
squamous carcinomas of the uterine cervix. Pathology 52163-169.

Seidman H, Stellman SD, Mushinski MH: 1982. A different perspective on breast cancer
risk factors: some implications of the non-attributable risk. CA: A Cancer Journal of
Clinic 32:301-313.

Semba K, Karnata N, Toyoshima K, et a1: 1985. A v-erbB-related protooncogene, c-
erbB-2, is distinct from the c-erbB-l/epidermal grth factor receptor family: evidence
for overexpression in a subset of human mammary tumors. Proceedings of National
Academy of Science of the United States of America 8226497-6501.

Sentman CL, Shutter JR, Hockenbery D et al: 1991. Bel-2 inhibits multiple forms of
apoptosis by not negative selection in thymocytes. Cell 682879-888.

Seto M, Jaeger U, Hockett RD, et al: 1988. Alternative promoters and exons, somatic
mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO Journal
7(1):]123-131.

Shaulsky G, Golfinger N, Ben Ze’ev A, et al: 1990. Nuclear accumulation of p53 protein
is mediated by several nuclear localization signals and plays a role in tumorgenesis.
Molecular and Cellular Biology 1026565-65 77.

Shaw P, Bovey R, Tardy S, et al: 1992. Induction of apoptosis by wild-type p53 in a
human colon tumor-derived cell line. Proceedings of National Academy of Science of the
United States of America 89:4495-4499.

Shibata D, Brynes RK, Nathwani B, et al: 1989. Human immunodeficiency viral DNA is
readily found in lymph node biopsies from seropositive individuals. Analysis of fixed
tissue using the polymerase chain reaction. American Journal of Pathology 1352697-702.

Shih C, Padhy LC, Murray M, et al: 1981. Transforming genes of carcinomas and
neuroblastomas introduced into mouse fibroblasts. Nature 290:261-264.

Sierra A, Lloveras B, Castellsague X, et al: 1995. Bel-2 expression is associated with
lymph node metastasis in human ductal breast carcinoma. International Journal of
Cancer 60:54-60.

Sickles EA: 1984. Breast cancer detection with transillurnination and mammography.
American Journal of Radiology 1422841 -844.

Silvestrini R, Benini E, Daidone MG, et al: 1993. P53 as an independent prognostic
marker in lymph node-negative breast cancer patients. Journal of National Cancer
Institute 85:965-970.

149

Siziopikou KP, Prioleau JE, Harris JR, et al: 1996. Bcl-2 expression in the spectrum of
preinvasive breast lesions. Cancer 772499-506

Sjalander A, Birgander R, Hallmans G, et al: 1996. p53 polymorphisms and haplotypes in
breast cancer. Carcinogenesis 17:1313-1316.

Slamon DJ, Godolphin W, Jones LA, et al: 1989a. Studies of the HER-2/neu proto-
oncogene in human breast and ovarian cancer. Science 244:707-712.

Slamon DJ, Press MF, Godolphin W, et al: 1989b. Studies of the HER-Z/neu proto-
oncogene in human breast cancer. I_n Cancer Cell . Edited by Fourth M and Greaves M.
COLD SPRING HARBOR LABORATORY PRESS. pp371-340

Slamon DJ, Clark GM, Wong SG, et al: 1987. Human breast cancer: correlation of
relapse and survival with amplification of the HER-Z/neu oncogene. Science 2352177-
182.

Smith CA: 1993. Monoclonal antibodies in pathological investigations. In Molecular
and Antibody Probes in Diagnosis. Edited by Walker MR and Rapley R. JOHN
WUKEY & SONS Chichester, New York, Brisbane, Toronto, Singapore. pp215-228.

Sobin LH, Hermanek P, Hutter RVP: 1988. TNM classification of malignant tumors. A
comparison between the new (1987) and old editions. Cancer 61 :23 10-14.

Soomro S, Taylor P, Shepard H, et al: 1993. c-erbBZ oncoprotein in screen-detected
breast carcinoma: an immunohistological study. International Journal of Cancer 55:63-
65.

Soto D, Sukumar S: 1992 Improved detection of mutations in the p53 gene in human
tumors as single-stranded conformation polymorphs and double-stranded heteroduplex
DNA. PCR Methods and Application 2296-98.

Soussi T, Caron de Fromentelc, May P: 1990. Structural aspects of the p53 protein in
relation to gene evolution. Oncogene 52945-953.

Soussi T, Legros Y, Lubin 1L et al: 1994. Multifactorial analysis of p53 alteration in
human cancer: a review. International Journal of Cancer 5721-9.

Spadoro JP, Dragon EA: 1993. Quality control of the polymerase chain reaction. I_n
Molecular Biology and Pathology A Guidebook for Quality Control Edited by F arkas,
D.H. Academic Press, Inc. pp149-158.

Spinardi L, Mazars R, Theillet C: 1991. Protocols for an improved detection of point
mutations by SSCP. Nucleic Acids Research 1924009.

150

Spriggs A1: 1984. The architecture of tumor cell clusters in serious effusions. In
Advances in Clinical Cytology vol. 2. Edited by Koss LG and Coleman DV. New York,
Masson. pp267-290.

Staecker H, Cammer M, Rubinstein R, et al: 1994. A procedure for RT-PCR
amplification of mRNAs on histological specimens. BioTechniques 16:76-80.

Stephens LC, Ang KK, Schultheiss TE, et al: 1991. Apoptosis in irradiated murine
tumors. Radiation Research 127:308-316.

Stern DF, Heffernan PA, Weinberg RA: 1986. p185, a product of the neu proto-
oncogene, is a receptor like protein associated with tyrosine kinase activity. Molecular
and Cellular Biology 6: 1729-1740.

Stern DF, Kamps Mp, Cao H: 1988a. Oncogenic activation of p185 neu stimulates
tyrosine phosphorylation in vivo. Molecular and Cellular Biology 823969-3973.

Stern DF, Kamps MP2 1988b. EGF-stimulated tyrosine phosphorylation of pl 85: a
potential model for receptor interactions. Engineering in Medicine and Biology Journal
72995-1001.

Strasser A, Harris AW, Bath ML, et al: 1990. Novel primitive lymphoid tumors induced
in transgenic mice by cooperation between myc and Bel-2. Nature 348:331-333.

Szabo CI, King MC: 1995. Inherited breast and ovarian cancer. Human Molecular
Genetics 42 1811-1817.

Szende B, Srkalovic G, Groot K, et al: 1990. Growth inhibition of mouse MXT
mammary tumor by the luteinizing horrnone-releasing hormone antagonist SB-75.
Journal of National Cancer Institute 822513-517.

Takahashi T, D’Amico D, Chiba I, et a1: 1990. Identification of intronic point mutations
as a altermative mechanism for p53 inactivation in lung cancer. Journal of Clinical
Investigation 86:363-369.

Takeshima Y, Seyama T, Bennett WP, et al: 1993. PS3 mutations in lung cancers fi'om
non-smoking atomicbomb survivors. Lancet 342:1520-1521.

Tal M, Wetzler M, Josefberg Z, et al: 1988. Sporadic amplification of the HER2/neu
protooncogene in adenocarcinomas of various tissues. Cancer Research 4821517-1520.

Tammenbaum, A: 1942. The genesis and growth of tumors, III effects of high fat diet.
Cancer Research 22468-475.

151

Tandon AK, Clark GM, Chamness GC, et al: 1989. HER/neu oncogene protein and
prognosis in breast cancer. Journal of Clinical Oncololgy 721120-1128.

Tavassoli FA, Norris H] 2 1990. A comparsion of the results of long-term follow-up for
atypical intraductal carcinma of the breast. Cancer 652518-529.

Tavassoli FA: 1992. Part 2. General Considerations In Pathologr of the Breast.
Appleton & LANGE. Norwalk, Connecticut. pp25-55.

Tetu B, Fradet Y, Allard P, et al: 1996. Prevalence and clinical significance of
HER/Zneu, p53 and Rb expression in primary superficial bladder cancer. Journal of
Urology 155:1784-1788.

Thomas DB: 1984. Do hormones cause breast cancer? Cancer 532595-604.

Thompson AM, Anderson TJ, Condie A, et al: 1992. PS3 allele losses, mutation and
expression in breast cancer and their relationship to clinicopathologic parameters
International Journal of Cancer 50:528-532.

Tomita Y, Bilim V, Kawasaki T, et al: 1996. Frequent expression of Bel-2 in renal-cell
carcinomas carrying wil-type p53. International Journal of Cancer 662322-3 25.

Tsuda H, Hirohashi S, Shimosato Y, et al: 1990. Immunohistochemical study on
overexpression of c-erbB-2 protein in human breast cancer: its correlation with gene
amplification and long-term survival of patients. Japanese Journal of Cancer Research.
81:327-332.

Tsujimoto Y, Finger LR, Yunis J, et al: 1984. Cloning of the chromosome break point of
neoplastic B cell with the t(l4,18) chromosome translocation. Science 226:1097-1099.

Tubiana M, Pejovic MH, Renaud A, et al: 1981. Kinetic parameters and the course of the
disease in breast cancer. Cancer 472937-943.

van de Vijver M, van de Bersselaar R, Devillee P, et al: 1987. Amplification of the neu
(c-erbB-Z) oncogene in human mammary tumors is relatively frequent and is often
accompanied by amplification of the linked c-erbA oncogene. Molecular and Cellular
Biology 722019-2023.

Van den Berg F, Schipper M, et al: 1989. Implausibility of an etiological association
between cytomegalovirus and Kaposi's sarcoma shown by four techniques. Journal of
Clinical Pathology 422128-131.

Vanhaesebroeck B, Reed JC, Valck DD, et al: 1993. Effect of Bel-2 proto-oncogene
expression on cellular sensitivity to tumor necrosis factor-mediated cytotoxicity.
Oncogene 821075-1081.

152

Varley JM, Swallow JE, Brammar WJ, et al: 1987. Alterations to either c-erbB-2(neu) or
c-myc proto-oncogenes in breast carcinomas correlate with poor short-term prognosis.
Oncogene 12423-430.

Vaux DL, Aguila HL, Weissman IL: 1992. Bel-2 prevents death of factor-deprived cells
but fails to prevent apoptosis in stargets of cell mediated killing. International
Immunology 42821-824.

Vaux DL, Cory S, Adams JM: 1988. Bel-2 gene promoters haemopoietic cell survival
and cooperates with c-myc to immortalize pre-B cells. Nature 3352440-442.

Veach SR, Ghosh BC: 1987. Biologic markers in breast cancer. I_n Tumor Markers and
T umor-Associated Antigens . Edited by Ghosh BC and Ghosh L. McGRAW-HILL
BOOK COMPANY. New York, et a1. pp87-101.

Venter DJ, Tuzi NL, Kumar S, et al: 1987. Overexpression of the cerbB-2 oncoprotein
in human breast carcinomas: immunohistological assessment correlates with gene
amplification. Lancer. July 69-71.

Vernon SW, Tilley BC, Neale V, et al: 1985. Ethnicity, survival, and delay in seeking
treatment for symptoms of breast cancer. Cancer 5521563-15711.

Vindelov LL, Christensen II: 1990. A review of techniques and results obtained in one
laboratory by an integrated system of methods designed for routine clinical flow
cytometric DNA analysis. Cytometry 11: 753-70.

Vogel VG, Love RR: 1991. High risk groups and cost strategies. I_n Approaches to
Breast Cancer Prevention. Edited by Stoll BA. Kluwer Academic Publishers.
Dordrecht, Boston, London. pp207-220.

Vogelsteinie B, Kinzler KW: 1992. p53 function and dysfunction. Cell 70:523-526.

Vorherr H: 1980. Breast cancer in relation to diet, ovemutrition, and obesity. I_n Breast
Cancer: Epidemiology Endocrinology Biochemistry and Pathobiology. Urban &
Schwarzenberg, Inc. Baltimore-Munich pp3 7-53.

Wagner FB Jr.2 1991. History of breast disease and its treatment. I_n The Breast. Edited
by Bland KI and Copeland EM 111. W.B. Saunders Company. pp1-16.

Walker RA, Senior PV, Jones JL, et al: 1989. An immunohistochemical and in situ
hybridization study of c-myc and c-erbB2 expression in primary human breast carcinomas.
Journal of Pathology 158297-105.

Waterhouse J, Muir C, Correa P, et al: 1976. Cancer incidence in five continents. In
Scientific Publication No.15, Lyon, IARC.

153

Waters JJ, Long SG: 1994. Interphase cytogenetics. In Molecular Biology in
Histopathology Edited by Crocker, J. JOHN WILEY & SONS. pp57-70.

Wazer DE, Chu Q, Liu SL, et al: 1994. Loss of p53 protein during radiation
transformation of primary human mammary epithelial cells. Molecular and Cellular
Biology 1422468-2478.

Weinberg RA: 1991. Tumor suppressor genes. Science 245:1138-1145.

Weinberg RA: 1994. You can’t get there from here: the tortuous road to basic research.
academic Medicine 69(6):441-444.

Weiner DB, Kokai Y, Wada T, et al 2 1989. Linkage of tyrosine kinase activity with
transforming ability of the p185 neu oncoprotein. Oncogene 421175-1183.

Weiner DB, Nordberg J, Robinson R, et al: 1990. Expression of the neu gene-encoded
protein p185neuP in human non-small cell carcinomas of the lung. Cancer Research
502421-425.

Weinstein JN, Myers TG, O’Connor PM, et al: 1997. An information -intensive approach
to the molecular pharmacology of cancer. Science 275:343-349.

Wels W, Moritz D, Schmidt M, et al: 1995. Biotechnological and gene therapeutic
strategies in cancer treatment. Gene 159273-80.

Whittemore AS, Wu-Williams AH, Lee M, et al: 1990. Diet, physical activity, and
colorectal cancer among Chinese in North America and China. Journal of national
Cancer Institute 82:915-926.

WHO: 1982. The World Health Organization Histological Typing of Breast Tumors--
Second Edition. American Journal of Clinical Pathologr 78: 806-816

Wilkinson DG: 1992. The theory and practice of in situ hybridization. I_n in situ
Hybridization A Practical Approach Edited by Wilkinson DG. IRL PRESS at Oxford
University Press Oxford, New York, Tokyo. pp1-14.

Wilkinson EJ, F ranzini DA, Masood S: 1991. Cytological needle sampling of the breast:
techniques and end results. I_n The Breast Comprehensive management of benign and
malignant diseases. Edited by Bland KI and Copeland III EM. W.B. SAUNDERS
COMPANY Philadelphia, London, Toronto, Montreal, Sydney, Tokyo. pp475-498.

Willet WC, Browne ML, Bain C, et al: 1985. Relative weight and risk of breast cancer
among premenopausal women. American Journal of Epidemiology 1222731-740.

Willett WC, Stampfer MJ, Colditz GA, et al: 1987. Moderate alcohol consumption and

154

the risk of breast cancer. New England Journal of Medicine 31621174-1179.

Winer DB, Liu J, Cohen JA, et al: 1989. A point mutation in the neu oncogene minces
lignd induction of receptor aggregation. Nature 3392230-231.

Wooster R, Bignell G, Lancaster J, et al: 1995. Indentification of the breast cancer
susceptibility gene BRCA2. Nature 3782789-792.

Wyllie AH, Rose KA, Morris RG, et a1: 1987. Rodent fibroblast tumours expressing
human myc and ras genes: growth, metastasis and endogenous oncogene expression.
British Journal of Cancer 562251-259.

Wyllie AH: 1985. The biology of cell death in tumours. Anticancer Research 52131-136.

Wyllie AH, Ker JFR, Currie AR: 1980. Cell death: the significance of apoptosis.
International Review of Cytology 68:251-306.

Yamamoto T, Ikawa S, Akiyarna T, et al: 1986. Similarity of protein encoded by the
human c-erbB-Z gene to epidermal grth factor. Nature 319:230-234.

Yang E, Zha JP, Jockel J, et al: 1995. Bad, a heterodimeric partner for Bcl-xl and Bel-2,
displaces Bax and promotes cell-death. Cell 802285-291.

Yap EPH., Martinez-Montero JC, McGee JO'D: 1992. mRNA detection in clinical
samples by non-isotopic in situ hybridization. In Diagnostic Molecular Pathology A
Practical Approach Volume I Edited by Herrington CS and McGee JO'D. IRL PRESS
at Oxford University Press. Oxford, New York, Tokyo. pp187-204.

Yap EP, McGee J02 1992. Nonisotopic SSCP detection in PCR products by ethidium
bromide staining. Trends in Genetics 8249.

Yarden Y, Ullrich A: 1988. Growth factor receptor tyrosine kinases. Annual Review of
Biochemistry 572443-478.

Yeatman TJ, Bland K12 1991. Clinical and pathological staging of breast cancer and
prognostic factors. In The Breast Comprehensive Management of Benign and
Malignant Diseases. Edited by Bland KI and Copeland III EM. W.B. SAUNDERS
COMPANY Harcourt Brace Jovanovich, Inc. Philadelphia, London, Toronto, Montreal,
Sydney, and Tokyo. pp313-350.

Yew PR, Berk AJ 2 1992. Inhibition of p53 transactivation required for transformation by
adenovirus early 1B protein. Nature 357282-85.

155

Yokota J, Yamamoto T, Miyalima N, et al: 1988. Genetic alterations of the c-erbB-2
oncogene occur fi'equently in tubular adenocarcinoma of the stomach and are often
accompanied by amplification of the v-erbA homologue. Oncogene 22283-287.

Yonisch-Rouach E, Resnitzky D, Lotem J, et al: 1991. Wild-type p53 induces apoptosis
of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352;345-347.

Yunis JJ, Oken MM, Kaplan ME, et a1: 1982. Distinctive chromosomal abnormalities in
histologic subtypes of non-Hodgkin’s lymphoma. New England Journal of Medicine
307:1231-1236.

Yu MC, Gerkins VR, Henderson BE, et al: 1981 Elevated levels of prolactin in
nulliparous women. British Journal of Cancer 43 2826-83 1.

Zeillinger R, Kury F, Czerwenka K, et al: 1989. HER-2 amplification, steroid receptors
and epidermal grth factor receptor in primary breast cancer. Oncogene 42109-114.

Zhang X, Siva E, Gershenson D, et a1: 1989. Amplification and rearrangement of c-erbB
proto-oncogene in cancer of human female genital tract. Oncogene 42985-989.

Zhou D, Battifora H, Yokota J, et al: 1987. Association of multiple copies of the c-erbB-
2 oncogene with spread of breast cancer. Cancer Research 47 26123-6125.

156

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