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DMEIWE DATE DUE DATE DUE NOV 2 4 1998 :jL__ I:— T‘ fire—I MSU Is An Affirmative ActiorVEqual Opportunity Institution chS-pd TECHNIQUES IN THE DETECTION AND TREATMENT OF BREAST CANCER: TISSUE SAMPLING METHODS AND THE EFFECTS OF TISSUE DESTRUCTION DIRECT ELECTRIC CURRENT BY XiaoTan Qiao A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1992 ABSTRACT TECHNIQUES IN DETECTION AND TREATMENT OF BREAST CANCER: TISSUE SAMPLING METHODS AND THE EFFECTS OF TISSUE DESTRUCTION BY DIRECT ELECTRIC CURRENT XiaoTan Qiao This study had two objectives. The first objective was to compare the fine needle aspiration biopsy (FNA) and Rotex needle biopsy techniques (RNB). The quantity and the quality of cells and the tissue fragments of specimens obtained with FNA and RNB were analyzed statistically. This study demonstrated that the RNB produces a greater quantity and a higher quality sample for interpretation. than does FNA. Correlations between cell numbers and fragment numbers varied with the different tissue types. The second objective was to observe the tissue changes during and.after the application of direct electrical current (DC). The tissue damage included necrosis. These experiments indicated that 10 - 20 volts is the optimal voltage needed to produce the desired degree of tissue damage if this technique were to be used in treatment of cancer or other unwanted tissues. ACKNOWLEDGMENTS I express my sincere appreciation to my advisor, Dr. Charles D. Mackenzie, for his guidance and encouragement throughout this study, for his invaluable assistance in the whole process of broadening my perspectives on experimentation in breast disease research and in the preparation of the manuscript" .Appreciation, is also extended to my other committee members, Dr. Stuart Sleight and Dr. James E. Potchen for serving on the guidance committee and for advice on pathology and radiology. Acknowledgement is made to my colleagues in the laboratories, Dr. Li Chang, Dr. Chuanguo Xu, Mark K. Huntington, and Jose I. Santiago, who have frequently helped in many ways. I am grateful to Dir. Arlene Sierra showed the biopsy techniques and Dr. Tim Cooper helped on MRI study. My personal thanks go to my family, especially to my parents, who have always given me support and encouragement through all my endeavors. On a professional level, I wish to express gratitude to Dr. Raymond H. Murray, Dr. David S. Greenbaum and Dr. James E. Malye, who first introduced me to the field of medical science in the United States and provided me with extensive educational opportunities. iii TABLE OF CONTENTS List of Tables .... ....... . ..... .. ...... ......... ......... v List of Figures .... .................... ................. vi IntrOduCtion .........OOOOOOO....OOOOOOOOO000......0...... 1 References 0.0....0.........OOCOOOOOOOOOOOOOOO0...... 5 Chapter One. Tissue sampling: a comparison of the weight and the cellular nature of the material obtained by fine needle aspiration and Rotex needle biopsy techniques ............. 7 BaCkground O O O O O O O O O O O O O O O O O O O O O O O O O O ..... 7 Materials and Methods ..... ........... ... 11 Animal Experiments ........... ....... 11 Human Material ...................... 17 Results ................................. 18 Animal Studies ...................... 18 Human Patients Observation .......... 33 Discussion ....... . ...................... 36 References . ............................ . 48 Chapter Two. The experimental use of direct electric current for the destruction of the tissue, and a discussion of its potential in cancer treatment .......................... 52 Background .............................. 52 Materials and Methods ................... 56 Results and Discussion .................. 59 References .............................. 76 iv LIST OF TABLES Table 1-1. The specimens used with the two sampling techniques for tissue weight comparison ................. 15 Table 1-2. The specimens used with the two sampling techniques in cytologic evaluation ...................... 15 Table 1-3. The weights (mg) of samples obtained from animal muscle, connective and adipose tissues ........... 19 Table 1-4. The average numbers of cells and cellular fragments obtained from animal connective tissue ........ 23 Table 1-5. The average numbers of cells and cellular fragments obtained from animal adipose tissue ........... 25 Table 1-6. Probability of results using Student's t test or Student's t' test on data concerning material obtained with the different needle biopsy techniques ............. 28 Table 1-7. Probability of correlation tests ............ 30 Table 1-8. The results of averaged cell numbers judged for quality of connective and adipose tissues with the fine needle aspiration and Rotex needle biopsy techniques, and the percentage of the "impaired" and "intact" cells .......................................... 31 Table 1-9. Number of samples used to examine the cellular material for diagnosis with human tissues ............... 34 LIST OF FIGURES Figure 1-1. The weights (mg) of samples obtained by the fine needle aspiration and the Rotex needle biopsy techniques from different tissue types ........ . ......... Figure 1-2. The means of the weight (mg) from all type tissues obtained using the fine needle aspiration and the Rotex needle biopsy techniques ............ ... ....... Figure 1-3. The numbers of cells obtained from 21 22 connective tissue with the fine needle aspiration and the Rotex needle biopsy Figure 1-4. The numbers of tissue with the fine needle needle biopsy techniques .. Figure 1-5. The numbers of techniques ........ ..... ..... 24 cells obtained from adipose aspiration and the Rotex fragments obtained from adipose and connective tissues with the fine needle aspiration and the Rotex needle biopsy techniques . ...... 27 Figure 1-6. The percentage of diagnosable cell groups obtained with the fine aspiration needle and the Rotex needle sampling of connective tissues and adipose tissues Figure 1-7. The results of biopsy reports indicating "sufficient material" for cytopathologic diagnosis for the fine needle aspiration and the Rotex needle biopsy 26 techniques on human derived samples ... Figure breast Figure breast Figure breast Figure breast 1-8. sample under DIC 1-9. sample under DIC 1-10. An object sample under DIC 1-11. An object sample under DIC An object seen in unstained slides of human Nomarski Optics microscopy ...... 39 An object seen in unstained slides of human Nomarski Optics microscopy ...... 4O seen in unstained slides of human Nomarski Optics microscopy ...... 41 seen in unstained slides of human Nomarski Optics microscopy ...... 42 vi Figure 1-12. The extraneous material fragments originating from needle and contaminating biopsy samples ................................................. 43 Figure 1-13. The appearance of material obtained from a Rotex needle biopsy unstained specimen .............. .... 44 Figure 2-1. The fluid produced at the positive electrode region with different coulomb and voltage levels ........ 62 Figure 2-2. The time required for development of coulomb With different veltages 0.0.00.0...00 ........ 0.0.0.000... 63 Figure 2-3-1. Correlation between coulomb and the diameter of tissue damage at the positive electrode ..... 64 Figure 2-3-2. Correlation between coulomb and the diameter of tissue damage at the negative electrode ..... 65 Figure 2-4-1. Relationship between voltage applied and the diameter of affected tissues at the positive electrode ............................................... 67 Figure 2-4-2. Relationship between voltage applied and the diameter of affected tissues at the negative electrode ................ . .............................. 68 Figure 2-5-1. Relationship between the distance separating the two electrodes and the diameter of the tissue damage at the positive and the negative electrodes ..................... ..... .................... 70 Figure 2-5-2. Relationship between the distance separating the two electrodes and the depth of the tissue damage at the positive and the negative electrodes .............................................. 71 Figure 2-6. The characteristics of Magnetic Resonance Imaging (MRI) at the anode and the cathode regions ...... 74 vii INTRODUCTION Breast cancer is the most common malignant disease in women of the western world (Factors, 1991), and is the leading cause of death from cancer in female between 40 to 45 years of age (Stockdale, 1990). Approximately 1 in every 10 American women will develop breast cancer (Gold, Bassett and Kimme- Smith, 1987), and only about one half of this group can be successfully treated (Factors, 1991). Thus, efforts are underway to find, more effective :methods for prevention, detection, and treatment of breast cancer (Oakar, 1992; Harness, 1988; Smalley and Oldham, 1988). One of the more recently accepted approaches to the treatment of bmeast disease incorporates stereotactic mammographic localization of lesions. This latter procedure particularly offers the advantage of an early diagnosis and local treatment through its incorporation of mammography and fine needle aspirations (FNA) and Rotex needle biopsy (RNB). Another potentially useful approach which uses these needle techniques, incorporated the application of local direct electric current (DC) therapy to destroy cancerous tissue (Bolmgren, Jacobson and Nordenstrfim, 1977; NordenstréSm, 1933). The concept of mammography was developed in 1913 by Salomon, a pathologist, to define more precisely the morphologic characterizations of tumors in the breast (quoted in Dodd, 1989). In 1927, mammography was performed by Kleinschmidt in vivo (quoted in Pennes and Adler, 1988). Three years later, 119 cases which included 58 malignant tumors were reported by Warren (Warren, 1930; Gold, Bassett, and.Kimme-Smith, 1987). Early mammography images were of poor quality, but by 1960 mammographic techniques were much improved (Egan, 1960). In 1964 Payr was the first one to perform mammograms on clinical patients (Payr, 1964). Thus mammography became an important technique for detecting breast disease and a unique method for detecting nonpalpable breast masses (Weiss and Wayrynen, 1976; Gold, Bassett and Kimme- Smith, 1987). an the past decade, mammographic techniques have made possible the early detection of nonpalpable, early breast cancers and have resulted in a reduction in the mortality and morbidity from breast cancer (Holleb, 1992). Although a presumptive diagnosis of breast cancer can be made from a patient's history, and a physical examination, or from radiological studies, one of the major problems with the mammographic diagnosis of breast cancer is its variable and sometimes subtle radiographic appearance. Therefore, the biopsy of breast abnormalities is necessary and pathological examination still essential for the confirmation of a 3 diagnosis (Souba and Bland, 1991). The use of specialized techniques, such as magnification and conedown radiography, as well as FNA of nonpalpable lesions, frequently allows the preoperative diagnosis of malignancy to be of a high degree of certainty. In addition to the use of FNA, Nordenstrdm reported using a Rotex needle to biopsy mammary tumors in 1975. Two years later, Bolmgren and his colleagues reported the use of a stereotactic instrument for assisting FNA and RNB. In 1989, Azavedo and his colleagues reported using stereotactic instruments for fine needle aspiration biopsy (SFNA) and Rotex needle biopsy (SRNB) in 2594 nonpalpable breast lesions (Azavedo, Svane and Auer, 1989) with the biopsy material used for morphological evaluation, and for assessment of tumors' biological properties, such as tumor nuclear DNA. This group also reported that in 77.3% of patients, surgery was avoided.because SFNA and SRNB technique had been performed (Azavedo, 1989). In 1983, Nordenstrom published his work on the use of direct current electrodes, positioned by the stereotactic instrument, to treat breast cancer locally, and reported remarkably successful results. In 1989, Quan used DC on 62 patients with malignant tumors (including 13 with breast cancers), and Xin reported 127 patients with malignant tumors (including 17 with breast cancers), treated with DC with an effectiveness of 93.5% and 77.1% respectively. DC local treatment was proposed as a new method for the treatment of malignant tumors. Potchen, Sierra and Mackenzie introduced the use of stereotactic Rotex needle biopsy technique specifically on breast tissue into the United States in 1990 (Potchen, et al, 1991). Stereotactic localization for fine needle aspiration and the "Biopsygun" biopsy techniques in the detection of breast disease has gained much attention in recent times. Stereotactic fine needle aspiration and Rotex needle biopsy techniques with direct current (DC) treatment have the potential not only for prevention and detection, but also for the treatment of and research into breast cancer (Azavedo, 1989; Azavedo, Svane and Auer, 1989; Nordenstrém, 1983; Taubes, 1986). REFERENCES Azavedo, E.: 1989. Thesis: Non palpable breast cancers. Detection, diagnostic and prognostic aspects. Stockholm, Sweden. pp 7-33. Azavedo, E., Svane, G. and Auer, G.: 1989. Stereotaxic fine needle biopsies in 2594 non-palpable breast lesions: A comparative mammographic, cytologic and histopathologic study. The Lancet pp 1033-1036. Bolmgren, J., Jacobson, B. and Nordenstrém, B.: 1977. Stereotaxic instrument for needle biopsy of the mamma. American Journal of Roentgenology 129:121-125. Dodd, G.: 1989. Mammography. In Breast cancer. Edited by Kennedy, B. Alan R. Liss, Inc. New York. pp 25-46. Egan, R.: 1960. Experience with mammography in a tumor institution. Evaluation of 1000 studies. Radiology 75:894-900. Factors, R.: 1991. Breast cancer. Scientific American. 91:1-16. Gold, R., Bassett, L. and Kimme-Smith, C.: 1987. Introduction to breast imaging: state of the art and future directions. In Breast Cancer Detection: Mammography and Other Methods in Breast Imaging 2nd Edition. Edited by Bassett, L. and Gold, R.. pp 3- 13. Harness, J. K.: 1988. Organizing for collaborative management: what are the options? In Breast Cancer. Collaborative Management. Edited by Harness, J., Oberman, H.. Lichter, A., Adler, D., and Cody, R. Lewis Publishers, Inc. pp:3-9. Holleb, A.: 1992. Review of breast cancer screening guidelines. Cancer 69:1911-1912. Nordenstrbm, B.: 1975. New instruments for biopsy. Radiology 117:474-475. Nordenstrbm, B.: 1983. Tissue transformations over BCEC in cancer of the breast. In Biologically Closed Electric Circuits. Clinical, Experimental and Theoretical .Evidence for'an Additional Circulatory'System. ZEdited.by Nordenstrfim, B. Nordic Medical Publication. Sweden. pp 203-266. 6 Oakar, M.: 1992. Legislative effect of the 102nd congress. Cancer Prevention, detection, treatment, and research. Cancer 69:1954-1956. Payr, E.: 1964. Classic Descriptions in Diagnostic. Q Radiology. Edited by Bruwer, A. Vol 1. Springfield, IL. pp 414-433. Pennes, D. and Adler, D.: 1988. Mammography: changing role and concepts. In Breast Cancer. Collaborative Management. Edited by Harness, J., Oberman, H., Lichter, A., Adler, D. and Cody, R.. Lewis Publishers, Potchen,J., Sierra, A., Mackenzie, C., and Osuch, J.: 1991. Svane localisation of non-palpable breast lesions. The Lancet 338:816. Smalley, R. and Oldham, R.: 1988. Newer methods of cancer screening and treatment with biologic. (In Controversies in Breast Disease Diagnosis and Management. Edited by Grundfest-Broniatowski, S. and Esselstyn, Jr. C. MarcelDekker, Inc. New York, Basel. pp 481-505. Souba, W. and Bland, K.: 1991. Indications and techniques for biopsy. In The Breast. Comprehensive Management of Benign and Malignant Diseases. Edited by Blud, K. and Copeland, III. E. W. B. Saunders Company. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo. pp 527-538. Stockdale, F.: 1990. Breast cancer. Scientific American 90:1-16. Taubes, G.: 1986. An electrifying possibility. Discover April:22-37. Warren S.: 1930. Roentgenologic study of the breast. American Journal Radiology 24:113-124. Weiss, J. and Wayrynen, R.: 1976. Imaging system for low- dose mammography; Journal of .Applied Photographic Engineering 2:7-10. Quan, K.: 1989. Direct current therapy on malignant tumor. 62 cases analyses. Translation: Shaanxi Medical Journal 18(11):19-21. Xin, Y.: 1990. Result of 127 cases of malignant tumor treated with direct current therapy. Journal of China— Japan Friendship Hospital 4(3):145-149. CHAPTER ONE TISSUE SAMPLING: A COMPARISON OF THE WEIGHT AND THE CELLULAR NATURE OF THE MATERIAL OBTAINED BY FINE NEEDLE ASPIRATION AND ROTEX NEEDLE BIOPSY TECHNIQUES BACKGROUND The use of needle aspiration for the diagnosis of breast diseases has had more than 60 years of tortuous history. As early as 1926, Martin and Ellis used an 18 gauge needle with a syringe for sampling tumors from patients with cancer and related diseases. In 1930, they published their data for 65 malignant tumors and this heralded the introduction of the needle aspiration biopsy technique to diagnostic medicine (Martin and Ellis, 1930; Schondorf and Schneider, 1978). In 1933, Stewart, a pathologist, published his experiences with cytopathologic diagnosis (Rosen et a1, 1972). One year later, Martin and Ellis reported results from 1,400 needle aspiration biopsies. They emphasized that this method offered the advantages of shortened anesthesia time and the establishment of a preoperative diagnosis. As a result, the needle aspiration biopsy became a widely utilized diagnostic procedure (Martin and Ellis, 1934; Feldman and.Covell, 1985). 8 About 13 years later, in 1947, needle aspiration was criticized by Ochsner and DeBakey when they found tumor implants had developed in three patients after needle aspiration biopsies. Their conclusion was that this procedure should not be used in operable patients (Ochsner and DeBakey, 1947; Feldman and Covell, 1985). Similar concerns about the needle aspiration biopsy were published by others, all of which resulted in restricted use of this procedure with a reduction in the number of the needle aspiration biopsy procedures in the United States at that time (Allbritten, Nealon and Gibbon, 1952; Schachter and Basta, 1973; Feldman and Covell, 1985). However, these conclusions were based on the 18 gauge needle aspiration biopsy procedure. ILater, these worries were found not to be valid for biopsies when the 22 gauge needle was used in tissue sampling (Wilkinson, Franzini and Masood, 1991). In the following years, investigators reviewed the results of over 22,000 biopsies (Godwin, 1956; Franzen and Zajicek, 1968; Zajicek, 1972; Feldman and Covell, 1985; Zajdela et al, 1975; Kline, 1979) and recommended the reintroduction of needle aspiration biopsies, but with the finer (22 gauge) needle replacing the larger needle for this procedure. Fine needle aspiration biopsy (FNA) is now used frequently in the United States to diagnose palpable lesions in the breast (Kern, 1979; Vetrani et a1, 1992). A major function of FNA biopsy is to separate benign 9 diseases from malignant tumors via pathological and biological studies (Strum, Phelps and McAtee, 1983; Azavedo, Svane and Auer, 1989). FNA combined with stereotactic mammographic localization has become the most reliable method for the diagnosis of nonpalpable breast cancer and other abnormalities in recent years (Kreula, 1990; Malberger et al, 1991; Vetrani et al, 1992; Mitnick et a1, 1992). The "Rotex" needle is not presently used as frequently as bevelled needles for obtaining samples for the interpretation of breast masses. Nordenstrbm first used the Rotex needle to obtain cytologic material from mammary tumors in 1975 (Nordenstrdm, 1975). Two years later, he performed biopsies by combining this instrument with stereotactic localization of mammographic lesions (Nordenstrbm, 1977). This combined procedure was then adopted by the Karolinska Institute and Hospital of Sweden as a part of their standard biopsy procedure for the detection of nonpalpable breast cancers. This group, Azavedo and colleagues, reported their experiences with 2594 mammographically detected nonpalpable breast lesions. Stereotactic Rotex needle biopsy technique (SRNB) was considered by them to be a highly sensitive procedure for early diagnosis of breast cancer when used in diagnostic combination with mammography (Azavedo, Svane and Auer, 1989) . In 1990, SRNB was first used with stereotactic fine needle aspiration biopsy in the United States for the detection of breast nonpalpable lesions by Potchen, Sierra and Mackenzie in 10 a joint program with the Swedish.group (Potchen et al, 1991). A successful needle biopsy sampling depends on three components being performed correctly: the biopsy procedure, the preparation of sample material, and the interpretation are all essential for the success of both FNA (Cohen et al, 1987) and for RNB. FNA biopsy performance has been thoroughly discussed by Ross, Fox and others (Koss, 1980; Fox, 1979; Feldman and Covell, 1985), as has the Rotex needle biopsy technique (Nordenstrfim, 1977). Both needle types have now been used for many thousands of patients. The fixation of the cellular material currently depends largely on the preference of the person carrying out the cytological examinations. Air drying fixation, followed by May—Grunwald Giemsa staining, is the most common method of preparation for cellular smears. Ninety-five percent ethyl alcohol is standard for use with the Papanicolaou stain and is also commonly used. Four percent formaldehyde with Feulgen stain is the method of choice for single cell cytophotometry, and 95% ice cold ethanol is the cellular fixation used for flow cytophotometry (Azavedo, et al, 1982; Verma and Kapila, 1989). Other techniques that have been used for specimen fixation include the wet fixation technique in which there is immediate submersion of freshly obtained cells in fixative. Spray fixation technique incorporating ethanol, polyethylene glycol, and distilled water, is sometimes substituted as a 11 coating fixative for the smear fixation. This procedure results in better cellular morphology than air-dry fixation does (Sheehan and Hrapchak, 1980; Danos and Keebler, 1975). Procedures which use May-Grunwald-Giemsa, Papanicolaou or Diff-Quik stain are commonly used for FNA or Rotex needle biopsies (Vilaplana and Ayala, 1975; Schondorf, 1978; Azavedo, Svane and Auer, 1989). MATERIALS AND METHODS ANIMAL EXPERIMENTS SPECIMENS Three-month-old female CBA/J mice were used as donors of connective and muscle tissues. Four-month-old female Sprague Dawley rats were used for the adipose tissue studies. Subdermal tissues were obtained from the dorsal, abdominal and iliac crest skin of the animal to provide representative connective and muscle tissues; the abdominal omenta of rats were sampled to provide adipose tissues. EQUIPMENT AND SOLUTIONS A CONTROL II INRAD analytical balance was used to weigh the samples. The biopsy needles used were: a) 22 gauge bevelled aspiration needles obtained from Becton Dickinson and Company, 12 these were 115 mm in length, b) Rotex needles (172 mm long) made up of a 0.8 mm thick cannula with an inner 15 mm long groove, and 0.55 mm thick; they were obtained from URSUS KONSULT AB, Sweden. A 10 cc syringe with syringe-control II supplied by INRAD was used. The instrument holder was supplied by URSUS KONSULT AB, Sweden. The glass slides used were precleaned and had a frosted end for labelling. Standard May-Grunwald Giemsa stain solutions (Harleco product) and Diff-Quik solution (Baxter product) were supplied by the histopathological laboratory of the Michigan State University Clinical Center. An OLYMPUS AHBS/AHBT research photomicrographic microscope system manufactured by Olympus (Japan) was used for examination of the stained slides. An HFX-DX Labophot-2 three dimension microscope system manufactured by Nikon was used to observe the unstained slides. The photographic film used was Polaroid 52 and 94 supplied by Polaroid Resource Center. EXPERIMENTAL PROTOCOL The animals were euthanatized by either cervical dislocation or ethyl ether overdose, and then immobilized on a cork board. The 22 gauge bevelled aspiration needle was inserted at least 15 mm into the sampling area, and moved forward and back between 35 to 70 times whilst maintaining negative pressure, 13 i.e. holding the piston at the 8-10 cc mark on the carrier 10 cc syringe. The negative pressure was released slowly, and the aspiration needle removed from the specimen. Ten cc syringes with a syringe-control II, which helps to hold a negative constant pressure, was used for this procedure. For sample weight determination, the 22 gauge bevelled needle was reweighed after taking the biopsy and compared with the weight before the procedure. To compare the quantity and quality of the cellular material, a drop of collected material was forced out of the aspiration needle onto a slide, another slide placed on the top of this drop, and the slides pulled apart to make smears. Slides were air dried. The Rotex needle was used in some cases with a custom instrument holder and at other times without the holder. The needle was rotated to introduce it into the sampling area with the inner screw component completely covered by the cannula. The inner screw part was then rotated out of the cannula into the tissue, rotated clockwise and counterclockwise several times. The cannula was then moved forward over the screw needle completely by a rotating movement, and both components of needle were removed together in one movement. Sample weights were obtained by the same methods as used to with the 22 gauge bevelled needle samples. To retrieve the Rotex needle sample for evaluation of sample quantity and quality, the inner part of the needle was l4 pushed.out of the cannula immediately, and.the tissue material obtained in the grooves of the Rotex needle was rotated clockwise and counterclockwise whilst "smearing" the needle along the glass slide. To remove all of the material from a groove, the edge of a glass slide was placed in the groove, and the screW'needle portion rotated counterclockwise onto the glass slide from the one side to the other. The material could be removed completely up to the point of the grooves. The material was then generally air dried. The slides were stained.with.May-Grunwald-Giemsa stain (Sheehan and Hrapchak, 1980) for cytomorphological evaluation and comparison. MEASUREMENTS AND OBSERVATIONS Samples were obtained from 12 different biopsies each of muscle, connective, and adipose tissues from 6 different animals for both FNA and RNB. The specimen sample weights used with the two biopsy techniques are shown in Table 1-1. The specimens observed from FNA and RNB techniques and tissues for the cytologic evaluations are shown in Table 1-2. As it was difficult to distinguish single myocytes from myofibers, this investigation did not include muscle specimens. 15 Table 1-1. The specimens used with the two sampling techniques for tissue weight comparison FNA RNB Connective Tissue 10 10 Adipose Tissue 8 10 Muscle 10 10 Table 1-2. The specimens used with the two sampling techniques in cytologic evaluation FNA RNB T I F T I F Connective Tissue 10 10 10 10 10 10 Adipose Tissue 8 8 8 10 10 10 Muscle - - 10 - - 10 FNA = fine needle aspiration biopsy technique. RNB = Rotex needle biopsy technique. T = total number of the cells present. I = number of cells identifiable as a particular type. F = number of the fragments of tissues or supporting tissue material. - = not counted. 16 For evaluation of quantity of sample, the cellular material obtained with the FNA and the RNB from connective and adipose tissue were divided into three groups. The "total cells" group was estimated total number of cells present by the number of nucleated cells, excluding leukocytes, regardless of whether or not their type was apparent. The "identifiable cells" group included the number of cells identifiable as a particular cell type. The "tissue fragments" group consisted of the number of conglomerated cells or tissue materials. The specimens were assessed three times for these three groups on each slide to limit counting error. The cells obtained with FNA and RNB were also observed for their morphologica1.quality, and were classified into five groups: group 0 - nothing found on slides; group 1 - some host material, but with no cells; group 2 -ncells present but not diagnosable; group 3 - cells recognizable, but with impaired cellular structures; group 4 - cells present with good sharp images of intact structure. Only cells in groups 3 and 4 usable for diagnosis. The Student's t test was used to analyze the sample weights obtained from the different tissues with the two types of needle biopsy techniques. In assessing cellular material for quality and quantity, the average and the mean numbers of the total cells present, the identifiable cells, and the tissue fragments were calculated and compared. The Student's 17 t or Student's t' test was used for assessing significant differences between the numbers of cells or the tissue fragments obtained with FNA or RNB. The numbers of total and identifiable cells were analyzed by Student's t' test. A correlation test was used to analyze the relationship between the number of obtained cells and the number of cellular fragments acquired with FNA and RNB. The Chi-square test was used to analyze the data concerning the quality of cells obtained from the two types of needle biopsy techniques (Steel and Torrie, 1980). HUMAN MATERIAL Two hundred nonpalpable breast lesions were examined by mammography, sampled by SFNA and/or SRNB, submitted for pathological diagnosis under standard CAP accredited procedures as part of a regular diagnostic program in the Clinical Center Michigan State University, and were examined retrospectively. The type of needle best providing sufficient cellular material for pathological diagnosis (for both benign and malignant breast lesions) was determined. Sixteen out of the 48 benign lesions, and 6 out of 26 "suspicious for malignancy" lesions did not have clear information about what type of needle biopsy gave sufficient cytopathologic tissues for diagnosis. Therefore, they were not included in this current comparison. All pathological diagnoses were made and reports prepared by board registered pathologists. 18 Classification of human tissue observations has three groups: "Aspiration only" - the diagnostic material obtained by SFNA; "Rotex only" - the diagnostic material obtained by SRNB; "Both needles" - the diagnostic material obtained by SFNA and SRNB“ The Chi-square test was used for analyzing the results obtained from cytopathologic reports under the hypothesis: "Are there significant differences of the sufficient material for diagnosis obtained by SFNA and SRNB" (Steel and Torrie, 1980). RESULTS ANIMAL STUDIES The cytological samples were found to consist mainly of single cells or aggregates of supporting tissues and cells with a varying mixture of blood cells, fibroblasts and tissue fragments. All samples were appropriately stained with May- Grunwald-Giemsa which allowed assessment. THE WEIGHT OF TISSUES OBTAINED BY FNA AND RNB TECHNIQUES The weights obtained from muscle, connective tissue and adipose tissue with FNA and RNB are presented in Tables 1-3. FNA samples compared with those of RNB, using the Student's t test, differed.significantly for muscle, connective tissue and adipose tissue types (P < 0.05). This indicates that the results of tissue weight obtained are significantly different between FNA and RNB for all types of tissues in this study. 19 Table 1-3. The weights (mg) of samples obtained from animal muscles, connective and adipose tissues Sample Muscle Tissue Connective Tissue Adipose Tissue Number FNA RNB FNA RNB FNA RNB 1 0.4 1.4 1.4 0.8 0.8 1.4 2 0.9 1.5 1.3 1.3 0.4 1.4 3 0.2 1.7 0.9 2.0 0.9 2.2 4 0.3 1.3 0.8 0.6 0.5 1.9 5 0.7 1.2 0.8 1.3 0.8 2.6 6 0.6 1.0 1.1 2.0 1.2 2.6 7 0.6 0.6 1.1 1.8 2.1 2.3 8 0.8 1.0 1.0 1.4 1.8 0.9 9 0.7 0.6 0.5 1.2 1.1 1.8 10 0.7 0.7 1.3 1.0 0.6 1.9 11 0.2 1.8 0.7 2.1 1.1 1.1 12 0.4 1.4 0.4 2.1 0.7 2.0 Means 0.541 1.183 0.942 1.467 1 1.842 SD 0.235 0.409 0.318 0.525 0.510 0.552 SE 0.068 0.118 0.092 0.151 0.147 0.159 p 0.0025 0.0228 0.0056 SD = Standard deviation . SE = standard error . P = probability value. 20 The median, maximum and minimum weights obtained from different tissues with FNA and RNB are presented in Figure 1- 1. The mean of the weights for all tissues obtained are shown in Figure 1-2. THE NUMBER OF CELLS AND CELLULAR FRAGMENTS OBTAINED FROM THE DIFFERENT TISSUE TYPES USING FINE NEEDLE ASPIRATION AND ROTEX NEEDLE BIOPSY TECHNIQUES The averaged total numbers of cells, of the identifiable cells and of tissue fragments obtained in connective and adipose tissue with FNA and RNB present on Table 1-4 and 1-5. Figures 1-3 and 1-4 show graphically the numbers of cells obtained from adipose tissue and connective tissue with FNA and RNB. Figures 1-5 graphically shows the numbers of ‘material fragments from.adipose and connective tissues by both needle biopsy techniques. Results of Student's t test and Student's t' tests analyzing these numbers obtained FNA and RNB from connective tissue and adipose tissue are shown in Table 1-6. All probability values are significant at confidence limits of alpha < 0.05 level. 21 The Weight of Samples (mg) 0 M-Asp. M-Rtx. A-Asp. A-Rtx. C-Asp. C-Rtx. Types of Tissues and Needles Figure 1—1. The weights (mg) of specimens obtained by the fine needle aspiration biopsy and Rotex needles bioPSY technique from different tissue types. (M = muscle, A = aglpose, C = connective tissue, FNA = fine needle aspiration biopsy technique, and RNB = Rotex needle biopsy technique). RNB provides higher weight of muscle, connective and adipose tissues (P< 0.05 on Students's t tests). 22 -L _s ...-L N -§ a) l .5 .0 a) The Weight of Samples (mg) 0 O is 'oo .0 N Asp. Rtx. Types of Tissues and Needles Figure:1-2. The means of the weights (mg) from all types of tissues obtained with the fine needle aspiration and the Rotex needle biopsy techniques. (FAN = fine needle aspiration biopsy, and RNB = Rotex needle biopsy). RNB provides higher mean weights (P< 0.05 on Students's t tests). 23 Table 1-4. The average numbers of cells and cellular fragments obtained from animal connective tissue FNA RNB No. Total Identifiable Fragments Total Identifiable Fragments 1 5071 2467 5 11683 7949 6 2 32 26 0 8158 6299 15 3 172 58 5 1665 1245 14 4 91 0 2 6371 5887 7 5 763 188 3 3615 2274 2 6 185 49 7 396 315 3 7 541 162 6 4509 3234 4 8 1707 785 20 5199 2545 28 9 811 349 19 5399 3812 33 10 593 344 0 3202 2411 15 Note: FNA = fine needle aspiration biopsy. RNB = Rotex needle biopsy. Total = total number of cells present. Identifiable = numbers of cells identifiable as a particular type. Fragments = the number of tissue fragments obtained. 24 5.02 01 A The Number of Cells (Thousand) h) (D —L A-T R-T A-R R-R Types of Needles and Cell Groups Figure 1-3. The numbers of cells obtained from connective tissue with the fine needle aspiration and the Rotex needle biopsy techniques. (A-T = the total number of cells obtained with the fine aspiration needle biopsy, R-T = the total number of total cells obtained with Rotex needle biopsy, A-I = the number of identifiable cells obtained with fine aspiration needle biopsy, and R-I = the number of identifiable cells obtained with Rotex needle biopsy). 25 Table 1-5. The average numbers of cells and cellular fragments obtained from animal adipose tissue Sample FNA RNB No. Total Identifiable Fragments Total Identifiable Fragments 1 0 0 3 130 72 2 2 0 0 1 6644 4335 19 3 1382 1 8 1632 760 6 4 0 0 O 1985 818 21 5 O 0 3 1039 478 18 6 O 0 5 4626 2817 7 7 0 O 9 6713 4060 41 8 0 0 5 195 130 6 9 - - - 1551 816 10 10 - - - 344 207 4 Note: FNA = fine needle aspiration biopsy. RNB = Rotex needle biopsy. Total = total numbers of cells presented. Identifiable = numbers of cells identifiable as a particular type obtained. Fragments. = number of tissue fragments obtained. 26 I 3,000 2,487 2,500 N o o o 1 ,500 The Number of Cells '0 o o 500 A-T R-T A-R R-R Types of Needles and Tissues . Figure 1-4. The numbers of cells obtained from adipose t1$SUe with the fine aspiration needle and Rotex needle biopsy teChniques. (A-T = the total number of cells presented with the fine aspiration needle biopsy, R-T = the total number of cells presented with Rotex needle biopsy, A-I = the number of cells identifiable as a particular type with fine aspiration l“eedle biopsy, and R-I = the number of cells identifiable as a Particular type obtained with Rotex needle biopsy). 27 The Number of Tissue Fragments A-C R-C A-A Fl-A Types of Needles and Cell Groups Figure 1-5. The numbers of material fragments obtained from adipose and connective tissues with the fine aspiration needle and the Rotex needle biopsy techniques. (A-C = the fine aspiration needle biopsy on connective tissue, R—C = the Rotex needle biopsy on connective tissue, A-A = fine aspiration needle biopsy on adipose tissue, and R-A = Rotex needle biopsy on adipose tissue). 28 Table 1-6. IProbability'of results using Student's t test or Student's t' test on data concerning material obtained with the fine needle aspiration. and the iRotex needle biopsy techniques Connective Tissue Adipose Tissue Total FNA:RNB 0.0006 0.0131 Identifiable FNA:RNB 0.0010 0.0145 Fragment FNA:RNB 0.0286* 0.0319* *The numbers of material fragments analyzed by Student's t test. The others analyzed by Student's t' test (because of the requirement of the Student's t test assumptions: variances in two group should equal). Total FNA:RNB = analysis of the total numbers of cells obtained with fine aspiration needle biopsy and Rotex needle biopsy. Identifiable FNA:RNB = analysis of the numbers of identifiable cells obtained with fine aspiration needle biopsy and Rotex needle biopsy techniques. Fragment FNA:RNB = analysis of the numbers of tissue fragments obtained with fine aspiration needle biopsy and Rotex needle biopsy techniques. 29 CORRELATION BETWEEN THE NUMBER OF CELLS AND FRAGMENTS OBTAINED BY FINE ASPIRATION NEEDLE AND ROTEX NEEDLE BIOPSY TECHNIQUES The results of the correlation analysis for the results of total, identifiable cells and material fragments obtained are presented in Table 1-7. The correlation between the number of total cells and identifiable cells are significant and linearly related (P < 0.05) for all tissue patterns by both needle biopsy techniques. The correlations between the number of total cells, identifiable:cells and tissue fragments are significant for adipose tissue by RNB, but not for connective tissue by either FNA or RNB (Steel and Torria, 1980). THE UALITY OF CELLULAR STRUCTURE IN SAMPLES FROM FINE NEEDLE ASPIRATION AND ROTEX NEEDLE BIOPSIES Results of the cell structure differences observed between FNA and RNB are shown in Table 1-8 and Figure 1-6 for different tissues. Chi-square test analysis for diagnosable cell groups (group 3 and 4) show FNA and RNB to be significantly different (P < 0.05). 30 Table 1-7. Probability of correlation test Connective Tissue Adipose Tissue FNA RNB FNA RNB Total & Fragment 0.649 0.815 - 0.023* Identifiable & Fragment 0.681 0.959 - 0.037* Total & Identifiable 0.030* <.0001* - <.0001* Note: Total & Fragment = analysis on the total number of cells compared with the number of material fragments. Identifiable & Fragment = analysis on the number of identifiable cells compared. with the number of ‘material fragments. FNA = fine needle aspiration biopsy. RNB = Rotex needle biopsy. * = significant result with a linearly relationship. 31 Table 1-8. The results of averaged cell numbers judged for quality with connective and adipose tissues with the fine needle aspiration and Rotex needle biopsy techniques Type of Tissue Groups 0 1 2 3 4 Needles Total # FNA-Connective 20 1 2 8 7 2 RNB-Connective 20 0 2 1 3 14 FNA-Adipose 16 4 11 o 1 o RNB-Adipose 20 4 o o 4 12 Notes: FNA-Connective = the material obtained with fine needle aspiration biopsy from connective tissue. RNB-Connective = the material obtained with Rotex needle biopsy from connective tissue. FNA-Adipose = the material obtained with the fine needle aspiration biopsy from adipose tissue. RNB-Adipose = the material obtained with Rotex needle biopsy. 32 —L C) C) I a) C) O) C) .h CD 20 The Percentage (%) of Slides in Each Biopsy Group A-C R-C A—A R-A Types of Needles and Cell Groups Figure 1-6. The percentages of diagnosable cell groups (group 3 - cells with impaired structure, and 4 - cells with intact structure) obtained with the fine aspiration needle biopsy and the Rotex needle biopsy techniques from connective and adipose tissue. (A-C = the fine needle aspiration biopsy on connective tissue, R-C = the Rotex needle biopsy on connective tissue, A-A = the fine needle aspiration biopsy on adipose tissue, and R-A = the Rotex needle biopsy on adipose tissue). RNB provides better quality of cells than does FNA. 33 HUMAN PATIENT OBSERVATIONS Forty-eight benign lesions and 26 suspicious malignant lesions from a base group of 200 non-palpable breast lesions were assessed by SFNA and SRNB. Sixteen of 48 benign lesions and 6 of 26 suspicious malignant lesions were not included in this analysis because of lack of information. Therefore, 32 benign and 20 suspicious lesions were examined. The results of the type of needle producing sufficient information for pathologic diagnosis are presented in Table 1-9 and the percentages are shown in Figure 1-7. Results of Chi-square analysis show a significant difference (P < 0.05) for sufficient cellular material for pathologic diagnosis with both benign and suspicious malignant lesions with either FNA and RNB. 34 Table 1-9. Number of samples used to examine the cellular material for diagnosis with human tissues Benign Lesions Suspicious Lesions Total No. Lesions examined 32 20 Aspirate Only 5 2 Rotex Only 14 9 Both Needles 13 9 35 100* 95 The Percentage (%) of Observations A-B R-B A-M R-M Types of Needles and Tissue Chang Figure 1-7. The results of biopsy reports indicating "sufficient material" for cytopathologic diagnosis for the fine needle aspiration and the Rotex needle biopsy techniques on human tissues. (A-B = the benign change in the breast was diagnosed with the fine aspiration needle biopsy, R-B = the benign change in the breast was diagnosed with the Rotex needle biopsy, A-M = the suspicious malignant change in the breast was diagnosed with the fine aspiration needle biopsy, R-M = the suspicious malignant change in the breast was diagnosed with the Rotex needle biopsy). 36 DISCUSSION This study addresses the questions: Why use the Rotex needle for sampling? Is RNB a better technique than FNA? How much tissue is enough for making a pathologic diagnosis from RNB? Stereotactic fine aspiration needle biopsy is now a well- established, reliable and safe method for the rapid diagnosis of non-palpable masses, but false negative (11.35 percent) and false positive results (0.17 percent) have been posed as two of the problems with this procedure (Wilkinson, Franzini and Masood, 1991). Results of Nordenstrdm and Azavedo who used fine aspiration and Rotex needles for breast biopsy are very encouraging (Nordenstrbm, 1975, 1977; Azavedo, 1989). In this study, fine needle aspiration biOpsy and Rotex needle biopsy were evaluated based on specimen weights, the number of cells obtained (from animal studies), and sufficiency of material for pathologic diagnosis (from the human tissue observations). Two of three essentials (performing, smearing and interpreting) which affect FNA and RNB results are discussed here. The weight of material obtained is used to evaluate and compare the capacity of cytological materials with FNA and RNB. The specimen data from muscle, connective and adipose tissues of animals, using the weight of obtained biopsy material, indicate that RNB provides significantly more cellular material than does FNA. 37 The numbers of cells and tissue fragments obtained by these two types of needle biopsy techniques are significantly different. The RNB provides greater quantity and higher quality biopsy material for diagnosis with connective tissue and adipose tissue of animals and mammary tissue of humans. Rotex needle biopsy technique not only gives more cells for diagnosis, but preserves the cells' morphology better as well. So this study shows that with the human mammary biopsies, SRNB can provide (44% more with benign lesions and 45% more with suspicious lesions, Figure 1-7 illustrates this showing), more material adequate for diagnosis than does SFNA in this study. The advantages of RNB over FNA are as follows: 1. Provides a more effective biopsy: RNB with one can obtain a 15 mm long biopsy whereas FNA collects sample only from the tip of needle and its trajectory tract. 2. Rotex needle produces not only single cells, but also some organized structure fragments because of its long cutting edge and the protective cannula cover. In the component of this study addressing adipose tissue biopsy, the FNA does not obtain intact cells as easily as the RNB. 3. A wider range of material can be obtained by RNB, eg. fibrous tissues (elastic fibrous or collagenous fibers); these tissues are hardly obtained by FNA” However, there are some problems inherent to the needle: 1. It cannot be easily used for cysts because it can not hold fluid. 2. Some cellular fragments are too 38 thick to be evaluated in a smear, reducing the needle's effectiveness. Interestingly, under specialized microscopy (Nikon HFX-DX or DIC Nomarski Optics) with the unstained fresh biopsy material, many extra findings.can.beiobtained, those shown in, Figures. 1-8, 1-9, 1-10 and 1-11. However, after staining, some of these structures cannot be observed anymore on the slide. These unstained structures have not been used, either for characterization of a diseases or for providing information useful for histopathological diagnosis. Indeed, further studies on three dimensional unstained biopsy materials are necessary. From our experience, we found some extraneous material (or adherent) fragments that may originate come from the needle and be confused with the host material (see Figure 1- 12). To avoid these artifacts, precleaning the biopsy needle, and gently smearing the needle on the slides are recommended. These are structures that may be useful for making a diagnosis. Fer example, round structures (macrophage like cells) often occur with cyst lesions (Figure 1-13). 39 Figure 1-8. An object seen in unstained slide of human breast sample under DIC Nomarski Optics microscopy. After staining the structure can not be observed. 40 I I .-": 3. :1 '( . ‘ L .‘e .47 Figure 1-9. An object seen in unstained slide of human breast sample under DIC Nomarski Optics microscopy. After staining the structure can not be observed. 41 Figure 1-10. An object seen in unstained slide of human breast sample under DIC Nomarski Optics microscopy. After staining the structure can not be observed. 42 An object seen in unstained slide of human Figure 1-11. breast sample under DIC Nomarski Optics microscopy. staining the structure can not be observed. After 43 _ Figure 1-12. The extraneous material fragments orlginating from needle and contaminating biopsy samples. 44 Figure 1-13. The appearance of material from a Rotex needle biopsy unstained specimen. It is often found in the fluid from cysts. 45 How much tissue from a rotex needle biopsy is enough for making a diagnosis? We assessed the correlation between the number of tissue fragments and the number of identifiable cells on a slide, because cellular fragments can be seen at the gross level and may give some indications of the presence of a diagnostic level of cells before the actual microscopic evaluation take place. Results from this analysis shows that the greater the number of total cells obtained, the higher the number of identifiable cells present in connective tissue and adipose tissue samples; this is true for both types of needle biopsy. The numbers of total and identifiable cells obtained are linearly related; however, the correlation between the number of identifiable cells and the number of tissue fragments is variable: with adipose tissue, they are linearly related, but with connective tissue the numbers of cells and tissue fragments obtained are not related to each other. An explanation for this may lie in differences in the character of individual tissue types” 'The number of fragments that will give an adequate number of cells for diagnosis seems to be 3 - 10 fragments, but the size of fragments, the thickness of specimens and the amount of blood in the background should also be considered (Schnitt and Wang, 1989). The technique for smearing a Rotex needle specimen was used as Nordenstrom first introduced it (Nordenstrdm, 1975). This method appears reliable from our experience. However, in this current study, we eventually made smears by when turning 46 the Rotex needle whilst smearing on the slide. This action involved.turning the inner part.of the needle by rotating back and forward for the first slides, then turning the needle completely counterclockwise to extrude all the material; this differs from Nordenstrbm (1975) who only turned the needle counterclockwise to make smears. Our approach gave a thinner and more identifiable sample. When using the edge of the glass slide to help to take tissue from the tip of the groove, the lesser the pressure used, the lesser the extraneous contamination from the Rotex needle itself. There are a number of differing opinions as to how best to make a slide preparation from the needle aspirated material (Schondorf and Schneider, 1978). We chose air-drying with the May-Grunwald-Giemsa stain for animal studies; and air-drying with Diff-Quik staining for human mammary tissue. All slides appeared appropriately stained. These methods are commonly used, simple and reliable. Our experiences with using the instrument holder with the Rotex needle to perform the biopsy showed that it can hold the Rotex needle in a stable position, allow for easy coverage of the cannula over the inner part of the needle, and allow the whole needle to be easily removed. These advantages shorten sampling time, offer good stability of handling and prevent seeding or implantation of tumor cells into the tissues around the needle track. On the other hand, using the instrument holder requires more working space and limits the angle of 47 insertion of the needle because of the large size of the instrument. Some experience is also necessary to successfully insert the needle with.the instrument holder guiding the Rotex needle. In conclusion, this study indicated.that the Rotex needle biopsy technique is a more suitable, more reliable, and more effective technique for tissue sampling because RNB provides higher quantity and quality of biopsy tissue material than does FNA. It is also diagnostically helpful and confirmatory when used in conjunction with FNA. SRNB can provide 44% on benign lesion and 45% on suspicions lesion more sufficient material for pathologic diagnosis compared with SFNA alone. The analyses in this study predict that the number of fragments could used as a reference for presence of an adequate the number of total and identifiable cells for cellular evaluation on adipose tissue; 'this may give the SFNA and SRNB user a rough idea of sufficiency of material for pathologic diagnosis. .Air-drying and.May-Grunwald-Giemsa and Diff-Quik stains are suitable for both needle biopsy techniques. The instrument holder for the Rotex needle could be helpful for the procedure when the user is familiar with its specific characteristics. REFERENCES Allbritten, F. Jr., Nealon, T. and Gibbon J. Jr.: 1952. Primary cancer of the lung. Surgical Clinical of North America 32:1657-1672. Azavedo, E. , Svane, G. and Auer, G. : 1989. Stereotactic fine-needle biopsy in 2594 mammographically detected non- palpable lesions. The Lancet May 13: 1033-1036. Azavedo, E., Tribukait, B., Konaka, C. and Auer, G.: 1982. Reproducibility of the cellular DNA-distribution patterns in multiple fine needle aspirates from human malignant tumors. Acta Pathologica, Microbiologica, et Immunologica Scandinavica. Section A 90:79-83. Cohen, M., Rodgers, C., Hales, M., Gonzales, J., Ljung, B., Beckstead, J., Bottles, K. and. Miller, T.: 1987. Influence of training and experience in fine-needle aspiration biopsy of breast. Archives of Pathology and Laboratory Medicine 111:518-520. Danos, M. and Keebler, C.: 1975. Fixation. In Manual of Cytotechnol ogy . Edited by Oertol , Y . Iganchu-Shoin Publisher. pp 262-265. Feldman, P. and Covell, J.: 1985. Introduction. In Fine Needle Aspiration Cytology and Its Clinical Applications: Breast & Lung. Edited by Feldman, P. and Covell, J. Library of Congress Cataloging in Publication Data. pp 3-10. Fox C. 1979.: Innovation in medical diagnosis; The Scandinavian Curiosity. The Lancet 1:1387-1388. Franzen, S. and Zajicek, J.: 1968. Aspiration biopsy in diagnosis of palpable lesions of the breast. Critical review of 3479 consecutive biopsies. Acta Radiologica 7:241-262. Godwin, T.: 1956. Aspiration biopsy. Technique and application. Annals New York Academy of Sciences 63:1348-1373. Kern W.: 1979. The diagnosis of breast cancer by fine- needle aspiration smears . Journal American Medical Association 241:1125-1127. Kline, T., Joshi, L. and Neal, H.: 1979. Fine needle aspiration of the breast: Diagnosis and pitfalls : a review of 3545 cases. Cancer 44:1458-1464. 48 49 Kline, T.: 1988. Fixation. In Handbook of Fine Needle Aspiration Biopsy cytology. Edited by Kline, T. pp 14- 15. Koss, L.: 1980. Thin needle aspiration biopsy. Acta Cytologica 24:1-3. Kreula, J.: 1990. A.new'method for investigating the sampling technique of fine needle aspiration biopsy. Investigative Radiology 3:245-249. Malberger, E., Edoute, Y., Toledano, O. and Sapir, D.: 1991. Fine-needle aspiration and cytologic finding of surgical scar lesions in 'women ‘with breast cancer. Cancer 69:148-152. Martin, H. and Ellis, E.: 1930. Biopsy by needle procedure and aspiration. Annals of Surgery 92:169-181. Martin, H. and Ellis, E.: 1934. .Aspiration biopsy; Surgery, Gynecology and Obstetrics 59:578-589. Mitnick, J., Vazquez, M., Roses, D., Harris, M. and Schechter, 8.: 1992. Recurrent breast cancer: Stereotaxic localization for Fine-needle aspiration biopsy. Work in progress. Radiology 182:103-106. Nordenstrbm, B.: 1975. New instruments for biopsy. Radiology 117:447-475. Nordenstrbm, B.: 1977. Stereotaxic screw needle biopsy of non-palpable breast lesions. In Breast Carcinoma: The Radiologist's Expanded Role. Edited by Westinghouse, M. John Wiley & Sons. New York. pp 313-318. Ochsner, A., DeBakey, M. and Dixon, J.: 1947. Primary cancer of the lung. Journal American Medical Association 135:321-327. Potchen, J., Sierra, A., Mackenzie, C. and Osuch, J.: 1991. Svane localisation of non-palpable breast lesions. The Lancet 338:816. Rosen, P., Hajdu, S., Robblns, G. and Foote, Jr. F.: 1972. Diagnosis of carcinoma of the breast by aspiration biopsy. Surgery, Gynecology and Obstetrics 134:837- 838. Schachter, E. and Basta, W.: 1973. Subcutaneous metastasis of an adenocarcinoma following a percutaneous pleural biopsy. American Review Respiratory Disease 107:283- 285. (I) Ve Vei Vil wi 11 50 Schnitt, S. and Wang, H.: 1989. Histologic sampling of grossly benign breast biopsies. How much is enough? American Journal Surgical Pathology 13(6):505-512. Schondorf, H. and Schneider, V.: 1978. Historical and regional development. In Aspiration Cytology of the Breast. Edited by Schondorf, H.. Translated by Schneider, V. W. B. Saunders Company. Philadelphia, London, Toronto. pp 4-14. Sheehan, D. and Hrapchak, B.: 1980. General staining considerations. In Theory and Practice of Histotechnology. The C. V. Mosby Company. Columbus. pp 157-159. Steel, R. and Torrie, J.: 1980. Chapter 3 Probability. In Principles and Procedures of Statistics A Biometrical Approach. Second Edition. Edited by Steel, R. and Torrie, J.. McGraw-Hill Book Company. New York, St. Louis, San Francisco, Auckland, Bogota, Hamburg, London, Madrid, Mexico, Montreal, New Delhi, Panama, Paris, Sao Paulo, Singapore, Sydney, Tokyo, Toronto. pp 39-65. Stewart, F.: 1933. The diagnosis of tumors by aspiration. American Journal of Pathology 9:801-812. Strum, J., Phelps P. and McAtee, M.: 1983. Resting human female breast tissue produces iodinated protein. Journal of Ultrastructure Research 84:130-139. Vetrani, A., Fulciniti, F., Benedetto, G., Zeppa, P., Troncone, G., Boscaino, A., Rosa, G. and Palmbini, L.: 1992. Fine-needle aspiration biopsies of breast masses. An additional experience with 1153 cases (1985 to 1988) and a Meta-analysis. Cancer 69:736-740. Verma, K. and Kapila, K.: 1989. The role of fine needle aspiration cytology of breast lumps in the management of patients. Indian Journal of Medical Research 90:135- 139. Vilaplana, V. and Ayala, M.: 1975. The cytologic diagnosis of breast lesions. Acta Cytologica 19:519-526. Wilkinson, E., Franzini, D. and Masood, S.: 1991. Cytological needle sampling of the breast: techniques and end results. Q1 The Breast. Comprehensive Management of Benign and Malignant Diseases. Edited by Bland, K. and Copeland, E. III. W. B. Saunders Company. Philadelphia, London, Toronto, Montreal, Sydney, Tokyo. pp 475-497. 51 Zajdela, A., Ghossein, N. and Pilleron, J.: 1975. The value of aspiration cytology'in.the:diagnosis of breast cancer: Experience at the foundation curie. Cancer 35:499-506. Zajicek, J.: 1972. Aspiration biopsy cytology. Part 1: In Cytology of Supradiaphragmatic Organs. Edited by Wied, G. Monographs in Clinical Cytology, vol 4. New York, Karger. pp 5-18. CHAPTER TWO THE EXPERIMENTAL USE OF DIRECT ELECTRIC CURRENT FOR THE DESTRUCTION OF TISSUE AND A DISCUSSION OF ITS POTENTIAL IN CANCER TREATMENT BACKGROUND Surgical, radiological and chemical therapies are the major treatments for breast cancer. Unfortunately, there are many limitation to these therapies. Extensive efforts are underway to develop better methods for cancer treatment. Endocrine, immunological and other approaches have been used experimentally to treat breast malignancy (Fournier and Kubli, 1989; Swain and Lippman, 1991). Nordenstrbm advanced a new theory which he named the Biological Closed. Electric: Circuits (BCEC) and. developed direct electric current therapy (DC) for treatment of malignant diseases (Nordenstrbm, 1978, 1983 and 1985). The basic principle of the BCEC theory is that the body electric can be compared to a battery, in which the circuit is driven by the separation of oppositely charged ions. In the body, electrical circuits are switched on by an injury, an infection, or a tumor, or even by the normal activity of the 52 53 body's organs. Electric currents course through arteries and veins and across capillary walls, drawing white blood cells and metabolic compounds into and out of surrounding tissues and thereby balancing the activity of internal organs and provides the foundation of the healing process. DC treatment can enhance the body's ability to fight tumors. From June 1978, Nordenstrbm began to successfully treat malignancies in patients with DC. This new theory and DC therapy have been followed with interest (Nordenstrdm, 1965, 1977, 1978, and 1983). The use of DC in medical research has a long history. In 1895 the electrophysiologist Golsinger inserted electrodes with 20 to 40 mA direct current into the brains of dogs and produced focal injuries of tissue (Golsinger, 1908). Horsley and Clarke (1908) reported "for that a unit of time, e.g., 1 minute, there will be about a 1 mm breadth of destruction for each unit of current employed". Ingvar (1920) briefly described reactions of cells to galvanic current using tissue cultures. Louchs (1953) found that cells were more resistant to electrolytic destruction by platinum electrodes than with other metal electrodes. Yasuda used 10 mA to enhance healing of injured bone (Yasuda, 1953). In 1965 the Chinese employed a jprocedure called "acupuncture narcosis" in a surgical operation which involved the use of a needle carrying a weak DC (Xin, 1990). Malzack described a mechanism for pain control that used.an 0.05). The probable explanation is that the major element affecting the depth of tissue damage is the depth of the electrode inserted in the specimen. 67 _5 O) l _L b _5 N —I 04 Diameter of Affected Tissue (cm) 9 (X) P N 0 5 1O 20 24.43 Voltage (at 30 coulomb) Figure 2-4-1. Relationship between voltage applied and the diameter of the affected tissues at the positive electrode with 30 coulomb level. The lowest voltage (5 V) did not provide the smallest diameter of tissue damage. 68 Diameter of Affected Tissue (cm) .0 .0 .0 h 0) m .0 m 0 5 10 20 24.43 Voltage (at 30 coulomb) Figure 2-4-2. Relationship between voltage and the diameter of affected tissues at the negative electrode with 30 coulomb level. The highest voltage (24.43 V) did not necessarily produce the greatest tissue damaged at the negative electrode. 69 The relationship between the distance ( 1.5, 3.0 and 8.0 cm) separating the two electrodes and the diameter of damaged tissue induced by 10 volt, 20 coulomb at are shown in Figure 2-5. Figure 2-5-1 and 2-5-2 present the diameter and depth of affected tissues respectively. These results imply that under the condition of the same applied voltage, varying’ the distance between the positive and.negative electrodes.does not produce significantly different amount of affected tissue area with Student's t test analysis results (P > 0.05). The gross morphological observation indicated that the affected area around the positive electrode turned grey brown with a white center and a dark red jelly-like margin; this affected tissue became firm and had less elastic. The area around the negative electrode turned to a dark red jelly-like substance but remained soft, and no white core was observed. 70 A I .0 a: Diameter of Affected Tissue (cm) 0 O 1:. b) .0 n3 O 1.5 3.0 8.0 Distance of Anode to Cathode (cm) '- Anode - Cathode Figure 2-5-1. Relationship between the distance separating the two electrodes and the diameter of the damaged tissue at the positive and the negative electrodes. (All measurements were done at the 10 volt and 20 coulomb level). There are no significant differences in the tissue damage caused by changing the distance of the two electrodes. 71 —L 0) fl 1.36 1.37 1.37 1.37 Depth of Affected Tissue (cm) .0 .0 .0 7‘ r‘ b O) (I) -* N A .0 N 1 .5 3.0 8.0 Distance of Anode to Cathode (cm) I Anode I Cathode Figure 2-5-2. Relationship between the distance separating the two electrodes and the depth of damaged tissue at the positive and the negative electrodes. (All measurements were done at the 10 volt and 20 coulomb level). There are no significant differences in the tissue damage caused by changing the distance of the two electrodes alone. 72 The histologic changesiin1regions at the positive and the negative electrodes were distinctly different. Both affected regions had signsiof necrosis“ .Around the positive electrode, the affected muscle fibers were shrunken and fractured, the cytoplasm lacked homogeneous staining, vacuolations developed, and the striations and the nuclei were absent" The epithelium of blood vessels has degenerated, and the epithelium layer was irregular. Around the negative electrode, the myofibrils were swollen and a homogeneous change appeared in the cytoplasm. The striations and nuclei had disappeared. The mesenchymal space and the blood vessels were narrowed. The morphological changes suggested that necrosis in tissues occurs at both electrodes. There is no morphological change found between the area of the electrode induced damage. The tissue damage may be caused by local joule heating, severe change in pH, and ions redispersing during the DC treatment. The occurrence of local edema and narrowed blood vessels an changes that could cause the obstruction of blood and oxygen supply to tumor tissue and result in further damage to the local tumor and thereby reduce the risk of metastasis (Xin, 1990; Pasachoff and Kutner 1981). 73 The MRI image is shown in Figure 2-6. On the anode side, a 1.1 x 0.8 x 0.8 cm, triangle shaped (with the tip downward), increased signal intensity area was noted. The signal intensity was not homogeneous, a vertical low signal line was seen in the middle of the lesion, which probably represented the pathway of the initial insertion of the needle and/or tissue dehydration. The superior part of the increased signal area was protruded out of the surface of the adjacent normal tissue. The changes on MRI represents severe edema with higher protein fluid and/ or a higher concentration of hydrogen ion in the tissue of this area. On the cathode side, a 0.6 x 1.0 x 0.6 cm rectangle shaped high signal intensity area was obtained at the superior aspect of the tissue. The increased signal intensity of this area was less prominent (less brighter) than at the anode. There was a very low signal intensity band which appeared at the center or the lower aspect of the lesion. A low signal linear line extended from the dark band to the surface of the lesion, which is almost as flat as the adjacent normal tissue. These changes on MRI indicate: the high signal intensity region represents an area with more fluid than normal. The low signal intensity region represents an area with severe dehydration. 'The exact areas of damage caused by DC treatment could be observed. 74 , 1.0-I 00055 UUl Ul II'H1‘~td Int ('01 00* UNI-[F II .1 ll;l"' 'lf'e' [Jill r WW I I {III .... 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