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Cytogenetic findings in a primary leiomyosarcoma of the prostate
Cytogenetic Findings in a Primary Leiomyosarcoma of the Prostate J. Limon, P. Dal Cin, and A. A. Sandberg
ABSTRACT: Cytogenetic analysis of a primary leiomyosarcoma of the prostate revealed a clonal chromosomal rearrangement involving chromosomes #2, #3, #9, #11, and #19. The results are discussed in relation to the cytogenetic findings in other solid tumors, especially those of the prostate and leiomyosarcoma at other sites. INTRODUCTION Sarcoma of the prostate is an infrequent lesion, difficult to diagnose, and even more difficult to treat [1-6]. The incidence of sarcoma compared with all other primary malignancies of the prostate ranges from 0.1% to 0.5% [2, 6]. Sarcomas arise from stromal tissue consisting of m u s c u l a r and fibrous elements, or from interstitial lymphoid foci of mesodermal tissue of the genital cord. The intraprostatic origin of myosarcomas is related to smooth muscle in the case of leiomyosarcoma and to striated muscle in that of rhabdomyosarcoma. These two types of myosarcoma compose the majority of primary sarcomas of the prostate [5]. Embryonal rhabdomyosarcoma of the prostate occurs most frequently in young boys and leiomyosarcoma in older age groups [1,5]. Chromosome analyses of h u m a n prostatic cancers and leiomyosarcomas are still very sparse [7, 8]. At present, five cases of adenocarcinoma of the prostate and one cell line of prostatic carcinoma have been analyzed with b a n d i n g techniques [911]. Only two cases (both intestinal) of leiomyosarcoma and n o n e at other sites have been analyzed by these techniques [12, 13]. We report herein a case of primary leiomyosarcoma of the prostate in which a clonal karyotype, 45,XY, - 2, - 6 - 9 - 11 - 19,t(2;19)(q22;q13.3),del(3)(p13p21),r(9;?), t(2;11;19)(q2;p13;q13.3), was found in 25 metaphases.
CASE REPORT The patient, a 38 year old man, was in good health until 1979 w h e n he passed a kidney stone. In 1980 he had hematuria and weakened urinary stream. He was hospitalized and a transurethral resection of a urethral polyp arising from the veru m o n t a n u m was carried out. In August 1983, a second urethral polyp was excised from the posterior urethra. The patient was healthy u n t i l November 1984, w h e n he was hospitalized again because of intermittent hematuria and weakened stream.
From the RoswellPark MemorialInstitute, Buffalo, NY. Address requests for reprints to Dr. Avery A. Sandberg, Depts. of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263. Received August 12, 1985; accepted September 30, 1985.
159 ©1986Elsevier Science PublishingCo., Inc. 52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet22:159-167(1986) 0165-4608/86/$3.50
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J. Limon, P. Dal Cin, and A. A. Sandberg Intravenous p y e l o g r a p h y s h o w e d a n o r m a l u p p e r urinary tract, a normal bladder, and no evidence of enlargement of the prostate. Cytoscopy was done and a transurethral resection of a p o l y p was performed. Pathological e x a m i n a t i o n showed p o l y p o i d urethritis. In December 1984 the patient was found to have a very large prostate. Intravenous p y l o g r a p h y s h o w e d a huge mass in the prostate, p u s h i n g the bladder neck and floor of the b l a d d e r u p w a r d s . A s u p r a p u b i c cystostomy was done and a necrotic tumor was f o u n d in the prostate at the base of the bladder, which was b i o p s i e d and s h o w n to be a leiomyosarcoma. F o l l o w i n g a negative work-up, the patient u n d e r w e n t a cystoprostatectomy with ileal loop diversion in January 1985. A s p e c i m e n of this t u m o r was sent for pathologic and cytogenetic examinations. Microscopic diagnosis i n d i c a t e d an unclassified sarcoma of the prostate with extension to the b l a d d e r neck (Figs. 1 and 2); electron m i c r o s c o p y revealed ceils with myofilaments and, hence, the t u m o r cells were interpreted as compatible with those of a l e i o m y o s a r c o m a of the prostate (Fig. 3).
MATERIALS AND METHODS Fresh tumor tissue was obtained from the Department of Urologic Oncology of the Roswell Park Memorial Institute. The tumor s a m p l e was w a s h e d in Hanks' solution with antibiotics and disaggregated m e c h a n i c a l l y by m i n c i n g w i t h a scalpel in a Petri dish. The pieces of tissue were transferred to culture flasks containing 3.5 ml of growth m e d i u m , 0.5 ml of collagenase (2000 U/ml) (Gibco) at a final concentration of 200 U/ml and 0.5 ml of cholera toxin (Schwarz) at a final concentration of 500 ~g/ml [14]. The culture m e d i u m used was McCoy's 5A (Gibco) with 20% of
Figure 1 Histologic appearance of tumor, showing interlacing bundles of spindle cells, with hyperchromatic nuclei with blunt ends. Some mitotic figures are present. ( × 196)
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Figure 2 Histologic appearance of tumor, showing interlacing bundles of spindle cells, with hyperchromatic nuclei with blunt ends. Some mitotic figures are present. ( × 196) fetal calf serum, L-glutamine, and antibiotics; penicillin (100 IU/ml), streptomycin (100 ~g/ml), gentamycin (50 Ixg/ml), and Fungizone (25 p.g/ml) (Gibco). Tumor pieces were incubated at 37°C for 16 hours without agitation. After incubation the cells were disaggregated by pippetting; fast growing primary monolayer cultures in the abovementioned growing medium supplemented with 10 p~g/ml of cholera toxin were established. The cultures were harvested 24 hours later, after 4 hours of treatment with colcemid (0.02 p~g/ml) (Gibco). The cells were exposed to hypotonic shock according to Gibas et al. [15] and the swollen cells detached by a scraper and transferred into centrifuge tubes. The cells were fixed with a mixture of methanol/ glacial acetic acid (3:1). Slides were prepared in a conventional way; G-banding was obtained by the method of Yunis [16]. RESULTS
Detailed banding analysis was performed on 25 metaphases. The modal chromosome number was 45. Five marker chromosomes were consistently observed in all metaphases (Figs. 4 and 5): MI: t(2;19)(q22;q13.3}2pter--,cen--~2q22 : : 19q13.3--~19qter) M2: del(3)(p13p21) (Fig. 6) M3: ring(9;?). This chromosome probably arose from a rearrangement between two chromosomes, because C-banding revealed two heterochromatic blocks. One of them was chromosome #9, whereas, the origin of the second one was an unknown.
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Figure 3 An electron micrograph of a representative elongated tumor cell showing the bulk of the cytoplasm to be filled with microfilaments arranged in a long axis is ill-defined. There were two types of filaments: numerous thin filaments and much smaller but thicker ones. The latter were distributed randomly and no precise relation to the former. The cell surface was also coated with filamentous materials. These features are characteristic of smooth muscle cells and, thus, the tumor is a leiomyosarcoma. Uranyl acetate-lead staining. ( × 11,340)
M4: t(2;11)(q22;p13) (2qter-2q22::11p13-cen-11qter) M5:t(11;19)(p13 ;q13.3)(19pter-cen-19q13.3::11p13-11pter) Almost all metaphases presented i n the following karyotype: 45 ,XY, - 2, - 6, - 9, - 11 - 19,T(2;19)(q22;q13.3),del(3)(p13p21),r(9;?),t(2;11;19)(q22;p13;q13.3).
DISCUSSION Primary prostatic malignant tumors arising from muscle are rare [1-6]. The presenl cytogenetic study of such a tumor revealed clonal chromosomal abnormalities in. volving chromosomes #2, #3, #9, #11, and #19. To date, only two cases of leio. myosarcoma, both intestinal, have been analyzed with b a n d i n g techniques [12, 13] In both cases, chromosomes #1, #11, and #21 were involved i n clonal abnormal ities [i.e., del(1)(p11),del(ll)(q11) and t(1;21)(p32;q22)]. In our case chromosomet other than these were involved in rearrangements, with the exception of chromo some #11. In one intestinal tumor the marker derived from chromosome #11 ap parently consisted of only the short arm [12], whereas, i n our case the breakpoin occurred in the short arm, with the p l 3 - p t e r fragment being translocated to the lonl arm of chromosome #19. It is impossible to reach any conclusion on the specificitI
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20
....
22:
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Figure 4 G-banded karyotype from a primary leiomyosarcoma of the prostate. Arrows point to structural abnormalities. See text for description of marker chromosomes.
of these c h r o m o s o m a l abnormalities on the basis of only three cases of leiomyosarcoma which, in addition, arose in different anatomical sites. The most c o m m o n t u m o r arising in the prostate is adenocarcinoma. In the few p u b l i s h e d cytogenetic studies of this cancer n o r m a l karyotypes were found in most cases [9, 17], probably due to overgrowth by stromal cells. Thus, o n l y five cases of a d e n o c a r c i n o m a a n d one h u m a n prostatic carcinoma line (LNCaP) have been analyzed successfully using b a n d i n g techniques [9-11]. In four cases and in the LNCaP line one c o m m o n marker c h r o m o s o m e was seen, del(10)(q24}. It has been suggested that a deletion of c h r o m o s o m e # 1 0 represents a specific change in prostatic carcinoma [11]. In the case of l e i o m y o s a r c o m a presented by us the structural karyotypic aberrations were different from those seen in a d e n o c a r c i n o m a of the prostate. There are m a n y reports on c h r o m o s o m e # 3 abnormalities in h u m a n tumors, but most of them a p p a r e n t l y lack specificity [18]. Recently, however, frequent aberrations of the short arm of the c h r o m o s o m e # 3 have been found in rhabdomyosarcoma [19], small cell cancer of the lung [20], in some ovarian cancers [21], and in mixed parotid gland tumors [22]. Some of the breakpoints occurred in the fragile site segment of this c h r o m o s o m e at 3p14 [23]. The del(3)(p13p21) in our case confirms the p r e d i s p o s i t i o n to nonspecific breakage of this c h r o m o s o m e in cancer cells. Though ring c h r o m o s o m e s have been observed in cancer cells [7], only some
164
a
b
C
2
11
19
22
Figure 5 Partial karyotype from three metaphases showing translocation between chromosomes #2, #11, and #19. Arrows indicate breakpoints.
Figure 6 Interstitial deletion of a chromosome #3 from four different metaphases (a-d Arrows indicate deleted region p13p21.
a
~b
c
d
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have been identified with b a n d i n g techniques. In the present s t u d y we found a ring c h r o m o s o m e that arose from a rearrangement of two chromosomes, one of w h i c h was a c h r o m o s o m e # 9 with breakpoints in pter and qter. The nature of the second c h r o m o s o m e i n v o l v e d is unknown; p o s s i b l y it is a fragment of c h r o m o s o m e #6. We are unaware of any reports of the presence of a r(9) c h r o m o s o m e in solid tumors [24]. The correlation between the c h r o m o s o m a l loci of oncogenes and c h r o m o s o m e breakpoints in cancer cells has recently received m u c h attention [25-29]. Involvement of c h r o m o s o m e #11 in t(11;19)(p13;q13.3) in the present study is especially interesting because the c-ras oncogene is located in band p14 [30] or distal to p15.115.5 on c h r o m o s o m e #11 [31]. The c - r a s oncogene is activated in some b l a d d e r cancer lines [32] and the p r o d u c t of this gene is expressed in b l a d d e r and lung carcinomas [33]. A second i m p o r t a n t p h e n o m e n o n is that c h r o m o s o m e #11 is frequently involved in structural aberrations (deletions) in the a n i r i d i a - W i l m ' s tumor s y n d r o m e [34, 35], carcinoma of the b l a d d e r [36, 37], and familial renal cell carcinoma [38]. Many more prostatic tumors, adenocarcinomas, and sarcomas will have to be e x a m i n e d before they can be characterized cytogenetically. As in the case of leukemias and some tumors in w h i c h the c h r o m o s o m e findings have led to a subclassification of k n o w n entities into several subentities, prostatic cancer m a y be shown to contain a n u m b e r of subentities defined karyotypically, however similar the histology of the tumors. The 1 0 q - a n o m a l y referred to above m a y characterize one of these subentities. In the case of leiomyosarcoma, the question is even more complicated. Not only is such a t u m o r very rare in the prostate and, hence, difficult to characterize cytogenetically, but it will also be necessary to ascertain whether leiomyosarcomas at different sites express similar karyotypic events, regardless of their site of origin, or w h e t h e r there is some specificity in the c h r o m o s o m e changes related to the organ or tissue from w h i c h they originate. Supported in part by Grant CA-15436 from the National Cancer Institute. The authors thank Dr. Annamma Isac for the histalogic photographs, and Dr. Untae Kim for the electronmicrographs of the tumor and Dr. Claude Turc-Carel and Dr. Herman Van Den Bergne for their helpful discussion of the karyotypes. ADDENDUM
After this p a p e r was submitted for publication, Atkin and Baker [Hum Genet 70:359-364, 1985] described the cytogenetic findings in a leiomyosarcoma of the prostate, w h i c h contained m a n y c h r o m o s o m e changes.
REFERENCES
1. Fitzpatrick TJ, Stump G (1960): Leiomyosarcoma of the prostate: Case report and review of the literature. J Urol 83:80-83. 2. Siegel J (1963): Sarcoma of the prostate: A report of four cases and a review of current therapy. J Urol 89:78-63. 3. Torres LF, Estrada JY, Esquivel EL [1966): Sarcoma of the prostate gland: Report of four cases. J Urol 96:380-384. 4. Vishwa NP, Tripathi P, Dick VS (1969): Primary sarcoma of the urogenital system in adult. J Urol 101:898-904. 5. Smith BH, Dehner LP (1972): Sarcoma of the prostate gland. Am J Clin Pathol 58:43-50.
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6. Schmidt JD, Wech MJ (1976): Sarcoma of the prostate. Cancer 37:1908-1912. 7. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier NorthHolland, NY. 8. Sandberg AA (1981): The prostatic cell: Chromosomal and DNA analyses. In: The Prostatic Cell: Structure and Function, Part A. Alan R. Liss, NY, pp. 75-92. 9. Gibas Z, Pontes JE, Sandberg AA (1985): Chromosome rearrangements in a metastatic adenocarcinoma of the prostate. Cancer Genet Cytogenet 16:301-304. 10. Gibas Z, Becher R, Kawinski E, Sandberg AA (1984): A high-resolution study of chromosome changes in a h u m a n prostatic carcinoma cell line (LNCaP). Cancer Genet Cytogenet 11:399-404. 11. Atkin NB, Becher MC (1985): Chromosome 10 deletion in carcinoma of the prostate. N Engl J Med 312:315. 12. Mark J (1976): G-band analyses of a h u m a n intestinal leiomyosarcoma. Acta Path Microbiol Scand 84:538-540. 13. Becher R, Wake N, Gibas Z, Ochi H, Sandberg AA (1984): Chromosome changes in soft tissue sarcomas. J Natl Cancer Inst 72:823-831. 14. Peehl DM, Stamey TA (1984): Serial propagation of adult h u m a n prostatic epithelial cells with cholera toxin. In Vitro 20:981-986. 15. Gibas L, Gibas Z, Sandberg AA (1984): Technical aspects of cytogenetic analysis of human solid tumors. Karyogram 10:25-27. 16. Yunis JJ (1981): New chromosome techniques in the study of h u m a n neoplasia. Hum Pathol 12:540-549. 17. Jellinghaus W, Okada K, Ragg C, Gerhard H, Schoder FN (1976): Chromosomal studies of h u m a n prostatic tumors in vitro. Invest Urol 14:16-19. 18. Sandberg AA, Hecht BK, Ondreyco SM, Prieto F, Hecht F (1982): Translocations involving chromosomes 3 and 12: Hematologic diseases associates with abnormalities of these chromosomes. Cancer Genet Cytogenet 7:1-17. 19. Trent J, Casper J, Meltzer P, T h o m p s o n F, Fogh J (1985): Nonrandom chromosome alterations in rhabdomyosarcoma. Cancer Genet Cytogenet 16:189-197. 20. Whang-Peng J, Bunn PA, Kao-Shan CS, Carney DN, Gazdar A, M i n n a JD (1982): A nonrandom chromosomal abnormality, del 3p(14-23) in h u m a n small cell lung cancer (SCLC). Cancer Genet Cytogenet 6:119-134. 21. Whang-Peng J, Knutsen T, Douglass EC, Chu E, Ozols RF, Hogan WM, Young RC (1984): Cytogenetic studies in ovarian cancer. Cancer Genet Cytogenet 11:91-106. 22. Mark J, Dahlenfors R, Ekedahl C, Stenman G (1980): The mixed salivary benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genet Cytogenet 2:231-241. 23. De Braekeleer M, Smith B, Lin CC (1985): Fragile sites and structural rearrangement in cancer. Hum Genet 69:112-116. 24. Mitelman F (1985): Catalogue of Chromosome Aberrations in Cancer. Alan R. Liss, NY. 25. Yunis JJ (1983): The chromosomal basis of h u m a n neoplasia. Science 221:227-236. 26. Rowley JD (1983): Human oncogene locations and chromosome aberrations. Nature 301:290-291. 27. Pimentel E (1985): Oncogenes and h u m a n cancer. Cancer Genet Cytogenet 14:347-368. 28. Sandberg AA (1986): Role of chromosome changes in carcinogenesis. In: Mechanisms of Environmental Carcinogenesis, JC Barrett, ed. CRC Press, Inc. (in press) 29. Klein G (1982): The role of gene dosage and genetic transposition in carcinogenesis. Nature 294:313-318. 30. Jhanwar SC, Neel BG, Hayward WS, Chaganti RSK (1983): Localization of c-ras oncogene family on h u m a n germ-line chromosomes. Proc Natl Acad Sci USA 80:4794-4797. 31. Huerre C, Despoisse S, Gilgenkrantz S, Lenoir GM and Junien C (1983): c-Ha-rasl is not deleted in aniridia-Wilms' tumor association. Nature 305:638-641. 32. de Martinville B, Giacalone J, Shih C, Weinberg RA, Francke U (1983): Oncogene from h u m a n EJ bladder carcinoma is located on short arm of chromosome 11. Science 219:498501.
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33. Der CJ, Krontiris TG, Cooper GM (1982): Transforming genes of h u m a n bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses. Proc Natl Acad Sci USA 79:3637-23640. 34. Francke U, Holmes LB, Atkins L, Riccardi VM (1979): A n i r i d i a - W i l m s ' tumor association: Evidence for specific deletion of 11p13. Cytogenet Cell Genet 24:185-192. 35. Slater RM, de Kraker J, Voute PA, Delemarre JFM (1985): A cytogenetic study of Wilms' tumor. Cancer Genet Cytogenet 14:95-109. 36. Gibas Z, Prout GR, Pontes JE, Connoly JG, Sandberg AA (1985): A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet Cytogenet (in press). 37. Atkin NB, Baker MC (1985): Cytogenetic study of ten carcinomas of the bladder: Involvement of chromosomes 1 and 11. Cancer Genet Cytogenet 15:253-268. 38. Pathak S, Strong LC, Ferrell RE, Trindade A (1982): Familial renal cell carcinoma with 3;11 chromosome translocation limited to tumor cells. Science 217:939-941.
ABSTRACT: Cytogenetic analysis of a primary leiomyosarcoma of the prostate revealed a clonal chromosomal rearrangement involving chromosomes #2, #3, #9, #11, and #19. The results are discussed in relation to the cytogenetic findings in other solid tumors, especially those of the prostate and leiomyosarcoma at other sites. INTRODUCTION Sarcoma of the prostate is an infrequent lesion, difficult to diagnose, and even more difficult to treat [1-6]. The incidence of sarcoma compared with all other primary malignancies of the prostate ranges from 0.1% to 0.5% [2, 6]. Sarcomas arise from stromal tissue consisting of m u s c u l a r and fibrous elements, or from interstitial lymphoid foci of mesodermal tissue of the genital cord. The intraprostatic origin of myosarcomas is related to smooth muscle in the case of leiomyosarcoma and to striated muscle in that of rhabdomyosarcoma. These two types of myosarcoma compose the majority of primary sarcomas of the prostate [5]. Embryonal rhabdomyosarcoma of the prostate occurs most frequently in young boys and leiomyosarcoma in older age groups [1,5]. Chromosome analyses of h u m a n prostatic cancers and leiomyosarcomas are still very sparse [7, 8]. At present, five cases of adenocarcinoma of the prostate and one cell line of prostatic carcinoma have been analyzed with b a n d i n g techniques [911]. Only two cases (both intestinal) of leiomyosarcoma and n o n e at other sites have been analyzed by these techniques [12, 13]. We report herein a case of primary leiomyosarcoma of the prostate in which a clonal karyotype, 45,XY, - 2, - 6 - 9 - 11 - 19,t(2;19)(q22;q13.3),del(3)(p13p21),r(9;?), t(2;11;19)(q2;p13;q13.3), was found in 25 metaphases.
CASE REPORT The patient, a 38 year old man, was in good health until 1979 w h e n he passed a kidney stone. In 1980 he had hematuria and weakened urinary stream. He was hospitalized and a transurethral resection of a urethral polyp arising from the veru m o n t a n u m was carried out. In August 1983, a second urethral polyp was excised from the posterior urethra. The patient was healthy u n t i l November 1984, w h e n he was hospitalized again because of intermittent hematuria and weakened stream.
From the RoswellPark MemorialInstitute, Buffalo, NY. Address requests for reprints to Dr. Avery A. Sandberg, Depts. of Genetics and Endocrinology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263. Received August 12, 1985; accepted September 30, 1985.
159 ©1986Elsevier Science PublishingCo., Inc. 52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet22:159-167(1986) 0165-4608/86/$3.50
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J. Limon, P. Dal Cin, and A. A. Sandberg Intravenous p y e l o g r a p h y s h o w e d a n o r m a l u p p e r urinary tract, a normal bladder, and no evidence of enlargement of the prostate. Cytoscopy was done and a transurethral resection of a p o l y p was performed. Pathological e x a m i n a t i o n showed p o l y p o i d urethritis. In December 1984 the patient was found to have a very large prostate. Intravenous p y l o g r a p h y s h o w e d a huge mass in the prostate, p u s h i n g the bladder neck and floor of the b l a d d e r u p w a r d s . A s u p r a p u b i c cystostomy was done and a necrotic tumor was f o u n d in the prostate at the base of the bladder, which was b i o p s i e d and s h o w n to be a leiomyosarcoma. F o l l o w i n g a negative work-up, the patient u n d e r w e n t a cystoprostatectomy with ileal loop diversion in January 1985. A s p e c i m e n of this t u m o r was sent for pathologic and cytogenetic examinations. Microscopic diagnosis i n d i c a t e d an unclassified sarcoma of the prostate with extension to the b l a d d e r neck (Figs. 1 and 2); electron m i c r o s c o p y revealed ceils with myofilaments and, hence, the t u m o r cells were interpreted as compatible with those of a l e i o m y o s a r c o m a of the prostate (Fig. 3).
MATERIALS AND METHODS Fresh tumor tissue was obtained from the Department of Urologic Oncology of the Roswell Park Memorial Institute. The tumor s a m p l e was w a s h e d in Hanks' solution with antibiotics and disaggregated m e c h a n i c a l l y by m i n c i n g w i t h a scalpel in a Petri dish. The pieces of tissue were transferred to culture flasks containing 3.5 ml of growth m e d i u m , 0.5 ml of collagenase (2000 U/ml) (Gibco) at a final concentration of 200 U/ml and 0.5 ml of cholera toxin (Schwarz) at a final concentration of 500 ~g/ml [14]. The culture m e d i u m used was McCoy's 5A (Gibco) with 20% of
Figure 1 Histologic appearance of tumor, showing interlacing bundles of spindle cells, with hyperchromatic nuclei with blunt ends. Some mitotic figures are present. ( × 196)
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Figure 2 Histologic appearance of tumor, showing interlacing bundles of spindle cells, with hyperchromatic nuclei with blunt ends. Some mitotic figures are present. ( × 196) fetal calf serum, L-glutamine, and antibiotics; penicillin (100 IU/ml), streptomycin (100 ~g/ml), gentamycin (50 Ixg/ml), and Fungizone (25 p.g/ml) (Gibco). Tumor pieces were incubated at 37°C for 16 hours without agitation. After incubation the cells were disaggregated by pippetting; fast growing primary monolayer cultures in the abovementioned growing medium supplemented with 10 p~g/ml of cholera toxin were established. The cultures were harvested 24 hours later, after 4 hours of treatment with colcemid (0.02 p~g/ml) (Gibco). The cells were exposed to hypotonic shock according to Gibas et al. [15] and the swollen cells detached by a scraper and transferred into centrifuge tubes. The cells were fixed with a mixture of methanol/ glacial acetic acid (3:1). Slides were prepared in a conventional way; G-banding was obtained by the method of Yunis [16]. RESULTS
Detailed banding analysis was performed on 25 metaphases. The modal chromosome number was 45. Five marker chromosomes were consistently observed in all metaphases (Figs. 4 and 5): MI: t(2;19)(q22;q13.3}2pter--,cen--~2q22 : : 19q13.3--~19qter) M2: del(3)(p13p21) (Fig. 6) M3: ring(9;?). This chromosome probably arose from a rearrangement between two chromosomes, because C-banding revealed two heterochromatic blocks. One of them was chromosome #9, whereas, the origin of the second one was an unknown.
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Figure 3 An electron micrograph of a representative elongated tumor cell showing the bulk of the cytoplasm to be filled with microfilaments arranged in a long axis is ill-defined. There were two types of filaments: numerous thin filaments and much smaller but thicker ones. The latter were distributed randomly and no precise relation to the former. The cell surface was also coated with filamentous materials. These features are characteristic of smooth muscle cells and, thus, the tumor is a leiomyosarcoma. Uranyl acetate-lead staining. ( × 11,340)
M4: t(2;11)(q22;p13) (2qter-2q22::11p13-cen-11qter) M5:t(11;19)(p13 ;q13.3)(19pter-cen-19q13.3::11p13-11pter) Almost all metaphases presented i n the following karyotype: 45 ,XY, - 2, - 6, - 9, - 11 - 19,T(2;19)(q22;q13.3),del(3)(p13p21),r(9;?),t(2;11;19)(q22;p13;q13.3).
DISCUSSION Primary prostatic malignant tumors arising from muscle are rare [1-6]. The presenl cytogenetic study of such a tumor revealed clonal chromosomal abnormalities in. volving chromosomes #2, #3, #9, #11, and #19. To date, only two cases of leio. myosarcoma, both intestinal, have been analyzed with b a n d i n g techniques [12, 13] In both cases, chromosomes #1, #11, and #21 were involved i n clonal abnormal ities [i.e., del(1)(p11),del(ll)(q11) and t(1;21)(p32;q22)]. In our case chromosomet other than these were involved in rearrangements, with the exception of chromo some #11. In one intestinal tumor the marker derived from chromosome #11 ap parently consisted of only the short arm [12], whereas, i n our case the breakpoin occurred in the short arm, with the p l 3 - p t e r fragment being translocated to the lonl arm of chromosome #19. It is impossible to reach any conclusion on the specificitI
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19
20
....
22:
X
Figure 4 G-banded karyotype from a primary leiomyosarcoma of the prostate. Arrows point to structural abnormalities. See text for description of marker chromosomes.
of these c h r o m o s o m a l abnormalities on the basis of only three cases of leiomyosarcoma which, in addition, arose in different anatomical sites. The most c o m m o n t u m o r arising in the prostate is adenocarcinoma. In the few p u b l i s h e d cytogenetic studies of this cancer n o r m a l karyotypes were found in most cases [9, 17], probably due to overgrowth by stromal cells. Thus, o n l y five cases of a d e n o c a r c i n o m a a n d one h u m a n prostatic carcinoma line (LNCaP) have been analyzed successfully using b a n d i n g techniques [9-11]. In four cases and in the LNCaP line one c o m m o n marker c h r o m o s o m e was seen, del(10)(q24}. It has been suggested that a deletion of c h r o m o s o m e # 1 0 represents a specific change in prostatic carcinoma [11]. In the case of l e i o m y o s a r c o m a presented by us the structural karyotypic aberrations were different from those seen in a d e n o c a r c i n o m a of the prostate. There are m a n y reports on c h r o m o s o m e # 3 abnormalities in h u m a n tumors, but most of them a p p a r e n t l y lack specificity [18]. Recently, however, frequent aberrations of the short arm of the c h r o m o s o m e # 3 have been found in rhabdomyosarcoma [19], small cell cancer of the lung [20], in some ovarian cancers [21], and in mixed parotid gland tumors [22]. Some of the breakpoints occurred in the fragile site segment of this c h r o m o s o m e at 3p14 [23]. The del(3)(p13p21) in our case confirms the p r e d i s p o s i t i o n to nonspecific breakage of this c h r o m o s o m e in cancer cells. Though ring c h r o m o s o m e s have been observed in cancer cells [7], only some
164
a
b
C
2
11
19
22
Figure 5 Partial karyotype from three metaphases showing translocation between chromosomes #2, #11, and #19. Arrows indicate breakpoints.
Figure 6 Interstitial deletion of a chromosome #3 from four different metaphases (a-d Arrows indicate deleted region p13p21.
a
~b
c
d
Primary Leiomyosarcoma of the Prostrate
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have been identified with b a n d i n g techniques. In the present s t u d y we found a ring c h r o m o s o m e that arose from a rearrangement of two chromosomes, one of w h i c h was a c h r o m o s o m e # 9 with breakpoints in pter and qter. The nature of the second c h r o m o s o m e i n v o l v e d is unknown; p o s s i b l y it is a fragment of c h r o m o s o m e #6. We are unaware of any reports of the presence of a r(9) c h r o m o s o m e in solid tumors [24]. The correlation between the c h r o m o s o m a l loci of oncogenes and c h r o m o s o m e breakpoints in cancer cells has recently received m u c h attention [25-29]. Involvement of c h r o m o s o m e #11 in t(11;19)(p13;q13.3) in the present study is especially interesting because the c-ras oncogene is located in band p14 [30] or distal to p15.115.5 on c h r o m o s o m e #11 [31]. The c - r a s oncogene is activated in some b l a d d e r cancer lines [32] and the p r o d u c t of this gene is expressed in b l a d d e r and lung carcinomas [33]. A second i m p o r t a n t p h e n o m e n o n is that c h r o m o s o m e #11 is frequently involved in structural aberrations (deletions) in the a n i r i d i a - W i l m ' s tumor s y n d r o m e [34, 35], carcinoma of the b l a d d e r [36, 37], and familial renal cell carcinoma [38]. Many more prostatic tumors, adenocarcinomas, and sarcomas will have to be e x a m i n e d before they can be characterized cytogenetically. As in the case of leukemias and some tumors in w h i c h the c h r o m o s o m e findings have led to a subclassification of k n o w n entities into several subentities, prostatic cancer m a y be shown to contain a n u m b e r of subentities defined karyotypically, however similar the histology of the tumors. The 1 0 q - a n o m a l y referred to above m a y characterize one of these subentities. In the case of leiomyosarcoma, the question is even more complicated. Not only is such a t u m o r very rare in the prostate and, hence, difficult to characterize cytogenetically, but it will also be necessary to ascertain whether leiomyosarcomas at different sites express similar karyotypic events, regardless of their site of origin, or w h e t h e r there is some specificity in the c h r o m o s o m e changes related to the organ or tissue from w h i c h they originate. Supported in part by Grant CA-15436 from the National Cancer Institute. The authors thank Dr. Annamma Isac for the histalogic photographs, and Dr. Untae Kim for the electronmicrographs of the tumor and Dr. Claude Turc-Carel and Dr. Herman Van Den Bergne for their helpful discussion of the karyotypes. ADDENDUM
After this p a p e r was submitted for publication, Atkin and Baker [Hum Genet 70:359-364, 1985] described the cytogenetic findings in a leiomyosarcoma of the prostate, w h i c h contained m a n y c h r o m o s o m e changes.
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