- Email: [email protected]
Flow cytometric and genetic study of familial gonadal tumors
FLOW CYTOMETRIC AND GENETIC STUDY OF FAMILIAL GONADAL TUMORS ARNOLD M. LEVENTHAL, PATRICIA CAMUTO, M.D. JOHN HAJJAR, M.D.
M.D.
SANDRA R. WOLMAN, M.D. JANUSZ PLAWNER, M.D.
From the Departments of Urology and Pathology, New York University Medical Center, New York, New York ABSTRACT-Three cases of familial gonadal tumors are reported. Genetic studies were conducted in 2 cases: a brother and sister with embryonal carcinoma and a benign cystic teratoma, respectively, and a father and son with embryonal carcinoma. DNAflow cytometry of paraffin-embedded tumors and constitutional karyotyping were performed. Further genetic studies are emphasized.
Gonadal tumors, including both testicular and ovarian neoplasms, occasionally appear in familial settings. However, definitive constitutional genetic defects or tumor markers crucial to tumorigenesis have not been identified for tumors of these tissues. Testicular tumors are not common neoplasms and in general comprise less than 2 percent of malignant tumors in males, with an overall incidence of 0.8/100,000 white males in the United States.’ Development of testicular cancers is far more frequent with cryptorchism.2 Inguinal hernias also have been mentioned as a risk factor.3 Sporadic reports of testicular malignancies in closely related family members have appeared in the literature. In 1930, the first case of testicular tumors in twins was reported.4 Since then, authors have reported tumors in twin brothers,1.5.6 non-twin brothers,7,8 fathers and sons,g-11cousins, 12.i3and nephew-uncles. l3 A recent survey included over 100 patients with testicular tumors occurring in families. l3 The possibility of genetic susceptibility to the development of testicular tumor has been investigated. In a study by the National Institute of Health, the fathers and brothers of patients with testicular cancer had a sixfold elevated risk of a testicular malignancy developing compared with men in the general population.14 More recently, chromosomal studies have re-
558
vealed a common genetic abnormality of chromosome 12, i(12)p, in some tumors from unrelated patients. 15m18If a specific chromosomal change characterizes malignant testicular tumors, it could be hypothesized to play a role in malignant transformation of germ cells.ig HLA antigen typing in testicular tumor families has suggested some HLA antigens that are common.20-22 Alternatively, Havald and Hauge,23 reviewed a series of 7,000 twins in the Danish birth registry, and concluded that genetic mechanisms were unimportant in cancer development. Benign ovarian teratomas are common among ovarian neoplasms. Multiple occurrences in families are reported, albeit rarely, in twins, sisters, mother-daughters, and grandmother-daughter-granddaughters.24.25 Teratomas have several characteristics of inherited tumors, such as an early onset and bilaterality,24 and genetic bases for teratoma development have been postulated.26m28 One theory suggests that teratomas arise from undifferentiated germ cells and take part in a process of imperfect parthenogenesis, possibly by a genetic defect that is transmitted from one generation to the next. 25 However, one review of incidence of benign teratoma reported no differences between twins and offspring of single births.2g
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Neither the urologic nor gynecologic literature provides evidence of a genetic origin for either testicular tumors or teratomas. We have studied two families with familial gonadal tumors by means of clinical history, cytogenetic analysis, and flow cytometry. Genetic and DNA studies of a brother and sister with germ cell tumors and a father and son with testicular tumors will be presented. An additional family with testicular tumors will also be presented.
16
22
20
1 3oL
FICLW: 1.
29-
-78 tmbryonal [age 26:
;lr Terntoma (age 19)
Pedigree of jamily in Case 1.
Case Reports Case 1A: Brother A twenty-eight-year-old white man presented at age twenty-six years with a one-week history of a right painless testicular mass. There was no history of cryptorchism, hernias, mumps, trauma, or infections. Family histor) included a sister who had a benign cystic teratoma removed at age nineteen years. The family had lived two miles from an atomic power research laboratory for fifteen years. The physical examination revealed a left varicocele and a nontender l-cm nodule in the right testicle. Ultrasound showed an area that was hypoechoic in that testicle. Serum human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), and lactic dehydrogenase (LDH) were normal. Preoperative sperm banking was performed. Surgical procedures included a right radical orchiectomy, placement of testis prosthesis, and a left varicocelectomy. Pathologic examination revealed an embryonal cancer invading blood vessels and tunica albuginea. Case 1B: Sister A twenty-four-year-old white woman presented at age nineteen years with irregular menstrual periods. On examination, she had a left adnexal mass. Ultrasound revealed a heterogeneous mass. At laparotomy, the left ovary was replaced by a dermoid cyst and a left oophorectomy was performed. The lesion was diagnosed as a benign cystic teratoma. The pedigree of Case 1 family is illustrated in Figure 1. Case 2A: Son A thirty-year-old white man presented at age twenty-eight years with a right testicular mass causing discomfort. He denied any trauma, hernias, or cryptorchism. One vear prior, he had been treated for right epididymitis. The physical examination revealed a 2-cm nodule on the right testis near the epididymal tail. UlIIHOLO(:k
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FIGURE 2.
Pedigree oj family in Casr 2
trasound showed a hypoechoic area. Serum HCG, AFP, and LDH were normal. Preoperative sperm banking was performed. Surgery revealed a testis with a nodule at the lower pole, and right radical orchiectomy was performed. Embryonal carcinoma with arterial invasion was defined by pathologic examination. Metastatic workup with computerized tomography was negative. The patient refused a lymph node dissection. Chemotherapy included bleomycin, cisplatin, and vinblastine. Case 2B: Father A fifty-three-year-old white man presented at age forty-five years with a painless indurated right testicular mass of two weeks’ duration. He denied prior trauma, infections, or cryptorchism. Past history included a hernia repair and mumps as a child. Physical examination revealed an indurated nodule on his right testis. Serum HCG, AFP, and LDH were normal. Chest x-ray film was clear. He underwent a right radical orchiectomy. Seminoma with areas of embryonal carcinoma was diagnosed. Findings on computerized tomography were negative. He subsequently underwent a retroperitoneal lymphadenectomy. Preaortic and precaval lymph nodes were positive for poorly differentiated embryonal carcinoma. Chemotherapy was given with five courses of treatment .
A pedigree of Case 2 family is illustrated in Figure 2. Case 3A: Brother A thirty-two-year-old white man was seen at age twenty-seven years with a large left scrotal mass and a history of local discomfort for seven months. Physical examination revealed a left testicle three times normal size, that was indurated. Serum HCG was 2,178, LDH was 352, and AFP was normal. Chest x-ray film and computerized tomography showed no evidence of metastasis. A left radical orchiectomy was performed. Pathologic examination revealed teratocarcinoma, with components of embryonal and choriocarcinoma, confined to the testis with no vascular invasion. A retroperitoneal lymphadenectomy was performed, with the lymph nodes showing no tumor on microscopic examination. Postoperative serum markers normalized, and the patient is without recurrence. Case 3B: Brother At age twenty-nine years the patient had a right scrotal mass. Right inguinal orchiectomy revealed a seminoma. Radiation treatment was given. Case 3C: Paternal uncle Seminoma eight years.
of testis was found at age forty-
Material and Methods Chromosome
analysis
Peripheral blood was obtained in Cases 1A and 1B. Constitutional chromosome evaluation was performed on PHA-stimulated blood peripheral lymphocytes. intrachromosomal banding patterns on 32-33 metaphases per case, stained with trypsin-Giemsa were examined, Tumor tissue was not available for cytogenetic analysis. DNA flow cytometry Flow cytometry was performed on samples of paraffin-embedded tumor from Cases lA, lB, 2A, and 2B using a deparaffinizatiompronase-digestion technique. A single 50 p section was cut from one block from each case. A 3-5 p hematoxylin-eosin stain section from the blocks processed for flow cytometry confirmed that each was composed predominately of tumor, admixed with lo-20 percent normal tissue (stroma, normal gonad). The sections were deparaffinized and rehydrated through a series of
560
xylene and graded alcohol washes. The tissue was then washed twice with distilled water and phosphate-buffered saline (PBS) and treated with 0.5% pronase at 37°C for thirty minutes. A drop of the cell suspension formed in this manner was air-dried on a glass slide and stained with Hemacolor (Harleco, Gibbstown, NJ) to determine the adequacy of cell separation and preservation of nuclei. The suspension was then filtered through a 41 p nylon mesh, centrifuged, and washed twice with PBS. The washed pellet was resuspended in 750 PL of ribonuclease A and incubated for ten minutes at room temperature. Subsequently 750 PL of 0.005 % propidium iodide was added.30 Cell cycle analysis was performed on a Becton-Dickinson FACSCAN interfaced to a Hewlett-Packard 9153B computer (Mountainview, CA) using the 488 nm emission of a 15 mW argon laser. Ten thousand cells were evaluated for each sample. Studies were performed within three hours of addition of propidium iodide. Paraffin-embedded sections of normal tonsil were used as diploid control. Tumors were defined as aneuploid when two or more GOGl peaks were noted. One of these peaks was presumed to correspond to a nonneoplastic population of diploid cells consisting of stromal and inflammatory cells within the tumor as well as any normal glandular tissue that was present. The remaining peak(s) represented the tumor cell population. Gl phase and cell cycle statistics were analyzed using the sum of broadened rectangle (SOBR) method. S-phase calculations could be determined only for the histograms showing the clearest separation of diploid and aneuploid populations. DNA indices, defined as the ratio between the peak GO/G1 channel for each aneuploid population to that of the diploid cells, were also determined for each case. Results * Chromosomal analyses of peripheral blood lymphocytes were performed on members of the Case 1 family. The brother had a karyotype of 46,XY and the sister had a karyotype of 46,Xx. No marked deviations of G-banding patterns were noted at the 550 band level of resolution.
*Note: Family of Case 2 refused comtitutional karyotyping. Family of Case 3 refused constitutional karyotyping and tumor studies. Two members of the third family refused history taking.
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TABLEI. Case 1A 1B 2A 2B
---
Brother Sister Son Father
Results
of flozc
cytometry
of gonadal
tumors
Karyotype
Pathology
Ploidl
DNA Index
46,XY 46,Xx . . .
Embryonal Teratoma Embryonal Embryonal
Diploid Diploid Aneuploid Aneuploid
1.0 1.0 1.4 1.2, 1.3
flow cytometry was performed on families of Cases 1 and 2. The nuclear DNA histograms are recorded in Figures 3 and 4. The results are summarized in Table I. DNA
Comment We have encountered three examples of familial gonadal tumors. The first family presentation involved a brother and sister. The testis and ovary arise from the same bipotential gonad,31 and the occurrence of tumor in this setting suggests a possible common genetic predisposing factor. Both brother and sister had normal constitutional karyotypes. DNA analysis of the tumors by flow cytometry revealed a predominant diploid population for both the benign teratoma and the embryonal carcinoma, with an increased percentage of cells in synthetic phase (S-phase) in both cases. The ab-
_____.._ % S-Phase _____-. 24 31 30
Met astasis NO NO
Yes Yes
sence of a detectable structural or numerical chromosome abnormality common to these siblings does not rule out the possibility of a defect detectable at a molecular genetic level. Our flow cytometric data are partially in agreement with published results. Teratomas, in general, have been diploid. 26 However, most testicular tumors have been reported to be aneuploid.32m34 Tumors with diploid DNA populations may display less aggressive behavior and have prognostic significance.3s The diploid testicular tumor in Case 1 showed absence of metastasis. Longterm clinical follow-up and extended DNA studies are needed to test this hypothesis. The second family in this study includes a father and son with embryonal carcinoma. Both tumors demonstrated aneuploid clones with different DNA indices, and both patients had evidence of metastatic spread of the tumors. The father’s primary tumor sample
FIGURE3. togram tometry 1B.
Relative DNA
content
Relative DNA
Nuclear DNA hispatterns by flow cyof Case 1A and Case
content
h:uclear DNA hisFICUW 4. togram patterns by flow cytometry of Case 2A and Case 2B.
i Relative DNA
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showed two populations merging with the diploid peak with indices of 1.2 and 1.3, while the tumor sample from the son showed an aneuploid peak distinct from the diploid at an index of 1.4. Therefore, the DNA results do not show grossly similar genetic alterations within the father’s and son’s tumors. The DNA nuclear histograms do not support a common genetic defect in these tumors. Further studies on familial cases are needed to achieve conclusions with statistical significance. The third family could not be further investigated because of lack of patient cooperation. In fact, all the families in this study were investigated incompletely in that fresh tumor was not available for culture cytogenetic examination. Pedigrees in families of Cases 1 and 2 illustrate no clear genetic pattern. Constitutional and tumor karyotyping, and molecular genetics are tools that should be employed to elucidate the role of genetic factors in gonadal tumorigenesis, and the unexplained frequency of familial occurrence of such tumors. 10 Linden Street Woodmere, New York 11598 (DR. LEVENTHAL) References 1. Segal R, Reif R, Goren E, and Gil I: Malignant testicular tumors in identical twins, Eur Urol 12: 443 (1986). 2. Batata MA, et pl: Cryptorchidism and testicular cancer, J Urol 124: 382 (1980). 3. Mills PK, Newell GR, and Johnson DE: Familial patterns of testicular cancer, Urology 24: 1 (1984). 4. Champlin HW: Similar tumors of testis occurring in identical twins, JAMA 95: 96 (1930). 5. Martin WMC, and Dane TEB: Testicular germ cell tumors in monozygotic twins: case report and review of the literature, J Urol 134: 765 (1985). 6. Zevallos M, Snyder RN, Sadoff L, and Cooper JF: Testicular neoplasm in identical twins-a case report, JAMA 250: 645 (1983). 7. Shapiro CM, Otero N, Garcia J, and Yoon WJ: Testicular neoplasms in Hispanic brothers, J Surg Oncol 28: 257 (1985). 8. Kademian MT, and Caldwell WL: Testicular seminoma: a case report of 2 brothers with seminoma and a review of the literature of testicular malignancies occurring in closely related family members, J Urol 116: 380 (1976). 9. Musella R, Giaccone G, D’Ambrosio E, and Calciati A: Father-son testicular cancer, case report, Tumori 69: 269 (1983). 10. Fuller DB, and Plenk HP: Malignant testicular germ cell
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tumors in a father and two sons-case report and literature review, Cancer 58: 955 (1986). 11. Wobbes TH, Hoekstra HJ, Oldhoff J, and Koops HS: Malignant testicular germ cell tumors in father and son. A report on 2 families, J Urol 129: 152 (1983). 12. Copeland GP Sherman JM, Muranda C, and Griffiths D: Familial occurrence of testicular neoplasia: case report of testicular embryonal carcinoma in two brothers and a first cousin, J Urol 136: 676 (1986). 13. Weissbach L, and Widmann T: Familial tumor of the te.tis, Eur Urol 12: 104 (1986). 14. Tollerud DJ, et al: Familial testicular cancer and urogenital developmental anomalies, Cancer 55: 1849 (1985). 15. Gibas Z, Prout GR, Pontes JE, and Sandberg AA: Chromosome changes in germ cell tumors of the testis, Cancer Genet Cytogenet 19: 245 (1986). 16. Atkin NB, and Baker MC: Chromosome analysis of three seminomas, Cancer Genet Cytogenet 17: 315 (1985). 17. Oosterhuis JW, et al: Karyotyping and DNA flow cytometry of mature residual teratoma after intensive chemotherapy of disseminated nonseminomatous germ cell tumor of the testis: a report of two cases, Cancer Genet Cytogenet 22: 149 (1986). 18. Gibas Z, Prout GR Jr, and Sandberg AA: Malignant teratoma of the testis with an isochromosome No. 12, i(12p), as the sole structural cytogenetic abnormality, J Urol 131: 762 (1984). 19. Atkin NB, and Baker MC: i(12p): Specific chromosomal marker in seminoma and malignant teratoma of the testis?, Cancer Genet Cytogenet 10: 199 (1983). 20. Hayakawa M, Mukai K, Nagakura K, and Hata M: A case of simultaneous bilateral germ cell tumors arising from cryptorchid testes, J Urol 136: 470 (1986). 21. Carr BI, and Bach FA: Possible association between HLAAw24 and metastatic testicular germ ceil tumors, Lancet 1: 1346 (1979). 22. Kubelka J, Sarkar NN, and Belitsky P: Familial occurrence of testicular neoplasia: case report of testicular seminoma in father and son, J Urol 130: 140 (1983). 23. Havald B, and Hauge M: Hereditary of cancer elucidated by a study of unselected twins, JAMA 186: 749 (1963). 24. Brenner SH, and Wallach RC: Familial benign cystic teratomata, Int J Gynaecol Obstet 21: 167 (1983). 25. Plattner G, and Oxorn H: Familial incidence of ovarian dermoid cysts, Can Med Assoc J 108: 892 (1973). 26. Linder D, McCaw BK, and Hecht F: Parthenogenic origin of benign ovarian teratomas, N Engl J Med 292: 63 (1975). 27. McCaw BK, Hecht F, and Patil S: Ovarian teratomas and genetics of germ cell formation, Lancet 2: 1311 (1976). 28. Riley PA, and Sutton PM: Ovarian teratomas and genetics of germ cell formation, Lancet 1: 362 (1977). 29. Brown EH Jr: Identical twins with twisted benign cystic teratoma of the ovary, Am J Obstet Gynecol 134: 879 (1979). 30. Auer G, et al: Personal communication, 1987. 31. Smith DR: General Urology, 10th ed, Los Altos, California, Lange, 1981, p 522. 32. FOSS SD, et al: DNA flow cytometry in human testicular cancer, Cancer Lett 28: 55 (1985). 33. Sledge GW Jr, et al: Flow cytometry derived DNA content of the primary lesions of advanced germ cell tumors, Int J Androl 10: 115 (1987). 34. Kleinhans G, et al: Studies of the cell kinetics of human malignant testicular tumors, Urologe 25: 294 (1986). 35. Heppner GH: Perspectives in cancer research: tumor heterogeneity, Cancer Res 44: 2259 (1984).
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M.D.
SANDRA R. WOLMAN, M.D. JANUSZ PLAWNER, M.D.
From the Departments of Urology and Pathology, New York University Medical Center, New York, New York ABSTRACT-Three cases of familial gonadal tumors are reported. Genetic studies were conducted in 2 cases: a brother and sister with embryonal carcinoma and a benign cystic teratoma, respectively, and a father and son with embryonal carcinoma. DNAflow cytometry of paraffin-embedded tumors and constitutional karyotyping were performed. Further genetic studies are emphasized.
Gonadal tumors, including both testicular and ovarian neoplasms, occasionally appear in familial settings. However, definitive constitutional genetic defects or tumor markers crucial to tumorigenesis have not been identified for tumors of these tissues. Testicular tumors are not common neoplasms and in general comprise less than 2 percent of malignant tumors in males, with an overall incidence of 0.8/100,000 white males in the United States.’ Development of testicular cancers is far more frequent with cryptorchism.2 Inguinal hernias also have been mentioned as a risk factor.3 Sporadic reports of testicular malignancies in closely related family members have appeared in the literature. In 1930, the first case of testicular tumors in twins was reported.4 Since then, authors have reported tumors in twin brothers,1.5.6 non-twin brothers,7,8 fathers and sons,g-11cousins, 12.i3and nephew-uncles. l3 A recent survey included over 100 patients with testicular tumors occurring in families. l3 The possibility of genetic susceptibility to the development of testicular tumor has been investigated. In a study by the National Institute of Health, the fathers and brothers of patients with testicular cancer had a sixfold elevated risk of a testicular malignancy developing compared with men in the general population.14 More recently, chromosomal studies have re-
558
vealed a common genetic abnormality of chromosome 12, i(12)p, in some tumors from unrelated patients. 15m18If a specific chromosomal change characterizes malignant testicular tumors, it could be hypothesized to play a role in malignant transformation of germ cells.ig HLA antigen typing in testicular tumor families has suggested some HLA antigens that are common.20-22 Alternatively, Havald and Hauge,23 reviewed a series of 7,000 twins in the Danish birth registry, and concluded that genetic mechanisms were unimportant in cancer development. Benign ovarian teratomas are common among ovarian neoplasms. Multiple occurrences in families are reported, albeit rarely, in twins, sisters, mother-daughters, and grandmother-daughter-granddaughters.24.25 Teratomas have several characteristics of inherited tumors, such as an early onset and bilaterality,24 and genetic bases for teratoma development have been postulated.26m28 One theory suggests that teratomas arise from undifferentiated germ cells and take part in a process of imperfect parthenogenesis, possibly by a genetic defect that is transmitted from one generation to the next. 25 However, one review of incidence of benign teratoma reported no differences between twins and offspring of single births.2g
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Neither the urologic nor gynecologic literature provides evidence of a genetic origin for either testicular tumors or teratomas. We have studied two families with familial gonadal tumors by means of clinical history, cytogenetic analysis, and flow cytometry. Genetic and DNA studies of a brother and sister with germ cell tumors and a father and son with testicular tumors will be presented. An additional family with testicular tumors will also be presented.
16
22
20
1 3oL
FICLW: 1.
29-
-78 tmbryonal [age 26:
;lr Terntoma (age 19)
Pedigree of jamily in Case 1.
Case Reports Case 1A: Brother A twenty-eight-year-old white man presented at age twenty-six years with a one-week history of a right painless testicular mass. There was no history of cryptorchism, hernias, mumps, trauma, or infections. Family histor) included a sister who had a benign cystic teratoma removed at age nineteen years. The family had lived two miles from an atomic power research laboratory for fifteen years. The physical examination revealed a left varicocele and a nontender l-cm nodule in the right testicle. Ultrasound showed an area that was hypoechoic in that testicle. Serum human chorionic gonadotropin (HCG), alpha-fetoprotein (AFP), and lactic dehydrogenase (LDH) were normal. Preoperative sperm banking was performed. Surgical procedures included a right radical orchiectomy, placement of testis prosthesis, and a left varicocelectomy. Pathologic examination revealed an embryonal cancer invading blood vessels and tunica albuginea. Case 1B: Sister A twenty-four-year-old white woman presented at age nineteen years with irregular menstrual periods. On examination, she had a left adnexal mass. Ultrasound revealed a heterogeneous mass. At laparotomy, the left ovary was replaced by a dermoid cyst and a left oophorectomy was performed. The lesion was diagnosed as a benign cystic teratoma. The pedigree of Case 1 family is illustrated in Figure 1. Case 2A: Son A thirty-year-old white man presented at age twenty-eight years with a right testicular mass causing discomfort. He denied any trauma, hernias, or cryptorchism. One vear prior, he had been treated for right epididymitis. The physical examination revealed a 2-cm nodule on the right testis near the epididymal tail. UlIIHOLO(:k
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FIGURE 2.
Pedigree oj family in Casr 2
trasound showed a hypoechoic area. Serum HCG, AFP, and LDH were normal. Preoperative sperm banking was performed. Surgery revealed a testis with a nodule at the lower pole, and right radical orchiectomy was performed. Embryonal carcinoma with arterial invasion was defined by pathologic examination. Metastatic workup with computerized tomography was negative. The patient refused a lymph node dissection. Chemotherapy included bleomycin, cisplatin, and vinblastine. Case 2B: Father A fifty-three-year-old white man presented at age forty-five years with a painless indurated right testicular mass of two weeks’ duration. He denied prior trauma, infections, or cryptorchism. Past history included a hernia repair and mumps as a child. Physical examination revealed an indurated nodule on his right testis. Serum HCG, AFP, and LDH were normal. Chest x-ray film was clear. He underwent a right radical orchiectomy. Seminoma with areas of embryonal carcinoma was diagnosed. Findings on computerized tomography were negative. He subsequently underwent a retroperitoneal lymphadenectomy. Preaortic and precaval lymph nodes were positive for poorly differentiated embryonal carcinoma. Chemotherapy was given with five courses of treatment .
A pedigree of Case 2 family is illustrated in Figure 2. Case 3A: Brother A thirty-two-year-old white man was seen at age twenty-seven years with a large left scrotal mass and a history of local discomfort for seven months. Physical examination revealed a left testicle three times normal size, that was indurated. Serum HCG was 2,178, LDH was 352, and AFP was normal. Chest x-ray film and computerized tomography showed no evidence of metastasis. A left radical orchiectomy was performed. Pathologic examination revealed teratocarcinoma, with components of embryonal and choriocarcinoma, confined to the testis with no vascular invasion. A retroperitoneal lymphadenectomy was performed, with the lymph nodes showing no tumor on microscopic examination. Postoperative serum markers normalized, and the patient is without recurrence. Case 3B: Brother At age twenty-nine years the patient had a right scrotal mass. Right inguinal orchiectomy revealed a seminoma. Radiation treatment was given. Case 3C: Paternal uncle Seminoma eight years.
of testis was found at age forty-
Material and Methods Chromosome
analysis
Peripheral blood was obtained in Cases 1A and 1B. Constitutional chromosome evaluation was performed on PHA-stimulated blood peripheral lymphocytes. intrachromosomal banding patterns on 32-33 metaphases per case, stained with trypsin-Giemsa were examined, Tumor tissue was not available for cytogenetic analysis. DNA flow cytometry Flow cytometry was performed on samples of paraffin-embedded tumor from Cases lA, lB, 2A, and 2B using a deparaffinizatiompronase-digestion technique. A single 50 p section was cut from one block from each case. A 3-5 p hematoxylin-eosin stain section from the blocks processed for flow cytometry confirmed that each was composed predominately of tumor, admixed with lo-20 percent normal tissue (stroma, normal gonad). The sections were deparaffinized and rehydrated through a series of
560
xylene and graded alcohol washes. The tissue was then washed twice with distilled water and phosphate-buffered saline (PBS) and treated with 0.5% pronase at 37°C for thirty minutes. A drop of the cell suspension formed in this manner was air-dried on a glass slide and stained with Hemacolor (Harleco, Gibbstown, NJ) to determine the adequacy of cell separation and preservation of nuclei. The suspension was then filtered through a 41 p nylon mesh, centrifuged, and washed twice with PBS. The washed pellet was resuspended in 750 PL of ribonuclease A and incubated for ten minutes at room temperature. Subsequently 750 PL of 0.005 % propidium iodide was added.30 Cell cycle analysis was performed on a Becton-Dickinson FACSCAN interfaced to a Hewlett-Packard 9153B computer (Mountainview, CA) using the 488 nm emission of a 15 mW argon laser. Ten thousand cells were evaluated for each sample. Studies were performed within three hours of addition of propidium iodide. Paraffin-embedded sections of normal tonsil were used as diploid control. Tumors were defined as aneuploid when two or more GOGl peaks were noted. One of these peaks was presumed to correspond to a nonneoplastic population of diploid cells consisting of stromal and inflammatory cells within the tumor as well as any normal glandular tissue that was present. The remaining peak(s) represented the tumor cell population. Gl phase and cell cycle statistics were analyzed using the sum of broadened rectangle (SOBR) method. S-phase calculations could be determined only for the histograms showing the clearest separation of diploid and aneuploid populations. DNA indices, defined as the ratio between the peak GO/G1 channel for each aneuploid population to that of the diploid cells, were also determined for each case. Results * Chromosomal analyses of peripheral blood lymphocytes were performed on members of the Case 1 family. The brother had a karyotype of 46,XY and the sister had a karyotype of 46,Xx. No marked deviations of G-banding patterns were noted at the 550 band level of resolution.
*Note: Family of Case 2 refused comtitutional karyotyping. Family of Case 3 refused constitutional karyotyping and tumor studies. Two members of the third family refused history taking.
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TABLEI. Case 1A 1B 2A 2B
---
Brother Sister Son Father
Results
of flozc
cytometry
of gonadal
tumors
Karyotype
Pathology
Ploidl
DNA Index
46,XY 46,Xx . . .
Embryonal Teratoma Embryonal Embryonal
Diploid Diploid Aneuploid Aneuploid
1.0 1.0 1.4 1.2, 1.3
flow cytometry was performed on families of Cases 1 and 2. The nuclear DNA histograms are recorded in Figures 3 and 4. The results are summarized in Table I. DNA
Comment We have encountered three examples of familial gonadal tumors. The first family presentation involved a brother and sister. The testis and ovary arise from the same bipotential gonad,31 and the occurrence of tumor in this setting suggests a possible common genetic predisposing factor. Both brother and sister had normal constitutional karyotypes. DNA analysis of the tumors by flow cytometry revealed a predominant diploid population for both the benign teratoma and the embryonal carcinoma, with an increased percentage of cells in synthetic phase (S-phase) in both cases. The ab-
_____.._ % S-Phase _____-. 24 31 30
Met astasis NO NO
Yes Yes
sence of a detectable structural or numerical chromosome abnormality common to these siblings does not rule out the possibility of a defect detectable at a molecular genetic level. Our flow cytometric data are partially in agreement with published results. Teratomas, in general, have been diploid. 26 However, most testicular tumors have been reported to be aneuploid.32m34 Tumors with diploid DNA populations may display less aggressive behavior and have prognostic significance.3s The diploid testicular tumor in Case 1 showed absence of metastasis. Longterm clinical follow-up and extended DNA studies are needed to test this hypothesis. The second family in this study includes a father and son with embryonal carcinoma. Both tumors demonstrated aneuploid clones with different DNA indices, and both patients had evidence of metastatic spread of the tumors. The father’s primary tumor sample
FIGURE3. togram tometry 1B.
Relative DNA
content
Relative DNA
Nuclear DNA hispatterns by flow cyof Case 1A and Case
content
h:uclear DNA hisFICUW 4. togram patterns by flow cytometry of Case 2A and Case 2B.
i Relative DNA
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showed two populations merging with the diploid peak with indices of 1.2 and 1.3, while the tumor sample from the son showed an aneuploid peak distinct from the diploid at an index of 1.4. Therefore, the DNA results do not show grossly similar genetic alterations within the father’s and son’s tumors. The DNA nuclear histograms do not support a common genetic defect in these tumors. Further studies on familial cases are needed to achieve conclusions with statistical significance. The third family could not be further investigated because of lack of patient cooperation. In fact, all the families in this study were investigated incompletely in that fresh tumor was not available for culture cytogenetic examination. Pedigrees in families of Cases 1 and 2 illustrate no clear genetic pattern. Constitutional and tumor karyotyping, and molecular genetics are tools that should be employed to elucidate the role of genetic factors in gonadal tumorigenesis, and the unexplained frequency of familial occurrence of such tumors. 10 Linden Street Woodmere, New York 11598 (DR. LEVENTHAL) References 1. Segal R, Reif R, Goren E, and Gil I: Malignant testicular tumors in identical twins, Eur Urol 12: 443 (1986). 2. Batata MA, et pl: Cryptorchidism and testicular cancer, J Urol 124: 382 (1980). 3. Mills PK, Newell GR, and Johnson DE: Familial patterns of testicular cancer, Urology 24: 1 (1984). 4. Champlin HW: Similar tumors of testis occurring in identical twins, JAMA 95: 96 (1930). 5. Martin WMC, and Dane TEB: Testicular germ cell tumors in monozygotic twins: case report and review of the literature, J Urol 134: 765 (1985). 6. Zevallos M, Snyder RN, Sadoff L, and Cooper JF: Testicular neoplasm in identical twins-a case report, JAMA 250: 645 (1983). 7. Shapiro CM, Otero N, Garcia J, and Yoon WJ: Testicular neoplasms in Hispanic brothers, J Surg Oncol 28: 257 (1985). 8. Kademian MT, and Caldwell WL: Testicular seminoma: a case report of 2 brothers with seminoma and a review of the literature of testicular malignancies occurring in closely related family members, J Urol 116: 380 (1976). 9. Musella R, Giaccone G, D’Ambrosio E, and Calciati A: Father-son testicular cancer, case report, Tumori 69: 269 (1983). 10. Fuller DB, and Plenk HP: Malignant testicular germ cell
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