Source: http://cancerres.aacrjournals.org/content/63/18/6063
Timestamp: 2019-04-20 06:38:41+00:00

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Metastatic mature teratoma is often present in postchemotherapy surgical specimens of lymph nodes from patients with pathological stage II or III testicular germ cell tumors. The stromal cells in these lesions have generally been considered “fibrosis” secondary to the chemotherapy and the necrosis it causes, although the frequent cytological atypia of the stromal cells suggests that they may be neoplastic. We studied 25 patients with pathological stage II or III testicular cancer who were treated with platinum-based chemotherapy followed by surgical resection of retroperitoneal lymph nodes that contained metastatic mature teratoma with “fibrosis” to determine the reactive or neoplastic nature of the stromal cells. We compared the pattern of allelic loss using nine microsatellite DNA markers (D9S177, D9S303, D9S778, D9S171, D12S1015, D1S508, D2S156, D18S46, and D11S903) between the epithelial cells of the teratoma and the cells in the adjacent stroma. A laser capture microdissection technique facilitated preparation of genomic DNA from the epithelial components of teratoma, adjacent stromal cells, and normal lymph node tissue from each patient. Of the 25 patients, loss of heterozygosity was seen at a minimum of one focus in 22 (92%) of the teratoma specimens and 16 (64%) of the adjacent stroma. Of the 16 cases for which the stroma showed loss of heterozygosity, 8 cases showed the identical pattern of allelic loss in the epithelial cells of the adjacent teratoma at all nine DNA loci studied. The remaining eight cases showed similar allelic loss in at least one of the nine DNA loci analyzed. Interestingly, three cases showed loss of heterozygosity in the stroma that was not seen in the matching teratoma specimens. Our results indicate that the stromal cells adjacent to metastatic mature teratoma in postchemotherapy lymph node specimens frequently have genetic abnormalities similar to the metastatic teratoma. Concordant genetic alterations observed in teratoma and stroma suggest that both are derived from the same element of the original germ cell tumor or the same progenitor cell.
Testis cancer is the most common malignancy in men 15–35 years old, with an estimated 7500 new cases and 400 deaths expected in 2003 (1) . Testicular germ cell tumors are the most common type of testis cancer, and they are often composed of two or more different histological components. Whether or not these mixed germ cell tumors are derived from a single precursor cell is still a point of debate (2) . After primary chemotherapy in advanced disease, imaging confirms the persistence of a mass in 30% of cases (3) , and surgical resection of the residual mass is usually undertaken. A meta-analysis review of the histology of surgical specimens from 24 publications (996 patients) found that the specimens contained fibrosis or necrosis in 48% of cases, mature teratoma in 36% of cases, and viable elements of other types of malignancy in 16% of cases (4) .
The presence of fibrosis or necrosis and the lack of viable germ cell tumor in the postchemotherapy surgery specimen are favorable prognostic factors. Clinical research has sought to identify factors predictive of favorable pathological reports in postchemotherapy resections so that surgery may potentially be avoided (5 , 6) . However, the genetic characteristics of this fibrous stromal tissue have not been adequately investigated. The presence of teratoma in the primary testis tumor has been shown to reduce the complete response rates of primary chemotherapy for metastatic retroperitoneal disease (7) , but it is not clear whether the teratoma, the adjacent elements, or both have an impact on prognosis.
Oncogene activation and tumor suppressor gene inactivation are important mechanisms in the genesis, propagation, and spread of most cancers, and the role of these processes in germ cell tumors is being explored (2 , 8, 9, 10, 11, 12, 13) . Because these genetic changes can be identified by allelic typing at polymorphic chromosomal loci, we compared the frequency of loss of heterozygosity and analyzed the pattern of allelic loss between teratoma and adjacent stromal cells in postchemotherapy resections. Our goal was to investigate the genetic alterations of the fibrous stroma relative to the adjacent epithelial component of the teratoma to determine whether malignant teratoma and the adjacent fibrous stroma arise from a common progenitor element, thereby elucidating the neoplastic or reactive nature of the fibrous component of these lesions.
Of 110 testicular cancer patients treated at the Indiana University Cancer Center with metastatic mature teratoma in their postchemotherapy retroperitoneal lymph node dissection specimens between January 2000 and September 2000, 25 were chosen at random. Their average age was 28 years (Table 1) ⇓ . Each was initially diagnosed with stage II or III testis cancer after standard computed tomography imaging evaluation. All patients underwent orchiectomy at other institutions, and histological slides were re-evaluated at Indiana University before treatment. Pathological staging was performed according to the 1997 tumor-node-metastasis (TNM) system (14) . After radical orchiectomy, 1 patient was pTis, 14 were pT1, and 10 were pT2. Twenty-three patients had malignant mixed germ cell tumors of the testis, one had pure embryonal carcinoma, and one had regressed germ cell tumor with residual intratubular germ cell neoplasia. Of 23 patients who had malignant mixed germ cell tumors, 15 had components of mature and/or immature teratoma, 10 had components of yolk sac tumor, 9 had components of embryonal carcinoma, 4 had components of choriocarcinoma, and 5 had components of seminoma.
All patients were treated with platinum-based chemotherapy and underwent postchemotherapy surgery for a residual mass. Patients with metastatic mature teratoma in the postchemotherapy surgical pathology specimen were included in this study. The Indiana University Institutional Review Board approved this research.
Tumor Microdissection and Detection of Loss of Heterozygosity.
Histological sections were prepared from formalin-fixed, paraffin-embedded blocks and stained with H&E for histopathological review and microdissection (Fig. 1) ⇓ . Genomic DNA was prepared from epithelial cells of metastatic mature teratoma as well as adjacent cells in the fibrous stroma using a laser capture microdissection method as described previously (15, 16, 17) . Normal control DNA was prepared from uninvolved lymph nodes in all cases. The following oligonucleotide primer pairs for the microsatellite DNA markers were chosen on the basis of their locations near oncogenes or tumor suppressor genes potentially involved in the development of germ cell tumors: D9S177 (chromosome 9q32–33; Ref. 18 ); D9S303 (9q13–22.3; Ref. 19 ); D9S778 (9q32–33); D9S171 (9p21; Ref. 12 ); D12S1051 (12q14–24; Ref. 20 ); D1S508 (1p36.2; Ref. 21 ); D2S156 (2q22–32; Ref. 22 ); D18S46 (18q21.1; Ref. 23 ); and D11S903 (11p13; Ref. 23 ; Research Genetics, Huntsville, AL). PCR amplification and gel electrophoresis were performed as described previously (24 , 25) . The criterion for allelic loss was complete or nearly complete absence of one allele in tumor DNA as described previously (24 , 25) . PCRs for each polymorphic microsatellite marker were repeated at least twice from the same DNA preparations, and the same results were obtained. Results were reported as noninformative when visual inspection could not distinguish two distinct band forms in control DNA after PCR amplification.
Laser microdissection of tumor from a patient with mature teratoma after platinum-based chemotherapy. A and C, H&E-stained sections before laser microdissection showing epithelium of mature teratoma and adjacent stroma. B and D, teratoma and adjacent stroma sections after laser microdissection removal of representative tissues.
Of the 25 patients we studied, loss of heterozygosity was seen at a minimum of one focus in 22 (92%) of the teratoma specimens and 16 (64%) of the adjacent stroma. The frequency of loss of heterozygosity in the informative teratoma specimens was 44% for D9S177, 18% for D9S303, 45% for D9S778, 0% for D9S171, 0% for D12S1051, 21% for D1S508, 17% for D2S156, 16% for D18S46, and 45% for D11S903 (Fig. 2) ⇓ . The frequency of loss of heterozygosity in the informative stromal specimens was 25% for D9S177, 12% for D9S303, 18% for D9S778, 5% for D9S171, 0% for D12S1051, 17% for D1S508, 17% for D2S156, 16% for D18S46, and 23% for D11S903 (Fig. 2) ⇓ . Of the 16 cases for which the stroma showed loss of heterozygosity, 8 cases showed the same pattern of allelic loss (either upper allele loss or lower allele loss) in the teratoma at all nine DNA loci studied. The remaining eight cases showed similar allelic loss in one or more of the nine DNA loci analyzed (Table 1) ⇓ . For example, cases 12 and 19 showed loss of the upper allele at D9S303 locus in both the stroma and the epithelial cells of the teratoma (Table 1 ⇓ ; Fig. 3 ⇓ ). Interestingly, three cases (cases 8, 19, and 20) displayed loss of alleles at one or more different loci in the stroma, but not in the matched teratoma.
Comparison of frequency of loss of heterozygosity across nine polymorphic microsatellite markers between mature metastatic teratoma and matched adjacent stroma.
Representative results of loss of heterozygosity analysis. DNA was prepared from normal tissue, metastatic mature teratoma, and adjacent stroma in matched specimens; amplified by PCR using polymorphic markers D9S177, D9S303, D9S778, D9S171, D12S1051, D1S508, D2S156, D11S903, and D18S46; and separated by gel electrophoresis (A and B). Arrows, allelic bands; N, lymphoid tissue (control); T, teratoma; S, stroma.
Testicular cancer is a true success story in modern oncology. Five-year survival rates for metastatic testis cancer have risen from less than 50% in the 1950s (26) to well above 90% today (27) . This success is largely attributable to the multidisciplinary approach using both chemotherapy and surgery and the fact that testicular germ cell tumors are commonly chemotherapy or radiation sensitive. The sensitivity of germ cell tumors to cisplatin and other DNA-damaging agents suggests that there are inherent genetic factors that influence drug sensitivity (28) . For those metastatic tumors that do not completely respond to chemotherapy, surgical excision of residual masses is common, and these tumors frequently consist of teratoma, fibrosis, or a combination thereof.
The presence of teratoma has important clinical consequences. Teratoma in the retroperitoneum, left untreated, may grow without tumor marker elevation and compress important structures, a condition termed the “growing teratoma syndrome” (29) . Also, teratomas may develop secondary, high-grade malignant components, including sarcomas and primitive neuroectodermal tumor (30) . Teratoma is chemoresistant, and it is not surprising that teratoma in an orchiectomy specimen predicts for a residual mass that necessitates surgical intervention (7) .
The activation of oncogenes and the inactivation of tumor suppressor genes may play a role in the development of germ cell tumors (2 , 8, 9, 10, 11, 12, 13 , 31) . The progression of testicular germ cell tumors is often associated with loss of chromosomal material. This loss at key chromosomes can be elucidated by loss of heterozygosity studies using certain polymorphic chromosomal loci. In this way, tumor suppressor genes have been identified for several human cancers, including BRCA1 for breast cancer (32) and DCC for colon cancer (33) .
Whereas alterations of the p53 tumor suppressor gene have been identified in nearly half of all human cancers, the role of p53 in germ cell tumors is controversial. Mutated p53 has a longer half-life than wild-type p53, and research has been directed at elucidating these mutations. Several investigators have shown by immunohistochemical techniques that p53 protein expression is increased in invasive components of mixed germ cell tumors as well as intratubular germ cell neoplasia (34, 35, 36) , but not in mature teratoma (36 , 37) . Mutated p53 has been demonstrated in testicular carcinoma in situ by some (38) , but not by others (35 , 39 , 40) . The clinical impact of these investigations in malignant germ cell tumors continues to be debated at this (5 , 41) and other institutions (2 , 34 , 36 , 40) .
Loss of heterozygosity studies have been used to identify sites of candidate tumor suppressor genes in germ cell tumors on chromosomes 3, 5, 9, 11, 12, and 18, although the lack of availability of large families with multiple generations affected by germ cell tumors has slowed progress in mapping a testis cancer-predisposing gene (42) . Similar analyses have helped to identify genes that undergo frequent nonrandom deletion in teratomas. For example, the NME genes are notable for a high degree of genetic loss (>70%) in teratomas (43) .
One area of germ cell tumor investigation that has not been adequately explored is the role of tumor-stroma interaction in carcinogenesis. Tumor stroma may play a more active and important role in tumor biology than previously understood. For example, growth factors and cytokines produced by macrophages appear to be crucial in angiogenesis; lytic enzymes provided by stromal cells may play a vital role in tumor invasion, and tumor necrosis factors and other inflammatory mediators may be critical in promoting tumor growth and the systemic effects of tumors, such as cachexia (44) . There is evidence (17 , 45, 46, 47, 48) suggesting that stromal cells may be involved in carcinogenesis and tumor progression. Their potential role in these processes warrants further investigation.
Our study shows that the fibrous stroma associated with metastatic mature teratoma, often considered a reactive response or “fibrosis,” has genetic abnormalities similar to the adjacent teratoma. This concept has been explored in other human cancers such as prostate cancer (47 , 48) , where growth factors and androgen receptors in the adjacent stroma may influence prostate cancer development and progression. Genetic alterations detected by loss of heterozygosity studies in the stroma adjacent to breast carcinomas have been demonstrated, suggesting a field effect and a common transformation from the same progenitor cell element (17 , 45 , 49) . It has even been suggested that the stroma adjacent to breast carcinoma cells may play a role in inducing neoplastic transformation in adjacent epithelial cells (45) .
In most of the cases we analyzed, the genetic changes between the teratoma and adjacent stroma were concordant, suggesting that the teratoma and stroma are derived from the same original germ cell element or tumor progenitor cells. Emerging evidence suggests that the different histological components commonly seen in mixed germ cell tumors often have the same clonal origin (13 , 50, 51, 52) . Rothe et al. (13) found identical allelic loss at all five DNA loci studied in all 11 patients with mixed germ cell tumors of the testis, supporting a common clonal origin of the different histological components of mixed germ cell tumors. It has previously been recognized that stromal cells in retroperitoneal masses after chemotherapy for testicular germ cell tumors often display variable degrees of cytological atypia, suggesting that they derive from spindle cell elements of teratoma or yolk sac tumor (5 , 53) . Our data support this concept. Some so-called “fibrosis” may actually represent “the fibrous variant” of mature teratoma, derived from malignant elements that have responded to chemotherapy but that may undergo additional genetic changes to transform to high-grade sarcomas. In our opinion, therefore, residual masses after chemotherapy that consist solely of such fibrous lesions should be totally excised, if at all possible, just as for teratoma. It should not be the goal of predictive models for residual histologies after chemotherapy to identify those cases consisting solely of necrosis and “fibrosis,” as opposed to teratoma, but of necrosis alone. Previous work at this institution has focused on the genetic alterations seen after chemotherapy for testicular germ cell tumors and showed that more genetic rearrangements and deletions occurred overall in postchemotherapy tumors compared with those that were untreated (54) . Clearly, genetic alteration and more specifically allelic loss may be accelerated after exposure to these DNA-damaging compounds. Additionally, it may be problematic to distinguish between the chromosomal changes attributed to tumor progression and therapy-related differentiation (55) .
Mature teratomas consist of heterogenous mixtures of diverse epithelial and stromal components derived from pluripotential progenitor cells. Both components are relatively chemoresistant and persist after chemotherapy. Concordant genetic alterations seen in both teratoma and stroma after chemotherapy support that both are derived from the same element of the original germ cell tumor or the same progenitor cell. There were also several nonconcordant cases between the stroma and adjacent teratoma. These may have resulted from genetic alterations introduced after metastasis had occurred or could be examples of cell populations with different clonal origins.
We report for the first time that after chemotherapy, the fibrous stroma adjacent to teratoma has neoplastic genetic characteristics. Concordant genetic alterations in the spindle cells of fibrous lesions and the epithelial cells of the adjacent teratoma suggest a shared development and clonal evolution from the original germ cell component. Our findings of a high frequency of allelic imbalance in the stromal elements challenge the common concept of the benign nature of the cells in postchemotherapy fibrous lesions. However, with our current level of understanding, we do not advocate any deviation from the current standard recommendations regarding surgery and postchemotherapy masses.
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