Imaging, Diagnosis, Prognosis Germ Cell Origin of Testicular Carcinoid Tumors Phillip H. Abbosh,1Shaobo Zhang,1Gregory T.MacLennan,3 Rodolfo Montironi,4 Antonio Lopez-Beltran,5 Joseph P. Rank,6 LeeAnn Baldridge,1and Liang Cheng1, 2 Abstract Purpose: Carcinoids are neuroendocrine tumors and most frequently occur within tissues derived from the embryonic gut.These tumors can occur in any organ site but are rare in the testis. The cell type giving rise to testicular carcinoid is unknown.We hypothesized that testicular carci- noid may have a germ cell origin. Experimental Design: We describe our analysis of protein and genetic markers of germ cell neoplasia, using immunohistochemistry and fluorescence in situ hybridization, in four testicular carcinoid tumors. Results: All four cases of testicular carcinoid tumor arose in a background of mature teratoma. Isochromosome 12p was identified in carcinoid tumor cells in all four samples. 12p overrepresen- tation was also observed in three cases. Isochromosome 12p and 12p overrepresentation were present in cells of coexisting mature teratoma in three cases. Carcinoid tumors showed strong immunoreactivity for synaptophysin and chromogranin, but no immunoreactivity for OCT4, CD30, c-kit,TTF-1, and CDX2. Membranous and cytoplasmic staining for h-catenin was detected in three cases. Conclusion: Our findings suggest that testicular carcinoid represents a phenotypic expression of testicular teratoma and is of germ cell origin. Testicular carcinoid tumor is rare. It was originally reported in Materials and Methods 1954 by Simon (1) who described it as part of a cystic teratoma, and additional cases have been subsequently reported. All Patients. We analyzed four cases of testicular carcinoid tumor. All reported cases have occurred in peripubertal or postpubertal four cases were carcinoids arising in mature teratomas in postpubertal males (2, 3). The origin of testicular carcinoid is a matter of males (age range, 20-38 years; mean, 32 years). No patient had controversy. Some investigators have hypothesized that testicular symptoms or signs of excess hormone secretion or a history of familial carcinoid tumor arises from germ cells and represents a mono- cancer syndrome. dermal teratoma or simply a component of a mature teratoma (4), Fluorescence in situ hybridization. Sections of 4-Am thickness were but definitive proof of this hypothesis has not been provided stained with H&E and reviewed, and the original diagnosis was in previous studies. Others have postulated that testicular confirmed. Sections were prepared from buffered formalin-fixed, carcinoid tumors are derived from Leydig cells (5). In this study, paraffin-embedded tissue blocks. FISH analysis was done as previously described (6, 7). Dual-color FISH was done by using a mixture of we did fluorescence in situ hybridization (FISH) analysis to Spectrum Orange–labeled centromeric a satellite DNA probe (CEP12) evaluate several testicular carcinoid tumors for the presence of a and Spectrum Green–labeled subtelomeric (Tel12) DNA probes for chromosomal abnormality commonly present in testicular germ chromosome arm 12p (Vysis), which were diluted with tDenHyb2 cell tumors, and also evaluated the immunoreactivity of these (Insitus) in a ratio of 1:50 and 1:20, respectively. Five microliters of tumors to antibodies against a number of well-established diluted probes were added to the slide in the reduced light condition. neuroendocrine and testicular germ cell tumor markers. Slides were covered with a 22 Â 22 mm coverslip and sealed with rubber cement. Denaturation was achieved by incubating the slides at 75jC for 10 min. The slides were examined using a Zeiss Axioplan 2 microscope ¶ 1 with the following filters: SP-100 for DAPI 4 ,6-diamidino-2-phenyl- Authors’Affiliations: Departments of Pathology and Laboratory Medicine, and indole, MF-101 for Spectrum Green (12p), and Gold 31003 for Spectrum 2Urology, Indiana University School of Medicine, Indianapolis, Indiana; Orange (CEP12) from Chroma. The images were acquired with a charge- 3Department of Pathology, Case Western Reserve University, Cleveland, Ohio; 4Institute of Pathological Anatomy and Histopathology, School of Medicine, coupled device camera and analyzed with MetaSystem Isis software. Five A Polytechnic University of the Marche Region (Ancona), United Hospitals, Ancona, sequential focus stacks with 0.4- m intervals were acquired and then Italy; 5Department of Pathology, Cordoba University, Cordoba, Spain; and integrated into a single image to reduce thickness-related artifacts. 6CellNetix Pathology, Swedish Medical Center, Seattle,Washington From each tumor section, 100 nuclei were scored for signals from Received 9/10/07; revised 10/30/07; accepted 12/3/07. CEP12 (red) and 12p (green) under the fluorescence microscope with The costs of publication of this article were defrayed in part by the payment of page Â1,000 magnification, and the ratio between green and red signals was charges. This article must therefore be hereby marked advertisement in accordance subsequently calculated. We analyzed the spatial distribution of the with 18 U.S.C. Section 1734solely to indicate this fact. green and red signals to detect the specific patterns of signal aggregation Requests for reprints: Liang Cheng, Department of Pathology and Laboratory consistent with i(12p), as previously reported (6, 8, 9). The green (12p) Medicine, Indiana University School of Medicine, 350 West 11th Street, Clarian Pathology Laboratory, Room 4010, Indianapolis, IN 46202. Phone: 317-491- and red (12 centromeric signal) were counted for each tumor cell 6442; Fax: 317-491-6419; E-mail: [email protected]. nucleus, and the average number of each signal was recorded. We F 2008 American Association for Cancer Research. considered a 12p/12 centromeric ratio of >1.5 as evidence of 12p doi:10.1158/1078-0432.CCR-07-4146 overrepresentation. www.aacrjournals.org 13 9 3 Clin Cancer Res 2008;14(5) March 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Imaging, Diagnosis, Prognosis Fig. 1. Testicular carcinoid tumor. A to C, case 1; D to F, case 2; G to I, case 3. A, the interface between tumor and adjacent testis is sharply circumscribed (case 1). B, higher-power view of the same case (case 1). Carcinoid tumor growing in solid nests and forming small acini. C, immunostaining for chromogranin A is strongly positive. D, carcinoid tumor adjacent to coexisting mature teratoma (case 2). E, high-power view of the same case (case 2), showing insular architecture. F, a typical FISH image of a carcinoid cell with isochromosome12p. FISH displayed two centromere12signals (red)andthree12psignals(green), two of which were in close proximity to one centromeric signal, with an aggregation pattern consistent with an isochromosome12p. G, testicular carcinoid tumor showing insular and trabecular architecture, as well as gland formation (case 3). H, high-power view of the same case (case 3).Tumor cells of carcinoid tumor have modest amounts of eosinophilic cytoplasm; nuclei are uniform, with dispersed chromatin. I, FISH of a carcinoid cell showed three centromere signals (red) and numerous 12p signals (green), consistent with 12p overrepresentation. A classic seminoma specimen was used as a positive control for FISH (1:150 dilution, DAKO) for 30 min at room temperature, followed analyses. Lymphocytes within each tumor were used as negative by biotinylated secondary antibody (DAKO) and peroxidase-labeled controls for each tumor. streptavidin. 3,3-Diaminobenzidine was used as the chromogen in the Immunohistochemistry. A Sections were cut at 4- m thickness and presence of hydrogen peroxide. Positive and negative controls were used deparaffinized for immunohistochemical studies, which were done and stained appropriately. using an automated immunostainer. Antigen retrieval was carried out by heating sections in DAKO Target Retrieval Solution S1699 for 15 min. Endogenous peroxidase activity was inactivated by incubation in 3% Results H2O2 for 15 min. Nonspecific binding sites were blocked using Protein Block (DAKO Corporation) for 20 min. Tissue sections were then All four cases showed histologic features typical of carcinoid incubated with the purified rabbit polyclonal antibody against c-kit tumor. Tumors were composed of monomorphous polygonally (1:50 dilution; Oncogene), goat polyclonal antibody against OCT4 shaped cells with modest amounts of eosinophilic cytoplasm (1:500 dilution; Santa Cruz Biochemical), rabbit polyclonal antibody to and uniform bland nuclei with finely dispersed chromatin synaptophysin (prediluted, DAKO), rabbit polyclonal antibody to (Fig. 1). The architectural arrangement of tumor cells was chromogranin (prediluted, DAKO), mouse monoclonal antibody to CD30 (prediluted, DAKO), mouse monoclonal antibody to typical of that seen in carcinoid tumors in other sites, with thyroid transcription factor-1 (TTF-1; 1:100 dilution, clone 8G7G3/1, insular and trabecular structures and with gland formation in DAKO), mouse monoclonal antibody to CDX2 (clone CDX2-88, some tumors. One tumor was entirely insular, one was insular prediluted, Biogenex), or mouse monoclonal antibody to h-catenin with gland formation, one was insular with gland formation Clin Cancer Res 2008;14(5) March 1, 2008 1394 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Germ Cell Origin of Testicular