Cytologic Features of Extragonadal Germ Cell Tumors. BACKGROUND. The objective of the current study was to evaluate and describe

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1 504 Cytologic Features of Extragonadal Germ Cell Tumors A Study of 88 Cases With Aspiration Cytology Ruchika Gupta, MD Sandeep R. Mathur, MD Vinod K. Arora, MD Shree G. Sharma, MD Department of Pathology, All India Institute of Medical Sciences, New Delhi, India. BACKGROUND. The objective of the current study was to evaluate and describe the cytologic features of extragonadal germ cell tumors (GCTs), both primary and metastasis from gonadal sites, in fine-needle aspiration cytology. METHODS. Aspirates from 88 extragonadal GCTs were retrieved with their clinical features and cytologic smears. The smears were assessed for cellularity, cell patterns, and cytologic features, which were summarized. Histopathology was available in 47 cases, and cytohistologic correlation was done in all such cases. RESULTS. Of 88 cases, 57 with adequate cytologic material were analyzed. Each type of GCT, except for embryonal carcinoma, had characteristic morphologic features. Seminomas had dyscohesive tumor cells with well defined, vacuolated cytoplasm; prominent nucleolus; and background lymphocytes. Tigroid background was noted in only a minority of cases. Yolk sac tumors revealed papillary fragments of tumor cells with metachromatic basement membrane-like material in the fragments. Mixed germ cell tumors were difficult to diagnose except for cases in which more than 1 type of GCT was observed on cytologic smears. In 37% of cases with mixed GCT, only 1 component was observed on cytology. CONCLUSIONS. Fine-needle aspiration cytology can reliably offer a diagnosis of GCT at extragonadal sites, even in rare locations and in patients in whom metastatic tumor may be the first clinical presentation. Immature teratoma and mixed GCTs pose a significant problem in cytology because of sampling error on needle aspiration. At extragonadal sites, the list of differential diagnoses is wider than that for gonadal GCTs. Cancer (Cancer Cytopathol) 2008;114: Ó 2008 American Cancer Society. Address for reprints: Sandeep R. Mathur, MD, Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi , India; Fax: (011) ; drsunnymathur@ yahoo.com Received August 11, 2008; revision received September 8, 2008; accepted September 15, KEYWORDS: extragonadal, germ cell tumor, seminoma, yolk sac tumor, embryonal carcinoma, teratoma, aspiration cytology. Germ cell tumors (GCTs) are common tumors of gonads (ie, testis and ovaries). Their occurrence at extragonadal sites, either as primary tumors or as metastatic foci, is rare. Although the utility of fine-needle aspiration (FNA) in the diagnosis of testicular GCTs has been shrouded by controversy, 1,2 it is an accepted alternative to biopsy diagnosis at sites such as the mediastinum and retroperitoneum because of the surgical risk of biopsy. 3,4 An accurate diagnosis of GCT has important therapeutic and prognostic implications for the patient and, thus, should be as rapid and reliable as possible. Cytologic features of primary gonadal GCTs have been well documented in the available literature. However, to our knowledge there are very few case series or larger studies in the literature detailing the cytologic features of GCTs at extragonadal sites. 3 6 Moreover, the diagnostic challenges are different at extragonadal locations, for example, the mediastinum, in which thymoma and large cell lymphoma need to be excluded in a case of seminoma. In this ª 2008 American Cancer Society DOI /cncr Published online 3 November 2008 in Wiley InterScience (

2 Cytology of Extragonadal GCTs/Gupta et al 505 report, we describe the distinctive cytomorphologic features of 88 extragonadal GCTs, including both primary and metastatic lesions. MATERIALS AND METHODS A retrospective search was conducted for all cases diagnosed on aspiration cytology as GCTs in extragonadal locations (ie, excluding testes and ovaries). During an 11-year period, 88 cases were retrieved. Clinical details, including age, sex, site of aspiration, and history of gonadal tumor were recorded. Superficial masses such as cervical lymph nodes and chest wall masses were aspirated using a 23-gauge needle without negative pressure. Deep-seated lesions, including abdominal masses, retroperitoneum, mediastinum, liver, and lung, were aspirated under ultrasound or computed tomography guidance. Aspirates were fixed in alcohol (for Papanicolaou stain) and also were airdried (for May-Grunwald-Giemsa stain [MGG]). The smears were evaluated for cellularity, cell patterns, cytoplasmic and nuclear features, background, and additional features such as mitosis, apoptosis, and necrosis. An attempt was made to subtype the GCT in all cases. The corresponding histopathologic sections, which were available in 47 cases, also were reviewed with their routine hematoxylin and eosin-stained sections along with histochemical and immunohistochemical stains to aid in the final diagnosis. RESULTS The 88 patients who were included in this study had a wide age range (between 4 months and 60 years; mean age, 19.9 years) and a male predominance (men/boys:girls/women, 2.2:1). A variety of aspiration sites was noted, and the most common was an abdominal mass, as summarized in Table 1. Of 88 cases, smears from 31 cases were not analyzed further because of reasons that included scant cellularity or cytohistologic discordance (4 cases). Therefore, 57 cytologically diagnosed extragonadal GCTs (EGCTs) were analyzed in this study. The most common EGCT was seminoma/dysgerminoma (19 cases) followed by yolk sac tumor (YST) (10 cases), embryonal carcinoma (EC) (2 cases), mature teratoma (3 cases), immature teratoma (5 cases), mixed GCT (16 cases), and 2 cases of GCT for which further subtyping could not be performed. Seminoma/Dysgerminoma (19 Cases) The smears from 19 seminoma/dysgerminomas were moderately to highly cellular. The predominant pattern was singly dispersed cells and a few small, loosely cohesive clusters. The tumor cells were large TABLE 1 Summary of the Location of Extragonadal Germ Cell Tumors in 57 Cases Site of Aspiration No. of Cases (%) Primary/Metastasis Abdominal mass 27 (47.3) 27 M Lymph nodes 15 (26.3) 15 M Mediastinum 3 (5.2) 3 P Retroperitoneum 4 (7) 4 P Sacrococcygeal 4 (7) 4 P Liver 2 (3.5) 2 M Buttocks 2 (3.5) 2 M M indicates metastatic lesion; P, primary tumor. with scant to moderate amounts of fragile, vacuolated, but well defined cytoplasm. Cells had large central nuclei with prominent irregularities of nuclear membrane, coarsely clumped chromatin, marked anisonucleosis, and a single, prominent, eosinophilic nucleolus (Fig. 1a). Mitotic figures were noted in 10 of 19 cases (52.6%), and apoptotic bodies were noted in 13 cases (68.4%). Foci of necrosis were identified in only 3 of these cases. A distinctive tigroid background was appreciated in only 7 cases (36.8%) that also was focal in the smears (Fig. 1b). Prominent lymphocytes and/or polymorphs were observed in the background in majority of cases (n 5 16; 84.2%). Other helpful features that were noted included chromatin threads/cytoplasmic fragments (4 cases; 21%), granulomas (7 cases; 36.8%), giant cells (4 cases; 21%), and bare nuclei (3 cases; 15.7%). A histologic confirmation was available in 7 of these cases, and the diagnosis was seminoma/dysgerminoma in all 7 cases. Yolk Sac Tumor/Endodermal Sinus Tumor (10 Cases) Smears from 10 YST/endodermal sinus tumors demonstrated moderate to high cellularity and were composed of papillary/papillaroid fragments, and 3 tumors (30%) had cohesive tumor groups with traversing vessels. Hyaline basement membrane-like material (that was stained pink for MGG and green for Papanicolaou stain) was noted in 6 of 10 YSTs (60%) (Fig. 2). The characteristic Schiller-Duval body was observed in the cytologic smear in only 1 case, and hyaline globules were noted in 2 cases (20%). The individual tumor cells were large with scant to moderate amounts of cytoplasm, which were finely vacuolated in some cells. The nuclei showed a vesicular chromatin pattern with moderate anisonucleosis and 1 or 2 prominent eosinophilic nucleoli. Variable numbers of mitotic figures and apoptotic bodies were noted with small foci of necrosis. Syncytio-

3 506 CANCER (CANCER CYTOPATHOLOGY) December 25, 2008 / Volume 114 / Number 6 FIGURE 1. (a) Smear from a seminoma demonstrating loosely cohesive clusters of cells with moderate cytoplasm, a vesicular nucleus, and prominent nucleolus (Papanicolaou stain, original magnification 3400). Inset: Two cells with well defined, clear cytoplasm. (b) A tigroid background with singly dispersed cells also was noted (Papanicolaou stain, original magnification 3200). FIGURE 2. (a) Smear from a yolk sac tumor demonstrating papillary clusters of cuboidal cells (Papanicolaou stain, original magnification 3200). (b) Air-dried smear showing the metachromatic basement membrane-like material within the cell cluster (arrow) (May-Grunwald-Giemsa stain, original magnification 3200). trophoblastic giant cells were observed in 1 case. Histopathology was available in 4 cases, which showed features of YST in the multiple sections that were studied. Embryonal Carcinoma (2 Cases) Aspiration smears from 2 ECs were moderately to highly cellular with tightly cohesive clusters of tumor cells along with a focal acinar arrangement. Prominent hemorrhage and focal necrosis were evident in all cases. The tumor cells were large with scant cytoplasm, large nuclei with irregular nuclear membranes, coarse chromatin, and prominent nucleoli. There was moderate to marked anisonucleosis and the presence of bizarre forms with frequent binucleation and multinucleation (Fig. 3). Mitosis and apoptosis were observed prominently along with tumor giant cells. In 1 case, occasional intranuclear inclusions were observed. Of the 2 cases, 1 showed features of EC, and the second case, from a patient who received preoperative chemotherapy, showed no residual tumor in the resected specimen. Mature Teratoma (3 Cases) Smears from mature teratoma usually were paucicellular and consisted of mature squamous cells, columnar cells (Fig. 4a), amorphous material resembling sebum, glial tissue, and mesenchymal

4 Cytology of Extragonadal GCTs/Gupta et al 507 FIGURE 3. (a) Photomicrograph of an embryonal carcinoma demonstrating a cohesive cluster of pleomorphic tumor cells with a large nucleus and multiple prominent nucleoli (Papanicolaou stain, original magnification 3400). (b) May-Grunwald-Giemsa stained smear from the same patient demonstrating marked nuclear pleomorphism (original magnification 3400). FIGURE 4. (a) Photomicrograph showing mature squamous cells and a cluster of columnar cells in a background of histiocytes and dirty material (Papanicolaou stain, original magnification 3100). (b) A case of immature teratoma demonstrating aggregates of immature neuroepithelium (Papanicolaou stain, original magnification 3100). fragments in various combinations and proportions. Occasional histiocytic giant cells also were observed, possibly as a reaction to keratinous material. A histologic diagnosis of mature cystic teratoma was made in all 3 cases Immature Teratoma (5 Cases) The cellularity of smears in 3 of these 5 cases of immature teratoma was greater than that observed in mature teratoma. In addition to squamous and columnar cells, primitive neuroepithelial elements in the form of round cell component with rosettes and fibrillary material were noted (Fig. 4b). Primitive mesenchyme also was noted in 1 case. However, in 2 cases, no neuroepithelial component was observed in smears; hence, these were diagnosed as mature teratoma in cytology. Histologic correlation could be done in all 5 cases. Mixed Germ Cell Tumor (16 Cases) Of the 16 mixed GCTs, aspiration smears showed features of more than 1 GCT component in 6 cases. One case demonstrated features of a mature teratoma on aspiration cytology; however, this was accompanied by increased serum levels of alphafetoprotein (AFP) and human chorionic gonadotro-

5 508 CANCER (CANCER CYTOPATHOLOGY) December 25, 2008 / Volume 114 / Number 6 TABLE 2 Extragonadal Germ Cell Tumors: Cytohistologic Correlation in 33 Cases With Histology Available Histologic Diagnosis No. of Cases Cytologic Diagnosis Seminoma 7 Seminoma 7 YST 4 YST 4 EC 1 Embryonal carcinoma 1 Mature teratoma 3 Mature teratoma 3 Immature teratoma 5 Immature teratoma 3 Mature teratoma 2 Mixed GCT YST and EC 3 EC component 1 GCT unclassified 2 YST and dysgerminoma 1 YST component 1 YST and teratocarcinoma 1 YST component 1 YST and teratoma 3 YST component 2 YST and teratoma 1 Teratocarcinoma 2 EC component 1 GCT unclassified 1 Immature teratoma and EC 1 Immature teratoma and EC 1 No residual tumor 2 EC 1 (after chemotherapy) GCT unclassified 1 Total 33 Total 33 No. of Cases YST indicates yolk sack tumor; EC, embryonal carcinoma. GCT, germ cell tumor; EC, embryonal carcinoma. pin (hcg) and, hence, the possibility of mixed GCT was suggested. Of the remaining 9 cases, 4 showed the presence of only a YST component in the smears, whereas 2 revealed only an EC component. In 3 cases, a cytologic diagnosis of GCT was rendered; however, further subtyping was not possible on aspiration smears. The cytohistologic correlation of these cases is shown in Table 2. Germ Cell Tumor, Unclassified (2 Cases) In 2 cases, a cytologic diagnosis of GCT was made; however, subclassification was not possible on cytology alone. One of these cases had complete tumor necrosis caused by chemotherapeutic effects, and the second case could not be confirmed histologically. The cytologic features of all 57 cases are summarized in Table 3, and cytohistologic correlation is shown in Table 2. DISCUSSION GCTs are common neoplasms of the gonads (ie, ovary and testes) in both children and adults. Extragonadal locations, including the mediastinum, retroperitoneum, pineal gland, and sacrococcygeal regions, may be the primary site of a GCT. 7 Involvement of other extragonadal sites, including lymph nodes, usually is associated with metastatic disease. 8 In many cases, the metastatic site may be the first clinical presentation in a patient with primary gonadal GCT. In such patients, a timely diagnosis of GCT would assist the clinician in instituting the appropriate therapeutic modality at the initial stage and provide a greater chance of patient survival. In the current study, a variety of extragonadal sites of GCT, including primary tumors in the mediastinum, retroperitoneum, and sacrococcygeal region along with rarer locations such as the liver and buttocks (presenting as metastatic lesions from gonadal tumors), etc, were included. Hence, the diagnosis of GCT should be considered in an appropriate clinical setting, even for tumors in rare locations. FNA currently is the first-line diagnostic modality for superficial swellings. The use of FNA in the diagnosis of testicular GCTs has been debated because of the potential risk of needle-track tumor seeding leading to scrotal involvement and up-staging of the tumor. 1 However, most authors who use this technique reiterate the safety of this technique while using thin-bore needles (22 gauge) along with the advantage of a rapid and reliable diagnosis. 2,9 11 In extragonadal locations, FNA, especially under radiologic guidance, offers a suitable alternative to surgical biopsy, especially in sites such as the mediastinum and retroperitoneum. There are very few large series or studies that have shared their experience and cytologic findings in extragonadal GCTs in the available literature. 3 6 In this report, we have described the cytologic features of various subtypes of GCTs at extragonadal sites in 57 cases. Seminomas/dysgerminomas were the most common GCTs in the current series. The characteristic cytologic findings included a dispersed cell population with few small clusters; large tumor cells with fragile, vacuolated cytoplasm; and round nuclei with a single, prominent nucleoli. Lymphocytes, plasma cells, giant cells, and granulomas in the background and a tigroid (lace-like) appearance were other useful supportive features that were encountered in many cases. However, in contrast to what was reported in primary gonadal tumors, the smears in our series had the characteristic background in only a few cases (36.8%). An earlier study by Caraway et al. in which mediastinal and metastatic GCTs were included also revealed a dispersed population of large cells with round nucleus and prominent nucleoli. In their study, a tigroid background was noted in \50% of cases, 12 which is similar to what we observed in the current study. Hence, the differential diagnosis of seminoma also may be considered at extragonadal sites in the absence of the characteristic background. In an extragonadal site, the differential diagnosis of seminoma includes YST, EC, melanoma, adenocarci-

6 Cytology of Extragonadal GCTs/Gupta et al 509 TABLE 3 Summary of the Cytologic Characteristics Studied in Germ Cell Tumors Feature Seminoma/ Dysgerminoma (n 5 19) Yolk Sac Tumor (n 5 14) Embryonal Carcinoma (n 5 4) Mature Teratoma (n 5 5) Immature Teratoma (n 5 3) Mixed GCT (n 5 7) GCT, Unclassified (n 5 5) Cellularity Moderate to high Moderate to high Moderate to high Low Low to moderate Moderate to high Low to moderate Single cells Cohesive fragments (Variable) 11 Papillaroid/papillary fragments Cell size Large (4 5 times lymphocytes) Large (4 5 times lymphocytes) Cytoplasm Moderate, well-defined vacuolated, fragile Large (5 6 times lymphocytes) Variable, depending on the component Variable Large Moderate, fine vacuolation Scant Variable Variable Moderate Nuclear chromatin Irregularly clumped Vesicular Coarse Variable Hyperchromatic in neuroepithelium Variable Vesicular to hyperchromatic Nucleoli 1 2, Prominent, eosinophilic 1 2, Prominent Single, prominent Variable Anisonucleosis Tigroid background 11 (7 Cases) Traversing vessels 11 (4 Cases) 1 (1 case) 1 (In case with yolk sac tumor) Basement membrane-like material 11 (9 Cases) 1 (In case with yolk sac tumor) Inclusions/hyaline globules 1 (4 Cases) 1 (1 Case) Cytoplasmic fragments/ chromatin threads 111 GCT indicates germ cell tumor; 1111, prominent feature; 1, few;, not observed; 11, frequent;, may be observed; 111, high.

7 510 CANCER (CANCER CYTOPATHOLOGY) December 25, 2008 / Volume 114 / Number 6 noma, thymic carcinoma (in mediastinal lesions), and large cell lymphomas. ECs, YSTs, and adenocarcinomas demonstrate glandular or papillary arrangement of tumor cells, at least focally. 13 Melanoma may have features similar to those of seminoma; however, the sprinkling of lymphocytes, giant cells, and granulomas and the characteristic tigroid background usually is not observed in melanoma. In addition, nuclear pleomorphism is much more pronounced in melanoma along with frequent binucliation/multinucleation. The presence of melanin pigment in the tumor cells supports a diagnosis of melanoma. 12 Large cell lymphoma of the immunoblastic type also has a dispersed cell pattern with moderate cytoplasm, rounded nuclei, and prominent nucleoli. However, the presence of a tigroid background, combined with the lack of lymphoglandular bodies and nuclear cleaving/irregularities, favors a diagnosis of seminoma. 14 In mediastinal tumors, seminoma with prominent lymphoid component may be confused with thymic carcinoma, especially the lymphoepithelioma-like variant. The presence of lymphoepithelial lesions favors thymic carcinoma; whereas a tigroid background, granulomas, and round nuclei with prominent nucleoli point toward a diagnosis of seminoma. 14 Smears from YSTs in our series had a variety of patterns, including papillary and/or papillaroid fragments, cohesive clusters, acinar structures, and single tumor cells that contained moderate amounts of vacuolated cytoplasm and vesicular nuclei with prominent nucleoli. Basement membrane-like material (observed in 60% of cases) was the feature that assisted most consistently in the cytodiagnosis of YST in combination with hyaline globules (observed in only 20% of cases). The finding of basement membrane-like material was reported previously by Mizrak and Ekinci 15 ; however, it has not been emphasized in other cytologic reports. In our study, we observed that this material was a very helpful feature in the diagnosis of YST. The presence of tumor with a glandular appearance in an appropriate clinical setting should arouse the suspicion of YST in cytology. Among GCTs, the distinction between YST and EC may be difficult. Cells of EC exhibit anaplastic features with poorly differentiated malignant cells that have scant cytoplasm, irregular hyperchromatic nuclei with prominent nucleoli, and blurred nuclear membranes. 16 Seminoma may enter the list of differential diagnosis if smears from YST contain a prominent component of single cells. In such cases, the presence of mucoid background in contrast with tigroid background, hypervacuolated cells with intracytoplasmic hyaline globules, favors a diagnosis of YST. 16 In addition, metastatic adenocarcinoma needs to be excluded. The presence of intracytoplasmic hyaline globules and metachromatic basement membrane-like material and the lack of prominent nuclear pleomorphism favor a diagnosis of YST, although this also is noted in renal cell carcinoma. Radiologic investigations such as ultrasound to delineate the origin of the tumor and immunocytochemistry for AFP (favor YST), cytokeratin, and epithelial membrane antigen (positive in both) can help in this regard. In our study, aspirates from EC showed cohesive clusters of large tumor cells that had pleomorphic nuclei with binucleation and multinucleation. Prominent hemorrhage and large areas of necrosis were observed frequently in this variant of GCT. An earlier study by Collins et al. revealed similar findings in their case of EC. 17 In the presence of these cytologic features, metastatic adenocarcinoma has to be excluded. A clinical history of a gonadal GCT and measurement of serum tumor markers may be helpful in such cases, because the distinction on cellular features alone may be difficult. 5 Because of the variability of components, a definite diagnosis of mature teratoma may be difficult on aspiration cytology. However, the presence of squamous cells, columnar cells, stromal fragments, and adipose tissue suggests a component of mature teratoma. In all cases in which this diagnosis is being considered, the smears need to be searched carefully for any immature component and for the presence of another subtype of GCT. In the current study, 2 cases of teratoma showed only mature components on aspiration smears, whereas histopathologic examination also revealed an immature neuroepithelial component, which rendered a diagnosis of immature teratoma. An earlier report by Garcia-Solano et al. on testicular GCTs also concluded that a reliable cytologic diagnosis of pure teratoma is difficult. Also, a small focus of another GCT component may be missed because of sampling bias. 2 The most common immature component observed in aspirates from immature teratoma is in the form of a round cell component with rosettes and fibrillary material (primitive neuroepithelial component) and/or primitive mesenchyme. In our series, 6 patients had features of mixed GCT in their smears, whereas 1 patient had elevated serum AFP and hcg levels with the cytologic features of a mature teratoma. In another 9 patients who had only 1 component of GCT observed in their smears, histologic examination revealed mixed GCT, underscoring the need to consider mixed GCT in all

8 Cytology of Extragonadal GCTs/Gupta et al 511 patients for whom GCT is being considered as a diagnostic possibility. A similar inability to make a confident diagnosis of mixed GCT was reported in earlier studies. 2,5 This has been attributed to limitations of sampling, poor display of some components, and the overwhelming presence of 1 component. 2 To overcome these limitations, adequate sampling, a thorough search for another component, and correlation with serum tumor markers are advised. In 2 cases in our series, although a diagnosis of GCT was entertained, an accurate subclassification was not possible on cytology. This occurred because of limited cellularity in 1 case and the presence of extensive hemorrhage and necrosis in the smears in another case. Although no case of choriocarcinoma was included in our study, the cytologic diagnosis of this entity usually is difficult because of the frequently hemorrhagic and necrotic smears. Syncytiotrophoblastic giant cells also have been described in other GCTs. It is the presence of cytotrophoblasts (polygonal cells with basophilic cytoplasm, eccentric nuclei, and distinct nucleoli) that helps in considering a diagnosis of choriocarcinoma. In addition, the history of a trophoblastic tumor site helps in this diagnosis. 2 The accuracy of FNA for diagnosing mediastinal GCTs reportedly is high, with a sensitivity of 91% and specificity of 98.3%. 18 An earlier study of metastatic GCTs indicated that it is possible to establish a diagnosis of GCT by FNA cytology. 5 The series by Kapila et al. included metastatic GCT in effusion fluids, sputum, bronchial aspirates, urine, cerebrospinal fluids, lymph nodes, lung aspirates, and soft tissues from patients who had a primary tumor located in the gonads, mediastinum, or pineal gland. In our study, only 4 of 88 patients who were diagnosed with GCT on cytology had a discordant diagnosis on histopathologic examination. Thus, in the current study, FNA cytology offered a reliable diagnosis of GCT in the majority of patients. The current study included a variety of extragonadal sites, and both primary and metastatic extragonadal GCTs were included in the initial presentation. At sites such as the liver and buttocks, GCTs usually are not considered in the initial list of differential diagnoses. However, because the cytologic features in our patients suggested GCT, systematic investigations led to an accurate diagnosis. FNA cytology, compared with a biopsy, is a rapid, reliable, and less invasive technique with which to make a diagnosis of GCTs at extragonadal sites, including metastatic lesions, which may be seen as the first clinical presentation. Even in cases for which a definitive diagnosis cannot be offered on FNA alone, it may provide an important lead in the diagnosis of a GCT. 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