Fine-Needle Aspiration Biopsy of Nonteratomatous Germ Cell Tumors of the Mediastinum

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1 Anatomic Pathology / FINE-NEEDLE ASPIRATION BIOPSY OF MEDIASTINAL GERM CELL TUMORS Fine-Needle Aspiration Biopsy of Nonteratomatous Germ Cell Tumors of the Mediastinum David C. Chhieng, MD, 1 Oscar Lin, MD, 2 Cesar A. Moran, MD, 3 Isam A. Eltoum, MD, 1 Nirag C. Jhala, MD, 1 Darshana N. Jhala, MD, 1 and Aylin Simsir, MD 4 Key Words: Fine-needle aspiration; Mediastinum; Germ cell tumors Abstract We assessed the usefulness of fine-needle aspiration biopsy (FNAB) in the diagnosis of mediastinal germ cell tumors (GCTs). In the archives of 3 pathology departments, we found records of 7 patients with mediastinal GCTs who underwent mediastinal FNAB as part of the diagnostic workup. The FNAB smears, results of the immunocytochemical analysis, the corresponding histologic findings, and the clinical charts were reviewed. All patients were men (age range, years; mean, 32 years). One patient had a history of testicular mixed GCT 10 years earlier. The 6 primary mediastinal GCTs consisted of 3 seminomas and 3 yolk sac tumors. Based on the cytologic features and immunocytochemical findings, a cytologic diagnosis of GCT was made in 5 cases, including the case of metastatic GCT. In 2 cases, the differential diagnosis was between poorly differentiated carcinoma and GCT. Results of ancillary studies were noncontributory in 1 case, and the aspirate of the second case demonstrated extensive necrosis. Our findings demonstrate that a diagnosis of mediastinal GCT, primary or secondary, can be established with a high degree of accuracy on the basis of FNAB. Immunocytochemical analysis helps confirm the diagnosis. The mediastinum is the anatomic space in the thoracic cavity that is bound by the pleura, sternum, vertebral column, thoracic inlet, and diaphragm. The majority of mediastinal lesions are of epithelial and lymphoid origin. 1-3 Germ cell tumors (GCTs) account for 20% of all mediastinal tumors. 4 Anterior mediastinum is the most common extragonadal site of GCTs in adults. 5 It is important to differentiate GCTs from other epithelial and nonepithelial malignant neoplasms owing to important therapeutic and prognostic implications. Fineneedle aspiration biopsy (FNAB) has become a standard diagnostic procedure for the investigation of mediastinal masses. 2,3,6-8 There are only a few reports discussing the diagnosis of mediastinal GCTs on FNAB We reported the cytologic features in 7 cases of mediastinal GCTs. The differential diagnosis, the diagnostic pitfalls, and the role of immunocytochemical analysis in the workup are discussed. Materials and Methods Through comprehensive computer searches, cases with mediastinal FNABs from the archives of 3 academic institutions (University of Alabama at Birmingham; New York University Medical Center, New York; and Memorial Sloan Kettering Cancer Center, New York) were identified. Only cases of mediastinal GCTs were included in the study. All FNABs were performed percutaneously under computed tomography guidance using 22-gauge needles. Smears were air dried and stained with a modified Romanowsky stain or immediately fixed in alcohol and stained with the Papanicolaou method. Additional material was obtained for cell block preparations. 418 Am J Clin Pathol 2002;118: American Society for Clinical Pathology

2 Anatomic Pathology / ORIGINAL ARTICLE Immunocytochemical analysis was performed on 5-µm paraffin sections from cell blocks. Heat-induced epitope retrieval was done by microwave heating for 10 minutes in a 0.01-mol/L concentration of citrate buffer, ph 6. Immunostaining was performed using a modified avidin-biotin peroxidase technique. The primary monoclonal antibodies used were cytokeratin (CK), placental-like alkaline phosphatase (PLAP), alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin (hcg). Appropriate positive and negative controls were performed for each case. Follow-up consisted of review of clinical charts and evaluation of corresponding resected tissue. The tissue specimens were fixed in 10% buffered formalin, processed in the routine manner, and embedded in semisynthetic paraffin. Sections were cut and stained with H&E. Results The 7 cases of mediastinal GCTs were from 7 men. Their ages ranged from 24 to 44 years (mean, 32 years). One patient had a history of testicular mixed GCT composed of choriocarcinoma and teratoma treated 10 years earlier with orchiectomy and chemotherapy, and he had a posterior mediastinal mass. All others had a anterior mediastinal mass. The clinical features for the 7 cases are summarized in Table 1. The 6 primary mediastinal GCTs consisted of 3 seminomas and 3 yolk sac tumors. The FNAB smears of seminomas were cellular and demonstrated loosely cohesive and single, large, mononucleated cells with scant to moderate cytoplasm and round to oval nuclei Image 1. The nuclei were vesicular with finely granular chromatin, thick nuclear membrane, and a single macronucleolus. A variable number of lymphocytes were present in the background. In addition, a tigroid background, interwoven strips of cytoplasm, was noted and was best observed on air-dried, rapid Romanowsky stained preparations Image 2. Immunocytochemical analysis was performed in all 3 cases. In 1 case, the Table 1 Clinical Features and Cytologic-Histologic Correlation for Seven Cases neoplastic cells were positive for PLAP and negative for CK, supporting a diagnosis of seminoma. The results of immunostaining were noncontributory in the remaining 2 cases because of high background. The aspirates of 2 of the cases of yolk sac tumors were cellular and consisted of medium-sized epithelioid cells arranged singly and in clusters Image 3. The cytoplasm was moderate in amount and sometimes vacuolated. Cytoplasmic and extracellular hyaline globules were noted in 1 case. The nuclei were round with finely to coarsely granular chromatin and small nucleoli. Glomeruloid structures or Schiller-Duval bodies were noted in the cell block preparation in 1 case Image 4. Immunocytochemical analysis performed on 1 case demonstrated that the neoplastic cells were positive for AFP and PLAP and negative for betahcg Image 5. Immunostaining was not done in the other case. The aspirate of the third case revealed predominantly necrotic tissue with only a few atypical epithelioid cells that were positive for CK. The differential diagnosis included a poorly differentiated non small cell carcinoma and a GCT. The FNAB of the metastatic GCT to the posterior mediastinum was cellular and demonstrated large syncytial tissue fragments in a hemorrhagic background. The neoplastic cells had ill-defined cytoplasmic borders and round to oval nuclei. Nucleoli were prominent. Occasional isolated cells also were noted. There were no multinucleated giant cells. Immunocytochemical analysis was not performed. A correct diagnosis of GCT was made in 5 cases, including the case of metastatic posterior mediastinal lesion. In 2 cases, the FNAB diagnoses were inconclusive. In 1 case, the differential diagnosis was between a poorly differentiated carcinoma and a GCT; a more specific diagnosis could not be reached owing to scant cellularity. The corresponding histologic examination revealed seminoma. The second case was identified as a yolk sac tumor on surgical biopsy; however, the material obtained by FNAB was extensively necrotic. Case No./ Primary vs Sex/Age (y) Secondary Cytologic Diagnosis Immunocytochemical Findings Histologic Diagnosis 1/M/27 Primary Yolk sac tumor PLAP, AFP positive; hcg negative Yolk sac tumor 2/M/33 Primary Seminoma PLAP positive; LCA, CK, hcg negative Extensively necrotic tumor consistent with seminoma 3/M/29 Primary PD carcinoma vs germ cell tumor Noncontributory Anaplastic seminoma 4/M/24 Primary Extensively necrotic tumor CK positive Yolk sac tumor 5/M/34 Primary Germ cell tumor ND Yolk sac tumor 6/M/34 Primary Germ cell tumor Noncontributory Seminoma 7/M/27 Secondary Consistent with metastatic germ ND Metastatic germ cell tumor cell tumor AFP, alpha-fetoprotein; CK, cytokeratin; hcg, human chorionic gonadotropin; LCA, leukocyte common antigen; ND, not done; PD, poorly differentiated; PLAP, placental-like alkaline phosphatase. American Society for Clinical Pathology Am J Clin Pathol 2002;118:

3 Chhieng et al / FINE-NEEDLE ASPIRATION BIOPSY OF MEDIASTINAL GERM CELL TUMORS Image 1 Seminoma. Large atypical cells admixed with small lymphocytes. The large neoplastic cells demonstrated vesicular nuclei and prominent nucleoli (Papanicolaou, 400). Image 3 Yolk sac tumor. Cohesive group of pleomorphic tumor cells with a moderate amount of vacuolated cytoplasm (rapid Romanowsky, 400). Discussion Mediastinum is the most common extragonadal site of GCTs in adults. It is the second most common extragonadal site in children, preceded by tumors in the sacrococcygeal area. The histogenesis of mediastinal GCTs is still poorly understood. One school of thought is that primordial germ cells, during their migration in the first few weeks, are misplaced into midline extragonadal sites such as the thymus in the anterior mediastinum However, it is difficult to Image 2 Seminoma. Similar large atypical tumor cells with prominent nucleoli admixed with small lymphocytes in a tigroid background (rapid Romanowsky, 400). Image 4 Yolk sac tumor. Cell block showing a Schiller-Duval body (H&E, 400). explain why primary GCTs are not found in the posterior mediastinum since the primordial germ cells pursue a dorsal route along the midline. 17,18 Another theory is that the GCTs may originate from the multipotent stem cells of the thymus. 19 GCTs account for 20% of all mediastinal tumors. 4 They must be differentiated from other more common mediastinal tumors including thymoma, malignant lymphoma, and poorly differentiated carcinoma, either primary or metastatic. 15,20 Since the prognosis and treatment of these tumors differ surgery vs chemotherapy and/or radiation a 420 Am J Clin Pathol 2002;118: American Society for Clinical Pathology

4 Anatomic Pathology / ORIGINAL ARTICLE Image 5 Yolk sac tumor. The neoplastic cells stained positive for alpha-fetoprotein (alpha-fetoprotein, 400). simple and accurate diagnostic method is preferred. As such, FNAB has been demonstrated to be reliable in the evaluation of mediastinal lesions. 2,3,6,21 The sensitivity and specificity of FNAB in the diagnosis of mediastinal malignant neoplasms range from 87% to 90% and 88% to 100%, respectively. 2,6 To our knowledge, there are only a few studies in the literature on the cytology of mediastinal GCTs. 2,3,9,10,12,13 Motoyama et al 10 reported 6 primary mediastinal GCTs including 1 seminoma, 1 embryonal carcinoma, 1 yolk sac tumor, and 3 mixed GCTs. In another study, Powers et al 2 reported 5 GCTs in their series of 54 mediastinal FNABs. In the present study, we identified 7 cases of mediastinal GCTs evaluated by FNAB from 3 academic institutes. Therefore, GCTs are not frequently encountered in FNABs of mediastinal masses. The present series included 3 primary seminomas, 3 primary yolk sac tumors, and 1 metastatic mixed GCT. The histologic and cytologic features of mediastinal GCTs are similar to those of tumors originating from the gonads. 5,9,10,13,22-24 Typical seminomas or dysgerminomas consist of uniform, large, round neoplastic cells with prominent nucleoli admixed with small lymphocytes in a tigroid background. The tigroid background is characteristic of seminoma but may not be present in every case. It is reported that the tigroid background is noted only in highly cellular specimens and is absent in specimens with low cellularity. 25 In addition, the tigroid background is not specific for seminoma and has been reported in renal cell carcinoma, squamous cell carcinoma, synovial sarcoma, and melanoma. 13,26 Other features present at times on the smears of seminomas are granulomas or aggregates of epithelioid histiocytes. 26,27 Nonseminomatous GCTs include embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma. The aspirates of embryonal carcinoma often demonstrate cohesive groups of pleomorphic epithelioid cells with prominent nucleoli. The cytoplasm of the neoplastic cells usually is scant with indistinct cell borders. The latter results in a syncytial growth pattern. 28 In addition, the neoplastic cells also can demonstrate a papillary and/or glandular arrangement. 13,24 These findings can be confused with those of a poorly differentiated adenocarcinoma or large cell undifferentiated carcinoma. The presence of abundant necrotic debris and scant viable tumor cells can further complicate the differential diagnosis. Yolk sac tumors demonstrate similar cytomorphologic features, with single and clusters of pleomorphic epithelioid cells with abundant vacuolated cytoplasm. The tumor cells can also demonstrate a papillary growth pattern. Necrosis can be quite prominent, as in one of our cases. Schiller- Duval bodies or glomeruloid structures formed by invagination of neoplastic cells and capillaries into a small space are characteristic of yolk sac tumors in histologic preparations but are noted only occasionally in cytologic preparations. 11 Another feature of yolk sac tumors is the presence of cytoplasmic and extracellular hyaline globules. The latter is positive for periodic acid Schiff, resistant to diastase, positive for AFP. However, the globules are by no means pathognomonic or diagnostic of yolk sac tumors, since they can be found in many different neoplasms. 10 The finding of metachromatic basement membrane like extracellular material also has been suggested to be helpful in recognizing the presence of a yolk sac tumor component in aspirates. 29,30 Aspirates of choriocarcinoma also reveal single and cohesive groups of pleomorphic cells with bizarre nuclei and prominent nucleoli, often in a hemorrhagic background. The finding of multinucleated tumor giant cells (syncytiotrophoblasts) suggests choriocarcinoma. Occasional syncytiotrophoblast-like multinucleated giant cells also can be seen in seminoma; however, they are not associated with cytotrophoblasts. 31 We did not encounter any immature teratomas in our study. The cytologic diagnosis of immature teratoma can be difficult because of the rarity of this neoplasm, accounting for only 5% of all mediastinal GCTs, and the variability in the cytologic features from one tumor to another, as well as within the same tumor. Mixed GCTs are relatively frequent in the mediastinum and consist of 2 or more of the aforementioned histologic subtypes of GCTs. 5 Recognition of all the components of mixed GCTs based on FNAB material may not be possible because of sampling limitations. 9,25,26,28,32 Because of the diverse morphologic features of GCT, the differential diagnosis includes a wide variety of entities. For American Society for Clinical Pathology Am J Clin Pathol 2002;118:

5 Chhieng et al / FINE-NEEDLE ASPIRATION BIOPSY OF MEDIASTINAL GERM CELL TUMORS example, if the neoplastic cells in seminoma tend to appear singly, the differential diagnosis should include large cell lymphoma. The presence of loosely cohesive tumor cell groups and a tigroid background, as well as the lack of lymphoglandular bodies, favors a diagnosis of seminoma over that of lymphoma. In addition, nuclear atypia such as cleaved nuclei and nuclear projections in lymphomas is a helpful feature. However, the presence of lymphoglandular bodies has been reported rarely in the aspirate of seminoma. 33 When the lymphoid component of seminoma is conspicuous, a biphasic pattern results. Then the differential diagnosis should include thymoma. The rounded nuclei, the prominent nucleoli of the neoplastic cells, and a tigroid background favor a diagnosis of seminoma, whereas the presence of a mixture of epithelioid and spindled cells points to a thymoma. 34 As mentioned earlier, the presence of markedly atypical epithelioid cells arranged in papillary or glandular structures in a hemorrhagic and/or necrotic background in nonseminomatous germ cell tumors can be confused with a thymic carcinoma or metastatic carcinoma to the mediastinum. Metastatic carcinoma to the mediastinum is more common than GCT and frequently is of bronchogenic origin, followed by an esophageal primary site. 35 It may be difficult to differentiate these entities based on morphologic features alone unless diagnostic cytologic features such as Schiller-Duval bodies are present. Clinical and serologic findings may be helpful. For example, mediastinal GCTs typically occur during young adulthood or early middle age, 5 whereas thymic carcinomas seldom occur in patients younger than 30 years, 20 and metastatic carcinomas tend to occur in elderly patients. The finding of elevated serum levels of hcg and/or AFP, which are present in about 90% of patients with a GCT, also favors a diagnosis of GCT. 4 Ancillary studies, particularly immunocytochemical analysis, are helpful in the differential diagnosis. Table 2 summarizes the differential diagnosis of GCTs based on immunocytochemical findings. A battery of antibodies often Table 2 Immunophenotypes of Germ Cell Tumor and Their Differential Diagnosis is required because of the overlaps in the immunophenotypic pattern of various tumors. It is important to note that all GCTs, both seminomatous and nonseminomatous, can demonstrate variable degree of staining with certain antikeratin antibodies including CK7, CAM 5.2, and AE1/AE3. 36 According to 1 study, CK7, CAM 5.2, and AE1/AE3 were positive in 41%, 30%, and 36% of seminomas and in 100%, 88%, 94%, and 88% of embryonal carcinomas, respectively. 36 CD30, originally described as a marker for Hodgkin disease and anaplastic large cell lymphoma, is strongly expressed in embryonal carcinoma It also has been reported that CD30 can demonstrate rare positive staining in about a quarter of seminomas. 40 Since the majority of the metastatic tumors are of bronchogenic origin, the use of markers such as thyroid transcription factor-1 (TTF-1) that are thought to be specific for bronchogenic carcinomas may be helpful in the differential diagnosis. The staining pattern of TTF-1 in germ cell tumors is unknown. In addition, the percentage of primary lung adenocarcinomas that demonstrated positive staining with TTF-1 ranged from 20% to 70% Therefore, the finding of positive TTF-1 staining favors a metastatic bronchogenic carcinoma over a GCT, but the converse may not necessarily be true. Based on our experience, as well as that of others, 9,13,21 cytologic examination along with immunocytochemical analysis is reliable in the diagnosis of GCTs. In 1 study, the authors correctly diagnosed all 4 primary nonteratomatous GCTs, including one of mediastinal origin. 13 In another study that included 4 mediastinal GCTs evaluated by FNA, the cytologic diagnosis of a germ cell tumor was confirmed by subsequent histologic examination in 3 cases. 9 Herman et al 21 reported sensitivity and specificity of 91% and 98%, respectively, in the diagnosis of mediastinal germ cell tumor by FNAB. We correctly diagnosed 5 cases of mediastinal GCTs based on the examination of cytologic materials. In the remaining 2 cases, GCT was included in the differential diagnosis. Yolk Sac Embryonal Marker Seminoma Tumor Carcinoma Choriocarcinoma Carcinoma Lymphoma Melanoma CK (AE1/AE3) ± PLAP ± AFP + ± beta-hcg (except tumor + giant cells) S-100 protein + LCA + CD 30 (Ber-H2) (rare cells + ± ± positive) AFP, alpha-fetoprotein; CK, cytokeratin; hcg, human chorionic gonadotropin; LCA, leukocyte common antigen; PLAP, placental-like alkaline phosphatase; +, positive; ±, sometimes positive;, negative. 422 Am J Clin Pathol 2002;118: American Society for Clinical Pathology

6 Anatomic Pathology / ORIGINAL ARTICLE It is not possible to distinguish between primary and secondary mediastinal GCTs based on morphologic features alone. Some differences have been observed between mediastinal and testicular seminomas. 44 Mediastinal seminomas showed strong dot-like paranuclear positivity of the tumor cells with antibodies to CAM 5.2 in 80% of cases, as compared with only 20% positivity in testicular seminomas; PLAP also was expressed less commonly in testicular seminomas than in mediastinal seminomas; a similar pattern of expression also was observed for vimentin, which was present in scattered tumor cells in a higher proportion of mediastinal seminomas than in testicular seminomas. According to the same group of investigators, the staining pattern and distribution of these markers did not show significant differences between the 2 groups for the nonseminomatous GCTs, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The knowledge of a previous testicular germ cell tumor and the location of the lesion are helpful clues. For example, in 1 of our cases, the tumor was located in the posterior mediastinum, which is unusual for a primary mediastinal germ cell tumor. It is important to note that a negative FNAB in patients after chemotherapy for a germ cell tumor does not necessarily rule out persistent disease because the lesion may contain only microscopic foci of malignancy. 45 Our experience, as well as that of others, suggests that FNAB is a reliable and accurate diagnostic modality for the evaluation of mediastinal masses due to primary or secondary GCTs. GCTs need to be differentiated from a wide spectrum of mediastinal neoplasms. The use of immunocytochemical analysis is helpful in the differential diagnosis. 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