Malignant Epithelioid Gastrointestinal Stromal Tumors. Report of a Case with Cytologic and Immunohistochemical Studies DO NOT DUPLICATE

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1 Malignant Epithelioid Gastrointestinal Stromal Tumors Report of a Case with Cytologic and Immunohistochemical Studies Juan B. Laforga, M.D. Background Gastrointestinal stromal tumors (GISTs) may exhibit a fusiform, epithelioid or mixed pattern of growth. Only rare articles report the cytologic and immunohistochemical features of malignant epithelioid tumors. Case A 57-year-old woman presented with a tumor mass in the small intestine omentum measuring 8 7 cm; it was surgically removed. Five years later 2 mesenteric relapses were studied by fine needle aspiration biopsy and later surgically excised. Cytologically the smears contained small clusters of epithelioid and plasmacytoid cells with round nuclei. The presence of nucleoli and occasional nuclear grooves were prominent. Focally the background was myxoid. Histologically the tumor showed an epithelioid pattern with moderate pleomorphism and mitoses in 6 of 50 high-power fields. Immunohistochemical study showed positivity for c- kit (CD117), vimentin, smooth muscle actin and caldesmon, and focally for desmin and cytokeratin. Conclusion This case illustrates the difficulty in making a reliable diagnosis of the epithelioid variant of GIST by cytology alone. On morphologic and immunohistochemical grounds, we initially diagnosed an epithelioid LMS, but with the aid of FNAB and CD117, the final diagnosis was epithelioid GIST. Case Reports The immunohistochemical panel (apart from c-kit) should include smooth muscle markers and cytokeratins because they are more likely to be reactive. A complete cytoimmunohistochemical evaluation is mandatory to make an accurate diagnosis. (Acta Cytol 2005; 49: ) Keywords: cancer of gastrointestinal tract; aspiration biopsy, fine-needle; malignant epithelioid gastrointestinal stromal tumors. Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract, affecting the entire length, from the esophagus to the anus, with a predominance in the stomach (60 70% of cases), followed by the small intestine (20 30%), colon and rectum (5%) and esophagus (<5%). Less than 5% arises in the omentum, retroperitoneum and mesentery. GIST typically occurs in individuals > 50 years of age, with a slight male predominance. 1-4 The histogenesis of this tumor is the interstitial cell of Cajal or a precursor cell, which displays a characteristic phenotype positive for CD117, CD34 and smooth muscle actin (SMA). Histologically the tumors may display 3 patterns of From the Department of Pathology, Hospital Marina Alta, Denia, Spain. Dr. Laforga is Staff Pathologist. Address reprint requests to: Juan B. Laforga, M.D., Servicio de Anatomía Patológica, Hospital Marina Alta, Ptda. Plana Est, 4, Denia (Alicante), Spain (laforga_jua@gva.es). Financial Disclosure: The author has no connection to any companies or products mentioned in this article. Received for publication March 17, Accepted for publication October 4, /05/ /$19.00/0 The International Academy of Cytology ACTA CYTOLOGICA 435

2 Laforga growth: fusiform (70%), epithelioid (20%) and mixed (10%). In the diagnosis of GIST by fine needle aspiration biopsy (FNAB), it is very important to highlight the c-kit mutation by immunohistochemistry in order to administer the diane therapy with Glivec (Novartis, Basel, Switzerland). 5 Although the cytologic features of fusiform or spindle cell variant of GIST are well established, only rare articles describing the epithelioid variant have been published. In this article we report a case arising in the small intestine and briefly discuss the differential diagnosis. Case Report In 1995 a 57-year-old woman was admitted for an abdominal tumor mass. At laparotomy an intestinal tumor measuring 8 7 cm attached to the peritoneal surface of the small intestine was observed. A segmentary ileal resection was performed, and a diagnosis of epithelioid leiomyosarcoma (LMS) was initially made. A Five years later the patient presented with abdominal pain for 2 months. Abdominal ultrasonography showed a mesenteric mass measuring 2.5 cm in diameter. Fine needle aspiration (FNA) and core biopsy (FNAB) were performed, followed by a wide segmentary intestinal surgical resection including the tumor mass. There was no evidence of disease 3 years after the operation. Pathologic Features Primary Tumor Grossly the primary intestinal tumor mass showed a smooth and multilobulated surface measuring cm. On the cut surface it was firm, fascicular and grayish. Microscopically the tumor was formed by fascicles of large and epithelioid cells characterized by large, eosinophilic cytoplasm with occasional vacuoles. The nuclei were round and centrally placed, with vesicular chromatin containing conspicuous nucleoli (Figure 1D). Mitotic figures averaged 5 per 50 high-power B C D Figure 1 (A) FNAB showing a fusiform tumor infiltrating the omentum. (B) Detail showing large, ovoid nuclei containing intranuclear inclusions. (C) Cut section of the relapsed omental tumor. (D) Histologic appearance showing malignant epithelioid cells and a mitotic figure (hematoxylin-eosin; A, 250; B and D, 400). 436 ACTA CYTOLOGICA Volume 49 Number 4 July August 2005

3 Gastrointestinal Stromal Tumors A C Figure 2 (A) Tumor showing SMA positivity. (B) Epithelioid cells showing strong positivity for cytokeratin. (C) Focally the tumor showed positivity with desmin. (D) The tumor cells stained diffusely with c-kit (A, SMA, 400; B, AE1/AE3, 400; C, desmin, 400; D, CD117, 400). fields. The resection margins were free of tumor. Immunohistochemical study showed focal positivity for smooth muscle actin (HHF-35) and strong reactivity for desmin and cytokeratin (AE1/AE3) (Figure 2A C). The tumor cells were negative for CD34 and S-100 protein. Omental Relapse Cytologic Features. FNA was performed under endoscopic ultrasound guidance. The slides were air dried and alcohol fixed and stained with Giemsa and Papanicolaou stain, respectively. They showed scattered, small, 3-dimensional clusters of epithelioid cells containing abundant cytoplasm and round, centrally located, hyperchromatic nuclei (Figure 3A and B). The nuclei showed occasional macronucleoli, some of which contained grooves (Figure 3C). In some slides, small clusters of epithelioid cells were floating in a mucoid background (Figure 3D). A diagnosis of positive for malignancy, consistent with a relapse of epithelioid malignant tumor, was made. The gross specimen showed a round, well-defined, nodular mass in the omentum measuring 2.5 cm in diameter. On the cut section the tumor was whitish and firm (Figure 1C). FNAB and Immunohistochemical Study. The aspirate showed histologic and immunohistochemical features overlapping with those of the primary tumor, and the presence of intranuclear inclusions was striking (Figure 1A and B). Additional immunostaining performed on the retrieved primary and relapsed tumor with CD117 showed strong and diffuse cytoplasmic positivity (Figure 2D) and focal positivity with caldesmon. A diagnosis of mesenteric metastatic epithelioid GIST could be made. B D Discussion GISTs have been a subject of study for decades. Initially Stout considered them smooth muscle tumors with different designations, such as leiomyoma, leiomyoblastoma, bizarre leiomyoma and leiomyosarcoma. Later ultrastructural features were not recognized in every tumor, questioning their smooth mus- Volume 49 Number 4 July August 2005 ACTA CYTOLOGICA 437

4 Laforga A C Figure 3 (A) Group of epithelioid cells showing a nested appearance. The cells have an epithelioid appearance with round to oval nuclei. (B) Small group of epithelioid cells showing pleomorphic nuclei and large cytoplasm. (C) Discohesive group of epithelioid cells, some of which contain nuclear grooves (arrows). (D) Three-dimensional group of malignant cells showing a mucoid background (A and B, Diff-Quik, 400; C and D, Papanicolaou stain, 400). cle origin. With immunohistochemical study, a large number of cases showed positive staining with CD34 and SMA. The reactivity with the former antibody was considered a constant marker for this tumor, which was no longer true. The study of larger series of GISTs showed that CD34 was positive in 60 70% of the cases while SMA was positive in 30 40% of the cases and desmin in only 1% to 2%. Kindblom et al showed that 15% of GIST are positive for smooth muscle actin and ultrastructurally display actin-type filaments. 6 In recent years the mutation of c-kit has been a constant event in these neoplasms. CD117 (c-kit) highlights this molecular translocation in nearly all cases. Recently a group of experts outlined the diagnostic approach to making a diagnosis of GIST in order to standardize the immunohistochemical and morphologic features. 7 The presence of c-kit positivity showing strong and diffuse cytoplasmatic staining is a prerequisite of the diagnosis. However, a small subgroup of GISTs is c-kit negative by immunohistochemistry. In such cases surrogate markers, such as mutational and cytogenetic analysis, may make the diagnosis. Thus, a recent paper reported on 7 of 173 GISTs that exhibited the same morphologic, cytogenetic and molecular features as do c-kit positive tumors. 8 Less frequently these tumors may show cytokeratin expression, especially in those with an epithelioid pattern of growth. Our case exhibited a peculiar phenotype, lacking CD34 expression (30 40% of GISTs) and being positive for smooth muscle actin and desmin (like smooth muscle tumors) together with cytokeratin expression, which induced us to make a preliminary diagnosis of LMS. This might not be regarded as a misdiagnosis, taking into account that the B D 438 ACTA CYTOLOGICA Volume 49 Number 4 July August 2005

5 Gastrointestinal Stromal Tumors morphologic features and the immunophenotype, SMA+ (70%), desmin and cytokeratin+ (10%), fit epithelioid LMS. Our initial immunohistochemical panel (previous to c-kit stain) was completed recently when CD117 became commercially available. Application of this antibody to the retrieved tissue blocks from the primary tumor and the mesenteric relapse was uniformly and strongly positive. Therefore, it provided the definitive key feature to making the diagnosis. Recurrence in GISTs occurs in 40 80% of cases even though the initial resection was thought to be sufficient, as in our case. Another feature that separate GISTs from LMS is that the former recur nearly exclusively in the abdominal cavity (mainly mesentery and liver), while in LMS, lung metastases are frequent. The morphologic criteria for malignancy in GIST were recently addressed, analyzing tumor size and mitotic activity. 7 Briefly, tumors > 5 cm and with > 5 mitoses per 50 high-power fields have a high chance of metastasis. Some small tumors (< 3 cm) with a long period of follow-up developed metastases, so it is no longer justified to make a diagnosis of benign GIST. FNAB has been used to make the diagnosis of GIST, with CD117 stain a possible tool. 9,10 This is very important to confirm a clinical suspicion of relapse or metastatic tumor, as occurred in our case. From cytologic grounds alone, the diagnosis of GIST must be discouraged because of the lack of specific features. 11 Indeed, it should be diagnosed as a fusiform or epithelioid tumor, and additional smears for immunocytochemical study must be ordered. Cytologic separation of GIST from other fusiform neoplasms, such as leiomyoma, leiomyosarcoma, cellular schwannoma, solitary fibrous tumor, sarcomatoid carcinoma and malignant melanoma, may be difficult and require additional histochemical studies along with histologic evaluation. Solitary fibrous tumor (SFT) shows patternless growth, which may pose a diagnostic challenge in the evaluation of small biopsies and FNA cytology. The cells show positive staining for CD34 and CD99. Both GIST and SFT are immunoreactive for CD34 and CD99, but CD117 is negative in SFT. 12 Separation of GIST from cellular schwannoma may be difficult. Morphologic key features, such as the presence of lymphoid aggregates, clusters of xanthomatous cells, and diffuse and strong positivity for S-100 are useful for diagnosis, while in GIST 5% of cases show S-100 positivity Concerning the epithelioid variant of GIST, the tumor cells are round, with eosinophilic and clear cytoplasm. Their nuclei are uniform and round to oval, with vesicular chromatin. The cytologic features were recently addressed in a series of 9 epithelioid GISTs in which the most frequent features were the presence of prominent nucleoli and intranuclear inclusions in all cases followed by binucleation and irregular nuclear contour. 16 In our case, although intranuclear inclusions were lacking, the presence of nuclear grooves similar to those observed in papillary thyroid carcinoma were documented. In addition, some nests of tumor cells were floating in a myxoid stroma. These features may expand the cytological appearance of this variant of GIST. Very often the cell nests exhibit a nested pattern of growth, which may simulate an epithelial tumor morphologically because of the large, eosinophilic cytoplasm (especially when cytokeratins are positive). That may induce a misdiagnosis of carcinoma and malignant melanoma. In the former, coexpression of vimentin and cytokeratins and negativity for muscular markers should contribute to the diagnosis. In malignant melanoma, vimentin and S-100 should be positive, along with HMB-45. These potential pitfalls may be avoided particularly in small biopsies or FNA, applying a panel of antibodies including CD34, CD117, S-100 and SMA. Another challenging differential diagnosis is with a neuroendocrine tumor because of the epithelioid and occasional plasmacytic appearance of the cells along with round nuclei containing finely granular chromatin. 17 However, these tumors display intracytoplasmic granules reactive for chromogranin- A and synaptophysin stain. According to Miettinen et al, 5 10% of GISTs contain desmin-positive tumor cells, 4 and such positivity may be more common among epithelioid GISTs. Besides, epithelial keratin expression is more infrequent than observed in typical leiomyosarcomas and occurs especially in malignant epithelioid GISTs. 18 This feature is in keeping with our case. In summary, on morphologic and immunohistochemical grounds, we initially diagnosed an epithelioid LMS, but with the aid of FNAB and CD117, the final diagnosis was epithelioid GIST. Acknowledgments The author thanks Delfina Escrivá for her excellent technical assistance. References 1. Lerma E, Oliva E, Tugues D, Prat J: Stromal tumours of the gastrointestinal tract: A clinicopathological and ploidy analysis of 33 cases. Virchows Arch 1994;424: Ueyama T, Guo KJ, Hashimoto H, Daimaru Y, Enjoji M: A clinicopathologic and immunohistochemical study of gastrointestinal stromal tumors. Cancer 1992;69: Miettinen M, Sarlomo-Rikala J, Lasota J: Gastrointestinal stromal tumors: New findings on their biology: A review. Hum Pathol 1999;123: Miettinen M, Sarlomo-Rikala M, Sobin LH, Lasota J: Gas- Volume 49 Number 4 July August 2005 ACTA CYTOLOGICA 439

6 Laforga trointestinal stromal tumors and leiomyomas in the colon: A clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases. Am J Surg Pathol 2000;13: Cormier JN, Patel SR, Pisters PWT: Gastrointestinal stromal tumors: Rationale for surgical adjuvant trials with Imatinib. Curr Oncol Rep 2002;4: Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM: Gastrointestinal pacemaker cell tumor (GIPACT): Gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am J Pathol 1998;152: Fletcher CD, Berman JJ, Corless C, Gorstein F, Lasota J, Longley BJ, Miettinen M, O Leary TJ, Remotti H, Rubin BP, Shmookler B, Sobin LH, Weiss SW: Diagnosis of gastrointestinal stromal tumors: A consensus approach. Hum Pathol 2002;33: Debiec-Rychter M, Wasag B, Stul M, De Wever I, Van Oosterom A, Hagemeijer, Sciot R: Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity. J Pathol 2004;202: Cheuk W, Lee KC, Chan JKC: C-kit immunocytochemical staining in the cytologic diagnosis of metastatic gastrointestinal stromal tumor. Acta Cytol 2000;44: Lozano MD, Rodriguez J, Algarra SM, Panizo A, Sola JJ, Pardo J: Fine-needle aspiration cytology and immunohistochemisty in the diagnosis of 24 gastrointestinal stromal tumors: A quick, reliable diagnostic method. Diagn Cytopathol 2003;28: Kimura M, Satou T, Hashimoto S: Can GIST be diagnosed reliably by cytology? Acta Cytol 2002;46: Shidham VB, Chivukula M, Gupta D, Rao RN, Komorowski R: Immunohistochemical comparison of gastrointestinal stromal tumor and solitary fibrous tumor. Arch Pathol Lab Med 2002; 126: Woodruff JM, Godwin TA, Erlandson RA: Cellular schwannoma: A variety of schwannoma sometimes mistaken for a malignant tumor. Am J Surg Pathol 1981;5: Fletcher CDM, Davies SE, McKee PH: Cellular schwannoma: A distinct pseudosarcomatous entity. Histopathology 1987;11: Laforga J: Cellular schwannoma: Report of a case diagnosed intraoperatively with the aid of cytologic imprints. Diagn Cytopathol 2003;29: Dong Q, McKee G, Pitman M, Geisinger K, Tambouret R: Epithelioid variant of gastrointestinal stromal tumor: Diagnosis by fine-needle aspiration. Diagn Cytopathol 2003;29: Szyfelbein WM, Ross JS: Carcinoids, atypical carcinoids, and small cell carcinomas of the lung: Differential diagnosis of fineneedle aspiration biopsy specimens. Diagn Cytopathol 1988; 4: Miettinen M, Lasota J: Gastrointestinal stromal tumors: Definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch 2001;438: ACTA CYTOLOGICA Volume 49 Number 4 July August 2005