hgh and GHR Expression in Large Cell Neuroendocrine Carcinoma of the Colon and Rectum

hgh and GHR Expression in Large Cell Neuroendocrine Carcinoma of the Colon and Rectum ZORAN JUKIĆ 1, RINË LIMANI 2, LUMTURIJE GASHI LUCI 2, VIVIAN NIKIĆ 3, AUGUST MIJIĆ 4, DAVOR TOMAS 3,5 and BOŽO KRUŠLIN 3,5 1 Department of Surgery, General Hospital Nova Gradiška, Nova Gradiška, Croatia; 2 Institute of Anatomical Pathology, Faculty of Medicine and University Clinical Center of Kosova, Prishtina, Kosovo; 3 Ljudevit Jurak Department of Pathology, University Hospital Centre Sestre Milosrdnice, Zagreb, Croatia; 4 Department of Surgery, University Hospital Centre Sestre Milosrdnice, Zagreb, Croatia; 5 Department of Pathology, School of Medicine, University of Zagreb, Zagreb, Croatia Abstract. Large cell neuroendocrine carcinoma (LCNEC) is an aggressive neoplasm with a low frequency of occurrence in the digestive tract. We present a series of eight patients diagnosed with LCNEC of the colon and rectum. Grossly, tumors were presented as endophytic/ulcerative, annular and polypoid masses, with a gray-white color and necrosis in most cases. Histologically, they were high-grade tumors composed of large cells of organoid, nesting, trabecular, rosette-like and palisading patterns, with a high mitotic rate. Tumors were immunoreactive for neuroendocrine markers, including chromogranin A (2/8), synaptophysin (7/8), and neuron-specific enolase (8/8). Moreover, we analyzed the expression of growth hormone (hgh) and growth hormone receptor (GHR) in colorectal LCNECs and six tumors were immunoreactive for hgh, while five tumors were immunoreactive for GHR. To our knowledge hgh and GHR expression has not been previously analyzed in colorectal LCNEC. Their overexpression suggests a role of hgh and GHR in the development of colorectal LCNEC. Large cell neuroendocrine carcinoma (LCNEC) is a poorly differentiated malignant neoplasm. Although it most commonly arises in the lung (1), a few cases have been reported in the large intestine (2-6). Colorectal LCNECs are very rare neoplasms. They belong within the poorest prognostic subgroups among primary colorectal neoplasms, usually presented with unfavorable outcomes (2). Growth Correspondence to: Dr. Rinë Limani, Institute of Anatomical Pathology, Faculty of Medicine and University Clinical Center of Kosova, Bulevardi i dëshmorëve p.n., 10000, Prishtina, Kosovo. Tel: +381 38500600 ext: 2051, e-mail: rinalimani@yahoo.com Key Words: LCNEC, colon, rectum, hgh, GHR. hormone (hgh) is a metabolic hormone which promotes growth and differentiation of many mammalian cells. It exerts its effects on the cells through its receptor (GHR), which is a transmembrane glycoprotein. A few studies have reported hgh and GHR overexpression in colorectal adenocarcinoma (7, 8). However, whether hgh and GHR have any direct effects in the etiology and pathogenesis of colorectal cancers has not yet been established. Therefore, in the present study we analyzed the immunohistochemical expression of hgh and GHR in colorectal LCNEC, looking for a potential role of these markers in the development of colorectal LCNEC. Case Reports Between January 1st 2008 and December 31st 2011, eight patients were diagnosed with colorectal LCNEC in the Ljudevit Jurak Department of Pathology, University Hospital Centre Sestre Milosrdnice, Zagreb, Croatia. For the purpose of this study, cases were recovered from our pathology database system. The average patient age at diagnosis was 69.3 years (range=44-94 years). There were five females and three males. The average size of the tumors was 5.9 cm (range=3-11 cm). Three tumors were located in the colon (two ascending, one descending), three in the rectum and two in the cecum. Grossly, the tumors were presented as endophytic/ulcerative, annular and polypoid masses, with a gray-white color and necrosis in most cases. Immunohistochemistry. Routine formalin-fixed paraffinembedded tissue sections were analyzed under light microscopy (hematoxylin and eosin stained, H&E) and by immunohistochemistry. Antibodies against chromogranin A (DAK A3, 1:100), synaptophysin (SY38, 1:10), neuron-specific enolase (NSE; BBS/NC/VI-H14, 1:100), Ki67 (MIB-1, 1:75), 0250-7005/2012 $2.00+.40 3377

pancytokeratins (AE1/AE3, 1:50), cytokeratin 7 (CK7; OV-TL 12/30, 1:50), cytokeratin 20 (CK20; Ks20.8, 1:25), growth hormone (hgh, 1:400), growth hormone receptor (GHR; MAB263, 1:250), were used and visualized using the streptavidin detection protocol with the DAB chromogen stainer (DAKO, Copenhagen, Denmark). All antibodies were supplied by DAKO, Copenhagen, Denmark, except for GHR which was supplied by ABCAM, Cambridge, MA, USA. Results of immunohistochemical analysis were evaluated semi-quantitatively and classified according to the percentage of positive cells as 1-10%, negative ( ); 11-320%, weak (+); 31-70%, moderate (++); and 71-100%, strongly positive (+++). For hgh and GHR only clear cytoplasmic positivity was scored. Results Histologically, tumors had organoid, nesting, trabecular, rosette-like and palisading appearance in at least 70% of the tumor, and a high mitotic rate of greater than 20 per 10 high power field (HPF). The tumor cells were large, with vesicular nuclei, coarse chromatin, prominent nucleoli and a moderate to abundant cytoplasm. The intercellular stroma was scant (Figure 1A and B). All eight tumors were diffusely positive for at least one immunohistochemical neuroendocrine marker including chromogranin A (2/8), synaptophysin (7/8), and NSE (8/8). Two tumors were immunoreactive for AE1/AE3, whereas one tumor was focally positive for CK7. Staining for CK20 was negative in all cases. The Ki67 proliferation index was high, between 40% and 80% (average of 58.7%). In addition, we analyzed the expression of hgh and GHR, and six tumors were found to be immunoreactive for hgh, while five tumors were immunoreactive for GHR. The pattern of expression for hgh and GHR in colorectal LCNEC was cytoplasmic (Figure 2A and B). The adjacent normal colorectal mucosa showed no expression, or only weak expression for hgh and GHR. Tumors were advanced at the time of diagnosis, and all but one case was presented with metastases. Two cases had distant liver metastases, with American Joint Committee on Cancer (AJCC) (9) stage IV, whereas five cases were presented with local lymph node metastases of stage III. Clinicopathological features including patient age at diagnosis, gender, tumor location, median tumor size, tumor stage, lymph node metastasis and the distant metastatic site are outlined in Table I. The immunoprofile for each case is summarized in Table II. Discussion Neuroendocrine neoplasms originate from the neuroendocrine system and encompass a wide range of pathological entities with distinct biological behaviors. Some of the clinical and pathological features of these neoplasms are characteristic of the organ of origin and others are common for neuroendocrine neoplasms, irrespective of their origin. The nomenclature of neuroendocrine neoplasms in general presents a challenge to pathologists as different terms are used for tumors of similar histological appearance in different organs. The 2010 WHO classification of tumors of the digestive system classifies neuroendocrine neoplasms as NET G1 (neuroendocrine tumor/carcinoid), NET G2 and NEC G3 (neuroendocrine carcinoma, large cell or small cell type) (10). The majority of digestive tract neuroendocrine neoplasms are NET G1, with a relatively good prognosis. In contrast, colorectal neuroendocrine carcinomas are rare but very aggressive neoplasms (10). Most patients with earlystage neuroendocrine carcinomas develop metastatic diseases. The rate of distant metastases from colorectal neuroendocrine carcinoma (large cell and small cell) is reported to be between 38% and 73%, the rate of lymph node metastases ranges from 60% to 87%, with a reported one-year survival rate between 15% and 46% (2, 5, 6). LCNEC is a poorly differentiated neoplasm with a low frequency occurrence in the digestive tract. Bernick et al., in a large series of 6,495 patients with colorectal cancer, reported that 0.6% (38/6,495) were neuroendocrine carcinomas, and only 0.2% (16/6,495) were LCNECs (2). Histologically, colorectal LCNEC resembles its counterpart in other organs; it is composed of round or polygonal cells arranged in organoid, nesting, trabecular, rosette-like and palisading patterns, frequently with large patches of geographical necrosis (10). The tumor cells are generally large, with vesicular nuclei, coarse or fine chromatin, in most cases with prominent nucleoli and a moderate to abundant cytoplasm. A high mitotic rate of 20 or greater per 10 HPF and/or Ki67 proliferation index of >20% is seen (10). Tumors usually stain positively for one or more neuroendocrine immunohistochemical markers such as chromogranin A, synaptophysin, NSE, and CD56 (10). In addition, there have been reports of positive staining for CK20 in LCNEC of the colon (11), whereas most neuroendocrine carcinomas, except for Merckel cell carcinoma, do not express CK20 (12). Our cases showed no immunoreactivity for CK20. In difficult cases, evidence of neurosecretory granules in the cytoplasm of tumor cells, which is characteristic of neuroendocrine differentiation, may also be determined ultrastructurally using electron microscopy. The diagnosis of colorectal LCNECs, in particular cases, can be difficult because colorectal carcinomas can also exhibit neuroendocrine differentiation (10). The key features for a correct diagnosis of colorectal LCNEC are its distinctive architectural pattern within at least 70% of the tumor; large cells diffusely expressing general neuroendocrine markers, marked nuclear atypia, multifocal necrosis, and a high number of mitoses (>20 per 10 HPF) (10). The most recent WHO classification of 3378

Jukić et al: hgh and GHR in Colorectal LCNEC Figure 1. A: Colorectal large cell neuroendocrine carcinoma showing organoid and nesting arrangement of tumor cells with peripheral palisading (H&E, original magnification, 100). B: Large tumor cells with vesicular nuclei, coarse chromatin, prominent nucleoli and a moderate to abundant cytoplasm (H&E, original magnification, 400). Figure 2. Colorectal large cell neuroendocrine carcinoma cells expressing cytoplasmic immunoreactivity for growth hormone (A) and growth hormone receptor (B) (original magnification, 400). tumors of the digestive system has included the term mixed adenoneuroendocrine carcinoma (MANEC) for tumors that exhibit at least 30% of either component, whereas colorectal carcinomas with fewer than 30% neuroendocrine differentiation and carcinomas with scattered neuroendocrine cells identified by immunohistochemistry, are qualified as colorectal carcinoma with neuroendocrine differentiation (10). In the present study, we also analyzed the expression of hgh and GHR in colorectal LCNEC. Recent studies have reported immunopositivity for hgh and GHR in colorectal carcinoma, whereas there was no expression or only slight focal positive reaction in the adjacent normal colorectal mucosa (7, 8). Moreover, a correlation between poorly differentiated tumors and the expression of hgh and GHR has been reported (7, 8). The expression of hgh and GHR in colorectal LCNEC has not been previously analyzed. In our study 6/8 of colorectal LCNECs were immunoreactive for hgh and 5/8 were immunoreactive for GHR. Similarly to previous findings (7, 8), the adjacent normal colorectal mucosa showed no expression or only weak expression of hgh and GHR. This suggests a possible relation between hgh and GHR overexpression and colorectal LCNEC. Colorectal LCNECs may be associated with a secondary primary adenoma or adenocarcinoma (10). However, the coexistence of colorectal LCNEC with an adjacent colorectal adenocarcinoma is very rare and yet only few cases have been reported (13-16). The possibility of a link between LCNEC and primary colon adenocarcinoma, which is 3379

Table I. Clinicopathological features of colorectal large cell neuroendocrine carcinoma. Case Years at diagnosis Gender Location Mean diameter LN metastasis Distant metastasis TNM AJCC stage 1 74 M Cecum 4 cm 11/14 Liver T3N2M1 IV 2 94 M Ascending colon 6 cm 1/15 T2N1M0 III 3 58 F Descending colon 11 cm 4/7 Liver T3N2M1 IV 4 63 M Rectum 3 cm 5/5 T3N2M0 III 5 75 F Rectum 3.5 cm 2/18 T3N1M0 III 6 44 F Ascending colon 8 cm 0/10 T3N0M0 II 7 67 F Rectum 4 cm 1/11 T3N1M0 III 8 80 F Cecum 8 cm 8/8 T3N2M0 III F, Female; M, male; LN, lymph nodes; TNM, tumor, node, metastasis; AJCC, American Joint Committee on Cancer. Table II. Colorectal large cell neuroendocrine carcinoma immunoprofile. Case CgA Syn NSE AE1/AE3 CK7 CK20 Ki67 hgh GHR 1 +++ ++ ++ 50% ++ 2 +++ +++ 80% +++ 3 ++ +++ +++ ++ 60% +++ ++ 4 ++ ++ ++ (Focal) 50% +++ ++ 5 +++ +++ 40% ++ 6 ++ +++ 80% ++ 7 ++ +++ 60% ++ 8 +++ +++ 50% +++ ++ CgA, Chromogranin A; Syn, synaptophysin; NSE, neuron-specific enolase; AE1/AE3, pancytokeratin; CK, cytokeratin; hgh, growth hormone; GHR, growth hormone receptor;, no immunoreactivity; +, weak immunoreactivity; ++, moderate immunoreactivity; +++, strong immunoreactivity. CK20+/CK7 has been raised by some authors (12, 16). Surgical resection is the primary treatment modality for colorectal LCNEC (2, 17, 18). However, because of the low occurrence of colorectal LCNEC, advanced stage at diagnosis and its aggressive behavior, there is a lack of sufficient data on the treatment of LCNEC. Adjuvant chemotherapy is reported to prolong the survival of patients with colorectal LCNEC only in the early stages (17). A recent case report indicated the clinical benefit of postoperative chemoradiation in a patient with advanced rectal LCNEC (18). Worldwide, there is an increasing incidence of reported neuroendocrine carcinomas, which is most likely attributed to the more sensitive diagnostic tools and the increased awareness of physicians regarding the existence of this entity. In conclusion, our series of colorectal LCNEC is among the largest reported in the literature. To our knowledge, this is the first report examining the immunohistochemical expression of hgh and GHR in colorectal LCNEC. Our results suggest a role of hgh and GHR in the development of colorectal LCNEC. Further larger studies are required to establish the potential prognostic and therapeutic role of these markers. Acknowledgements Presented in part at the 21st Ljudevit Jurak Symposium on Comparative Pathology, Zagreb, Croatia 2010. This study was supported in part by the Ministry of Science, Education and Sports, Croatia, project number 108-1081870-1884 to K.B. References 1 Travis WD, Brambilla E, Muller-Hermelink HK and Harris CC (eds.): World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. IARC Press: Lyon, France, 2004. 2 Bernick PE, Klimstra DS, Shia J, Minsky B, Saltz L, Shi W, Thaler H, Guillem J, Paty P, Cohen AM and Wong WD: Neuroendocrine carcinomas of the colon and rectum. Dis Colon Rectum 47: 163-169, 2004. 3 Shia J, Tang LH, Weiser MR, Brenner B, Adsay NV, Stelow EB, Saltz LB, Qin J, Landmann R, Leonard GD, Dhall D, Temple L, Guillem JG, Paty PB, Kelsen D, Wong WD and Klimstra DS: Is non small cell type high-grade neuroendocrine carcinoma of the tubular gastrointestinal tract a distinct disease entity? Am J Surg Pathol 32: 719-731, 2008. 3380

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