Section 3 -

Molecular Targeted Therapy for Neuroendocrine Tumors (NETs) Robert Y. Osamura Department of Pathology Tokai University School of Medicine Isehara-city Kanagawa JAPAN

Introduction Neuroendocrine tumors(NETs) are the tumors which produce peptide hormones and are composed of the neoplastic cells which contain dense cored neuroendocrine secretory granules(SGs) in the cytoplasm. Morphologically, the NETs exhibit characteristic histological patterns which suggest pathological diagnosis in our daily pathology practice. The final pathological diagnosis of NETs is usually established with the aid of immunohistochemical detection of various neuroendocrine markers which include chromogranin A, synaptophysin, CD56(NCAM), SNAP25. The tumors have been designated as "carcinoid" for many years since Oberdorfer introduced the term. In order to correlate the terminology with biological behavior of the NETs in the pancreas, they have been classified into the following categories in the recent WHO classification(2004). (1)Well differentiated neuroendocrine tumors(WDETs) (2)Well differentiated neuroendocrine carcinomas(WDECs) (3)Poorly differentiated neuroendocrine carcinomas(PDNECs) (4)Mixed exocrine-endocrine carcinomas Among these tumor, NECs are the tumors in which the malignant behavior is apparent such as angio-lymphatic invasion, invasion into the adjacent tissues, and apparent metastases. The NECs are classified into WDNECs and PDNECs. It is our understanding that the WDNECs are the tumors with the morphological feature of PDNECs and which been designated as carcinoid tumors resulted in apparent metastatic spread. The frequent metastatic sites include liver and regional lymph nodes. In contrast, PDNECs are the tumors with less differentiated morphology and are frequently composed of small or large and markedly atypical cells which demonstrate apparent neuroendocrine markers. These tumors are designated as small or large cell neuroendocrine carcinoma, SCNEC or LCNEC. By electron microscopically, a few small secretory granules are identified in the cytoplasm. It is noteworthy, the secretory granules can be depicted in the formalin-fixed wet tissues. PDNECs are well known to occur most frequently in the lung, but extra-pulmonary PDNECs have been reported in the wide variety of the organs, such as uterine cervix, ovaries, prostate and esophagus. For the neuroendocrine tumors(NETs), the target molecules for therapy have been focused on somatostatin receptors(SSTRs). Somatostatin analogues can be expected to exert their effects to suppress hormone secretion and cell proliferation, when the tumors are equipped with SSTRs. For Octreotide which has been used as a popular therapeutic agent, SSTR2 and SSTR5 are particularly important. Octreotide has been used for the treatment of .various NETs such as carcinoids of the GI tract, pancreatic NETs and those of the other locations.. This lecture focuses on the tumors which we can expect the therapeutic effects of somatostatin analogues. Detection of somatostatin receptors(SSTRs) and therapy of metastatic PNETs Since its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. Its actions include inhibition of endocrine and exocrine secretions, modulation of neurotransmission, motor and cognitive functions, inhibition of intestinal motility, absorption of nutrients and ions and vascular contractility. In addition, the peptide controls the proliferation of normal and tumor cells. Its action is mediated by a family of G protein-coupled receptors [somatostatin receptor (SSTR)1-SSTR5] that are widely distributed in normal and cancer cells. Direct antitumor activities, mediated through SSTR expressed in tumor cells, include blockade of autocrine/paracrine growth-promoting hormone and growth factor production, inhibition of growth factor-mediated mitogenic signals and induction of apoptosis. Indirect antitumor effects include inhibition of growth-promoting hormone and growth factor secretion, and antiangiogenic actions. Many human tumors express more than one SSTR subtype, with SSTR2 being predominant. These receptors represent the molecular basis for the clinical use of somatostatin analogs in the treatment of endocrine tumors and their in vivo localization Papotti M.et reported that t he vast majority expressed SSTR types 1,2,3,5 and SSTR4 was detected in a small minority. There was a good correlation between RT-PCR and IHC. By immunohistochemistry on35 GEP tumors, pancreatic insulinomas showed heterogeneous exhibited SSTR expression, 100% of somatostatinomas:SSTR5 and 100% of gastrinomas and glucagonomas:possessed SSTR2. The liver is the most frequent metastatic site of NECs. Nasir et al. reported that SSTR subtype expression needs to be correlated to somatostatin analog therapy. They studied the expression of SSTRs of the various metastatic lesions in the liver. Eleven (61%) of 18 hepatic metastases from small intestinal and pancreatic ECAs were positive for SSTR-1, 15 (83%) for SSTR-2, 13 (72%) for SSTR-3, 10 (56%) for SSTR-4, and 15 (83%) for SSTR-5. Among 11 hepatic ECA metastases from small intestinal ECAs (carcinoids), 7 (63%) expressed SSTR-1, 9 (81%) expressed SSTR-2, 8 (72%) expressed SSTR-3, 6 (54%) expressed SSTR-4, and 10 (91%) expressed SSTR-5. Of 7 hepatic ECA metastases from pancreatic ECAs, 4 expressed SSTR-1, 6 expressed SSTR-2, and 5 expressed SSTR-3 and SSTR-5 each. SSTR subtype expression needs to be correlated to somatostatin analog therapy. Immunohistochemical profiling of various SSTR subtypes as a part of routine surgical pathologic analysis of enteropancreatic ETs may become a useful predictor of responsiveness of ETs to various SSTR analogs. Immunohistochemistry and RT-PCR using formalin-fixed paraffin embedded (FFPE) materials Formalin-fixed paraffin embedded(FFPE) sections can be applied to immuno- histocheical staining of SSTR1,2a,3,4,5 and RT-PCR to analyze SSTR1,2a,3,4,5 mRNA. For the therapy by Octreotide, the detection of SSTR2a is particularly important. FFPE materials have used successfully for not only DNA studies but mRNA studies. It is particularly important for pathologists, because the paraffin blocks which have been stored in the pathology departments are the target to perform genetic analysis. Gjerdrum et al. found a marked decrease in the mRNA yield from 500 microdissected cells from frozen and paraffin sections after immunostaining. Recovery of mRNA decreased by up to 89%, comparing the immunostained with the routinely stained sections. Slides mounted with paraffin sections could be stored at room temperature for up to 90 days with no significant decrease in mRNA recovery. Abrahamsen et al. have reported that the polymerase chain reaction product from FFPE tissues could be increased up to 100-fold amplifying short (<136 bp) compared with long amplicons. Variations in time before tissue processing and in fixation length seem to be less important sources of imprecision than previously assumed. Their findings suggested that quantitative analysis of mRNA in archive and routine diagnostic tissues may be possible. Immunohistochemical detection of SSTR2a on the cell memberane of the GHoma cells. Immunohistochemical detection of SSTR2a on the cell members of the WDNEC (carcinoid) of the small intestine and pancreas. Non-neoplastic pancreatic islets also show SSTR2a. References: Guillermet-Guibert J, Lahlou H, Cordelier P, Bousquet C, Pyronnet S, Susini C： Physiology of somatostatin receptors J Endocrinol Invest 28(11 Suppl):5-9,2005 Papotti M, Croce S, Bello M, Bongiovanni M, Allia E, Schindler M, Bussolati G: al.Expression of somatostatin receptor types 1-5 in 81cases of gastrointestinal and pancreatic endocrine tumors.A correlative immunohistochemical and reverse-transcriptase polymerase chain analysis. Virchow Arch 440:461-75, 2002 Nasir A, Stridsberg M, Strosberg J, Su PH, Livingston S, Malik HA, Kelley ST, Centeno BA, Coppola D, Malafa ME, Yeatman TJ, Kvols LK. Somatostatin receptor profiling in hepatic metastases from small intestinal and pancreatic neuroendocrine neoplasms: immunohistochemical approach with potential clinical utility. 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