Analysis of 320 gastroenteropancreatic neuroendocrine tumors identifies TS expression as independent biomarker for survival

Abstract

Thymidylate synthase (TS), a critical enzyme for DNA synthesis and repair, is both a potential tumor prognostic biomarker as well as a tumorigenic oncogene in animal models. We have now studied the clinical implications of TS expression in gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) and compared these results to other cell cycle biomarker genes. Protein tissue arrays were used to study TS, Ki-67, Rb, pRb, E2F1, p18, p21, p27 and menin expression in 320 human GEP-NETs samples. Immunohistochemical expression was correlated with univariate and multivariate predictors of survival utilizing Kaplan Meier and Cox proportional hazards models. Real time RT-PCR was used to validate these findings. We found that 78 of 320 GEP-NETs (24.4%) expressed TS. NETs arising in the colon, stomach and pancreas showed the highest expression of TS (47.4%, 42.6% and 37.3%, respectively), whereas NETs of the appendix, rectum and duodenum displayed low TS expression (3.3%, 12.9% and 15.4%, respectively). TS expression in GEP-NETs was associated with poorly differentiated endocrine carcinoma, angiolymphatic invasion, lymph node metastasis and distant metastasis (p < 0.05). Patients with TS-positive NETs had markedly worse outcomes than TS-negative NETs as shown by univariate (p < 0.001) and multivariate (p 5 0.01) survival analyses. Expression of p18 predicted survival in TS-positive patients that received chemotherapy (p 5 0.015). In conclusion, TS protein expression was an independent prognostic biomarker for GEP-NETs. The strong association of increased TS expression with aggressive disease and early death supports the role of TS as a cancer promoting agent in these tumors.Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) represent a heterogeneous disease entity with different biological, histopathological and clinical features. 1 Recent epidemiological studies suggest a rising incidence in GEP cases world-wide which may be due to improvements in diagnostic techniques as well as unknown environmental factors. 2 There are no universally accepted standards for the classification and treatment of these tumors. 3 In 2000, the World Health Organization (WHO) classified GEP-NETs into welldifferentiated (WD) NETs with benign behavior, WD NETs with uncertain behavior, WD neuroendocrine carcinomas and poorly differentiated (PD) carcinomas. 4 The most recent WHO classification in 2010 revised the nomenclature and provided a grading classification based on morphological criteria and the assessment of the proliferative fraction. 5 This, in turn, was based on mitotic count and Ki-67 staining and, to date, the proliferative fraction is the most important criteria for determining the grade classification in GEP-NETs. 5 Although there are promising advances in the treatment of malignant pancreatic NETs with recently approved drugs such as sunitinib and everolimus, these drugs primarily stabilize rather than cure the disease and there are few treatment options for nonpancreatic NETs. 6 5-Fluorouracil (5-FU), a thymidylate synthase ...

Expression Analysis of Genes Involved in DNA Repair or Synthesis in Mixed Neuroendocrine/Nonneuroendocrine Carcinomas

“…This finding is of particular interest as it again supports the concept that mixed carcinomas have a distinct molecular profile and possibly clinical behavior, and that molecular biomarkers might be predictive of the response to chemotherapy also in this group of neoplasms. In fact, referring to the knowledge available about colorectal cancer (which was the most prevalent location in our series), our data are in line with some previous studies on the unfavorable prognostic role of low TS gene expression in colon cancer patients, with special reference to those treated with antifolate drugs [24,25], even though at the same time our results are in sharp contrast to recent data on the adverse prognostic role of high TS protein expression in gastroenteropancreatic neuroendocrine neoplasms [16]. However, due to the limited number of cases, it was not possible to analyze the impact of TS gene expression levels on survival in a homogeneous group of chemotherapy-treated patients.…”

“…However, scarce data are available on the expression of DNA repair and synthesis genes in neuroendocrine tumors. In a previous paper by our group, high expression levels of the thymidylate synthase (TS) gene have been described to be associated with a lower response to antifolate drugs [15], and TS protein overexpression has recently been associated with aggressive behavior [16]. Ribonucleotide reductase, large subunit 1 (RRM1), excision repair cross-complementation group 1 (ERCC1) and topoisomerase IIa (TOPOIIa) enzymes have been investigated exclusively in high-grade neuroendocrine carcinomas (namely, small-cell lung carcinomas) [17,18], but no data are available on well-differentiated neuroendocrine tumors or on other molecules involved in the DNA repair/synthesis pathway.…”

Original paper Morphological and immunohistochemical profile of pancreatic neuroendocrine neoplasms

“…It has a dual function, including proliferation inhibition and positive modulation, and can play anti-and pro-apoptotic function, depending on the nature of the apoptotic stimulus [62]. Although strong expression of p21 in gastroenteropancreatic neuroendocrine neoplasms has been previously associated with poor outcome [6], in our study, positive p21 expression showed limited correlations, possibly due to this duality. In vitro, induction of p21 has been associated with aspirin-induced inhibition of neuroendocrine tumour cell viability [63], further emphasising the controversies in the interpretation of p21 presence.…”

“…Alternatively, p27 alterations can be late event in NEN development, related to high tumour grade. Loss of p27 in gastroenteropancreatic NENs has been associated with worse prognosis in some [7,67], but not all [6], studies. Disappearance of p27 was characteristic in poorly differentiated neuroendocrine carcinomas and metastatic well-differentiated endocrine carcinomas (by 2004 WHO classification), as Grabowski et al [67] reported.…”

“…Here, we present an integrated molecular and morphological study of PNENs. The proteins that hypothetically can be involved in PNEN development include proliferation markers, notably Ki-67 [4], tumour suppressor p53 [5], cell cycle regulators p21, p27 and cyclin D1 [6][7][8], anti-apoptotic protein Bcl-2 [5], cell adhesion molecule E-cadherin [9] and cancer stem cell marker CD44 [10,11]. The cytokeratin (CK) spectrum, in addition to intestinal and squamous markers, is widely used in the assessment of tumour histogenesis; however, cytokeratin-associated cell plasticity or differentiation can confer a pathogenetic role that should be evaluated as well [2,12,13].…”