Clinical Neuroendocrinology and Neuroendocrine Tumors

Purpose: Ileal carcinoids are gut epithelial tumors originating from serotonin-containing enterochromaffin (EC) cells. Therapeutic options for effectively inhibiting the growth and spread of metastatic carcinoids are still limited. We aimed to identify the role of matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) during tumor development and metastasis. Patients and Methods: Tissue samples were obtained from surgically treated patients. Expression of the EC-cell marker, vesicular monoamine transporter-1 (VMAT-1), was used to verify ileal carcinoids. We investigated the differential expression of MMP-2, 7, 9, 11, and 13 and their endogenous inhibitors (TIMP-1, 2, and 3) by quantitative real-time RT-PCR in 25 primary tumors, their corresponding lymph node metastases and/or liver metastases and matched normal mucosa. Results: Significantly increased expression of VMAT-1, MMP-2, MMP-11, TIMP-1 and TIMP-3 was determined by quantitative RT-PCR in EC-cell carcinoids compared to normal intestinal mucosa (p ! 0.05). In contrast, MMP-2 and MMP-9 as well as TIMP-1, TIMP-2, and TIMP-3 expression in primary tumors of patients with liver metastases (M1) was significantly lower than in paReceived: November 11, 2007 Accepted after revision: May 9, 2008 Published online: August 18, 2008 Christian Prinz II. Medical Department, Technical University of Munich Ismaninger Strasse 22 DE–81675 Munich (Germany) Tel. +49 89 4140 5973, Fax +49 89 4140 4905, E-Mail [email protected] © 2008 S. Karger AG, Basel 0028–3835/09/0891–0066$26.00/0 Accessible online at: www.karger.com/nen Prognostic Importance of MMPs and TIMPs in Ileal Carcinoids Neuroendocrinology 2009;89:66–78 67 correlate with the presence of metastases in the liver which depends on the size of the primary tumor. If the tumor is ! 1 cm, only 20–30% metastasize into the sentinel lymph nodes and eventually into the liver. If the size exceeds 2 cm, there is a 80% probability of metastases in the lymph nodes and a 50% probability of metastases in the liver [4, 5] . Therefore, during the course of tumor development, there might be a step of proteolytic dysregulation causing deranged ratios of proteases and their inhibitors which we sought to discover in the current project. A family of proteases possibly involved in tumor invasion are matrix metalloproteinases (MMPs), which are physiologically expressed at low levels in chromaffin cells and other neuroendocrine cells, e.g. in the thyroid [6] . There are about 25 subtypes of MMPs, which are membrane-bound or secreted comprising collagenases (MMP1, 8, 13, and 18), gelatinases (MMP-2 and 9), stromelysins (MMP-3, 10, and 11), matrilysins (MMP-7 and 26), and membrane-type MMPs (MMP-14–17, 24, and 25) [7] . MMPs are involved in the physiological remodeling of tissues and they also play a role in tumor progression and metastasis [6, 8] . Various MMPs have been shown to influence the initiation, invasion and metastasis of tumors [8, 9] . In the human organism, there are endogenous tissue inhibitors of MMPs, the TIMPs. Four TIMPs, found in almost all tissues and body fluids, have currently been characterized in humans and designated TIMP-1, 2, 3, and 4. TIMP-1, 2, and 4 are present in soluble forms, while TIMP-3 is bound to the extracellular matrix (ECM). The expression pattern of TIMP-4 differs from that of the other TIMPs. TIMP-4 mRNA is localized in the brain and heart of adult humans, as well as the ovary and skeletal muscle, suggesting a role as an important tissue-specific regulator of ECM remodeling [10] . The balanced activities of MMPs and TIMPs are involved in normal and pathological events such as wound healing, tissue remodeling, angiogenesis, invasion, tumorigenesis and metastasis. TIMPs turn out to be multifunctional proteins which regulate different processes through MMP-dependent as well as MMP-independent pathways that might even be paradoxical or controversial [11, 12] . Until now, only little is known about the role of MMPs and TIMPs in endocrine tumors. Carcinoids of the small intestine are a relatively rare tumor disease. This fact may cause a possible bias in the data achieved, thus presenting a limiting aspect to studies about this tumor entity. However, we have used quantitative real-time RT-PCR analysis of primary EC-cell carcinoids, lymph node metastases and liver metastases to examine the level and pattern of the expression of MMPs and TIMPs exactly. We investigated the association of these factors with the progression of EC-cell carcinoids and examined their usefulness as prognostic markers. Patients and Methods Patient Characteristics and Specimens This retrospective study included patients first diagnosed and treated between 1992 and 2004 at the Klinikum rechts der Isar, Technical University of Munich, Germany. Tissue samples from 28 patients with ileal carcinoids with lymphatic and/or hepatic metastases were histological evaluated by 2 different histopathologists and were investigated after obtaining informed consent. To confirm tumor origin an evaluation of the expression of the marker protein for EC-cells, vesicular monoamine transporter-1 (VMAT-1), in the primary tumor was performed. As a negative control, two duodenal carcinoids were evaluated but not included in the study and further analysis. In our study, the cutoff value was set at 1,000 VMAT-1 mRNA copies/10 6 GAPDH mRNA copies in the primary tumor since this level corresponded to the 99.9% confidence interval. Three patients with ileal carcinoids were removed from the study because we did not find a VMAT-1 expression level above 1,000 VMAT-1 mRNA copies/10 6 GAPDH, in parallel with negative staining for VMAT-1, and negative staining for chromogranin and/or serotonin. Consequently, these 3 patients were removed from the study since the origin of those tumors could not be determined accurately as ileal EC-cell carcinoids. Thus, the work compares a homogenous group of 25 patients with ileal EC-cell carcinoids. The included patients with primary tumors of ileal carcinoids were surgical treated, all of them showed lymphatic infiltration (n = 25 lymph node positive). 10 patients had liver metastases, but liver tissue samples were only obtained in 8 patients by biopsy or during surgical removal (n = 10, liver positive). All tumors were found to be well-differentiated according to the histological reports. 15 patients were female and 10 male. The average age at first diagnosis and surgery was 59 8 14 (range 35–87) years. Further characteristics, e.g. additional therapy, TNM status, grading, etc., of the patients included in the study are summarized in table 1 . TNM staging was performed following current guidelines for endocrine ileal tumors [13] . T1 was defined as tumors invading the mucosa or submucosa with diameters of ̂ 1 cm. T2 was defined as tumors invading the muscularis propria or showing diameters of 1 1 cm; T3 = tumors invading the subserosa, and T4 = tumors invading the peritoneum and/or other organs; m = multiple tumors. As all patients presented lymph node metastases at the time of diagnosis, disease stage was at least IIIB for any T, N1, and M0 or stage IV for any T and any N or M1. Ki-67 staining was performed, positive cells were counted, and corresponding grading stages are listed in table 1 . Grading distinguishes between three grades: G1 = Ki-67 index ̂ 2%; G2 = Ki-67 index between 1 2 and 20%, and G3 = Ki-67 index 1 20%. Most tumors had a Ki-67 index of ! 2% and were thus well-differentiated. Three patients were lost during follow-up and defined as dropouts. Complete follow-up was available for 22 patients, of whom 8 died and 14 were still alive