Dieter Hörsch

Sunitinib malate for the treatment of pancreatic neuroendocrine tumors

BACKGROUND The multitargeted tyrosine kinase inhibitor sunitinib has shown activity against pancreatic neuroendocrine tumors in preclinical models and phase 1 and 2 trials. METHODS We conducted a multinational, randomized, double-blind, placebo-controlled phase 3 trial of sunitinib in patients with advanced, well-differentiated pancreatic neuroendocrine tumors. All patients had Response Evaluation Criteria in Solid Tumors-defined disease progression documented within 12 months before baseline. A total of 171 patients were randomly assigned (in a 1:1 ratio) to receive best supportive care with either sunitinib at a dose of 37.5 mg per day or placebo. The primary end point was progression-free survival; secondary end points included the objective response rate, overall survival, and safety. RESULTS The study was discontinued early, after the independent data and safety monitoring committee observed more serious adverse events and deaths in the placebo group as well as a difference in progression-free survival favoring sunitinib. Median progression-free survival was 11.4 months in the sunitinib group as compared with 5.5 months in the placebo group (hazard ratio for progression or death, 0.42; 95% confidence interval [CI], 0.26 to 0.66; P<0.001). A Cox proportional-hazards analysis of progression-free survival according to baseline characteristics favored sunitinib in all subgroups studied. The objective response rate was 9.3% in the sunitinib group versus 0% in the placebo group. At the data cutoff point, 9 deaths were reported in the sunitinib group (10%) versus 21 deaths in the placebo group (25%) (hazard ratio for death, 0.41; 95% CI, 0.19 to 0.89; P=0.02). The most frequent adverse events in the sunitinib group were diarrhea, nausea, vomiting, asthenia, and fatigue. CONCLUSIONS Continuous daily administration of sunitinib at a dose of 37.5 mg improved progression-free survival, overall survival, and the objective response rate as compared with placebo among patients with advanced pancreatic neuroendocrine tumors. (Funded by Pfizer; ClinicalTrials.gov number, NCT00428597.).

Everolimus plus octreotide long-acting repeatable for the treatment of advanced neuroendocrine tumours associated with carcinoid syndrome (RADIANT-2): a randomised, placebo-controlled, phase 3 study

BACKGROUND Everolimus, an oral inhibitor of the mammalian target of rapamycin (mTOR), has shown antitumour activity in patients with advanced pancreatic neuroendocrine tumours. We aimed to assess the combination of everolimus plus octreotide long-acting repeatable (LAR) in patients with low-grade or intermediate-grade neuroendocrine tumours (carcinoid). METHODS We did a randomised, double-blind, placebo-controlled, phase 3 study comparing 10 mg per day oral everolimus with placebo, both in conjunction with 30 mg intramuscular octreotide LAR every 28 days. Randomisation was by interactive voice response systems. Participants were aged 18 years or older, with low-grade or intermediate-grade advanced (unresectable locally advanced or distant metastatic) neuroendocrine tumours, and disease progression established by radiological assessment within the past 12 months. Our primary endpoint was progression-free survival. Adjusted for two interim analyses, the prespecified boundary at final analysis was p≤0·0246. This study is registered at ClinicalTrials.gov, number NCT00412061. FINDINGS 429 individuals were randomly assigned to study groups; 357 participants discontinued study treatment and one was lost to follow-up. Median progression-free survival by central review was 16·4 (95% CI 13·7-21·2) months in the everolimus plus octreotide LAR group and 11·3 (8·4-14·6) months in the placebo plus octreotide LAR group (hazard ratio 0·77, 95% CI 0·59-1·00; one-sided log-rank test p=0·026). Drug-related adverse events (everolimus plus octreotide LAR vs placebo plus octreotide LAR) were mostly grade 1 or 2, and adverse events of all grades included stomatitis (62%vs 14%), rash (37%vs 12%), fatigue (31%vs 23%), and diarrhoea (27%vs 16%). INTERPRETATION Everolimus plus octreotide LAR, compared with placebo plus octreotide LAR, improved progression-free survival in patients with advanced neuroendocrine tumours associated with carcinoid syndrome. FUNDING Novartis Pharmaceuticals.

Telotristat ethyl, a tryptophan hydroxylase inhibitor for the treatment of carcinoid syndrome

Purpose Preliminary studies suggested that telotristat ethyl, a tryptophan hydroxylase inhibitor, reduces bowel movement (BM) frequency in patients with carcinoid syndrome. This placebo-controlled phase III study evaluated telotristat ethyl in this setting. Patients and Methods Patients (N = 135) experiencing four or more BMs per day despite stable-dose somatostatin analog therapy received (1:1:1) placebo, telotristat ethyl 250 mg, or telotristat ethyl 500 mg three times per day orally during a 12-week double-blind treatment period. The primary end point was change from baseline in BM frequency. In an open-label extension, 115 patients subsequently received telotristat ethyl 500 mg. Results Estimated differences in BM frequency per day versus placebo averaged over 12 weeks were -0.81 for telotristat ethyl 250 mg ( P < .001) and ‒0.69 for telotristat ethyl 500 mg ( P < .001). At week 12, mean BM frequency reductions per day for placebo, telotristat ethyl 250 mg, and telotristat ethyl 500 mg were -0.9, -1.7, and -2.1, respectively. Responses, predefined as a BM frequency reduction ≥ 30% from baseline for ≥ 50% of the double-blind treatment period, were observed in 20%, 44%, and 42% of patients given placebo, telotristat ethyl 250 mg, and telotristat ethyl 500 mg, respectively. Both telotristat ethyl dosages significantly reduced mean urinary 5-hydroxyindole acetic acid versus placebo at week 12 ( P < .001). Mild nausea and asymptomatic increases in gamma-glutamyl transferase were observed in some patients receiving telotristat ethyl. Follow-up of patients during the open-label extension revealed no new safety signals and suggested sustained BM responses to treatment. Conclusion Among patients with carcinoid syndrome not adequately controlled by somatostatin analogs, treatment with telotristat ethyl was generally safe and well tolerated and resulted in significant reductions in BM frequency and urinary 5-hydroxyindole acetic acid.

Integrative Mitogenic Role of Protein Kinase B/Akt in β‐Cells

Abstract: Protein kinase B/Akt (PKB/Akt) is activated by phosphatidylinositol 3‐kinase (PI 3‐K) and is a central mediator of cellular proliferation and protection against apoptosis. Insulin, insulin‐like growth factor (IGF‐1), and glucagon‐like peptide‐1 (GLP‐1) act as glucose‐dependent growth factors for pancreatic β‐cells. We assessed signaling pathways and stimulation patterns of PKB/Akt activation by these ligands in the β‐cell line INS‐1. Insulin, IGF‐1, and GLP‐1 induced distinctive time dependent, dose dependent, and glucose dependent phosphorylation of PKB/Akt. Insulin and IGF‐1 stimulated PI 3‐K activity was mainly associated with insulin receptor substrate (IRS) isoforms IRS‐1 and IRS‐2 and less so with the IRS‐isoform Grb‐2 associated binder‐1 (Gab‐1). In contrast, GLP‐1 induced PI 3‐K activity mainly in Gab‐1 and also in IRS‐2 immunoprecipitates, although in an attenuated kinetic. Thus, activation pathways of PKB/Akt by insulin, IGF‐1, and GLP‐1 converge at the level of IRS‐isoforms and PI 3‐K inducing differential activation of PKB/Akt. These data indicate an essential role of PKB/Akt in regulation of β‐cell proliferation.

BRAF Gene Mutations Are Rare Events in Gastroenteropancreatic Neuroendocrine Tumors

The BRAF gene, one of the human isoforms of RAF, is activated by ras, leading to cooperative effects in cells responsive to growth factor signals. We studied the frequency of BRAF and k-ras-2 mutations in primary neuroendocrine gastroenteropancreatic (GEP) tumors. Mutation analysis of the BRAF and k-ras-2 genes was performed in 40 primary neuroendocrine tumors of the GEP system. The expression of extracellular signaling-related kinase (ERK) 1/2, an important downstream point of convergence in the ras-RAF-mitogen-activated protein-ERK pathway was analyzed immunohistochemically. We detected one 1796 T-->A BRAF mutation that led to a substitution of valine by glutamic acid at position 599 (V599E) in 40 primary neuroendocrine GEP tumors (3%). We failed to detect specific mutation of the k-ras-2 gene. We identified constitutively activated ERK in almost all neuroendocrine tumor tissues tested irrespective of BRAF mutation or localization or functional activity. These results suggest that BRAF mutations do not have a role in tumorigenesis of neuroendocrine tumors. Nevertheless, activation of the RAF/mitogen-activated protein kinase pathway might have a causative role in the development of neuroendocrine tumors, independent of BRAF or k-ras-2 mutation.

Nuclear Survivin Is a Powerful Novel Prognostic Marker in Gastroenteropancreatic Neuroendocrine Tumor Disease

Gastroenteropancreatic neuroendocrine tumors represent a heterogeneous tumor entity. The growth pattern ranges from very slowly to fast growing, aggressive types of tumors. Survivin, a member of the family of apoptosis inhibitors, is a bifunctional protein that suppresses apoptosis and regulates cell division. In this study we determined the prognostic value of survivin in this tumor entity. Tumor specimens from 104 patients (38 foregut, 53 midgut, 13 hindgut) were studied immunohistochemically for cytoplasmic and nuclear survivin expression as well as for ki-67 antigen expression. 5-year-follow-up was complete in 89 patients. 29 patients with localized, well-differentiated gastroenteropancreatic neuroendocrine tumors (WDET, WHO class 1) had been curatively treated by surgical or endoscopic tumor resection. 50 patients suffered from well-differentiated endocrine carcinomas (WDEC, WHO class 2), 10 patients were diagnosed with poorly differentiated neuroendocrine carcinomas (PDEC, WHO class 3). Survivin expression was correlated with survival for the 50 patients with metastatic WDEC disease. All 29 WDETs were negative for nuclear survivin, whereas all 10 PDECs stained positive for nuclear survivin. In the 50 patients with metastatic WDECs, 5/50 (10%) tumors were nuclear survivin positive. Those 5 patients had a statistically significant worse prognosis (survival of 41 vs. 103 months, p = 0.01). ki-67 was not a prognostic factor in this subgroup of patients. Nuclear survivin expression thus appears to be upregulated during progression of gastroenteropancreatic neuroendocrine tumors. The analysis of nuclear survivin expression identifies subgroups in metastatic disease (WHO class 2) with good (survivin–) or with less favorable prognosis (survivin+). We propose that the determination of nuclear survivin expression could be used to individualize therapeutic strategies in this tumor entity in the future.

Loss of Nuclear p27 Expression and Its Prognostic Role in Relation to Cyclin E and p53 Mutation in Gastroenteropancreatic Neuroendocrine Tumors

Purpose: Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are classified by the WHO, yet its prognostic value needs to be confirmed. Therefore, we aimed to determine the prognostic role of cell cycle key regulatory genes p53, p27kip1 (p27), and cyclin E in this tumor entity. Experimental Design: Tumor specimen from 89 patients with a complete follow-up were studied immunohistochemically for p27 and cyclin E expression and for p53 mutations. The functional relevance of p27 was evaluated in the neuroendocrine cell lines BON1 (human) and INS1 (rat) by the use of small interfering RNA. Results: Twenty-six of 29 benign, well-differentiated endocrine tumors (WHO class 1) showed a high expression (>50%) of p27, whereas all 10 poorly differentiated endocrine carcinomas (WHO class 3) displayed a low expression of p27. Metastatic well-differentiated endocrine carcinomas (WHO class 2) showed a low p27 expression in 20 of 50 (40%) patients, which conferred a poor prognosis (median survival, 57 versus 140 months; P = 0.037). This prognostic dichotomy was improved by the use of a combination of p27 and cyclin E (high cyclin E/low p27 versus low cyclin E/high p27: median survival 53 months versus not reached; P = 0.0044). p53 mutations were rare (1 of 10 poorly differentiated endocrine carcinomas). Conclusions: Loss of p27 and overexpression of cyclin E play a critical role in the aggressiveness of gastroenteropancreatic neuroendocrine tumors. This coincides with increased cell cycle progression. We propose a discussion whether to incorporate the immunohistochemical expression of p27 into a revised classification to individualize therapeutic strategies in this tumor entity.

Rap1/B-raf signaling is activated in neuroendocrine tumors of the digestive tract and raf kinase inhibition constitutes a putative therapeutic target

Objective: Molecular pathogenesis of digestive neuroendocrine tumors (dNETs) is largely unknown. Recently, the serine-threonine kinase B-Raf was identified as an oncogene in endocrine cancer such as thyroid carcinoma. In endocrine cells, the small G-protein Rap1 stimulates mitogen-activated protein kinase (MAPK) signaling by activating B-Raf. We examined the expression of Rap1 and B-Raf in dNETs and their contribution to MAPK signaling in neuroendocrine cell lines. In addition, we explored the effect of suppressing B-Raf kinase by the recently developed inhibitor BAY43-9006 (Sorafinib) on growth, apoptosis and MAPK activation neuroendocrine cell lines. Methods and Results: Expression of Rap1 and B-Raf in dNETs (19 insulinomas, 15 carcinoid tumors and 10 gastrinomas) was examined by immunohistochemistry, which revealed that Rap1 and B-Raf were highly prevalent in the majority of dNETs. Overexpression of Rap1 and B-Raf activated MAPK extracellular dependent kinase (ERK) ERK-2 and ERK-dependent transcription factor Elk-1 in neuroendocrine cell lines Bon and INS-1. Suppression of B-Raf by BAY43-9006 inhibited growth and induced apoptosis in Bon and INS-1 cells. In addition, BAY43-9006 suppressed phosphorylation of MAPK ERK1/2 and its upstream kinase MEK1/2 in Bon and INS-1 cells. Conclusion: These results indicate that Rap1-B-Raf signaling may contribute to pathogenesis of dNETs and provides a molecular target for treatment of dNETs.

Effects of the tyrosine kinase inhibitor imatinib on neuroendocrine tumor cell growth.

Aim: We investigated the effects of the tyrosine kinase inhibitor imatinib (Gleevec®) on neuroendocrine tumor cells. Methods: Neuroendocrine tumor cells were incubated without and with imatinib. The effects on growth were examined by methylthiazoletetrazolium (MTT) assay. The c-Kit expression in human endocrine tumor tissue and cell lines was determined by immunohistochemistry and Western blot analysis, respectively. Cytotoxicity assay was performed by fluorescence-activated cell sorting. The telomerase activity was determined using the telomeric repeat amplification protocol. Results: 28% of the insulinomas, 100% of the gastrinomas, and 38% of the carcinoids expressed c-Kit. Imatinib at concentrations >5 µM inhibited cell proliferation and induced apoptosis in both c-Kit-positive and c-Kit-negative cell lines. The PI-3K inhibitor wortmannin did not enhance the effects of imatinib. Imatinib did not sensitize endocrine tumor cells to doxorubicin and 5-fluorouracil. Imatinib inhibited the telomerase activity. Conclusion: Imatinib inhibits neuroendocrine tumor cell growth independently of c-Kit by inhibition of other tyrosine kinases or through tyrosine kinase independent pathways.

Distribution and chemical phenotypes of neuroendocrine cells in the human anal canal

The presence, morphology and distribution of anal neuroendocrine cells were investigated with a panel of antisera and antibodies for neural markers, biogenic amines, and neuropeptides by the sensitive streptavidin-biotin-peroxidase immunocytochemistry, and coexistence patterns of neurochemically characterized neuroendocrine cells were examined by double immunofluorescence cytochemistry. In the colorectal zone, endocrine-like cells were immunoreactive for chromogranin A (CGA), serotonin (5-HT), pancreastatin (PST), peptide tyrosine tyrosine (PYY), glucagon-like peptide-1 (GLP-1), and somatostatin (SOM). Coexistence patterns of endocrine-like cell phenotypes with CGA and GLP-1 were heterogeneous. In the anal transitional zone (ATZ), endocrine-like cells were immunoreactive for CGA, 5-HT and PST. Furthermore, six new phenotypes of endocrine-like cells were characterized by their immunoreactivity for PYY, GLP-1, protein gene product 9.5 (PGP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and SOM. All endocrine-like cell types in the ATZ were immunoreactive for CGA. In the squamous zone and perianal skin, CGA-immunopositive Merkel cells were also immunoreactive for CGRP, PST, NT and PGP. Neuroendocrine cells in the anal canal exhibit epithelial zone-related diversities in their neurochemical phenotypes and coexistence patterns, which may indicate specific regulatory functions. In the epithelium of the ATZ, which is regarded as metaplastic, endocrine-like cells expressed phenotypes characteristic of the neuroendocrine cells of the colorectal zone and the squamous zones, indicating a possible metaplastic origin of these cells.