Inhibition of ribonucleotide reductase subunit M2 enhances the radiosensitivity of metastatic pancreatic neuroendocrine tumor.

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

Should temozolomide be used on the basis of O6-methylguanine DNA methyltransferase status in patients with advanced neuroendocrine tumors? A systematic review and meta-analysis.

BACKGROUND: Temozolomide (TEM) is an active treatment in metastatic neuroendocrine tumors (NETs). Patients affected by glioblastoma multiforme or advanced melanoma treated with TEM who have deficiency of O6-methylguanine DNA methyltransferase (MGMT) have a better responses and survival. However, the predictive role of MGMT in patients with NETs treated with TEM is still debated. METHODS: We conducted a systematic review of the literature and meta-analysis, based on PRISMA methodology, searching in the main databases (PubMed, Embase, Scopus, Web of Science, Cochrane Library and clinical trial.gov) and the proceedings of the main international congresses, until April 26, 2021. RESULTS: Twelve out of 616 articles were selected for our analysis, regarding a total of 858 NET patients treated with TEM-based chemotherapy. The status of MGMT had been tested in 513 (60%) patients, using various methods. The pooled overall response rate (ORR) was higher in MGMT-deficient compared with MGMT-proficient NETs, with a risk difference of 0.31 (95% confidence interval, CI: 0.13-0.50; p < 0.001; I2: 73%) and risk ratio of 2.29 (95% CI: 1.34-3.91; p < 0.001; I2: 55%). The pooled progression free survival (PFS) (hazard ratio, HR = 0.56; 95% CI: 0.43-0.74; p < 0.001) and overall survival (OS) (HR = 0.41; 95% CI: 0.20-0.62; p = 0.011) were longer in MGMT-deficient versus MGMT-proficient NETs. CONCLUSIONS: Our meta-analysis suggested that MGMT status may be predictive of TEM efficacy. However, due to the high heterogeneity of the evaluated studies the risk of biases should be considered. On this hypothesis future homogeneous prospective studies are warranted.

Association between MGMT status and response to alkylating agents in patients with neuroendocrine neoplasms: a systematic review and meta-analysis.

BACKGROUND: O6-methylguanine-DNA methyltransferase (MGMT) is a specific DNA damage reversal repair protein. The influence of MGMT status on alkylating agent sensitivity in patients with neuroendocrine neoplasms (NENs) is controversial. We conducted a meta-analysis to assess the influence of MGMT status on the therapeutic sensitivity of alkylating agents in patients with NENs. METHODS: We searched PubMed, EmBase, and Cochrane library public databases through 3 July 2019. The objective response rate (ORR) was the outcome data of interest. Subgroup analysis was performed according based on MGMT methylation and expression of MGMT protein. RESULTS: Eleven studies were included in the meta-analysis. The proportion of patients with NENs that achieved an ORR after alkylating agent treatment was higher in the MGMT-deficient group than the non-deficient group (OR: 5.00; 95% CI: 3.04-8.22; P < 0.001; I2: 3%). Similar results were noted in the MGMT methylation and MGMT protein expression subgroups. CONCLUSION: Patients with NENs and MGMT methylation or low protein expression had a higher ORR proportion than patients without MGMT methylation or high protein expression. The MGMT status can be used as a biological indicator of the response to alkylating agent treatment in patients with NENs.

Pharmacodynamics, clinical findings and approval status of current and emerging tyrosine-kinase inhibitors for pancreatic neuroendocrine tumors.

Introduction: Pancreatic neuroendocrine tumors (panNETs) represent a rare group of malignancies. For decades, chemotherapy, somatostatin analogs and interferon represented the only systemic therapies; however, over the latest years, new options were registered, including Everolimus, Sunitinib (SUN), and Peptide Receptor Radionuclide Therapy.Areas covered: This review discusses the role of tyrosine kinase inhibitors (TKIs) in advanced panNETs.Expert opinion: TKIs showed an antiangiogenic and antiproliferative impact on advanced panNETs. Sunitinib is the only TKI currently available in clinical practice, having been approved on the basis of relevant results of a specific panNET phase III trial. New TKIs, such as Cabozantinib, Lenvatinib, Pazopanib, Surufatinib are still on investigation in panNETs. Although some phase II studies with the new TKIs yielded better PFS and RR compared with SUN, different study designs and tumor populations may have induced selection biases. However, it was reported that panNETs resistant to SUN could respond to a new TKI, indicating a possible further therapeutic line in this context. The global investigation plan of TKIs in panNETs is not homogeneous and it is difficult to understand what kind of development this can have in the near future for clinical practice.

Rare BRAF mutations in pancreatic neuroendocrine tumors may predict response to RAF and MEK inhibition.

The clinical significance of BRAF alterations in well-differentiated (WD) metastatic pancreatic neuroendocrine tumor (panNET) is unknown, but BRAF-mutated panNET could represent a subset characterized by an identifiable and clinically actionable driver. Following the identification of two patients with WD metastatic panNET whose tumors harbored BRAF mutations, we queried the MSK-IMPACT series of 80 patients with WD metastatic panNET for additional mutations in BRAF, and in other genes involved in RAS/ RTK/ PI3K signaling pathways. BRAF mutations were identified in six samples (7.5%): two tumors harbored V600E mutations, one tumor each expressed K601E, T599K, and T310I mutations, and one tumor expressed both G596D and E451K BRAF. Few additional actionable driver alterations were identified. To determine the ERK activating capability of four BRAF mutations not previously characterized, mutant constructs were tested in model systems. Biochemical characterization of BRAF mutations revealed both high and low activity mutants. Engineered cells expressing BRAF K601E and V600E were used for in vitro drug testing of RAF and MEK inhibitors currently in clinical use. BRAF K601E demonstrated reduced sensitivity to dabrafenib compared to BRAF V600E, but the combination of RAF plus MEK inhibition was effective in cells expressing this mutation. Herein, we describe the clinical course of a patient with BRAF K601E and a patient with BRAF V600E WD metastatic panNET, and the identification of four mutations in BRAF not previously characterized. The combined clinical and biochemical data support a potential role for RAF and MEK inhibitors, or a combination of these, in a selected panNET population.

Anaplastic lymphoma kinase expression in small-cell lung cancer.

AIMS: Anaplastic lymphoma kinase (ALK) immunohistochemistry has shifted from being a screening tool to being a sole determinant for ALK-targeted therapy. Recent articles have referred to small-cell lung cancer (SCLC) transformation as a resistance mechanism after ALK inhibitor treatments, but few reports have addressed ALK expression in treatment-naive SCLC in a comprehensive manner. Therefore, we examined ALK expression and the mechanisms in treatment-naive SCLCs. METHODS AND RESULTS: We examined ALK expression in a consecutive series of SCLC tumours, and the expression mechanism was analysed regarding gene rearrangement, copy number changes, and point mutations. We also examined whether SCLC with ALK expression can be suppressed by crizotinib treatment in vitro. Immunohistochemical results revealed that ALK was expressed in 16 of 142 (11.3%) SCLCs. The expression was focal and less intense, which is in contrast to strong and uniform expression in adenocarcinoma with ALK rearrangement. Two combined SCLCs showed a positive reaction restricted to the SCLC component. None of the known genetic alterations, including rearrangement, amplification, copy number gain, or point mutations, were associated with ALK expression. A SCLC cell line, SKLC2, which expressed ALK without known genetic alterations, was not inhibited by a practically achievable serum concentration of crizotinib. CONCLUSIONS: Anaplastic lymphoma kinase immunohistochemistry for treatment-naive SCLCs should not be used as a predictive biomarker for ALK inhibitor therapy, because the positive reactions were due to intrinsic expression of normal ALK transcript.

mTOR kinase inhibition reduces tissue factor expression and growth of pancreatic neuroendocrine tumors.

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

Expression and Molecular Regulation of the Cox2 Gene in Gastroenteropancreatic Neuroendocrine Tumors and Antiproliferation of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs).

BACKGROUND Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has had a significant increase over the past 4 decades. The pathophysiological role of the cyclooxygenase-2 (cox-2) gene and factors responsible for the expression in GEP-NETs is of clinical value. Current study determined the expression of cox-2 gene in human GEP-NET tissues and corresponding cell lines, investigated the molecular mechanisms underlying the regulation of cox-2 gene expression and assessed the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on both anchorage-dependent and independent growth of GEP-NET cells. MATERIAL AND METHODS GEP-NET tissues and QGP-1, BON, and LCC-18 GEP-NET cell lines were used. The expression of cox-2 gene was analyzed by immunohistochemistry, western blot, RT-PCR, and enzyme immunoassay. Transient transfection and luciferase assays along with electrophoretic mobility shift assays were conducted to explore the regulation of cox-2 gene expression. The effect of COX-inhibitors on GEP-NET cell growth was determined by proliferation assays and colony growth assessment. RESULTS We found 87.8% of GEP-NET tissues stained positive for COX-2. QGP-1 and LCC-18 cells expressed cox-2 gene. PGE2 (prostaglandin E2) amounts quantified in the supernatants of NET cells matched to cox-2 expression level. The CRE-E-box element (-56 to -48 bp) and binding of USF1, USF2, and CREB transcription factors to this proximal promoter element were essential for cox-2 promoter activity in GEP-NET cells. COX-2-specific inhibitor NS-398 potently and dose-dependently inhibited PGE2 release from QGP-1 cells. Interestingly, both NS-398 and acetylic salicylic acid effectively suppressed proliferation of QGP-1 and BON cells in a dose-dependent manner. CONCLUSIONS The majority of GEP-NETs over express cox-2 gene. The binding of CREB and USF-1/-2 transcription factors to a proximal, overlapping CRE-Ebox element is the underlying mechanism for cox-2 gene expression. NSAIDs potently suppressed the proliferations and may offer a novel approach for chemoprevention and therapy of GEP-NETs.

Serum Elastase 1 Level as a Risk Factor for Postoperative Recurrence in Patients with Well-Differentiated Pancreatic Neuroendocrine Neoplasms.

PURPOSE: This study was designed to assess the potential role of the preoperative serum level of elastase 1 as a risk factor for recurrence in patients with resectable well-differentiated pancreatic neuroendocrine neoplasms (PanNETs). METHODS: Preoperative serum elastase 1 levels were measured in 53 patients with PanNETs who underwent complete tumor resection in two tertiary referral centers between January 2004 and June 2017. The preoperative elastase 1 levels were correlated with clinicopathological characteristics, including tumor recurrence and recurrence-free survival. RESULTS: The median elastase 1 level was 96 ng/dL (range: 21-990 ng/dL). Preoperative serum elastase 1 levels were significantly higher in those with tumors ≥ 20 mm in diameter (vs. < 20 mm, P = 0.018), WHO grade 2 (vs. grade 1, P = 0.035), and microscopic venous invasion (vs. without venous invasion, P = 0.039). The median preoperative serum level of elastase 1 was higher in patients with recurrence than in those without recurrence (251 vs. 80 ng/dL, P = 0.004). Receiver operating characteristic analysis of elastase 1 levels showed that a cutoff level of 250 ng/dL was associated with postoperative recurrence, with 63% sensitivity, 100% specificity, and 94% overall accuracy. Patients with higher elastase 1 levels showed significantly worse recurrence-free survival than that of those with lower levels (2-year recurrence-free survival rate: 25% and 92%, respectively, P < 0.001). CONCLUSIONS: Our data provide the first evidence that high preoperative elastase 1 levels may be a risk factor for postoperative recurrence in patients with resectable PanNETs.

New insights into hypoxia-related mechanisms involved in different microvascular patterns of bronchopulmonary carcinoids and poorly differentiated neuroendocrine carcinomas. Role of ribonuclease T2 (RNASET2) and HIF-1α.

Ribonuclease T2 (RNASET2) is a pleiotropic and polyfunctional protein, which exerts several different activities in neoplastic cells since the early steps of tumor development. Besides having an antitumorigenic activity, RNASET2 inhibits both bFGF-induced and VEGF-induced angiogenesis and has a role as a stress-response, alarmin-like, protein. In this study, we investigated RNASET2 expression in well-differentiated and poorly differentiated neuroendocrine neoplasms of the lung (Lu-NENs), which are known to show clear-cut differences in morphology, biology and clinical behavior. In addition, we explored possible relationships between RNASET2 expression and a series of immunohistochemical markers related to hypoxic stress, apoptosis, proliferation and angiogenesis. Our results showed a significantly higher expression of RNASET2, HIF-1α, and its target CA IX in poorly differentiated than in well-differentiated Lu-NENs, the former also showing higher proliferation and apoptotic rates, as well as a lower microvessel density (MVD) than the latter. Moreover, we were able to demonstrate in vitro an overexpression of RNASET2 in consequence of the activation of HIF-1α. In conclusion, we suggest that in poorly differentiated Lu-NENs, RNASET2 expression may be induced by HIF-1α, behaving as an alarmin-like molecule. In this aggressive group of cancers, which have highly deregulated proliferation pathways, RNASET2 fails to exert the growth-inhibiting effects described in other types of neoplasms. Its increased expression, however, may contribute to the typical phenotypic alterations seen in poorly differentiated Lu-NENs, such as the high apoptotic rate and the extensive necrosis, and may also enhance the low MVD observed in these neoplasms.

Carbonic anhydrase 9 expression in well-differentiated pancreatic neuroendocrine neoplasms might be associated with aggressive behavior and poor survival.

Well-differentiated pancreatic neuroendocrine neoplasms/tumors (PanNETs) are rare neoplasms with diverse clinical behavior. Biomarker discovery is important for predicting clinical course and prognosis of PanNET patients. Carbonic anhydrase 9 (CA9) and vimentin are hypoxia and epithelial-mesenchymal transition-related proteins of which expression in many carcinomas has been associated with poor prognosis, but their significance in PanNET has yet to be determined. We assessed CA9 and vimentin expression in 164 PanNETs and compared this with clinicopathologic characteristics. CA9 expression was observed in normal islets, while neuroendocrine microadenomas and small (< 1 cm) PanNETs showed loss of CA9 expression. CA9 and vimentin expression was observed in 38 (23%) and 36 (22%) of PanNETs, respectively. CA9 expression was associated with larger size (p = 0.001), higher grade (p < 0.001), higher pT category (p < 0.001), lymph node (p = 0.003) and distant (p = 0.047) metastases, higher AJCC stage (p < 0.001), and lymphovascular (p < 0.001) and perineural (p = 0.002) invasion. PanNET patients with CA9 expression had a shorter recurrence-free survival (5-year survival rate 47%) than those without CA9 expression (76%) by univariate (p = 0.001) but not multivariate analysis. Vimentin expression correlated with CA9 expression (p < 0.001) but not with other clinicopathologic factors. In conclusion, CA9 expression was observed in normal islets, while neuroendocrine microadenomas and small (< 1 cm) PanNETs showed CA9 expression loss. CA9 expression gradually reappeared in larger PanNETs, and this was associated with clinical progression and decreased patient survival by univariate but not multivariate analysis.

Letter to the Editor.

BACKGROUND: Neuroendocrine tumors (NETs) are uncommon type of cancers, also known as APUD (amine precursor uptake decarboxylation) tumors, which are becoming increasingly prevalent. Alkaline phosphatase (ALP) is a poor prognosis factor in a number of hepatic diseases. However, its distribution and prognostic value in primary hepatic neuroendocrine tumors (PHNETs) are still not clear. In this study, our aim is to investigate the correlations between ALP and clinicopathological features and prognostic factors of PHNETs. METHODS: The clinical data of 22 patients with PHNETs were retrospectively reviewed to investigate whether ALP affects the prognosis of PHNETs. RESULTS: In this study, ALP is correlated to γ-glutamyl transpeptidase (GGT; p = 0.002) and the tumor location in the liver (p = 0.007), and increased levels of ALP had poor effects on overall survival (p = 0.006) and progression-free survival (p = 0.022). CONCLUSION: ALP was identified as an independent prognostic factor for overall survival of PHNETs.

The Novel Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) Alone and in Dual-Targeting Approaches Demonstrates Antitumoral Efficacy in Neuroendocrine Tumors in vitro.

BACKGROUND/AIM: Cyclin-dependent kinases (CDKs) are crucial for cell cycle regulation, and alterations in the cell cycle are often observed in human cancer. CDK4/6 in particular orchestrates G1 phase progression and the G1/S transition. Here, we investigated the in vitro effects of the CDK4/6 inhibitor LEE011 in human neuroendocrine tumor cells. METHODS: The human neuroendocrine tumor cell lines BON1, QGP1, NCI-H727 and GOT1 were treated with different concentrations of LEE011 alone and in combination with 5-fluorouracil and everolimus. RESULTS: Cell viability decreased in a time- and dose-dependent manner in BON1, QGP1, and NCI-H727 cells upon LEE011 treatment, whereas GOT1 cells were treatment resistant. Treatment sensitivity towards LEE011 was associated with the high expression of cyclin D1 and Rb. LEE011 caused the dephosphorylation of Rb and a subsequent G1 phase cell cycle arrest. Combined treatment with LEE011 and 5-fluorouracil or everolimus showed a significant enhancement in the inhibition of cell viability when compared to single-substance treatments due to PI3K-Akt-mTOR and Ras-Raf-MEK-ERK pathway downregulation and cooperative downregulation of cell cycle components. However, LEE011 also exhibited antagonizing effects with 5-fluorouracil, protecting NET cells from DNA-damaging chemotherapy by blocking PARP cleavage and caspase-3/7 activity. CONCLUSIONS: Our data demonstrate that the CDK 4/6 inhibitor LEE011 exhibits promising anti-tumoral properties alone and in combination treatment approaches with 5-fluorouracil or everolimus in human neuroendocrine tumor cell lines.

O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

OBJECTIVES: Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. METHODS: We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. RESULTS: Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. CONCLUSIONS: We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

An elevated serum alkaline phosphatase level in hepatic metastases of grade 1 and 2 gastrointestinal neuroendocrine tumors is unusual and of prognostic value.

BACKGROUND: In our clinical practice we have observed that despite a high hepatic metastatic tumor burden, serum alkaline phosphatase (AP) levels are frequently normal in cases of metastatic neuroendocrine tumor (NET). PATIENTS AND METHODS: We retrospectively reviewed the records of patients with grade 1 and 2 NETs with liver metastases but without bone metastases seen at our institution in 2013. In total, 49 patients were included (22 female), with a median age of 60 years (range: 28 to 84 years). The primary tumors were located in the duodenum/pancreas (n = 29), small bowel (n = 17) or colon/rectum (n = 3); 10 cases were grade 1 and 39 grade 2. Hepatic involvement was bulky, with more than 10 lesions in 23 patients and a tumor burden above 10% of the liver volume in 26 patients. RESULTS: Serum AP levels were elevated (≥ upper limit of normal (ULN)) in 16 patients. In multiparametric analysis, elevated serum AP levels were not associated with the primary site, grade, or number or volume of metastases. In multiparametric analysis, progression-free survival was only correlated with grade (p = 0.010) and AP level (p = 0.017). CONCLUSIONS: Serum AP levels are frequently normal in liver metastases from NET, even in the event of a major tumor burden, and the serum AP level can be of prognostic value.

Critical focus on mechanisms of resistance and toxicity of m-TOR inhibitors in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (pNETs) are rare neoplasms representing less than 2% of all pancreatic malignancies. The PI3K-AKT-mTOR pathway is often deregulated in pNETs and seems to play a key role in tumorigenesis. Everolimus, an inhibitor of the mTOR pathway, has demonstrated efficacy in the treatment of pNETs. Nevertheless de novo or acquired drug resistance is responsible for disease progression and represents a major obstacle to overcome by clinicians. Blocking the PI3K/AKT/mTOR pathway may cover the supposed main mechanisms of resistance to everolimus. Therefore, BEZ-235, a potent oral dual PI3K/mTOR inhibitor was investigated in clinical trials. Globally more than 250 patients with different types of solid tumors were treated. Two studies were conducted in pNETs with BEZ-235 as single agent. The former was a phase 2 trial conducted in pNETs resistant to everolimus while the latter a randomized trial comparing everolimus and BEZ-235. Unfortunately, both the studies disappointed the expectations and were prematurely halted mainly due to severe toxicity. On this basis we reviewed m-TOR inhibitors in pNETs, focusing on their mechanisms of resistance and toxicity.

Short article: Evaluation of O6-methylguanine-DNA methyltransferase as a predicting factor of response to temozolomide-based chemotherapy in well-differentiated metastatic pancreatic neuroendocrine tumors.

OBJECTIVE: Temozolomide (TMZ) is an alkylating agent frequently used in well-differentiated metastatic pancreatic neuroendocrine tumors (PNETs) with very variable responses. O-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme whose loss of expression has been suggested to be predictive of response to TMZ in various human tumors. We evaluated the predictive value of MGMT status, assessed by immunohistochemistry (IHC) and methylation-specific PCR (MS-PCR), in well-differentiated metastatic PNETs treated by a TMZ-based chemotherapy. PATIENTS AND METHODS: All patients with metastatic PNETs treated with TMZ-based chemotherapy between 2010 and 2016 in two academic centers, for whom the tumor samples were available, were included. Clinical data were collected and the MGMT status of the tumors was analyzed using MS-PCR and IHC. RESULTS: Twenty-two patients (nine men, median age 61 years) were included. The loss of MGMT protein expression detected by IHC was observed in 13 (59%) patients and MGMT promoter hypermethylation was detected by MS-PCR in three (15%) out of 20 interpretable cases. MGMT status did not correlate significantly with the best radiological response according to the Response Evaluation Criteria In Solid Tumors criteria or with progression-free survival. There was no correlation between MGMT protein expression and MGMT gene promoter methylation. CONCLUSION: These results indicate that a deficient MGMT status in PNETs, determined by loss of protein expression in IHC or by the presence of MGMT gene promoter methylation measured by MS-PCR, is not associated with a better response to TMZ-based chemotherapy and cannot be used as a predictive marker to lead treatment decisions.

Comprehensive immunohistochemical analysis of histone deacetylases in pancreatic neuroendocrine tumors: HDAC5 as a predictor of poor clinical outcome.

Epigenetic factors contribute to carcinogenesis, tumor promotion, and chemoresistance. Histone deacetylases (HDACs) are epigenetic regulators that primarily cause chromatin compaction, leading to inaccessibility of promoter regions and eventually gene silencing. Many cancer entities feature overexpression of HDACs. Currently, the role of HDACs in pancreatic neuroendocrine tumors (pNETs) is unclear. We analyzed the expression patterns of all HDAC classes (classes I, IIA, IIB, III, and IV) in 5 human tissue microarrays representing 57 pNETs resected between 1997 and 2013 and corresponding control tissue. All pNET cases were characterized clinically and pathologically according to recent staging guidelines. The investigated cases included 32 (56.1%) female and 25 (43.9%) male pNET patients (total n=57, 47.4% immunohistochemically endocrine positive). Immunohistochemical profiling revealed a significant up-regulation of all HDAC classes in pNET versus control, with different levels of intensity and extensity ranging from 1.5- to >7-fold up-regulation. In addition, expression of several HDACs (HDAC1, HDAC2, HDAC5, HDAC11, and Sirt1) was significantly increased in G3 tumors. Correlation analysis showed a significant association between the protein expression of HDAC classes I, III, and IV and rate of the pHH3/Ki-67-associated mitotic and proliferation index. Furthermore, especially HDAC5 proved as a negative predictor of disease-free and overall survival in pNET patients. Overall, we demonstrate that specific members of all 4 HDAC classes are heterogeneously expressed in pNET. Moreover, expression of HDACs was associated with tumor grading, proliferation markers, and patient survival, therefore representing interesting new targets in pNET treatment.

Increased Activity of the Immunoregulatory Enzyme Indoleamine-2,3-Dioxygenase with Consecutive Tryptophan Depletion Predicts Death in Patients with Neuroendocrine Neoplasia.

BACKGROUND/AIMS: Data from a considerable number of malignancies demonstrate that depletion of the essential amino acid tryptophan via induction of the immunoregulatory enzyme indoleamine-2,3-dioxygenase (IDO) serves as an important tumour escape strategy and is of prognostic importance. Here we investigate the predictive value of the activity of IDO as well as levels of tryptophan and respective downstream catabolites in a large cohort of patients with neuroendocrine neoplasms (NEN). METHODS: 142 consecutive Caucasian patients (62 male, aged 60.3 ± 11.9 years) with histologically confirmed NEN were systematically analysed in a retrospective blinded end point analysis. Patients were followed up for a mean period of about 3.9 ± 1.9 years. Clinical outcome, levels of established biomarkers, and tryptophan degradation markers (assessed using tandem mass spectrometry) including estimated IDO activity were recorded. Cox proportional hazards regression models were performed for the assessment of prognostic power. RESULTS: We found that baseline tryptophan levels were significantly lower and IDO activity was significantly increased in non-survivors. The risk for death inclined stepwise and was highest in patients in the upper tertile of IDO activity. Cox proportional regression models identified IDO activity as an independent predictor of death. CONCLUSIONS: In this retrospective analysis, we observed that baseline activity of the immunoregulatory enzyme IDO was significantly increased in non-survivors. IDO activity was identified as an independent predictor of death in this cohort of NEN patients. Whether IDO activity or tryptophan depletion serves to guide future therapeutic interventions in NEN remains to be established.