Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Transcriptomic analysis of micropapillary high grade T1 urothelial bladder cancer.

No consensus currently exist on the optimal treatment of patients with high-risk nonmuscle invasive (HGT1) micropapillary variant of bladder cancer (MPBC). Transcripsome analysis may allow stratification of MPBC-HGT1 enabling prediction of recurrence and guide therapeutic management for individual patients. Whole transcriptome RNA-Sequencing of tumors from 23 patients with MPBC-HGT1 and 64 conventional urothelial carcinomas (cUC) (reference set) was performed. Differentially expressed genes between MPBC-HGT1 and cUC-HGT1 were explored. Cox proportional hazard models and Kapplan-Meier methods were used to assess the relation between time to progression (TTP) and individual gene expression adjusting for clinical covariates. Over 3000 genes were differentially expressed in MPBC-HGT1 as compared with cUC-HGT1 and a 26-gene signature is characteristic of MPBC within HGT1. A set of three genes; CD36, FAPB3 and RAETE1; were significantly associated with TTP. High expression of FABP3 and CD36 were associated with shorter TTP (p = 0.045 and p = 0.08) as was low expression of RAET1E (p = 0.01). Our study suggest that a 26-gene signature can define MPBC-HGT1 within conventional urothelial carcinomas. A prognostic risk index of three genes (FABP3, CD36 and RAET1E) was found to be associated with shorter TTP and may help classify a group of patients with MPBC-HGT1 with high-risk of early progression. These observations might have implications in terms of radical cystectomy recommendation in MPBC patients.

Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tumor Tissue to Identify Single-Cell-Level PI3K Pathway Activation.

PURPOSE: Identifying cancers with high PI3K pathway activity is critical for treatment selection and eligibility into clinical trials of PI3K inhibitors. Assessments of tumor signaling pathway activity need to consider intratumoral heterogeneity and multiple regulatory nodes. EXPERIMENTAL DESIGN: We established a novel, mechanistically informed approach to assessing tumor signaling pathways by quantifying single-cell-level multiplex immunofluorescence using custom algorithms. In a proof-of-concept study, we stained archival formalin-fixed, paraffin-embedded (FFPE) tissue from patients with primary prostate cancer in two prospective cohort studies, the Health Professionals Follow-up Study and the Physicians' Health Study. PTEN, stathmin, and phospho-S6 were quantified on 14 tissue microarrays as indicators of PI3K activation to derive cell-level PI3K scores. RESULTS: In 1,001 men, 988,254 tumor cells were assessed (median, 743 per tumor; interquartile range, 290-1,377). PI3K scores were higher in tumors with PTEN loss scored by a pathologist, higher Gleason grade, and a new, validated bulk PI3K transcriptional signature. Unsupervised machine-learning approaches resulted in similar clustering. Within-tumor heterogeneity in cell-level PI3K scores was high. During long-term follow-up (median, 15.3 years), rates of progression to metastases and death from prostate cancer were twice as high in the highest quartile of PI3K activation compared with the lowest quartile (hazard ratio, 2.04; 95% confidence interval, 1.13-3.68). CONCLUSIONS: Our novel pathway-focused approach to quantifying single-cell-level immunofluorescence in FFPE tissue identifies prostate tumors with PI3K pathway activation that are more aggressive and may respond to pathway inhibitors.

Genomic Predictors of Good Outcome, Recurrence, or Progression in High-Grade T1 Non-Muscle-Invasive Bladder Cancer.

High-grade T1 (HGT1) bladder cancer is the highest risk subtype of non-muscle-invasive bladder cancer with unpredictable outcome and poorly understood risk factors. Here, we examined the association of somatic mutation profiles with nonrecurrent disease (GO, good outcome), recurrence (R), or progression (PD) in a cohort of HGT1 patients. Exome sequencing was performed on 62 HGT1 and 15 matched normal tissue samples. Both tumor only (TO) and paired analyses were performed, focusing on 95 genes known to be mutated in bladder cancer. Somatic mutations, copy-number alterations, mutation load, and mutation signatures were studied. Thirty-three GO, 10 R, 18 PD, and 1 unknown outcome patients were analyzed. Tumor mutational burden (TMB) was similar to muscle-invasive disease and was highest in GO, intermediate in PD, and lowest in R patients (P = 0.017). DNA damage response gene mutations were associated with higher TMB (P < 0.0001) and GO (P = 0.003). ERCC2 and BRCA2 mutations were associated with GO. TP53, ATM, ARID1A, AHR, and SMARCB1 mutations were more frequent in PD. Focal copy-number gain in CCNE1 and CDKN2A deletion was enriched in PD or R (P = 0.047; P = 0.06). APOBEC (46%) and COSMIC5 (34%) signatures were most frequent. APOBEC-A and ERCC2 mutant tumors (COSMIC5) were associated with GO (P = 0.047; P = 0.0002). pT1b microstaging was associated with a genomic cluster (P = 0.05) with focal amplifications of E2F3/SOX4, PVRL4, CCNE1, and TP53 mutations. Findings were validated using external public datasets. These findings require confirmation but suggest that management of HGT1 bladder cancer may be improved via molecular characterization to predict outcome. SIGNIFICANCE: Detailed genetic analyses of HGT1 bladder tumors identify features that correlate with outcome, e.g., high mutational burden, ERCC2 mutations, and high APOBEC-A/ERCC2 mutation signatures were associated with good outcome.

Defining tumor resistance to PD-1 pathway blockade: recommendations from the first meeting of the SITC Immunotherapy Resistance Taskforce.

As the field of cancer immunotherapy continues to advance at a fast pace, treatment approaches and drug development are evolving rapidly to maximize patient benefit. New agents are commonly evaluated for activity in patients who had previously received a programmed death receptor 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitor as standard of care or in an investigational study. However, because of the kinetics and patterns of response to PD-1/PD-L1 blockade, and the lack of consistency in the clinical definitions of resistance to therapy, the design of clinical trials of new agents and interpretation of results remains an important challenge. To address this unmet need, the Society for Immunotherapy of Cancer convened a multistakeholder taskforce-consisting of experts in cancer immunotherapy from academia, industry, and government-to generate consensus clinical definitions for resistance to PD-(L)1 inhibitors in three distinct scenarios: primary resistance, secondary resistance, and progression after treatment discontinuation. The taskforce generated consensus on several key issues such as the timeframes that delineate each type of resistance, the necessity for confirmatory scans, and identified caveats for each specific resistance classification. The goal of this effort is to provide guidance for clinical trial design and to support analyses of emerging molecular and cellular data surrounding mechanisms of resistance.

Publisher Correction: Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Loss of PTEN Expression Detected by Fluorescence Immunohistochemistry Predicts Lethal Prostate Cancer in Men Treated with Prostatectomy.

BACKGROUND: PTEN deletion is associated with relapse after therapy for localized prostate cancer. There are limited data on PTEN loss as detected by immunohistochemistry (IHC) and the risk of lethal disease after surgery. OBJECTIVE: To determine whether PTEN loss as detected by quantitative fluorescence IHC (FIHC) predicts lethal disease outcomes after surgery for prostate cancer. DESIGN, SETTING AND PARTICIPANTS: We used formalin-fixed, paraffin-embedded radical prostatectomy specimens to construct tissue microarrays and perform dual FIHC for PTEN and AMACR for masking tumor epithelium, plus semi-quantitative multispectral imaging analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The association of PTEN status analyzed continuously and dichotomously (low [expression in the lowest quartile] vs higher [expression >lowest quartile]) with disease outcomes (metastasis and death) was assessed with adjustment for age, Gleason score, and stage in multivariable analyses. The prognostic ability of PTEN was assessed using logistic regression models. RESULTS AND LIMITATIONS: Low PTEN expression was associated with a higher risk of metastatic disease as both a continuous (hazard ratio [HR] 1.49, 95% confidence interval [CI] 1.14-1.92; p<0.003) and dichotomous (HR 1.92, 95% CI 1.02-3.63; p=0.04) variable. A significant association between low PTEN expression and poorer overall survival was observed (continuous: HR 1.89, 95% CI 1.37-2.63; p<0.001; dichotomous: HR 2.66, 95% CI 1.34-5.28; p=0.005). Addition of PTEN status to clinicopathologic factors (age, Gleason score, and stage) incrementally improved a prognostic model assessing 10-yr outcomes for metastatic disease (area under the curve [AUC] 0.76 vs 0.80) and death (AUC 0.70 vs 0.75). CONCLUSIONS: Low PTEN expression detected by FIHC in primary prostate cancer is an independent prognostic biomarker for metastatic disease and death after definitive therapy. FIHC for PTEN is a viable clinical diagnostic assay in this context. PATIENT SUMMARY: We looked at loss of the PTEN protein in prostate tumors from men treated with surgery. Men with PTEN loss were at higher risk of metastasis and death. Assessing PTEN status may be useful in better determination of the risk of poorer outcomes.

Enhancer signatures stratify and predict outcomes of non-functional pancreatic neuroendocrine tumors.

Most pancreatic neuroendocrine tumors (PNETs) do not produce excess hormones and are therefore considered 'non-functional'1-3. As clinical behaviors vary widely and distant metastases are eventually lethal2,4, biological classifications might guide treatment. Using enhancer maps to infer gene regulatory programs, we find that non-functional PNETs fall into two major subtypes, with epigenomes and transcriptomes that partially resemble islet α- and ß-cells. Transcription factors ARX and PDX1 specify these normal cells, respectively5,6, and 84% of 142 non-functional PNETs expressed one or the other factor, occasionally both. Among 103 cases, distant relapses occurred almost exclusively in patients with ARX+PDX1- tumors and, within this subtype, in cases with alternative lengthening of telomeres. These markedly different outcomes belied similar clinical presentations and histology and, in one cohort, occurred irrespective of MEN1 mutation. This robust molecular stratification provides insight into cell lineage correlates of non-functional PNETs, accurately predicts disease course and can inform postoperative clinical decisions.

Circulating inflammation markers and prostate cancer.

BACKGROUND: Chronic inflammation is thought to influence the risk of prostate cancer. The purpose of this population-based case-control study was to evaluate the association of 48 circulating inflammation markers with prostate cancer, to identify candidate markers for further investigation. METHODS: Serum samples collected from 235 prostate cancer patients and 198 population-based controls recruited in Örebro County, Sweden, in 1989-1991, were assessed using a multiplex bead-based immunoassay to determine concentrations of 48 circulating inflammation markers. Logistic regression was first used to evaluate the association between individual markers (highest vs lowest concentration quartile) and prostate cancer in unadjusted and mutually adjusted models. Second, patients with inflammatory conditions, metastatic or advanced prostate cancer, were excluded to address the possible influence of systemic disease on inflammation markers. RESULTS: Individual analyses first identified 21 markers associated with prostate cancer (P < .05), which after mutual adjustment were reduced to seven markers. After the exclusion of men with conditions linked with systemic inflammation, associations between prostate cancer and deviant levels of C-X3-C motif chemokine ligand 1, platelet-derived growth factor subunit B homodimer, interleukin 10, C-C motif chemokine ligand (CCL) 21, and CCL11 remained statistically significant. CONCLUSIONS: In this explorative study, we identified candidate inflammation markers of possible importance for prostate cancer pathophysiology, for further evaluation in prospective studies.

Transcriptome Deconvolution of Heterogeneous Tumor Samples with Immune Infiltration.

Transcriptome deconvolution in cancer and other heterogeneous tissues remains challenging. Available methods lack the ability to estimate both component-specific proportions and expression profiles for individual samples. We present DeMixT, a new tool to deconvolve high-dimensional data from mixtures of more than two components. DeMixT implements an iterated conditional mode algorithm and a novel gene-set-based component merging approach to improve accuracy. In a series of experimental validation studies and application to TCGA data, DeMixT showed high accuracy. Improved deconvolution is an important step toward linking tumor transcriptomic data with clinical outcomes. An R package, scripts, and data are available: https://github.com/wwylab/DeMixTallmaterials.

3D microfluidic ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade.

Microfluidic culture has the potential to revolutionize cancer diagnosis and therapy. Indeed, several microdevices are being developed specifically for clinical use to test novel cancer therapeutics. To be effective, these platforms need to replicate the continuous interactions that exist between tumor cells and non-tumor cell elements of the tumor microenvironment through direct cell-cell or cell-matrix contact or by the secretion of signaling factors such as cytokines, chemokines and growth factors. Given the challenges of personalized or precision cancer therapy, especially with the advent of novel immunotherapies, a critical need exists for more sophisticated ex vivo diagnostic systems that recapitulate patient-specific tumor biology with the potential to predict response to immune-based therapies in real-time. Here, we present details of a method to screen for the response of patient tumors to immune checkpoint blockade therapy, first reported in Jenkins et al. Cancer Discovery, 2018, 8, 196-215, with updated evaluation of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS), including evaluation of the requirement for 3D microfluidic culture in MDOTS, demonstration of immune-checkpoint sensitivity of PDOTS, and expanded evaluation of tumor-immune interactions using RNA-sequencing to infer changes in the tumor-immune microenvironment. We also examine some potential improvements to current systems and discuss the challenges in translating such diagnostic assays to the clinic.

BET Bromodomain Inhibition Cooperates with PD-1 Blockade to Facilitate Antitumor Response in Kras-Mutant Non-Small Cell Lung Cancer.

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS-mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS-mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Treg) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1-inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras+/LSL-G12D ; Trp53L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared with either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Cancer Immunol Res; 6(10); 1234-45. ©2018 AACR.

Characterization of the Neuroendocrine Tumor Immune Microenvironment.

OBJECTIVES: The immune environment and the potential for neuroendocrine tumors (NETs) to respond to immune checkpoint inhibitors remain largely unexplored. We assessed immune checkpoint marker expression, lymphocytic infiltrate, and associated mutational profiles in a cohort of small intestine and pancreatic NETs. METHODS: We assessed expression of PDCD1 (PD-1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) in archival tissue from 64 small intestine (SINETs) and 31 pancreatic NETs (pNET). We additionally assessed T-cell infiltrates, categorizing T-cell subsets based on expression of the T-cell markers CD3, CD8, CD45RO (PTPRC), or FOXP3. Finally, we explored associations between immune checkpoint marker expression, lymphocytic infiltrate, and tumor mutational profiles. RESULTS: Expression of PD-1 or PD-L1 in small intestine or pancreatic NET was rare, whereas expression of PD-L2 was common in both NET subtypes. T-cell infiltrates were more abundant in pNET than in SINET. We found no clear associations between immune checkpoint marker expression, immune infiltrates, and specific mutational profile within each tumor type. CONCLUSIONS: Our findings provide an initial assessment of the immune environment of well-differentiated NETs. Further studies to define the immunologic differences between pNET and SINET, as well as the role of PD-L2 in these tumors, are warranted.

Calcium intake and risk of colorectal cancer according to expression status of calcium-sensing receptor (CASR).

OBJECTIVE: Although evidence suggests an inverse association between calcium intake and the risk of colorectal cancer, the mechanisms remain unclear. The calcium-sensing receptor (CASR) is expressed abundantly in normal colonic epithelium and may influence carcinogenesis. We hypothesized that calcium intake might be associated with lower risk of CASR-positive, but not CASR-negative, colorectal cancer. DESIGN: We assessed tumour CASR protein expression using immunohistochemistry in 779 incident colon and rectal cancer cases that developed among 136 249 individuals in the Nurses' Health Study and Health Professionals Follow-Up Study. Duplication method Cox proportional hazards regression analysis was used to assess associations of calcium intake with incidence of colorectal adenocarcinoma subtypes by CASR status. RESULTS: Total calcium intake was inversely associated with the risk of developing colorectal cancer (ptrend=0.01, comparing ≥1200 vs <600 mg/day: multivariable HR=0.75, 95% CI 0.60 to 0.95). For the same comparison, higher total calcium intake was associated with a lower risk of CASR-positive tumours (ptrend=0.003, multivariable HR=0.67, 95% CI 0.51 to 0.86) but not with CASR-negative tumours (ptrend=0.67, multivariable HR=1.15, 95% CI 0.75 to 1.78; pheterogeneity=0.06 between the CASR subtypes). The stronger inverse associations of calcium intake with CASR-positive but not CASR-negative tumours generally appeared consistent regardless of sex, tumour location and source of calcium. CONCLUSIONS: Our molecular pathological epidemiology data suggest a causal relationship between higher calcium intake and lower colorectal cancer risk, and a potential role of CASR in mediating antineoplastic effect of calcium.

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

High grade serous ovarian carcinomas originate in the fallopian tube.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian cancer and has a poor outcome. It has been proposed that fallopian tube cancers may be precursors of HGSOC but evolutionary evidence for this hypothesis has been limited. Here, we perform whole-exome sequence and copy number analyses of laser capture microdissected fallopian tube lesions (p53 signatures, serous tubal intraepithelial carcinomas (STICs), and fallopian tube carcinomas), ovarian cancers, and metastases from nine patients. The majority of tumor-specific alterations in ovarian cancers were present in STICs, including those affecting TP53, BRCA1, BRCA2 or PTEN. Evolutionary analyses reveal that p53 signatures and STICs are precursors of ovarian carcinoma and identify a window of 7 years between development of a STIC and initiation of ovarian carcinoma, with metastases following rapidly thereafter. Our results provide insights into the etiology of ovarian cancer and have implications for prevention, early detection and therapeutic intervention of this disease.

Profiling of metastatic small intestine neuroendocrine tumors reveals characteristic miRNAs detectable in plasma.

BACKGROUND: Current diagnostic and prognostic blood-based biomarkers for neuroendocrine tumors are limited. MiRNAs have tumor-specific expression patterns, are relatively stable, and can be measured in patient blood specimens. We performed a multi-stage study to identify and validate characteristic circulating miRNAs in patients with metastatic small intestine neuroendocrine tumors, and to assess associations between miRNA levels and survival. METHODS: Using a 742-miRNA panel, we identified candidate miRNAs similarly expressed in 19 small intestine neuroendocrine tumors and matched plasma samples. We refined our panel in an independent cohort of plasma samples from 40 patients with metastatic small intestine NET and 40 controls, and then validated this panel in a second, large cohort of 120 patients with metastatic small intestine NET and 120 independent controls. RESULTS: miRNA profiling of 19 matched small intestine neuroendocrine tumors and matched plasma samples revealed 31 candidate miRNAs similarly expressed in both tissue and plasma. We evaluated expression of these 31 candidate miRNAs in 40 independent cases and 40 normal controls, and identified 4 miRNAs (miR-21-5p, miR-22-3p, miR-29b-3p, and miR-150-5p) that were differently expressed in cases and controls (p<0.05). We validated these 4 miRNAs in a separate, larger panel of 120 cases and 120 controls. We confirmed that high circulating levels of miR-22-3p (p<0.0001), high levels of miR 21-5p, and low levels of miR-150-5p (p=0.027) were associated with the presence of metastatic small intestine NET. While levels of 29b-3p were lower in cases than in controls in both the initial cohort and the validation cohort, the difference in the validation cohort did not reach statistical significance. We further found that high levels of circulating miR-21-5p, high levels of circulating miR-22-3p and low levels of circulating miR-150-5p were each independently associated with shorter overall survival. A combined analysis using all three markers was highly prognostic for survival (HR 0.47, 95% CI 0.27-0.82). CONCLUSIONS: Our study suggests that elevated circulating levels of miR-21-5p and miR-22-3p and low levels of miR-150-5p are characteristic in patients with metastatic small intestine neuroendocrine tumors, and further suggests that levels of these miRNAs are associated with overall survival. These observations provide the basis for further validation studies, as well as studies to assess the biological function of these miRNAs in small intestine neuroendocrine tumors.

Tumor expression of calcium sensing receptor and colorectal cancer survival: Results from the nurses' health study and health professionals follow-up study.

Although experimental evidence suggests calcium-sensing receptor (CASR) as a tumor-suppressor, the prognostic role of tumor CASR expression in colorectal carcinoma remains unclear. We hypothesized that higher tumor CASR expression might be associated with improved survival among colorectal cancer patients. We evaluated tumor expression levels of CASR by immunohistochemistry in 809 incident colorectal cancer patients within the Nurses' Health Study and the Health Professionals Follow-up Study. We used Cox proportional hazards regression models to estimate multivariable hazard ratio (HR) for the association of tumor CASR expression with colorectal cancer-specific and all-cause mortality. We adjusted for potential confounders including tumor biomarkers such as microsatellite instability, CpG island methylator phenotype, LINE-1 methylation level, expressions of PTGS2, VDR and CTNNB1 and mutations of KRAS, BRAF and PIK3CA. There were 240 colorectal cancer-specific deaths and 427 all-cause deaths. The median follow-up of censored patients was 10.8 years (interquartile range: 7.2, 15.1). Compared with patients with no or weak expression of CASR, the multivariable HRs for colorectal cancer-specific mortality were 0.80 [95% confidence interval (CI): 0.55-1.16] in patients with moderate CASR expression and 0.50 (95% CI: 0.32-0.79) in patients with intense CASR expression (p-trend = 0.003). The corresponding HRs for overall mortality were 0.85 (0.64-1.13) and 0.81 (0.58-1.12), respectively. Higher tumor CASR expression was associated with a lower risk of colorectal cancer-specific mortality. This finding needs further confirmation and if confirmed, may lead to better understanding of the role of CASR in colorectal cancer progression.