Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma.

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Immune biomarkers are more accurate in prediction of survival in ulcerated than in non-ulcerated primary melanomas.

INTRODUCTION: Ulcerated melanomas may have a unique biology and microenvironment. We test whether markers of immune infiltration correlate with clinical outcome in ulcerated compared to non-ulcerated primary melanoma tumors. METHODS: Sixty-two stage II-III cutaneous melanomas, 32 ulcerated and 30 non-ulcerated, were analyzed for tumor-infiltrating lymphocytes (TILs). Immunohistochemistry (IHC) was performed for CD2, a marker previously shown to correlate with overall survival (OS) and recurrence-free survival (RFS) in this patient population. IHC using antibody, VE1, to BRAF V600E was also performed on a subset of 41 tumors to assess the relationship of BRAF mutation to immune markers. RESULTS: We found, using Cox regression models, that the presence of TILs was associated with improved OS (p = 0.034) and RFS (p = 0.002) in ulcerated melanoma tumors, but not in non-ulcerated melanoma (p = 0.632, 0.416). CD2 expression also was correlated with improved OS (p = 0.021) and RFS (p = 0.001) in ulcerated melanoma, but no relationship was seen in non-ulcerated melanoma (p = 0.427, 0.682). In this small population, BRAF status did not correlate with TILs or CD2+ count. CONCLUSION: Our data show that immune markers including TILs and CD2 count correlate more closely with survival in ulcerated melanomas than that in non-ulcerated melanomas. We propose that immune biomarkers may be particularly relevant to ulcerated, as compared to non-ulcerated, melanomas and that this merits study in larger populations.

Successful Treatment of Eosinophilic Enterocolitis in an Adult Patient With Adalimumab.

Eosinophilic gastrointestinal diseases are increasing in prevalence, but understanding of their causes and effective treatments remain elusive, especially in adults. We present a case of eosinophilic gastroenteritis and colitis with extraintestinal manifestations that was successfully treated with a tumor necrosis factor α inhibitor, adalimumab.

Structure and reversible pyran formation in molybdenum pyranopterin dithiolene models of the molybdenum cofactor.

The syntheses and X-ray structures of two molybdenum pyranopterin dithiolene complexes in biologically relevant Mo(4+) and Mo(5+) states are reported. Crystallography reveals that these complexes possess a pyran ring formed through a spontaneous cyclization reaction of a dithiolene side-chain hydroxyl group at a C═N bond of the pterin. NMR data on the Mo(4+) complex suggest that a reversible pyran ring cyclization occurs in solution. These results provide experimental evidence that the pyranopterin dithiolene ligand in molybdenum and tungsten enzymes could participate in catalysis through dynamic processes modulated by the protein.

Genetic Evaluation of Pancreatitis.

Hereditary pancreatitis (HP) is a rare inherited chronic pancreatitis (CP) with strong genetic associations, with estimated prevalence ranging from 0.3 to 0.57 per 100,000 across Europe, North America, and East Asia. Apart from the most well-described genetic variants are PRSS1, SPINK1, and CFTR, many other genes, such as CTRC, CPA1, and CLDN2 and CEL have been found to associate with HP, typically in one of the 3 main mechanisms such as altered trypsin activity, pancreatic ductal cell secretion, and calcium channel regulation. The current mainstay of management for patients with HP comprises genetic testing for eligible individuals and families, alcohol and tobacco cessation avoidance, pain control, and judicious screening for complications, including exocrine and endocrine insufficiency and pancreatic cancer.

Epidemiology of community-acquired and recurrent Clostridioides difficile infection.

Clostridioides difficile infection is a leading cause of healthcare-associated infections with significant morbidity and mortality. For the past decade, the bulk of infection prevention and epidemiologic surveillance efforts have been directed toward mitigating hospital-acquired C. difficile. However, the incidence of community-associated infection is on the rise. Patients with community-associated C. difficile tend to be younger and have lower mortality rate. Rates of recurrent C. difficile infection overall have decreased in the United States, but future research and public health endeavors are needed to standardize and improve disease detection, stratify risk factors in large-scale population studies, and to identify regional and local variations in strain types, reservoirs and transmission routes to help characterize and combat the changing epidemiology of C. difficile.

Relationship Between Dietary Fiber Intake and Short-Chain Fatty Acid-Producing Bacteria During Critical Illness: A Prospective Cohort Study.

BACKGROUND: Dietary fiber increases short-chain fatty acid (SCFA)-producing bacteria yet is often withheld in the intensive care unit (ICU). This study evaluated the safety and effect of fiber in ICU patients with gut microbiome sampling. METHODS: This was a retrospective study nested within a prospective cohort. Adults were included if newly admitted to the ICU and could receive oral nutrition, enteral feedings, or no nutrition. Rectal swabs were performed at admission and 72 hours later. The primary exposure was fiber intake over 72 hours, classified in tertiles and adjusted for energy intake. The primary outcome was the relative abundance (RA) of SCFA producers via 16S RNA sequencing and the tolerability of fiber. RESULTS: In 129 patients, median fiber intake was 13.4 g (interquartile range 0-35.4 g) over 72 hours. The high-fiber group had less abdominal distension (11% high fiber vs 28% no fiber, P < .01) and no increase in diarrhea (15% high fiber vs 13% no fiber, P = .94) or other adverse events. The median RA of SCFA producers after 72 hours was 0.40%, 0.50%, and 1.8% for the no-, low-, and high-fiber groups (P = .05 for trend). After correcting for energy intake, the median RA of SCFA producers was 0.41%, 0.32%, and 2.35% in the no-, low-, and high-corrected-fiber categories (P < .01). These associations remained significant after adjusting for clinical factors including antibiotics. CONCLUSIONS: During the 72 hours after ICU admission, fiber was well tolerated, and higher fiber intake was associated with more SCFA-producers.

Validation of Melanoma Immune Profile (MIP), a Prognostic Immune Gene Prediction Score for Stage II-III Melanoma.

PURPOSE: Biomarkers are needed to stratify patients with stage II-III melanoma for clinical trials of adjuvant therapy because, while immunotherapy is protective, it also confers the risk of severe toxicity. We previously defined and validated a 53-immune gene melanoma immune profile (MIP) predictive both of distant metastatic recurrence and of disease-specific survival (DSS). Here, we test MIP on a third independent population. EXPERIMENTAL DESIGN: A retrospective cohort of 78 patients with stage II-III primary melanoma was analyzed using the NanoString assay to measure expression of 53 target genes, and MIP score was calculated. Statistical analysis correlating MIP with DSS, overall survival, distant metastatic recurrence, and distant metastasis-free interval was performed using ROC curves, Kaplan-Meier curves, and standard univariable and multivariable Cox proportional hazards models. RESULTS: MIP significantly distinguished patients with distant metastatic recurrence from those without distant metastatic recurrence using ROC curve analysis (AUC = 0.695; P = 0.008). We defined high- and low-risk groups based on the cutoff defined by this ROC curve and find that MIP correlates with both DSS and overall survival by ROC curve analysis (AUC = 0.719; P = 0.004 and AUC = 0.698; P = 0.004, respectively). Univariable Cox regression reveals that a high-risk MIP score correlates with DSS (P = 0.015; HR = 3.2). CONCLUSIONS: MIP identifies patients with low risk of death from melanoma and may constitute a clinical tool to stratify patients with stage II-III melanoma for enrollment in clinical trials.

Quantitative Analysis of Immune Infiltrates in Primary Melanoma.

Novel methods to analyze the tumor microenvironment (TME) are urgently needed to stratify melanoma patients for adjuvant immunotherapy. Tumor-infiltrating lymphocyte (TIL) analysis, by conventional pathologic methods, is predictive but is insufficiently precise for clinical application. Quantitative multiplex immunofluorescence (qmIF) allows for evaluation of the TME using multiparameter phenotyping, tissue segmentation, and quantitative spatial analysis (qSA). Given that CD3+CD8+ cytotoxic lymphocytes (CTLs) promote antitumor immunity, whereas CD68+ macrophages impair immunity, we hypothesized that quantification and spatial analysis of macrophages and CTLs would correlate with clinical outcome. We applied qmIF to 104 primary stage II to III melanoma tumors and found that CTLs were closer in proximity to activated (CD68+HLA-DR+) macrophages than nonactivated (CD68+HLA-DR-) macrophages (P < 0.0001). CTLs were further in proximity from proliferating SOX10+ melanoma cells than nonproliferating ones (P < 0.0001). In 64 patients with known cause of death, we found that high CTL and low macrophage density in the stroma (P = 0.0038 and P = 0.0006, respectively) correlated with disease-specific survival (DSS), but the correlation was less significant for CTL and macrophage density in the tumor (P = 0.0147 and P = 0.0426, respectively). DSS correlation was strongest for stromal HLA-DR+ CTLs (P = 0.0005). CTL distance to HLA-DR- macrophages associated with poor DSS (P = 0.0016), whereas distance to Ki67- tumor cells associated inversely with DSS (P = 0.0006). A low CTL/macrophage ratio in the stroma conferred a hazard ratio (HR) of 3.719 for death from melanoma and correlated with shortened overall survival (OS) in the complete 104 patient cohort by Cox analysis (P = 0.009) and merits further development as a biomarker for clinical application. Cancer Immunol Res; 6(4); 481-93. ©2018 AACR.

Requirement for innate immunity and CD90⁺ NK1.1⁻ lymphocytes to treat established melanoma with chemo-immunotherapy.

We sought to define cellular immune mechanisms of synergy between tumor-antigen-targeted monoclonal antibodies and chemotherapy. Established B16 melanoma in mice was treated with cytotoxic doses of cyclophosphamide in combination with an antibody targeting tyrosinase-related protein 1 (αTRP1), a native melanoma differentiation antigen. We find that Fcγ receptors are required for efficacy, showing that antitumor activity of combination therapy is immune mediated. Rag1(-/-) mice deficient in adaptive immunity are able to clear tumors, and thus innate immunity is sufficient for efficacy. Furthermore, previously treated wild-type mice are not significantly protected against tumor reinduction, as compared with mice inoculated with irradiated B16 alone, consistent with a primarily innate immune mechanism of action of chemo-immunotherapy. In contrast, mice deficient in both classical natural killer (NK) lymphocytes and nonclassical innate lymphocytes (ILC) due to deletion of the IL2 receptor common gamma chain IL2γc(-/-)) are refractory to chemo-immunotherapy. Classical NK lymphocytes are not critical for treatment, as depletion of NK1.1⁺ cells does not impair antitumor effect. Depletion of CD90⁺NK1.1⁻ lymphocytes, however, both diminishes therapeutic benefit and decreases accumulation of macrophages within the tumor. Tumor clearance during combination chemo-immunotherapy with monoclonal antibodies against native antigen is mediated by the innate immune system. We highlight a novel potential role for CD90⁺NK1.1⁻ ILCs in chemo-immunotherapy.

Blood mRNA expression profiling predicts survival in patients treated with tremelimumab.

PURPOSE: Tremelimumab (ticilimumab, Pfizer), is a monoclonal antibody (mAb) targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Ipilimumab (Yervoy, BMS), another anti-CTLA-4 antibody, is approved by the U.S. Federal Drug Administration (FDA). Biomarkers are needed to identify the subset of patients who will achieve tumor control with CTLA-4 blockade. EXPERIMENTAL DESIGN: Pretreatment peripheral blood samples from 218 patients with melanoma who were refractory to prior therapy and receiving tremelimumab in a multicenter phase II study were measured for 169 mRNA transcripts using reverse transcription polymerase chain reaction (RT-PCR). A two-class latent model yielded a risk score based on four genes that were highly predictive of survival (P < 0.001). This signature was validated in an independent population of 260 treatment-naïve patients with melanoma enrolled in a multicenter phase III study of tremelimumab. RESULTS: Median follow-up was 297 days for the training population and 386 days for the test population. Expression levels of the 169 genes were closely correlated across the two populations (r = 0.9939). A four-gene model, including cathepsin D (CTSD), phopholipase A2 group VII (PLA2G7), thioredoxin reductase 1 (TXNRD1), and interleukin 1 receptor-associated kinase 3 (IRAK3), predicted survival in the test population (P = 0.001 by log-rank test). This four-gene model added to the predictive value of clinical predictors (P < 0.0001). CONCLUSIONS: Expression levels of CTSD, PLA2G7, TXNRD1, and IRAK3 in peripheral blood are predictive of survival in patients with melanoma treated with tremelimumab. Blood mRNA signatures should be further explored to define patient subsets likely to benefit from immunotherapy.

Activation of toll-like receptor-2 by endogenous matrix metalloproteinase-2 modulates dendritic-cell-mediated inflammatory responses.

Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-κB activation, OX40L upregulation on DCs, and ensuing TH2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity.

Dissection of immune gene networks in primary melanoma tumors critical for antitumor surveillance of patients with stage II-III resectable disease.

Patients with resected stage II-III cutaneous melanomas remain at high risk for metastasis and death. Biomarker development has been limited by the challenge of isolating high-quality RNA for transcriptome-wide profiling from formalin-fixed and paraffin-embedded (FFPE) primary tumor specimens. Using NanoString technology, RNA from 40 stage II-III FFPE primary melanomas was analyzed and a 53-immune-gene panel predictive of non-progression (area under the curve (AUC)=0.920) was defined. The signature predicted disease-specific survival (DSS P<0.001) and recurrence-free survival (RFS P<0.001). CD2, the most differentially expressed gene in the training set, also predicted non-progression (P<0.001). Using publicly available microarray data from 46 primary human melanomas (GSE15605), a coexpression module enriched for the 53-gene panel was then identified using unbiased methods. A Bayesian network of signaling pathways based on this data identified driver genes. Finally, the proposed 53-gene panel was confirmed in an independent test population of 48 patients (AUC=0.787). The gene signature was an independent predictor of non-progression (P<0.001), RFS (P<0.001), and DSS (P=0.024) in the test population. The identified driver genes are potential therapeutic targets, and the 53-gene panel should be tested for clinical application using a larger data set annotated on the basis of prospectively gathered data.