Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

Cell-Free Tumor DNA Dominant Clone Allele Frequency Is Associated With Poor Outcomes in Advanced Biliary Cancers Treated With Platinum-Based Chemotherapy.

PURPOSE: This investigation sought to evaluate the prognostic value of pretreatment of circulating tumor DNA (ctDNA) in metastatic biliary tract cancers (BTCs) treated with platinum-based first-line chemotherapy treatment. MATERIALS AND METHODS: We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis, we considered the detected gene with highest variant allele frequency as the dominant clone allele frequency (DCAF). Results of ctDNA analysis were correlated with patients' demographics, progression-free survival (PFS), and overall survival (OS). RESULTS: The median age of patients was 67 (27-90) years. Fifty-four (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma, and six gallbladder cancers. Forty-six (68.6%) of the patients were treated with cisplatin plus gemcitabine, and 16.4% of patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations, whereas 15% of patients were treated on a clinical trial with gemcitabine and cisplatin plus additional agents (CX4945, PEGPH20, or nab-paclitaxel). TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1 were the genes with highest frequency as DCAF. The median DCAF was 3% (0%-97%). DCAF > 3% was associated with worse OS (median OS: 10.8 v 18.8 months, P = .032). Stratifying DCAF in quartiles, DCAF > 10% was significantly related to worse PFS (median PFS: 3 months, P = .014) and worse OS (median OS: 7.0 months, P = .001). Each 1% increase in ctDNA was associated with a hazard ratio of 13.1 in OS when adjusting for subtypes, metastatic sites, size of largest tumor, age, sex, and CA19-9. CONCLUSION: DCAF at diagnosis of advanced BTC can stratify patients who have worse outcomes when treated with upfront platinum-based chemotherapy. Each increase in %ctDNA decreases survival probabilities.

Synergistic combination of cytotoxic chemotherapy and cyclin-dependent kinase 4/6 inhibitors in biliary tract cancers.

BACKGROUND AND AIMS: Biliary tract cancers (BTCs) are uncommon, but highly lethal, gastrointestinal malignancies. Gemcitabine/cisplatin is a standard-of-care systemic therapy, but has a modest impact on survival and harbors toxicities, including myelosuppression, nephropathy, neuropathy, and ototoxicity. Whereas BTCs are characterized by aberrations activating the cyclinD1/cyclin-dependent kinase (CDK)4/6/CDK inhibitor 2a/retinoblastoma pathway, clinical use of CDK4/6 inhibitors as monotherapy is limited by lack of validated biomarkers, diffident preclinical efficacy, and development of acquired drug resistance. Emerging studies have explored therapeutic strategies to enhance the antitumor efficacy of CDK4/6 inhibitors by the combination with chemotherapy regimens, but their mechanism of action remains elusive. APPROACH AND RESULTS: Here, we report in vitro and in vivo synergy in BTC models, showing enhanced efficacy, reduced toxicity, and better survival with a combination comprising gemcitabine/cisplatin and CDK4/6 inhibitors. Furthermore, we demonstrated that abemaciclib monotherapy had only modest efficacy attributable to autophagy-induced resistance. Notably, triplet therapy was able to potentiate efficacy through elimination of the autophagic flux. Correspondingly, abemaciclib potentiated ribonucleotide reductase catalytic subunit M1 reduction, resulting in sensitization to gemcitabine. CONCLUSIONS: As such, these data provide robust preclinical mechanistic evidence of synergy between gemcitabine/cisplatin and CDK4/6 inhibitors and delineate a path forward for translation of these findings to preliminary clinical studies in advanced BTC patients.

FGFR2-IIIb Expression by Immunohistochemistry Has High Specificity in Cholangiocarcinoma with FGFR2 Genomic Alterations.

BACKGROUND: FGFR2 genomic alterations are observed in 10-20% of cholangiocarcinoma (CCA). Although FGFR2 fusions are an important actionable target, FGFR2 protein expression has not been thoroughly characterized. AIMS: To evaluate FGFR2 protein expression in cholangiocarcinoma harboring FGFR2 genomic alterations. METHODS: FGFR2 protein expression was evaluated in 99 CCA cases with two different antibodies. FGFR2 genomic alterations were confirmed via next-generating sequencing (NGS) or FISH. Primary objective was to determine the specificity and sensitivity of FGFR2 immunohistochemistry staining for detecting FGFR2 genomic alterations. Secondary objectives included overall FGFR2 immunohistochemistry staining in CCA patients, and evaluation of whether FGFR2 expression correlates with clinical outcomes including overall survival (OS), progression-free survival (PFS), and time-to-tumor recurrence (TTR). RESULTS: Immunohistochemistry staining with two antibodies against FGFR2, FPR2-D, and clone 98706 showed high accuracy (78.7% and 91.9%) and specificity (82.9% and 97.7%), and moderate sensitivity (53.9% and 57.1%), respectively, when compared with the standard methods for detecting FGFR2 genomic alterations. In a median follow-up of 72 months, there were no statistically significant differences in OS, PFS, and TTR, for patients with positive or negative FGFR2 staining. CONCLUSION: FGFR2 protein expression by immunohistochemistry has high specificity and therefore could be used to imply the presence of FGFR2 genomic alterations in the context of a positive test. In the case of a negative test, NGS or FISH would be necessary to ascertain cases with FGFR2 genomic alterations.

Ipilimumab and Radiation in Patients with High-risk Resected or Regionally Advanced Melanoma.

PURPOSE: In this prospective trial, we sought to assess the feasibility of concurrent administration of ipilimumab and radiation as adjuvant, neoadjuvant, or definitive therapy in patients with regionally advanced melanoma. PATIENTS AND METHODS: Twenty-four patients in two cohorts were enrolled and received ipilimumab at 3 mg/kg every 3 weeks for four doses in conjunction with radiation; median dose was 4,000 cGy (interquartile range, 3,550-4,800 cGy). Patients in cohort 1 were treated adjuvantly; patients in cohort 2 were treated either neoadjuvantly or as definitive therapy. RESULTS: Adverse event profiles were consistent with those previously reported with checkpoint inhibition and radiation. For the neoadjuvant/definitive cohort, the objective response rate was 64% (80% confidence interval, 40%-83%), with 4 of 10 evaluable patients achieving a radiographic complete response. An additional 3 patients in this cohort had a partial response and went on to surgical resection. With 2 years of follow-up, the 6-, 12-, and 24-month relapse-free survival for the adjuvant cohort was 85%, 69%, and 62%, respectively. At 2 years, all patients in the neoadjuvant/definitive cohort and 10/13 patients in the adjuvant cohort were still alive. Correlative studies suggested that response in some patients were associated with specific CD4+ T-cell subsets. CONCLUSIONS: Overall, concurrent administration of ipilimumab and radiation was feasible, and resulted in a high response rate, converting some patients with unresectable disease into surgical candidates. Additional studies to investigate the combination of radiation and checkpoint inhibitor therapy are warranted.

Establishment of normative ranges of the healthy human immune system with comprehensive polychromatic flow cytometry profiling.

Existing normative flow cytometry data have several limitations including small sample sizes, incompletely described study populations, variable flow cytometry methodology, and limited depth for defining lymphocyte subpopulations. To overcome these issues, we defined high-dimensional flow cytometry reference ranges for the healthy human immune system using Human Immunology Project Consortium methodologies after carefully screening 127 subjects deemed healthy through clinical and laboratory testing. We enrolled subjects in the following age cohorts: 18-29 years, 30-39, 40-49, and 50-66 and enrolled cohorts to ensure an even gender distribution and at least 30% non-Caucasians. From peripheral blood mononuclear cells, flow cytometry reference ranges were defined for >50 immune subsets including T-cell (activation, maturation, T follicular helper and regulatory T cell), B-cell, and innate cells. We also developed a web tool for visualization of the dataset and download of raw data. This dataset provides the immunology community with a resource to compare and extract data from rigorously characterized healthy subjects across age groups, gender and race.

Immune Activation in Early-Stage Non-Small Cell Lung Cancer Patients Receiving Neoadjuvant Chemotherapy Plus Ipilimumab.

Purpose: To determine the immunologic effects of neoadjuvant chemotherapy plus ipilimumab in early-stage non-small cell lung cancer (NSCLC) patients.Experimental Design: This is a single-arm chemotherapy plus phased ipilimumab phase II study of 24 treatment-naïve patients with stage IB-IIIA NSCLC. Patients received neoadjuvant therapy consisting of 3 cycles of paclitaxel with either cisplatin or carboplatin and ipilimumab included in the last 2 cycles.Results: Chemotherapy alone had little effect on immune parameters in PBMCs. Profound CD28-dependent activation of both CD4 and CD8 cells was observed following ipilimumab. Significant increases in the frequencies of CD4+ cells expressing activation markers ICOS, HLA-DR, CTLA-4, and PD-1 were apparent. Likewise, increased frequencies of CD8+ cells expressing the same activation markers, with the exception of PD-1, were observed. We also examined 7 resected tumors and found higher frequencies of activated tumor-infiltrating lymphocytes than those observed in PBMCs. Surprisingly, we found 4 cases of preexisting tumor-associated antigens (TAA) responses against survivin, PRAME, or MAGE-A3 present in PBMC at baseline, but neither increased frequencies nor the appearance of newly detectable responses following ipilimumab therapy. Ipilimumab had little effect on the frequencies of circulating regulatory T cells and MDSCs.Conclusions: This study did not meet the primary endpoint of detecting an increase in blood-based TAA T-cell responses after ipilimumab. Collectively, these results highlight the immune activating properties of ipilimumab in early-stage NSCLC. The immune profiling data for ipilimumab alone can contribute to the interpretation of immunologic data from combined immune checkpoint blockade immunotherapies. Clin Cancer Res; 23(24); 7474-82. ©2017 AACR.