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Abstract Background CTLA4 blocking monoclonal antibodies provide a low frequency but durable tumor responses in patients with metastatic melanoma, which led to the regulatory approval of ipilimumab based on two randomized clinical trials with overall survival advantage. The similarly fully human anti-CTLA4 antibody tremelimumab had been developed in the clinic at a fixed rate infusion, resulting in very prolonged infusion times. A new formulation of tremelimumab allowed testing a shorter infusion time. Methods A phase 1 multi-center study to establish the safety and tolerability of administering tremelimumab as a 1-hour infusion to patients with metastatic melanoma. Secondary endpoints included pharmacokinetic and clinical effects of tremelimumab. Results No grade 3 or greater infusion-related adverse events or other adverse events preventing the administration of the full tremelimumab dose were noted in 44 treated patients. The overall side effect profile was consistent with prior experiences with anti-CTLA4 antibodies. Objective tumor responses were noted in 11% of evaluable patients with metastatic melanoma, which is also consistent with the prior experience with CTLA4 antagonistic antibodies. Conclusions This study did not identify any safety concerns when tremelimumab was administered as a 1-hour infusion. These data support further clinical testing of the 1-hour infusion of tremelimumab. (Clinical trial registration number NCT00585000).

Abstract Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

Abstract Background T cell activation is associated with a rapid increase in intracellular fructose-2,6-bisphosphate (F2,6BP), an allosteric activator of the glycolytic enzyme, 6-phosphofructo-1-kinase. The steady state concentration of F2,6BP in T cells is dependent on the expression of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) and the fructose-2,6-bisphosphatase, TIGAR. Of the PFKFB family of enzymes, PFKFB3 has the highest kinase:bisphosphatase ratio and has been demonstrated to be required for T cell proliferation. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), recently has been shown to reduce F2,6BP synthesis, glucose uptake and proliferation in transformed cells. We hypothesized that the induction of PFKFB3 expression may be required for the stimulation of glycolysis in T cells and that exposure to the PFKFB3 antagonist, 3PO, would suppress T cell activation. Methods We examined PFKFB1-4 and TIGAR expression and F2,6BP concentration in purified CD3+ T cells stimulated with microbead-conjugated agonist antibodies specific for CD3 and the co-stimulatory receptor, CD28. We then determined the effect of 3PO on anti-CD3/anti-CD28-induced T cell activation, F2,6BP synthesis, 2-[1-14C]-deoxy-d-glucose uptake, lactate secretion, TNF-α secretion and proliferation. Finally, we examined the effect of 3PO administration on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice. Results We found that purified human CD3+ T cells express PFKFB2, PFKFB3, PFKFB4 and TIGAR, and that anti-CD3/anti-CD28 conjugated microbeads stimulated a >20-fold increase in F2,6BP with a coincident increase in protein expression of the PFKFB3 family member and a decrease in TIGAR protein expression. We then found that exposure to the PFKFB3 small molecule antagonist, 3PO (1–10 μM), markedly attenuated the stimulation of F2,6BP synthesis, 2-[1-14C]-deoxy-D-glucose uptake, lactate secretion, TNF-α secretion and T cell aggregation and proliferation. We examined the in vivo effect of 3PO on the development of delayed type hypersensitivity to methylated BSA and on imiquimod-induced psoriasis in mice and found that 3PO suppressed the development of both T cell-dependent models of immunity in vivo. Conclusions Our data demonstrate that inhibition of the PFKFB3 kinase activity attenuates the activation of T cells in vitro and suppresses T cell dependent immunity in vivo and indicate that small molecule antagonists of PFKFB3 may prove effective as T cell immunosuppressive agents.

Abstract Background We previously found that administration of an interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK) to stage IV melanoma patients depleted CD4+CD25HIFoxp3+ regulatory T cells and expanded melanoma-specific CD8+ T cells. The goal of this study was to assess the clinical efficacy of DAB/IL2 in an expanded cohort of stage IV melanoma patients. Methods In a single-center, phase II trial, DAB/IL2 (12 μg/kg; 4 daily doses; 21 day cycles) was administered to 60 unresectable stage IV melanoma patients and response rates were assessed using a combination of 2-[18 F]-fluoro-2-deoxy-glucose (FDG)-positron emission tomography (PET) and computed tomography (CT) imaging. Results After DAB/IL2 administration, 16.7% of the 60 patients had partial responses, 5% stable disease and 15% mixed responses. Importantly, 45.5% of the chemo/immuno-naïve sub-population (11/60 patients) experienced partial responses. One year survival was markedly higher in partial responders (80 ± 11.9%) relative to patients with progressive disease (23.7 ± 6.5%; p value < 0.001) and 40 ± 6.2% of the total DAB/IL2-treated population were alive at 1 year. Conclusions These data support the development of multi-center, randomized trials of DAB/IL2 as a monotherapy and in combination with other immunotherapeutic agents for the treatment of stage IV melanoma. Trial registration NCT00299689

Abstract Background Cognate immunity against neoplastic cells depends on a balance between effector T cells and regulatory T (Treg) cells. Treg cells prevent immune attack against normal and neoplastic cells by directly suppressing the activation of effector CD4+ and CD8+ T cells. We postulated that a recombinant interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; Ontak) may serve as a useful strategy to deplete Treg cells and break tolerance against neoplastic tumors in humans. Methods We administered DAB/IL2 (12 μg/kg; four daily doses; 21 day cycles) to 16 patients with metastatic melanoma and measured the effects on the peripheral blood concentration of several T cell subsets and on tumor burden. Results We found that DAB/IL2 caused a transient depletion of Treg cells as well as total CD4+ and CD8+ T cells (< 21 days). T cell repopulation coincided with the de novo appearance of melanoma antigen-specific CD8+ T cells in several patients as determined by flow cytometry using tetrameric MART-1, tyrosinase and gp100 peptide/MHC conjugates. Sixteen patients received at least one cycle of DAB/IL2 and five of these patients experienced regressions of melanoma metastases as measured by CT and/or PET imaging. One patient experienced a near complete response with the regression of several hepatic and pulmonary metastases coupled to the de novo appearance of MART-1-specific CD8+ T cells. A single metastatic tumor remained in this patient and, after surgical resection, immunohistochemical analysis revealed MART1+ melanoma cells surrounded by CD8+ T cells. Conclusion Taken together, these data indicate that transient depletion of T cells in cancer patients may disrupt the homeostatic control of cognate immunity and allow for the expansion of effector T cells with specificity against neoplastic cells. Several T cell depleting agents are clinically available and this study provides strong rationale for an examination of their efficacy in cancer patients. Trial registration NCT00299689 (ClinicalTrials.gov Identifier).

Abstract Background Neoplastic cells increase glycolysis in order to produce anabolic precursors and energy within the hypoxic environment of a tumor. Ras signaling is activated in several cancers and has been found to regulate metabolism by enhancing glycolytic flux to lactate. We examined the effects of sequential immortalization and H-RasV12-transformation of human bronchial epithelial cells on the anabolic fate of fully-labeled 13C-glucose-derived carbons using two-dimensional total correlated spectroscopic analysis-nuclear magnetic resonance spectroscopy (2D TOCSY-NMR). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells unexpectedly increased tricarboxylic acid cycle activity as measured by the direct conversion of 13C-glucose carbons into the anabolic substrates glutamate/glutamine, aspartate and uridine. We then observed that immortalization and H-RasV12-transformation of bronchial epithelial cells caused a stepwise increase in oxygen consumption, a global measure of electron transport chain activity. Importantly, ectopic expression of H-RasV12 sensitized immortalized cells to the ATP-depleting and cytotoxic effects of electron transport perturbation using the complex I inhibitor rotenone. Conclusion Taken together, these data indicate that the oncoprotein H-RasV12 increases mitochondrial metabolism and provide new rationale for the targeting of the tricarboxylic acid cycle and electron transport chain as anti-neoplastic strategies.

Abstract Background PCR-based detection and identification of viruses assumes a known, relatively stable genome. Unfortunately, high mutation rates may lead to extensive changes in viral nucleic acid sequences making dedicated PCR primer use problematic. Furthermore, in bioterrorism, viral consensus sequences can be genetically modified as a countermeasure to RT-PCR and DNA chip detection. Accordingly, there is a great need for the development of rapid and universal virus detection and identification technologies. Results We report herein that viral genomic DNA or RNA can be separated from host nucleic acids in plasma by filtration and nuclease digestion, and randomly amplified in a single PCR using a mixture of primers designed to be resistant to primer-dimer amplification (5'-VVVVVVVVAA-3', V = A, G or C; 38 or 6561 primers). We have termed this novel PCR method Random Multiplex (RT)-PCR since hundreds of overlapping PCR amplifications occur simultaneously. Using this method, we have successfullydetected and partially sequenced 3 separate viruses in human plasma without using virus-specific reagents (i.e., Adenovirus Type 17, Coxsackievirus A7, and Respiratory Syncytial Virus B). The method is sensitive to ~1000 genome equivalents/ml and may represent the fastest means of detection of unknown viruses. Conclusion These studies suggest that the further development of random multiplex (RT)-PCR may lead to a diagnostic assay that can universally detect viruses in donated blood products as well as in patients suffering with idiopathic disease states of possible viral etiology.

Background: Treatment options for immunotherapy-refractory melanoma are an unmet need. The MASTERKEY-115 phase II, open-label, multicenter trial evaluated talimogene laherparepvec (T-VEC) plus pembrolizumab in advanced melanoma that progressed on prior programmed cell death protein-1 (PD-1) inhibitors., Methods: Cohorts 1 and 2 comprised patients (unresectable/metastatic melanoma) who had primary or acquired resistance, respectively, and disease progression within 12 weeks of their last anti-PD-1 dose. Cohorts 3 and 4 comprised patients (resectable disease) who underwent complete surgery, received adjuvant anti-PD-1, and experienced recurrence. Cohort 3 were disease-free for < 6 months and cohort 4 for ≥ 6 months after starting the adjuvant anti-PD-1 therapy and before confirmed recurrence. The primary endpoint was objective response rate (ORR) per RECIST v1.1. Secondary endpoints included complete response rate (CRR), disease control rate (DCR) and progression-free survival (PFS) per RECIST v1.1 and irRC-RECIST, and safety., Results: Of the 72 enrolled patients, 71 were treated. The ORR (95% CI) was 0%, 6.7% (0.2-32.0), 40.0% (16.3-67.7), and 46.7% (21.3-73.4) in cohorts 1-4, respectively; iORR was 3.8% (0.1-19.6), 6.7% (0.2-32.0), 53.3% (26.6-78.7), and 46.7% (21.3-73.4). iCRR was 0%, 0%, 13.3%, and 13.3%. Median iPFS (months) was 5.5, 8.2, not estimable [NE], and NE for cohorts 1-4, respectively; iDCR was 50.0%, 40.0%, 73.3%, and 86.7%. Treatment-related adverse events (TRAEs), grade ≥ 3 TRAEs, serious AEs, and fatal AEs occurred in 54 (76.1%), 9 (12.7%), 24 (33.8%), and 10 (14.1%) patients, respectively., Conclusion: T-VEC-pembrolizumab demonstrated antitumor activity and tolerability in PD-1-refractory melanoma, specifically in patients with disease recurrence on or after adjuvant anti-PD-1., Trial Registration: ClinicalTrials.gov identifier - NCT04068181., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Caroline Robert: Consulting or advisory role: Bristol-Myers Squibb (BMS), Roche, Novartis, Pierre Fabre, MSD, Sanofi, AstraZeneca, Pfizer, Sunpharma; Scientific co-founder of Ribonexus. Brian Gastman: Consulting or advisory role: Quest Imaging, Castle Biosciences; Speakers' Bureau: Castle Biosciences; Stock and other ownership interests: Castle Biosciences; Research funding: Alkermes, NeoImmuneTech, Merck, Instil Bio. Helen Gogas: Advisory board: BMS, MSD, Pierre Fabre, Sanofi; Invited Speaker: BMS, MSD, Novartis, Pierre Fabre, Sanofi; Local PI: Amgen, Bayer, BMS, Iovance, MSD, Replimune; Research Grant: BMS, Lilly, Pfizer, Pierre Fabre; Steering Committee Member: Amgen, Replimune. Piotr Rutkowski: Consulting or advisory role: BMS, MSD, Novartis, Pierre Fabre, Sanofi, Merck, Philogen, Blueprint Medicine; Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Bristol Myers Squibb, MSD, Novartis, Pierre Fabre, Sanofi, Merck, Astra Zeneca, Philogen, Blueprint Medicine. Georgina V. Long: Consulting fees and advisory boards: Agenus, Amgen, Array Biopharma, AstraZeneca, Bayer, BioNTech, Boehringer Ingelheim, BMS, Evaxion, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., IOBiotech, Immunocore, Innovent Biologics USA, Merck Sharpe & Dohme, Novartis, PHMR Ltd, Pierre Fabre, Regeneron, Scancell; Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: BMS, Pierre Fabre. Marya F. Chaney: Employee: Employee of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA; Stock Ownership: Merck & Co., Inc., Rahway, NJ, USA. Harshada Joshi: Employee: Parexel. Yu-Lin Lin: Employee/stock ownership: Amgen. Wendy Snyder: Employee/stock ownership: Amgen. Jason A. Chesney: Research funding: Amgen., (Copyright © 2024. Published by Elsevier Ltd.)

Immune checkpoint inhibitor (ICI) therapy is effective against many cancers for a subset of patients; a large percentage of patients remain unresponsive to this therapy. One contributing factor to ICI resistance is accumulation of monocytic myeloid-derived suppressor cells (M-MDSCs), a subset of innate immune cells with potent immunosuppressive activity against T lymphocytes. Here, using lung, melanoma, and breast cancer mouse models, we show that CD73-expressing M-MDSCs in the tumor microenvironment (TME) exhibit superior T cell suppressor function. Tumor-derived PGE 2 , a prostaglandin, directly induces CD73 expression in M-MDSCs via both Stat3 and CREB. The resulting CD73 overexpression induces elevated levels of adenosine, a nucleoside with T cell-suppressive activity, culminating in suppression of antitumor CD8 + T cell activity. Depletion of adenosine in the TME by the repurposed drug PEGylated adenosine deaminase (PEG-ADA) increases CD8 + T cell activity and enhances response to ICI therapy. Use of PEG-ADA can therefore be a therapeutic option to overcome resistance to ICIs in cancer patients.

Melanoma is the fifth most common cancer in the United States and the deadliest of all skin cancers. Even with recent advancements in treatment, there is still a 13% two-year recurrence rate, with approximately 30% of recurrences being distant metastases. Identifying patients at high risk for recurrence or advanced disease is critical for optimal clinical decision-making. Currently, there is substantial variability in the selection of screening tests and imaging, with most modalities characterized by relatively low accuracy. In the current study, we built upon a preliminary examination of differential scanning calorimetry (DSC) in the melanoma setting to examine its utility for diagnostic and prognostic assessment. Using regression analysis, we found that selected DSC profile (thermogram) parameters were useful for differentiation between melanoma patients and healthy controls, with more complex models distinguishing melanoma patients with no evidence of disease from patients with active disease. Thermogram features contributing to the third principal component (PC3) were useful for differentiation between controls and melanoma patients, and Cox proportional hazards regression analysis indicated that PC3 was useful for predicting the overall survival of active melanoma patients. With the further development and optimization of the classification method, DSC could complement current diagnostic strategies to improve screening, diagnosis, and prognosis of melanoma patients.

Talimogene laherparepvec (T-VEC) plus ipilimumab has demonstrated greater antitumor activity versus ipilimumab alone, without additional toxicity, in patients with advanced melanoma. Here, we report the 5-year outcomes from a randomized phase II study. These data provide the longest efficacy and safety follow-up for patients with melanoma treated with a combination of an oncolytic virus and a checkpoint inhibitor.Eligible patients with unresectable stage IIIB‒IV melanoma were randomized 1:1 to receive T-VEC plus ipilimumab or ipilimumab alone. T-VEC was administered intralesionally at 10 6 plaque-forming units (PFU)/mL in week 1, followed by 10 8 PFU/mL in week 4 and every 2 weeks thereafter. Ipilimumab (3 mg/kg every 3 weeks; ≤4 doses) was administered intravenously starting at week 1 in the ipilimumab arm and week 6 in the combination arm. The primary end point was investigator-assessed objective response rate (ORR) per immune-related response criteria; key secondary end points included durable response rate (DRR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety.Overall, 198 patients were randomized to receive the combination (n=98) or ipilimumab (n=100). The combination improved the ORR versus ipilimumab (35.7% vs 16.0%; OR 2.9; 95% CI 1.5 to 5.7; p=0.003). DRR was 33.7% and 13.0% (unadjusted OR 3.4; 95% CI 1.7 to 7.0; descriptive p=0.001), respectively. Among the objective responders, the median DOR was 69.2 months (95% CI 38.5 to not estimable) with the combination and was not reached with ipilimumab. Median PFS was 13.5 months with the combination and 6.4 months with ipilimumab (HR 0.78; 95% CI 0.55 to 1.09; descriptive p=0.14). Estimated 5-year OS was 54.7% (95% CI 43.9 to 64.2) in the combination arm and 48.4% (95% CI 37.9 to 58.1) in the ipilimumab arm. Forty-seven (48.0%) and 65 (65.0%) patients in the combination and ipilimumab arms, respectively, received subsequent therapies. No new safety signals were reported.At the 5-year follow-up, the improved response rates observed with T-VEC plus ipilimumab were durable. This is the first randomized controlled study of the combination of an oncolytic virus and a checkpoint inhibitor that meets its primary end point.Trial registration number: NCT01740297., Competing Interests: Competing interests: JAC: research funding: Amgen, Replimune, Iovance Biotherapeutics, Bristol Myers Squibb; patents, royalties, other intellectual property: University of Louisville US Patents. IP: stock and other ownership interests: Celldex; consulting or advisory role: Amgen, Iovance Biotherapeutics, Merck, Roche, Nouscom, Seneca Therapeutics, Nektar, Oncorus. FAC: research funding: Amgen and Replimune. PS: consulting fees: Aveo, EMD Serono, Janssen, Bayer; payment or honoraria for lectures, presentations, speakers bureau, manuscript writing, or educational events: CURIO Sciences; participation on a Data Safety Monitoring Board or Advisory Board: Aveo, EMD Serono, Janssen, Bayer. MMM: consulting: Syneos, Exicure, Novartis, Immunocore, Biontech, Blueprints Medicine, Amgen, Array, Trieza. JG: funding for this trial: Amgen. OH: consultant advisor: Aduro, Akeso, Amgen, BeiGene, BioAtla, BMS, Roche Genentech, GSK, Immunocore, Idera, Incyte, Janssen, Merck, NextCure, Novartis, Pfizer, Sanofi/Regeneron, Seattle Genetics, Tempus, Zelluna; speaker bureau: BMS, Novartis, Pfizer, Sanofi/Regeneron; contracted research (for institution): Arcus, Aduro, Akeso, Amgen, BioAtla, BMS, CytomX, Exelixis, Roche Genentech, GSK, Immunocore, Idera, Incyte, Iovance, Merck, Moderna, Merck-Serono, NextCure, Novartis, Pfizer, Sanofi/Regeneron, Seattle Genetics, Torque, Zelluna. MR: honoraria: Merck and Amgen; advisory board: Merck and Amgen; research funding: Amgen and Provectus; travel funding: Merck, Amgen, Provectus, Novartis and Castle Biosciences. PF: consultant: DBV Technologies; travel to and participation on the advisory board: Castle Biosciences; stock or stock options: Gilead, Iovance. CG: personal fees: Amgen, MSD, Philogen; grants and personal fees: Novartis, NeraCare, BMS, Roche, Sanofi, outside of the submitted work. TL: funding for this trial: Amgen; grants or contracts: Abbott, Abraxis, Acceleron, Amgen, Argos, AstraZeneca, Aveo, Biovex, Bristol Myers Squibb, Eisai, Lilly, GlaxoSmithKline, Immatics, Roche, Merck, Novartis, Pfizer, Synta, Thershold, Millenium, Tracon, Cerulean, EMD Serono, Prometheus, Macrogenics, Peloton, Iovance, MedImmune, Dynavax, NiKang; consulting fees: Prometheus; payment or honoraria for lectures, presentations, speakers bureau, manuscript writing, or educational events: SITC Advances in Cancer Immunotherapy program, organizer and presented for local program in Indianapolis. Two separate programs. AH: grants and personal fees: Amgen, BMS, Eisai, Immunocore, Pfizer, MSD/Merck, Novartis Pharma, Philogen, Pierre Fabre, Regeneron, Replimune, Immunocore, Roche, Sanofi-Genzyme, Seagen, outside the submitted work. CL: Honoraria: Roche, Bristol Myers Squibb, Novartis, Amgen, MSD, Pierre Fabre, Pfizer, Incyte; consulting or advisory role: Bristol Myers Squibb, MSD, Novartis, Amgen, Roche, Merck Serono, Sanofi, Pierre Fabre; speakers bureau: Roche, Bristol Myers Squibb, Novartis, Amgen, MSD; research funding: Roche (Inst), Bristol Myers Squibb (Inst); travel, accommodations, expenses: Bristol Myers Squibb, MSD, Novartis, Sanofi, Pierre Fabre; other relationship: Avantis Medical Systems, InflaRx. HJ: employee: Parexel. WS: employee and stockholder: Amgen. JMM: stock and other ownership interests: Pfizer; honoraria: EMD Serono, Pfizer/EMD Serono; consulting or advisory role: Merck Sharp and Dohme, Celldey, Sanofi/Regeneron, Bristol Myers Squibb, Seattle Genetics, Eisai, Novartis; research funding: Amgen, AstraZeneca, Incyte, Kinnate, Macrogenics, Bristol Myers Squibb, Merck, Novartis, Regeneron; travel, accommodations, expenses: EMD Serono, Merck Sharp and Dohme, Array BioPharma, Bristol Myers Squibb., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: The combination of talimogene laherparepvec (T-VEC) and pembrolizumab previously demonstrated an acceptable safety profile and an encouraging complete response rate (CRR) in patients with advanced melanoma in a phase Ib study. We report the efficacy and safety from a phase III, randomized, double-blind, multicenter, international study of T-VEC plus pembrolizumab (T-VEC-pembrolizumab) versus placebo plus pembrolizumab (placebo-pembrolizumab) in patients with advanced melanoma., Methods: Patients with stage IIIB-IVM1c unresectable melanoma, naïve to antiprogrammed cell death protein-1, were randomly assigned 1:1 to T-VEC-pembrolizumab or placebo-pembrolizumab. T-VEC was administered at ≤ 4 × 10 6 plaque-forming unit (PFU) followed by ≤ 4 × 10 8 PFU 3 weeks later and once every 2 weeks until dose 5 and once every 3 weeks thereafter. Pembrolizumab was administered intravenously 200 mg once every 3 weeks. The dual primary end points were progression-free survival (PFS) per modified RECIST 1.1 by blinded independent central review and overall survival (OS). Secondary end points included objective response rate per mRECIST, CRR, and safety. Here, we report the primary analysis for PFS, the second preplanned interim analysis for OS, and the final analysis., Results: Overall, 692 patients were randomly assigned (346 T-VEC-pembrolizumab and 346 placebo-pembrolizumab). T-VEC-pembrolizumab did not significantly improve PFS (hazard ratio, 0.86; 95% CI, 0.71 to 1.04; P = .13) or OS (hazard ratio, 0.96; 95% CI, 0.76 to 1.22; P = .74) compared with placebo-pembrolizumab. The objective response rate was 48.6% for T-VEC-pembrolizumab (CRR 17.9%) and 41.3% for placebo-pembrolizumab (CRR 11.6%); the durable response rate was 42.2% and 34.1% for the arms, respectively. Grade ≥ 3 treatment-related adverse events occurred in 20.7% of patients in the T-VEC-pembrolizumab arm and in 19.5% of patients in the placebo-pembrolizumab arm., Conclusion: T-VEC-pembrolizumab did not significantly improve PFS or OS compared with placebo-pembrolizumab. Safety results of the T-VEC-pembrolizumab combination were consistent with the safety profiles of each agent alone.

Introduction: Metabolomics has emerged as a powerful method to provide insight into cancer progression, including separating patients into low- and high-risk groups for overall (OS) and progression-free survival (PFS). However, survival prediction based mainly on metabolites obtained from biofluids remains elusive., Objectives: This proof-of-concept study evaluates metabolites as biomarkers obtained directly from tumor core biopsies along with covariates age, sex, pathological stage at diagnosis (I/II vs. III/VI), histological subtype, and treatment vs. no treatment to risk stratify lung cancer patients in terms of OS and PFS., Methods: Tumor core biopsy samples obtained during routine lung cancer patient care at the University of Louisville Hospital and Norton Hospital were evaluated with high-resolution 2DLC-MS/MS, and the data were analyzed by Kaplan-Meier survival analysis and Cox proportional hazards regression. A linear equation was developed to stratify patients into low and high risk groups based on log-transformed intensities of key metabolites. Sparse partial least squares discriminant analysis (SPLS-DA) was performed to predict OS and PFS events., Results: Univariable Cox proportional hazards regression model coefficients divided by the standard errors were used as weight coefficients multiplied by log-transformed metabolite intensity, then summed to generate a risk score for each patient. Risk scores based on 10 metabolites for OS and 5 metabolites for PFS were significant predictors of survival. Risk scores were validated with SPLS-DA classification model (AUROC 0.868 for OS and AUROC 0.755 for PFS, when combined with covariates)., Conclusion: Metabolomic analysis of lung tumor core biopsies has the potential to differentiate patients into low- and high-risk groups based on OS and PFS events and probability., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Improving our understanding of the immunologic response to cancer cells within the sentinel lymph nodes (SLN) of primary tumors is expected to identify new approaches to stimulate clinically meaningful cancer immunity., Experimental Design: We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T-cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of patients with melanoma., Results: We found increased effector-memory αβ T cells, TCR clonality, and γδ T cells selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an antitumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells, which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared with non-melanoma-bearing SLNs include (i) reduced CD8+CD69+ T cell/T regulatory cell ratio, (ii) high PD-1 expression on CD4+ and CD8+ T cells, and (iii) high CTLA-4 expression on γδ T cells., Conclusions: Our data suggest that these immunologic changes compromise antimelanoma immunity and contribute to a high relapse rate. We propose the development of clinical trials to test the neo-adjuvant administration of anti-PD-1 antibodies prior to SLN resection in patients with stage III melanoma. See related commentary by Lund, p. 1996., (©2022 American Association for Cancer Research.)

Purpose: Effective treatment options are limited for patients with advanced (metastatic or unresectable) melanoma who progress after immune checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes has demonstrated efficacy in advanced melanoma. Lifileucel is an autologous, centrally manufactured tumor-infiltrating lymphocyte product., Methods: We conducted a phase II open-label, single-arm, multicenter study in patients with advanced melanoma who had been previously treated with checkpoint inhibitor(s) and BRAF ± MEK targeted agents. Lifileucel was produced from harvested tumor specimens in central Good Manufacturing Practice facilities using a streamlined 22-day process. Patients received a nonmyeloablative lymphodepletion regimen, a single infusion of lifileucel, and up to six doses of high-dose interleukin-2. The primary end point was investigator-assessed objective response rate (ORR) per RECIST, version 1.1., Results: Sixty-six patients received a mean of 3.3 prior therapies (anti-programmed death 1 [PD-1] or programmed death ligand 1 [PD-L1]: 100%; anticytotoxic T-lymphocyte-associated protein-4: 80%; BRAF ± MEK inhibitor: 23%). The ORR was 36% (95% CI, 25 to 49), with two complete responses and 22 partial responses. Disease control rate was 80% (95% CI, 69 to 89). Median duration of response was not reached after 18.7-month median study follow-up (range, 0.2-34.1 months). In the primary refractory to anti-PD-1 or PD-L1 therapy subset, the ORR and disease control rate were 41% (95% CI, 26 to 57) and 81% (95% CI, 66 to 91), respectively. Safety profile was consistent with known adverse events associated with nonmyeloablative lymphodepletion and interleukin-2., Conclusion: Lifileucel demonstrated durable responses and addresses a major unmet need in patients with metastatic melanoma with limited treatment options after approved therapy, including the primary refractory to anti-PD-1 or PD-L1 therapy subset., Competing Interests: Amod A. SarnaikHonoraria: Physicans' Education ResourceConsulting or Advisory Role: B4CC, Iovance Biotherapeutics, Guidepoint Global, Defined Health, Gerson Lehrman GroupResearch Funding: Provectus, Genentech, Iovance BiotherapeuticsPatents, Royalties, Other Intellectual Property: Compositions and methods for improving tumor-infiltrating lymphocytes for adoptive cell therapy, filed March 20, 2014 US Patent Application No. 61/955,970 and second Application No. 61/973,002Travel, Accommodations, Expenses: Iovance Biotherapeutics Omid HamidHonoraria: Bristol Myers Squibb, Novartis, Sanofi/Regeneron, PfizerConsulting or Advisory Role: Amgen, Novartis, Roche, Bristol Myers Squibb, Merck, Aduro Biotech, Akeso Biopharma, BeiGene, Genentech, GlaxoSmithKline, Immunocore, Incyte, Janssen, NextCure, Regeneron, Sanofi, Seattle Genetics, Tempus, Zelluna, BioAtla, Idera, PfizerSpeakers' Bureau: Bristol Myers Squibb, Novartis, Sanofi/Regeneron, PfizerResearch Funding: Bristol Myers Squibb, Genentech, Immunocore, Incyte, Merck, Merck Serono, Novartis, Pfizer, Roche, Amgen, CytomX Therapeutics, Iovance Biotherapeutics, NextCure, GlaxoSmithKline, Arcus Biosciences, Aduro Biotech, Akeso Biopharma, Exelixis, Moderna Therapeutics, Regeneron, Sanofi, Seattle Genetics, Torque, Zelluna, Bioatla, Idera Nikhil I. KhushalaniStock and Other Ownership Interests: Bellicum Pharmaceuticals, Mazor Robotics, Amarin Corporation, Asensus SurgicalHonoraria: SanofiConsulting or Advisory Role: Bristol Myers Squibb, AstraZeneca, Regeneron, Array BioPharma, Immunocore, Merck, Incyte, Jounce Therapeutics, Iovance Biotherapeutics, NCCN/PfizerResearch Funding: Bristol Myers Squibb, Merck, Novartis, GlaxoSmithKline, HUYA Bioscience International, Amgen, Regeneron, Celgene, Replimune Karl D. LewisHonoraria: Array BioPharma, Iovance BiotherapeuticsConsulting or Advisory Role: Array BioPharma, Merck, Roche, Regeneron, Sanofi, Iovance BiotherapeuticsResearch Funding: Roche/Genentech, Merck, Array BioPharma, Incyte, Nektar, Iovance Biotherapeutics, Bristol Myers Squibb, Kartos Therapeutics, OncoSec, Regeneron, Alkermes, Neon Therapeutics, Ultimovacs, Senhwa Biosciences, Replimune, AmgenTravel, Accommodations, Expenses: Merck, Roche/Genentech, Regeneron, Neon Therapeutics, AlkermesUncompensated Relationships: Roche/Genentech, Regeneron Theresa MedinaConsulting or Advisory Role: Bristol Meyer Squibb, Iovance BiotherapeuticsResearch Funding: Merck, Replimune, Bristol Myers Squibb, Iovance Biotherapeutics, Nektar, Immunocore, Checkmate Pharmaceuticals, BioAtla Harriet M. KlugerConsulting or Advisory Role: Nektar, Celldex, Iovance Biotherapeutics, Merck, Immunocore, Array BioPharma, ElevateBio, Instil Bio, Clinigen Group, Shionogi, Bristol Myers SquibbResearch Funding: Merck, Bristol Myers Squibb, ApexigenTravel, Accommodations, Expenses: Apexigen Sajeve S. ThomasSpeakers' Bureau: BMS, Merck, Genentech, Ipsen, Amgen, Pfizer, Novartis Evidio Domingo-MusibayHonoraria: RegeneronConsulting or Advisory Role: Sanofi/Regeneron, Castle BiosciencesSpeakers' Bureau: Sanofi/RegeneronResearch Funding: Clinigen Group, Iovance Biotherapeutics, Nektar Anna C. PavlickConsulting or Advisory Role: Bristol Myers Squibb, Merck, Regeneron, Sanofi/RegeneronResearch Funding: Bristol Myers Squibb, Merck, Millennium, Regeneron, Replimune Eric D. WhitmanConsulting or Advisory Role: Merck Sharp & Dohme, Bristol Myers Squibb, Castle Biosciences, Novartis, Eisai, PfizerSpeakers' Bureau: Bristol Myers Squibb, Merck Sharp & Dohme, Castle Biosciences, Sanofi/RegeneronResearch Funding: Bristol Myers Squibb, Merck Sharp & Dohme, Castle Biosciences, Genentech/Roche, Amgen, TRACON Pharma, AstraZeneca/MedImmune, Provectus, Oncolys BioPharma, Iovance Biotherapeutics, Dynavax Technologies, OncoSec, Toray Industries, Array BioPharmaPatents, Royalties, Other Intellectual Property: Nerve monitoring dissection device, Lighted Polyhedral surgical retractor Salvador Martin-AlgarraConsulting or Advisory Role: MSD Oncology, Sanofi, Regeneron, AstraZenecaSpeakers' Bureau: Bristol Myers Squibb, MSD Oncology, AstraZeneca, Novartis, Roche, Sanofi/RegeneronTravel, Accommodations, Expenses: Pierre Fabre, Roche, MSD Oncology Pippa CorrieHonoraria: Novartis, Merck Sharp & Dohme, Pierre Fabre, Bristol Myers SquibbConsulting or Advisory Role: Novartis, Bristol Myers Squibb, Merck Sharp & Dohme, Incyte, Pierre Fabre, Roche, MicrobioticaSpeakers' Bureau: Merck Sharp & Dohme, Novartis, Bristol Myers SquibbResearch Funding: MSD, Bristol Myers Squibb, Novartis, Array BioPharma, Celgene, Halozyme, Iovance Biotherapeutics, LillyTravel, Accommodations, Expenses: Bristol Myers Squibb, Merck Sharp & Dohme Brendan D. CurtiHonoraria: Clinigen Group, NektarConsulting or Advisory Role: MerckResearch Funding: Bristol Myers Squibb, Galectin Therapeutics, Clinigen GroupPatents, Royalties, Other Intellectual Property: Biomarkers for OX40 responseTravel, Accommodations, Expenses: Agonox Jose LutzkyConsulting or Advisory Role: Castle Biosciences, Iovance Biotherapeutics, Replimune, RegeneronResearch Funding: Bristol Myers Squibb, Novartis, Iovance Biotherapeutics, Immunocore, Regeneron, Replimune, Vyriad Jeffrey S. WeberStock and Other Ownership Interests: CytomX Therapeutics, Biond Biologics, Protean Biodiagnostics, NeximmuneHonoraria: Bristol Myers Squibb, Merck, Genentech, AstraZeneca, Daiichi Sankyo, GlaxoSmithKline, Amgen, Roche, Celldex, CytomX Therapeutics, Novartis, Sellas Life Sciences, WindMIL, Takeda, Moderna Therapeutics, Jounce Therapeutics, Kirin Pharmaceuticals, Regeneron, Idera, OncosecConsulting or Advisory Role: Celldex, Bristol Myers Squibb, Merck, Genentech, Roche, Amgen, AstraZeneca, GlaxoSmithKline, Daiichi Sankyo, CytomX Therapeutics, Novartis, Sellas Life Sciences, WindMIL, Jounce Therapeutics, Moderna Therapeutics, Kirin Pharmaceuticals, Protean Biodiagnostics, Idera, OncosecResearch Funding: Bristol Myers Squibb, Merck, GlaxoSmithKline, Genentech, Astellas Pharma, Incyte, Roche, Novartis, NextCure, Moderna TherapeuticsPatents, Royalties, Other Intellectual Property: Named on a patent submitted by Moffitt Cancer Center for an IPILIMUMAB biomarker, named on a patent from Biodesix for a PD-1 antibody biomarker, named on a patent for 41BB induced TIL by Moffitt Cancer CenterTravel, Accommodations, Expenses: Bristol Myers Squibb, GlaxoSmithKline, Roche, Celldex, Amgen, Merck, AstraZeneca, Genentech, Novartis James M. G. LarkinHonoraria: Eisai, Bristol Myers Squibb, MSD, GlaxoSmithKline, Pfizer, Novartis, Roche/Genentech, Pierre Fabre, EUSA Pharma, Achilles Therapeutics, AstraZeneca, Boston Biomedical, Ipsen, Imugene, Incyte, iOncologi, Merck Serono, Nektar, Vitaccess, Kymab, SecarnaConsulting or Advisory Role: Eisai, Bristol Myers Squibb, MSD, GlaxoSmithKline, Pfizer, Novartis, Roche/Genentech, Pierre Fabre, EUSA Pharma, Achilles Therapeutics, AstraZeneca, Boston Biomedical, Ipsen, Imugene, Incyte, iOncologi, Merck Serono, Nektar, Vitaccess, Secarna, KymabResearch Funding: Pfizer, Novartis, MSD, Bristol Myers Squibb, Achilles Therapeutics, Roche, Nektar, Covance, Immunocore, AVEOTravel, Accommodations, Expenses: Bristol Myers Squibb, Pfizer, Novartis, Roche/Genentech, AstraZeneca, Boston Biomedical, Incyte, GlaxoSmithKline, Pierre Fabre, Merck Serono Wen ShiEmployment: Iovance BiotherapeuticsStock and Other Ownership Interests: Iovance BiotherapeuticsTravel, Accommodations, Expenses: Iovance Biotherapeutics Toshimi TakamuraEmployment: Iovance Biotherapeutics Inc Madan JagasiaEmployment: Iovance BiotherapeuticsStock and Other Ownership Interests: Iovance BiotherapeuticsConsulting or Advisory Role: Kadmon Harry QinEmployment: Iovance BiotherapeuticsStock and Other Ownership Interests: Iovance Biotherapeutics Xiao WuEmployment: Iovance BiotherapeuticsStock and Other Ownership Interests: Iovance BiotherapeuticsTravel, Accommodations, Expenses: Iovance Biotherapeutics Cecile ChartierEmployment: Iovance BiotherapeuticsLeadership: Iovance BiotherapeuticsPatents, Royalties, Other Intellectual Property: PCT/US2019/059598 for Expansion of TILs Utilizing AKT Pathway Inhibitors. PCT/US2019/012729 for Processes for Generating TIL Products Enriched for Tumor Antigen-specific T-cells. PCT/US2019/029286 for Gene Editing of Tumor Infiltrating Lymphocytes and Uses of Same in Immunotherapy. PCT/US2019/065892 for Methods of Expanding Tumor Infiltrating Lymphocytes Using Engineered Cytokine Receptor Pairs and Uses Thereof. PCT/US2019/012733 for Processes for Generating TIL Products Enriched for Tumor Antigen-Specific T-Cells. PCT/US2020/013095 for System and Methods for Monitoring Adoptive Cell Therapy Clonality and Persistence. PCT/US2020/063767 for Processes for the Production of Tumor Infiltrating Lymphocytes (TILs) and Methods of Using the Same. PCT/US2020/057135 for Gene Editing of Tumor Infiltrating Lymphocytes and Uses of Same in Immunotherapy. One patent application is nonpublic, for which Iovance declines to furnish any informationTravel, Accommodations, Expenses: Iovance Biotherapeutics Friedrich Graf FinckensteinEmployment: Adverum, Iovance Biotherapeutics, Roche/GenentechLeadership: Iovance BiotherapeuticsStock and Other Ownership Interests: Roche/Genentech, Bristol Myers Squibb, Johnson & Johnson, Iovance Biotherapeutics, AdverumTravel, Accommodations, Expenses: Iovance Biotherapeutics, Roche/Genentech Maria FardisEmployment: Iovance Biotherapeutics, Acerta PharmaLeadership: Iovance Biotherapeutics, Acerta PharmaStock and Other Ownership Interests: Iovance Biotherapeutics, Acerta Pharma, Gilead Sciences, AbbVie, Kartos TherapeuticsPatents, Royalties, Other Intellectual Property: Iovance Biotherapeutics, Acerta PharmaTravel, Accommodations, Expenses: Iovance Biotherapeutics, Acerta Pharma John M. KirkwoodConsulting or Advisory Role: Bristol Myers Squibb, Novartis, Iovance Biotherapeutics, Elsevier, Amgen, Checkmate Pharmaceuticals, Harbour BioMed, Istari Oncology, OncoSec, Scopus BioPharma, PfizerSpeakers' Bureau: Bristol Myers SquibbResearch Funding: Amgen, Bristol Myers Squibb, Castle Biosciences, Checkmate Pharmaceuticals, Immunocore, Iovance Biotherapeutics, Novartis, Merck Jason A. ChesneyResearch Funding: Amgen, Replimune, Iovance Biotherapeutics, Bristol Myers SquibbPatents, Royalties, Other Intellectual Property: University of Louisville US PatentsNo other potential conflicts of interest were reported.

Background: Circulating tumor DNA (ctDNA) may complement radiography for interim assessment of patients with cancer., Objective: Our objective was to explore the relationship between changes in plasma ctDNA versus radiographic imaging among patients with metastatic melanoma., Methods: Using the Idylla system, we measured B-Raf proto-oncogene (BRAF) V600 ctDNA in plasma from 15 patients with BRAF V600E/K-positive primary tumors undergoing standard-of-care monitoring, including cross-sectional computed tomography (CT) imaging. BRAF V600 mutant allele frequency (%MAF) was calculated from the Idylla Cq values and directly measured using droplet digital polymerase chain reaction (ddPCR)., Results: The Idylla ctDNA assay demonstrated 91% sensitivity, 96% specificity, 91% positive predictive value, and 96% negative predictive value for the presence of > 93 mm metastatic disease. Qualitative ctDNA results corresponded to changes in RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 status determined by CT imaging in 11 of 15 subjects (73%). Calculated %MAF results correlated with ddPCR (R 2 = 0.94) and provided evidence of progressive disease 55 and 97 days in advance of CT imaging for two subjects with persistently positive qualitative results., Conclusions: Overall, interim ctDNA results provided evidence of partial response or progressive disease an average of 82 days before radiography. This pilot study supports the feasibility of using the Idylla plasma BRAF V600 ctDNA assay as a complement to CT scanning for routine monitoring of therapeutic response. Somatic mutation quantification based on Cq values shows promise for identifying disease progression and warrants further validation.

Purpose: The prognosis for patients with recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is poor, and only a minority of patients benefit from checkpoint immunotherapy. Talimogene laherparepvec (T-VEC), an oncolytic immunotherapy approved for advanced melanoma, in combination with pembrolizumab may yield enhanced antitumor activity over either agent alone., Patients and Methods: This was a phase Ib/III, multicenter trial testing intratumoral T-VEC combined with intravenous pembrolizumab in R/M HNSCC refractory to platinum-based chemotherapy. For phase Ib, primary endpoint was incidence of dose-limiting toxicity (DLT). Key secondary endpoints included objective response rate and progression-free survival per irRECIST, overall survival, and safety., Results: Thirty-six patients were enrolled into the phase Ib study. The data cut-off date was August 28, 2018. Median follow-up was 5.8 months (range, 0.3-24.2). One DLT of T-VEC-related fatal arterial hemorrhage was reported. Twenty (55.6%) and 21 (58.3%) patients experienced adverse events (AE) related to T-VEC and pembrolizumab, respectively. Besides the DLT, there were no treatment-related fatal AEs. A confirmed partial response was observed in 5 (13.9%) patients. Ten (27.8%) patients were unevaluable for response due to early death. Median PFS and OS were 3.0 months [95% confidence interval (Cl), 2.0-5.8] and 5.8 months (95% Cl, 2.9-11.4), respectively., Conclusions: The combination of T-VEC and pembrolizumab demonstrated a tolerable safety profile in R/M HNSCC. The efficacy with the combination was similar to that with pembrolizumab monotherapy in historical HNSCC studies. Phase III part of this study was not further pursued (ClinicalTrials.gov Identifier: NCT02626000)., (©2020 American Association for Cancer Research.)

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

Background: Talimogene laherparepvec (T-VEC) is an intralesionally delivered, modified herpes simplex virus type-1 oncolytic immunotherapy. The biodistribution, shedding, and potential transmission of T-VEC was systematically evaluated during and after completion of therapy in adults with advanced melanoma., Methods: In this phase 2, single-arm, open-label study, T-VEC was administered into injectable lesions initially at 10 6 plaque-forming units (PFU)/mL, 10 8 PFU/mL 21 days later, and 10 8 PFU/mL every 14 (±3) days thereafter. Injected lesions were covered with occlusive dressings for ≥1 week. Blood, urine, and swabs from exterior of occlusive dressings, surface of injected lesions, oral mucosa, anogenital area, and suspected herpetic lesions were collected throughout the study. Detectable T-VEC DNA was determined for each sample type; infectivity was determined for all swabs with detectable T-VEC DNA., Findings: Sixty patients received ≥1 dose of T-VEC. During cycles 1-4, T-VEC DNA was detected in blood (98·3% of patients, 36·7% of samples), urine (31·7% of patients, 3·0% of samples) and swabs from injected lesions (100% of patients, 57·6% of samples), exterior of dressings (80% of patients,19·5% of samples), oral mucosa (8·3% of patients, 2·5% of samples), and anogenital area (8·0% of patients, 1·1% of samples). During the safety follow-up period, T-VEC DNA was only detected on swabs from injected lesions (14% of patients, 5.8% of samples). T-VEC DNA was detected in 4/37 swabs (3/19 patients) of suspected herpetic lesions. Among all samples, only those from the surface of injected lesions tested positive for infectivity (8/740 [1·1%]). Three close contacts reported signs and symptoms of suspected herpetic origin; however, no lesions had detectable T-VEC DNA., Interpretation: Using current guidelines, T-VEC can be administered safely to patients with advanced melanoma and is unlikely to be transmitted to close contacts with appropriate use of occlusive dressings. FUND: This study was funded by Amgen Inc.: ClinicalTrials.gov, NCT02014441., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: In the precheckpoint inhibitor era, high-dose interferon was the only approved adjuvant therapy for high-risk melanoma. In this manuscript, we analyze the recurrence-free survival, overall survival and toxicity profile of adjuvant treatment with interleukin-2 (IL-2) and 5-(3,3-dimethyle-1-triazeno) imidazole-4-carboxamide (DTIC) for resected high-risk melanoma patients., Methods: All patients with stage IIB, IIC or stage III melanoma who were treated with DTIC/IL-2 combination therapy at a single institution from 2000 to 2010 were identified from the University of Louisville Hospital medical record. Patients received 6 months of subcutaneous IL-2 (12 × 10 6 units days 1-4) and intravenous DTIC (750 mg/m 2 day 1 of each cycle) every 28 days for 6 cycles. Individual medical records were accessed to collect the data., Results: Of the 112 patients treated, all underwent surgical resection and then received adjuvant treatment. A total of 58.7% of the patients were male, 42.2% female; 99% were Caucasian. A total of 79 (72.5%) of the patients were alive at the time of analysis and 57 (47.7%) patients were currently event free. A total of 69 (63.3%) patients completed all 6 months of adjuvant combination treatment with 13.8% of the patients requiring IL-2 and 21.1% of the patients requiring DTIC dose reduction. Five year overall survival was 75.57% with recurrence-free survival of 53.05%., Conclusions: For several decades, there has not been an ideal adjuvant treatment for patients with resected high risk melanoma. Our retrospective analysis suggests that combination therapy with DTIC/IL-2 is beneficial and relatively well tolerated as an alternative adjuvant treatment for patients with high-risk melanoma., (Copyright © 2018. Published by Elsevier Inc.)

Background: Differential Scanning Calorimetry (DSC) is a technique traditionally used to study thermally induced macromolecular transitions, and it has recently been proposed as a novel approach for diagnosis and monitoring of several diseases. We report a pilot study applying Thermal Liquid Biopsy (TLB, DSC thermograms of plasma samples) as a new clinical approach for diagnostic assessment of melanoma patients., Methods: Multiparametric analysis of DSC thermograms of patient plasma samples collected during treatment and surveillance (63 samples from 10 patients) were compared with clinical and diagnostic imaging assessment to determine the utility of thermograms for diagnostic assessment in melanoma. Nine of the ten patients were stage 2 or 3 melanoma subjects receiving adjuvant therapy after surgical resection of their melanomas. The other patient had unresectable stage 4 melanoma and was treated with immunotherapy. Two reference groups were used: (A) 36 healthy subjects and (B) 13 samples from 8 melanoma patients who had completed successful surgical management of their disease and were determined by continued clinical assessment to have no evidence of disease., Results: Plasma thermogram analysis applied to melanoma patients generally agrees with clinical evaluation determined by physical assessment or diagnostic imaging (~80% agreement). No false negatives were obtained from DSC thermograms. Importantly, this methodology was able to detect changes in disease status before it was identified clinically., Conclusions: Thermal Liquid Biopsy could be used in combination with current clinical assessment for the earlier detection of melanoma recurrence and metastasis., General Significance: TLB offers advantages over current diagnostic techniques (PET/CT imaging), limited in frequency by radiation burden and expense, in providing a minimally-invasive, low-risk, low-cost clinical test for more frequent personalized patient monitoring to assess recurrence and facilitate clinical decision-making., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that accumulate during pathologic conditions, such as cancer. Patients diagnosed with advanced metastatic cancers have an average survival of 12-24 mo, a survival time that hasn't changed significantly in the past 30 yr. Despite some encouraging improvements in response rates and overall survival in patients receiving immunotherapies, such as immune checkpoint inhibitors, most patients will ultimately progress. MDSCs contribute to immunotherapeutic resistance by actively inhibiting antitumor T cell proliferation and cytotoxic activity as well as by promoting expansion of protumorigenic T regulatory cells, thereby, dampening the host immune responses against the tumor. In addition, MDSCs promote angiogenesis, tumor invasion, and metastasis. Thus, MDSCs are potential therapeutic targets in cases of multiple cancers. This review focuses on the phenotypic and functional characteristics of MDSCs and provides an overview of the mono- and combinatorial-therapeutic strategies that target MDSCs with an objective of enhancing the efficacy of cancer immunotherapies., (© Society for Leukocyte Biology.)

Transforming growth factor β1 (TGFβ1) is a well-established inducer of the epithelial-mesenchymal transition (EMT) that is essential for the acquisition of malignant properties, such as invasion, in tumor cells. Although recent studies suggest that the EMT in tumor cells is associated with reprogramming of energy metabolism and TGFβ1 has been shown to stimulate glycolysis in multiple primary cell lines, little is known about TGFβ1's effect on glycolysis and glycolytic regulators in transformed cells. Given the known regulatory role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) in glycolysis and association of glycolytic activity with malignant features such as invasion, we sought to investigate whether TGFβ1 regulates PFKFB3 expression and if PFKFB3 is involved in the TGFβ1-mediated increase in the invasive ability of the Panc1 cell cline-a well-established model of TGFβ1-initiated EMT. Herein we demonstrate that TGFβ1 induces PFKFB3 expression and stimulates glycolysis in Panc1 cells. We also show that siRNA silencing of PFKFB3 prevents the stimulation of glycolysis and in vitro invasive ability of Panc1 cells by TGFβ1. Furthermore, PFKFB3 silencing suppresses the TGFβ1-mediated induction of the Snail protein, suggesting that PFKFB3 is required for the regulation of Snail expression by TGFβ1. Taken together, our study identifies PFKFB3 as a key TGFβ1 effector protein that mediates TGFβ1's effect on Snail expression, invasion, and glycolysis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Twenty-five years ago marked the publication of the first report describing a functional contribution by the cytokine, macrophage migration inhibitory factor (MIF), to tumor-associated angiogenesis and growth. Since first appearing, this report has been cited 304 times (as of this writing), underscoring not only the importance of this landmark study but also the importance of MIF in tumor neovascularization. Perhaps more importantly, this first link between MIF and stromal cell-dependent tumor angiogenesis presaged the subsequent identification of MIF in mediating protumorigenic contributions to several solid tumor stromal cell types, including monocytes, macrophages, T lymphocytes, NK cells, fibroblasts, endothelial progenitors and mesenchymal stem cells. This retrospective review will broadly evaluate both past and present literature stemming from this initial publication, with an emphasis on cellular sources, cellular effectors, signal transduction mechanisms and the clinical importance of MIF-dependent tumor vascularization.

Cancer therapeutics has seen an emergence and re-emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramide's essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre-clinical inhibitor ABT-263, this effect was augmented and production of pro-apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia., (© 2014 Wiley Periodicals, Inc.)

Purpose: To evaluate the usefulness of routine blood cultures in patients who develop temperatures of 38.5 degrees C or higher while treated with interleukin-2 (IL-2)., Methods: Retrospective chart review study. Charts of patients treated with high-dose IL-2 or biochemotherapy for metastatic melanoma were reviewed at the University of Louisville from 2005 to 2007. The study objective was to estimate the frequency of true and false positive blood cultures., Results: A total of 205 blood cultures in 46 patients (27 male, 19 female) were reviewed. The average age was 53 years (25-71 years). The patients had an average of 3 cycles of therapy. The mean temperature of the febrile episodes was 38.7 degrees C. The mean absolute neutrophil count was 5.1 K/microL. Of these 205 febrile episodes, only 1 blood culture was true positive. The patient had methicillin sensitive staphylococcus aureus bacteremia. There were 5 false positive blood cultures. Four hundred thirty-four further febrile episodes were documented without blood cultures drawn. None of these patients were found to be infected. The yield of true positive blood cultures in this setting was 0.5% (0%-3%, CI). There was, however, a higher number of false positive blood cultures, 2.4% (0.5%-4.5%, CI)., Conclusions: Blood cultures during IL-2 containing therapy are very inefficient to differentiate between infections versus IL-2-related fever.

The clinical benefit of digitalis for patients with heart disease is well established. However, recent studies have also suggested that digitalis has antineoplastic activities at clinically relevant serum concentrations. Much of the early evidence supporting the anticancer activity of digitalis has been circumstantial. Observational studies suggest a protective benefit and improved outcomes in patients who develop cancer while they are taking digitalis. The mechanism by which digitalis selectively affects the growth of malignant cells is complex, involving several important signaling pathways. Experiments to determine its mechanism of action have demonstrated that digitalis inhibits cell growth and angiogenesis and induces apoptosis in multiple cancer cell lines. Most, if not all, of these effects are mediated through its target enzyme, sodium- and potassium-activated adenosine triphosphatase. This article reviews the literature, which supports the use of digitalis in patients with malignancies with a discussion of the potential mechanisms of action. We hypothesize that sodium- and potassium-activated adenosine triphosphatase is an important new target for cancer therapy. It is reasonable to expect that the addition of digitalis to current cancer treatments will improve the clinical outcomes.

Background: The mediator known historically as macrophage migration inhibitory factor (MIF) has been identified recently as being released into the circulation by the anterior pituitary gland as a consequence of stress or during a systemic inflammatory response. Macrophages and T cells also secrete MIF, both in response to proinflammatory factors or upon stimulation with glucocorticoids. Once released, MIF "overrides" or counterregulates the immunosuppressive effects of steroids on cytokine production and immune cellular activation. To further investigate the biology of MIF and its role in the neuroendocrine system, we have studied the regional and cellular expression of MIF in brain tissue obtained from normal rats and rats administered LPS intracisternally., Materials and Methods: Rat brain sections were analyzed by immunohistochemistry utilizing an affinity-purified, anti-MIF antibody raised to recombinant MIF, and by in situ hybridization using a digoxigenin-labeled, antisense MIF cRNA probe. The kinetics of MIF mRNA expression in brain were compared with that of IL-1, IL-6, and TNF-alpha by RT-PCR of total brain RNA. The cerebrospinal fluid content of MIF and TNF-alpha proteins was analyzed by Western blotting and ELISA., Results: A strong baseline expression pattern for MIF was observed in neurons of the cortex, hypothalamus, hippocampus, cerebellum, and pons. By in situ hybridization, MIF mRNA was found predominantly in cell bodies whereas MIF protein was detected mostly within the terminal fields associated with neurons. There was a marked pattern of MIF immunoreactivity within the mossy fibers of the dentate gyrus and dendrites of the hippocampal CA3 field. These structures have been shown previously to be involved in glucocorticoid-induced tissue damage within the hippocampus, suggesting an association between MIF and targets of glucocorticoid action. The intracisternal injection of LPS increased MIF mRNA and protein expression in brain and MIF immunoreactivity was due in part to infiltrating monocytes/macrophages. MIF protein also was found to be rapidly released into the cerebrospinal fluid. This response corresponded with that of LPS-induced cytokine release and MIF mRNA expression increased in a distribution that colocalized in large part with that of TNF-alpha, IL-1 beta, and IL-6., Conclusion: The significant levels of baseline and inducible MIF expression in the brain and its regional association with glucocorticoid action underscore the importance of this mediator as a physiological regulator of the inflammatory stress response and further define its role within the neuroendocrine system.

Macrophage migration inhibitory factor (MIF) is an important constituent of the host response to stress and infection and is the first mediator that has been identified to be released from immune cells upon stimulation with glucocorticoids. MIF also has been shown to be secreted from the anterior pituitary gland, monocytes/macrophages, and T cells activated by various proinflammatory stimuli. Once released, MIF acts to counter-regulate the inhibitory effect of glucocorticoids on inflammatory cytokine production. To characterize more precisely the role of MIF in the host response to infection, we undertook a systematic analysis of MIF expression in various organs of the rat after endotoxin (lipopolysaccharide) administration. MIF protein and mRNA were analyzed by immunohistochemistry and in situ hybridization, respectively. MIF was found to be expressed constitutively in organs such as the lung, liver, kidney, spleen, adrenal gland, and skin. Significant quantities of MIF protein were detected preformed in various cell types and appeared to be released as a consequence of endotoxemia. In virtually all tissues examined, the loss of MIF protein 6 hours after lipopolysaccharide administration was accompanied by the induction of MIF mRNA and, at 24 hours, by the restoration of immunoreactive, intracellular MIF. The constitutive production of MIF by several cell and tissue types together with its rapid release from intracellular pools distinguishes MIF from other cytokines or hormonal mediators and significantly expands the physiological role of this unique counter-regulator of glucocorticoid action.

Aerobic organisms possess a number of often overlapping and well-characterized defenses against common oxidants such as superoxide and hydrogen peroxide. However, much less is known of mechanisms of defense against halogens such as chlorine compounds. Although chlorine-based oxidants may oxidize a number of cellular components, sulfhydrl groups are particularly reactive. We have, therefore, assessed the importance of intracellular glutathione in protection of Escherichia coli cells against hydrogen peroxide, hypochlorous acid, and chloramines. Employing a glutathione-deficient E. coli strain (JTG10) and an otherwise isogenic glutathione-sufficient E. coli strain (AB1157), we find that glutathione-deficient organisms are approximately twice as sensitive to killing by both hydrogen peroxide and chlorine compounds. However, the mode of protection by glutathione in these two cases appears to differ: exogenous glutathione added to glutathione-deficient E. coli in amounts equal to those which would be present in a similar suspension of the wild-type bacteria fully restored resistance of glutathione-deficient bacteria to chlorine-based oxidants but did not change resistance to hydrogen peroxide. Furthermore, in protection against chlorine compounds, oxidized glutathione is almost as effective as reduced glutathione, implying that the tripeptide and/or oxidized thiol undergo further reactions with chlorine compounds. Indeed, in vitro, 1 mol of reduced glutathione will react with approximately 3.5 to 4.0 mol of hypochlorous acid. We conclude that glutathione defends E. coli cells against attack by chlorine compounds and hydrogen peroxide but, in the case of the halogen compounds, does so nonenzymatically and sacrificially.

Unlabelled: BACKGROUND AND PURPOSE. Previous studies have shown that injection of the metalloproteinase collagenase directly into the caudate nucleus of rats causes an intracerebral hemorrhage. The purpose of the present study is to determine functional deficits associated with a collagenase-induced hemorrhagic lesion of the striatum., Methods: Twelve adult rats received a 2-microL infusion of bacterial collagenase (0.5 U in saline) into the right striatum. The rotational response to apomorphine (1 mg/kg SC) administration was then examined at 1, 4, 7, 21, 35, and 70 days after the surgery. In addition to the rotational asymmetry studies, the initiation of stepping movements in each forelimb was determined 8 weeks after the collagenase injections. In the assessment of rotational asymmetry and stepping ability, an additional six control animals received unilateral injections of saline alone. After behavioral testing, brains were processed for neuropathological evaluation., Results: A net ipsilateral rotation was noted at all posthemorrhage time periods. The average rotational asymmetries on these days were 14.57 +/- 2.9, 20.33 +/- 2.7, 19.99 +/- 4.4, 18.95 +/- 4.9, 17.03 +/- 4.9, and 14.4 +/- 4.7, respectively (data expressed as mean clockwise rotations per 5 minutes +/- SEM). The average number of steps initiated by the forelimb ipsilateral and contralateral to the lesion was 28.3 +/- 2.1 steps per minute and 13.6 +/- 1.5 steps per minute, respectively. This difference between left and right forelimb stepping was stable and reproducible for 3 consecutive days. Histological studies revealed a long-lasting hematoma cavity surrounded by dense reactive gliosis in the striatum., Conclusions: We conclude that collagenase-induced intrastriatal hemorrhage results in long-term locomotor deficits and is therefore a useful model for developing and assessing therapeutic approaches for the restoration of neurological function after intracerebral hemorrhage.

Determinations of hypochlorous acid and chloramine compounds are important in a number of areas. Several techniques are now available for such analyses, but most require unstable reagents and/or multiple steps in the analytical procedure. We have developed a simple, one-step spectrophotometric assay for reactive chlorine-containing compounds involving the oxidation of ascorbic acid by hypochlorous acid or chloramines. There is no interference from other nonhalide oxidants such as hydrogen peroxide or hypothiocyanous acid. Because small amounts of ascorbic acid will not damage biological materials, this method also allows continuous measurements of the generation of chlorine-containing compounds by activated neutrophils. This simple assay permits precise analysis of as little as 1 nmol of HOCl.