The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer Immunotherapy.

PURPOSE: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit for patients with cancer. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death (ICD). The approval of pemetrexed and carboplatin with anti-PD-1 (pembrolizumab) for treatment of non-small cell lung cancer represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggest a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown. EXPERIMENTAL DESIGN: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex, and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. ICD in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis. RESULTS: Pemetrexed treatment alone increased T-cell activation in mouse tumors in vivo, robustly induced ICD in mouse tumor cells and exerted T-cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T-cell activation in vitro. Increased antitumor efficacy and pronounced inflamed/immune activation were observed when pemetrexed was combined with anti-PD-L1. CONCLUSIONS: Pemetrexed augments systemic intratumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T-cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T-cell infiltration/activation along with modulation of innate immune pathways, which are significantly enhanced in combination with PD-1 pathway blockade.See related commentary by Buque et al., p. 6890.

Identification of hepatitis C virus 2k/1b intergenotypic recombinants in Georgia.

BACKGROUND AND AIMS: This study aimed to evaluate the prevalence of the hepatitis C virus intergenotype recombinant strain RF1_2k/1b in Georgia, confirm viral recombination by full genome sequencing, and determine a genetic relationship with previously described recombinant hepatitis C viruses. METHODS: We retrospectively analysed data from 1421 Georgian patients with chronic hepatitis C. Genotyping was performed with the INNO-LiPA VERSANT HCV Genotype 2.0 Assay. RESULTS: Virus isolates were assigned to nonspecific hepatitis C genotypes 2a/2c (n = 387) as performed by sequencing of core and NS5B genes. Subsequently, sequencing results classified the core region as genotype 2k and the NS5B region as genotype 1b for 72% (n = 280) of genotype 2 patients, corresponding to 19.7% of hepatitis C patients in Georgia. Eight samples were randomly selected for full genome sequencing which was successful in 7 of 8 samples. Analysis of the generated consensus sequences confirmed that all 7 viruses were 2k/1b recombinants, with the recombination breakpoint located within 73-77 amino acids before the NS2-NS3 junction, similar to the previously described RF1_2k/1b virus. Phylogenetic analysis revealed clustering of the Georgian 2k/1b viruses and RF1_2k/1b, suggesting that they are genetically related. CONCLUSIONS: The 19.7% prevalence of RF1_2k/1b in Georgia patients is far higher than has generally been reported to date worldwide. Identification of recombinants in low income countries with a high prevalence of HCV infection might be reasonable for choosing the most cost-effective treatment regimens.

Global epidemiology of HCV subtypes and resistance-associated substitutions evaluated by sequencing-based subtype analyses.

BACKGROUND & AIMS: HCV genotype, subtype, and presence of resistance-associated substitutions (RASs) are key determinants for the selection of direct-acting antiviral (DAA) treatment regimens. However, current HCV genotyping assays have limitations in differentiating between HCV subtypes, and RAS prevalence is largely undefined. The aim of this study was to investigate HCV epidemiology in 12,615 patient samples from 28 different countries across five geographic regions. METHODS: We compared HCV genotype and subtypes using INNO-LiPA 2.0 vs. amplicon sequencing among 8,945 patients from phase II/III clinical trials of DAAs. Global HCV molecular epidemiology in 12,615 patients was investigated. Subtype RAS prevalence was determined by population or deep sequencing, and phylogenetic analyses investigating subtype diversity were performed. RESULTS: Although there was high concordance between INNO-LiPA and sequencing for genotype determination, INNO-LiPA was insufficient for subtype determination for genotype 2, 3, 4, and 6. Sequencing provided subtype refinement for 42%, 10%, 81%, and 78% of genotype 2, 3, 4, and six patients, respectively. Genotype discordance (genotype 2-genotype 1) was observed in 28 of 950 (3%) genotype 2 patients, consistent with inter-genotype recombinants. Sequencing-based analyses demonstrated variations in regional subtype prevalence, notably within genotype 2, 4 and 6. RAS prevalence varied by subtype, with the clinically relevant NS3 RAS Q80K found in genotype 1a, 5a and 6a and the NS5A RAS Y93H in genotype 1b, 3a, 4b, 4r and 7. CONCLUSIONS: Together, these analyses provide an understanding of subtyping accuracy and RAS distribution that are crucial for the implementation of global HCV treatment strategies. LAY SUMMARY: Hepatitis C virus (HCV) is highly variable, with seven genotypes and 67 subtypes characterized to date. The aim of this study was to i) compare two different methods of discriminating between genotypes; ii) investigate the prevalence of HCV subtypes for each genotype around the world; iii) find the prevalence of resistance-associated substitutions (RASs) in different subtypes. We found that both methods showed high concordance in genotype discrimination, but specific subtypes were not always identified accurately. Sequencing-based analyses demonstrated variations in regional subtype prevalence for some genotypes, notably within GT2, 4 and 6. RAS prevalence also varied by subtype. These variations could determine how successful different drugs are for treating HCV.

Late Relapse Versus Hepatitis C Virus Reinfection in Patients With Sustained Virologic Response After Sofosbuvir-Based Therapies.

BACKGROUND: The development of direct-acting antivirals in recent years has dramatically enhanced rates of viral eradication to >90% in patients with chronic hepatitis C virus (HCV) infection. To determine true treatment efficacy and define the most appropriate retreatment, it is important to distinguish virologic relapse from reinfection when patients in whom HCV is eradicated during treatment become infected with a new HCV strain after treatment. METHODS: We investigated the prevalence of late recurrent viremia (patients with sustained virologic response 12 weeks after the end of treatment but detectable HCV RNA at follow-up week 24) and used refined phylogenetic analysis of multiple HCV genes to distinguish virologic relapse from reinfection. RESULTS: Across 11 phase 3 clinical trials of ledipasvir-sofosbuvir (SOF) and SOF, only 12 of 3004 patients had detectable HCV RNA following sustained virologic response 12 weeks after the end of treatment. Of these 12 patients with late recurrent viremia, 11 had the same HCV genotype/subtype at baseline and at recurrence. Phylogenetic analysis demonstrated that 58% (7 of 12) of these patients were successfully treated with the SOF-based regimen, with HCV eradication achieved, but became reinfected with a different HCV strain after treatment. The remaining 5 patients with late recurrent viremia had virologic relapse in which the HCV present at baseline persisted in the liver or another compartment and reemerged in the blood 24 weeks after treatment. CONCLUSIONS: The incidence of late recurrent viremia was low. Distinguishing reinfection from virologic relapse has implications for determining true treatment efficiency and selecting optimal retreatment strategies.

Treatment of a patient with genotype 7 hepatitis C virus infection with sofosbuvir and velpatasvir.

During a phase 3 study evaluating the combination of sofosbuvir-velpatasvir for 12 weeks in patients with genotype 1, 2, 4, 5, and 6 hepatitis C virus (HCV) infection, we enrolled a patient who was subsequently found to be infected with genotype 7 HCV. This patient tolerated the study regimen well and achieved sustained virological response 12 weeks after treatment (SVR12). (Hepatology 2016;64:983-985).

Genotype- and Subtype-Independent Full-Genome Sequencing Assay for Hepatitis C Virus.

Hepatitis C virus (HCV) exhibits a high genetic diversity and is classified into 6 genotypes, which are further divided into 66 subtypes. Current sequencing strategies require prior knowledge of the HCV genotype and subtype for efficient amplification, making it difficult to sequence samples with a rare or unknown genotype and/or subtype. Here, we describe a subtype-independent full-genome sequencing assay based on a random amplification strategy coupled with next-generation sequencing. HCV genomes from 17 patient samples with both common subtypes (1a, 1b, 2a, 2b, and 3a) and rare subtypes (2c, 2j, 3i, 4a, 4d, 5a, 6a, 6e, and 6j) were successfully sequenced. On average, 3.7 million reads were generated per sample, with 15% showing HCV specificity. The assembled consensus sequences covered 99.3% to 100% of the HCV coding region, and the average coverage was 6,070 reads/position. The accuracy of the generated consensus sequence was estimated to be >99% based on results from in vitro HCV replicon amplification, with the same extrapolated amount of input RNA molecules as that for the patient samples. Taken together, the HCV genomes from 17 patient samples were successfully sequenced, including samples with subtypes that have limited sequence information. This method has the potential to sequence any HCV patient sample, independent of genotype or subtype. It may be especially useful in confounding cases, like those with rare subtypes, intergenotypic recombination, or multiple genotype infections, and may allow greater insight into HCV evolution, its genetic diversity, and drug resistance development.

Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin.

UNLABELLED: To date, intergenotypic recombinant hepatitis C viruses (HCVs) and their treatment outcomes have not been well characterized. This study characterized 12 novel HCV recombinant strains and their response to sofosbuvir in combination with ribavirin (SOF/RBV) treatment. Across the phase II/III studies of SOF, HCV samples were genotyped using both the Siemens VERSANT HCV Genotype INNO-LiPA 2.0 Assay (Innogenetics, Ghent, Belgium) and nonstructural (NS)5B sequencing. Among these patient samples, genotype assignment discordance between the two methods was found in 0.5% of all cases (12 of 2,363), of which all were identified as genotype 2 by INNO-LiPA (12 of 487; 2.5%). HCV full-genome sequences were obtained for these 12 samples by a sequence-independent amplification method coupled with next-generation sequencing. HCV full-genome sequencing revealed that these viruses were recombinant HCV strains, with the 5' part corresponding to genotype 2 and the 3' part corresponding to genotype 1. The recombination breakpoint between genotypes 2 and 1 was consistently located within 80 amino acids of the NS2/NS3 junction. Interestingly, one of the recombinant viruses had a 34-amino-acid duplication at the location of the recombination breakpoint. Eleven of these twelve patients were treated with a regimen for genotype 2 HCV infection, but responded as if they had genotype 1 infection; 1 patient had received placebo. CONCLUSION: Twelve new HCV intergenotypic recombinant genotype 2/1 viruses have been characterized. The antiviral response to a 12- to 16-week course of SOF/RBV treatment in these patients was more similar to responses among genotype 1 patients than genotype 2 patients, consistent with their genotype 1 NS5B gene.