Biomarkers predictive of response to pembrolizumab in head and neck cancer.

BACKGROUND: We performed an integrated biomarker evaluation in pembrolizumab-treated patients with R/M HNSCC enrolled in KEYNOTE-012 or KEYNOTE-055. The relationship between biomarkers and HPV status was explored. METHODS: We evaluated PD-L1 (combined positive score [CPS]), TMB, T-cell-inflamed gene expression profile (Tcellinf GEP), and HPV status. Associations between biomarkers were evaluated by logistic regression (ORR) and Cox regression (PFS, OS). RESULTS: Two hundred and fifty-seven patients (KEYNOTE-012, n = 106; KEYNOTE-055, n = 151) had TMB data available; of these, 254 had PD-L1 and 236 had Tcellinf GEP. TMB, PD-L1, and Tcellinf GEP were each significantly associated with ORR (p < 0.01). Kaplan-Meier curves at prespecified cutoffs generally showed PFS and OS separation in the anticipated direction for these biomarkers, except for OS and TMB. TMB did not correlate with PD-L1 or Tcellinf GEP (Spearman ρ = -0.03 and ρ = -0.13, respectively); PD-L1 and Tcellinf GEP were moderately correlated (Spearman ρ = 0.47). In multivariate models, TMB, PD-L1, and Tcellinf GEP were each independently predictive for ORR (p < 0.001). ORR was higher in patients with high versus low levels of biomarkers when dichotomized using prespecified cutoffs; patients with higher versus lower levels of TMB and PD-L1 or TMB and Tcellinf GEP had the highest ORRs. Within HPV subgroups, higher versus lower distributions of biomarkers (PD-L1, TMB, and Tcellinf GEP) were associated with response. HPV detection by p16-immunohistochemistry and WES showed good concordance (81%); results were generally similar by HPV status, regardless of the detection method. CONCLUSIONS: TMB and the inflammatory biomarkers PD-L1 and Tcellinf GEP, assessed alone or together, may be useful for characterizing clinical response to pembrolizumab in R/M HNSCC.

Concordance between single-nucleotide polymorphism-based genomic instability assays and a next-generation sequencing-based homologous recombination deficiency test.

BACKGROUND: We evaluated the performance of single-nucleotide polymorphism (SNP) genotyping arrays OncoScan (Thermo Fisher Scientific, San Diego, CA) and Infinium CytoSNP-850K (CytoSNP; Illumina, Waltham, MA) for assessing homologous recombination deficiency (HRD) genomic instability. METHODS: DNA (pretreatment samples) across 20 tumor types was evaluated with OncoScan, CytoSNP, and the clinically validated HRD test. Copy number variation (CNV) and loss of heterozygosity (LOH) analyses were performed with ASCATv2.5.1. Aggregate HRD genomic metrics included LOH, telomeric-allelic imbalance number (TAI), and large-scale state transition (LST). Associations between BRCA mutation (BRCAm) status and the clinically validated HRD test metric (dichotomized at a clinical cut-off) were evaluated using area under the receiver operating characteristic (AUROC); Spearman ρ was calculated for continuous metrics. CNV segmentation and HRD genomic metrics were calculated (n = 120, n = 106, and n = 126 for OncoScan, CytoSNP and clinically validated HRD test, respectively). RESULTS: When assessed by SNP arrays, the genomic metric demonstrated good association with BRCAm (AUROC of HRD: OncoScan, 0.87; CytoSNP, 0.75) and the clinically validated test (cut-off, 42; AUROC of HRD: OncoScan, 0.92; CytoSNP, 0.91). The genomic metrics demonstrated good correlation with the clinically validated aggregate HRD test metric (ρ: OncoScan, 0.82; CytoSNP, 0.81) and for each component (ρ: OncoScan, 0.68 [LOH], 0.76 [TAI], and 0.78 [LST]; CytoSNP, 0.59 [LOH], 0.77 [TAI], and 0.82 [LST]). HRD assessed by SNP genotyping arrays and the clinically validated test showed good correlation. CONCLUSION: OncoScan and CytoSNP may potentially identify most HRD-positive tumors with appropriate clinically relevant cut-offs.

Tumor mutational burden predicts the efficacy of pembrolizumab monotherapy: a pan-tumor retrospective analysis of participants with advanced solid tumors.

BACKGROUND: Several studies have evaluated the relationship between tumor mutational burden (TMB) and outcomes of immune checkpoint inhibitors. In the phase II KEYNOTE-158 study of pembrolizumab monotherapy for previously treated recurrent or metastatic cancer, high TMB as assessed by the FoundationOne CDx was associated with an improved objective response rate (ORR). METHODS: We retrospectively assessed the relationship between TMB and efficacy in participants with previously treated advanced solid tumors enrolled in 12 trials that evaluated pembrolizumab monotherapy, including 3 randomized trials that compared pembrolizumab with chemotherapy. TMB was assessed in formalin-fixed, paraffin-embedded pretreatment tumor samples by whole-exome sequencing. High TMB was defined as ≥175 mutations/exome. Microsatellite instability (MSI) phenotype was based on whole-exome sequencing results. Programmed death ligand 1 (PD-L1) expression was assessed by immunohistochemistry. The primary end point was ORR assessed per RECIST V.1.1 by independent central review. Other end points included progression-free survival (PFS) assessed per RECIST V.1.1 by independent central review and overall survival (OS). RESULTS: Of the 2234 participants in the analysis, 1772 received pembrolizumab monotherapy and 462 received chemotherapy. Among the pembrolizumab-treated participants, ORR was 31.4% (95% CI 27.1 to 36.0) in the 433 participants with TMB ≥175 mutations/exome and 9.5% (95% CI 8.0 to 11.2) in the 1339 participants with TMB <175 mutations/exome. The association of TMB with ORR was observed regardless of PD-L1 expression and not driven by specific tumor types or participants with very high TMB or high MSI. In the 3 randomized controlled trials, TMB was associated with ORR (p≤0.016), PFS (p≤0.005), and OS (p≤0.029) of pembrolizumab but not of chemotherapy (p≥0.340, p≥0.643, and p≥0.174, respectively), and pembrolizumab improved efficacy versus chemotherapy in participants with TMB ≥175 mutations/exome. CONCLUSIONS: TMB ≥175 mutations/exome is associated with clinically meaningful improvement in the efficacy of pembrolizumab monotherapy and improved outcomes for pembrolizumab versus chemotherapy across a wide range of previously treated advanced solid tumor types. These data suggest TMB has broad clinical utility irrespective of tumor type, PD-L1 expression, or MSI status and support its use as a predictive biomarker for pembrolizumab monotherapy in participants with previously treated advanced solid tumors.

Influence of tumor mutational burden, inflammatory gene expression profile, and PD-L1 expression on response to pembrolizumab in head and neck squamous cell carcinoma.

BACKGROUND: To characterize genomic determinants of response to pembrolizumab in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) in the KEYNOTE-012 study. METHODS: Associations between biomarkers (tumor mutational burden (TMB), neoantigen load (NL), 18-gene T-cell-inflamed gene expression profile (TcellinfGEP), and PD-L1 combined positive score (CPS)) and clinical outcomes with pembrolizumab were assessed in patients with R/M HNSCC (n=192). Tumor human papillomavirus (HPV) status was also evaluated with the use of p16 immunohistochemistry and whole exome sequencing (WES; HPV+, mapping >20 HPV reads) in pretreatment tumor samples (n=106). RESULTS: TMB, clonality-weighted TMB, and TcellinfGEP were significantly associated with objective response (p=0.0276, p=0.0201, and p=0.006, respectively), and a positive trend was observed between NL and PD-L1 CPS and clinical response (p=0.0550 and p=0.0682, respectively). No correlation was observed between TMB and TcellinfGEP (Spearman ρ=-0.026) or TMB and PD-L1 (Spearman ρ=0.009); a correlation was observed between TcellinfGEP and PD-L1 (Spearman ρ=0.511). HPV status by WES and p16 immunohistochemistry showed concordance (84% Ò¡=0.573) among patients whose HPV results were available using both methods. CONCLUSIONS: TMB and inflammatory biomarkers (TcellinfGEP and PD-L1) may represent distinct and complementary biomarkers predicting response to anti-programmed death 1 therapies in HNSCC; further study of these relationships in randomized clinical trials is needed. TRIAL REGISTRATION NUMBER: NCT01848834.

Transcriptomic Determinants of Response to Pembrolizumab Monotherapy across Solid Tumor Types.

PURPOSE: To explore relationships between biological gene expression signatures and pembrolizumab response. EXPERIMENTAL DESIGN: RNA-sequencing data on baseline tumor tissue from 1,188 patients across seven tumor types treated with pembrolizumab monotherapy in nine clinical trials were used. A total of 11 prespecified gene expression signatures [18-gene T-cell-inflamed gene expression profile (TcellinfGEP), angiogenesis, hypoxia, glycolysis, proliferation, MYC, RAS, granulocytic myeloid-derived suppressor cell (gMDSC), monocytic myeloid-derived suppressor cell (mMDSC), stroma/epithelial-to-mesenchymal transition (EMT)/TGFß, and WNT] were evaluated for their relationship to objective response rate (per RECIST, version 1.1). Logistic regression analysis of response for consensus signatures was adjusted for tumor type, Eastern Cooperative Oncology Group performance status, and TcellinfGEP, an approach equivalent to evaluating the association between response and the residuals of consensus signatures after detrending them for their relationship with the TcellinfGEP (previously identified as a determinant of pembrolizumab response) and tumor type. Testing of the 10 prespecified non-TcellinfGEP consensus signatures for negative association [except proliferation (hypothesized positive association)] with response was adjusted for multiplicity. RESULTS: Covariance patterns of the 11 signatures (including TcellinfGEP) identified in Merck-Moffitt and The Cancer Genome Atlas datasets showed highly concordant coexpression patterns in the RNA-sequencing data from pembrolizumab trials. TcellinfGEP was positively associated with response; signatures for angiogenesis, mMDSC, and stroma/EMT/TGFß were negatively associated with response to pembrolizumab monotherapy. CONCLUSIONS: These findings suggest that features beyond IFNγ-related T-cell inflammation may be relevant to anti-programmed death 1 monotherapy response and may define other axes of tumor biology as candidates for pembrolizumab combinations. See related commentary by Cho et al., p. 1479.

Global Landscape of Clostridioides Difficile Phylogeography, Antibiotic Susceptibility, and Toxin Polymorphisms by Post-Hoc Whole-Genome Sequencing from the MODIFY I/II Studies.

INTRODUCTION: Clostridioides (Clostridium) difficile infection, the leading cause of healthcare-associated diarrhea, represents a significant burden on global healthcare systems. Despite being a global issue, information on C. difficile from a global perspective is lacking. The aim of this study is to model the global phylogeography of clinical C. difficile. METHODS: Using samples collected from the MODIFY I and II studies (NCT01241552, NCT01513239), we performed whole-genome sequencing of 1501 clinical isolates including 37 novel sequence types (STs), representing the largest worldwide collection to date. RESULTS: Our data showed ribotypes, multi-locus sequence typing clades, and whole-genome phylogeny were in good accordance. The clinical C. difficile genome was found to be more conserved than previously reported (61% core genes), and modest recombination rates of 1.4-5.0 were observed across clades. We observed a significant continent distribution preference among five C. difficile clades (Benjamini-Hochberg corrected Fisher's exact test P < 0.01); moreover, weak association between geographic and genetic distance among ribotypes suggested sources beyond healthcare-related transmission. Markedly different trends of antibiotic susceptibility among lineages and regions were identified, and three novel mutations (in pyridoxamine 5'-phosphate oxidase family protein: Tyr130Ser, Tyr130Cys, and a promoter SNP) associated with metronidazole-reduced susceptibility were discovered on a nim-related gene and its promotor by genome-wide association study. Toxin gene polymorphisms were shown to vary within and between prevalent ribotypes, and novel severe mutations were found on the tcdC toxin regulator protein. CONCLUSION: Our systematic characterization of a global set of clinical trial C. difficile isolates from infected individuals demonstrated the complexity of the genetic makeup of this pathogenic organism. The geographic variability of clades, variability in toxin genes, and mutations associated with antibiotic susceptibility indicate a highly complex interaction of C. difficile between host and environment. This dataset will provide a useful resource for validation of findings and future research of C. difficile.

The development and approval of tecoviromat (TPOXX®), the first antiviral against smallpox.

The classification of smallpox by the U.S. Centers for Disease Control and Prevention (CDC) as a Category A Bioterrorism threat agent has resulted in the U.S. Government investing significant funds to develop and stockpile a suite of medical countermeasures to ameliorate the consequences of a smallpox epidemic. This stockpile includes both vaccines for prophylaxis and antivirals to treat symptomatic patients. In this manuscript, we describe the path to approval for the first therapeutic against smallpox, identified during its development as ST-246, now known as tecovirimat and TPOXX®, a small-molecule antiviral compound sponsored by SIGA Technologies to treat symptomatic smallpox. Because the disease is no longer endemic, the development and approval of TPOXX® was only possible under the U.S. Food and Drug and Administration Animal Rule (FDA 2002). In this article, we describe the combination of animal model studies and clinical trials that were used to satisfy the FDA requirements for the approval of TPOXX ® under the Animal Rule.

Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy.

Programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) checkpoint blockade immunotherapy elicits durable antitumor effects in multiple cancers, yet not all patients respond. We report the evaluation of >300 patient samples across 22 tumor types from four KEYNOTE clinical trials. Tumor mutational burden (TMB) and a T cell-inflamed gene expression profile (GEP) exhibited joint predictive utility in identifying responders and nonresponders to the PD-1 antibody pembrolizumab. TMB and GEP were independently predictive of response and demonstrated low correlation, suggesting that they capture distinct features of neoantigenicity and T cell activation. Analysis of The Cancer Genome Atlas database showed TMB and GEP to have a low correlation, and analysis by joint stratification revealed biomarker-defined patterns of targetable-resistance biology. These biomarkers may have utility in clinical trial design by guiding rational selection of anti-PD-1 monotherapy and combination immunotherapy regimens.

IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade.

Programmed death-1-directed (PD-1-directed) immune checkpoint blockade results in durable antitumor activity in many advanced malignancies. Recent studies suggest that IFN-γ is a critical driver of programmed death ligand-1 (PD-L1) expression in cancer and host cells, and baseline intratumoral T cell infiltration may improve response likelihood to anti-PD-1 therapies, including pembrolizumab. However, whether quantifying T cell-inflamed microenvironment is a useful pan-tumor determinant of PD-1-directed therapy response has not been rigorously evaluated. Here, we analyzed gene expression profiles (GEPs) using RNA from baseline tumor samples of pembrolizumab-treated patients. We identified immune-related signatures correlating with clinical benefit using a learn-and-confirm paradigm based on data from different clinical studies of pembrolizumab, starting with a small pilot of 19 melanoma patients and eventually defining a pan-tumor T cell-inflamed GEP in 220 patients with 9 cancers. Predictive value was independently confirmed and compared with that of PD-L1 immunohistochemistry in 96 patients with head and neck squamous cell carcinoma. The T cell-inflamed GEP contained IFN-γ-responsive genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance, and these features were necessary, but not always sufficient, for clinical benefit. The T cell-inflamed GEP has been developed into a clinical-grade assay that is currently being evaluated in ongoing pembrolizumab trials.

Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples.

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Annexin 2: a novel human immunodeficiency virus type 1 Gag binding protein involved in replication in monocyte-derived macrophages.

Human immunodeficiency virus (HIV) replication in the major natural target cells, CD4+ T lymphocytes and macrophages, is parallel in many aspects of the virus life cycle. However, it differs as to viral assembly and budding, which take place on plasma membranes in T cells and on endosomal membranes in macrophages. It has been postulated that cell type-specific host factors may aid in directing viral assembly to distinct destinations. In this study we defined annexin 2 (Anx2) as a novel HIV Gag binding partner in macrophages. Anx2-Gag binding was confined to productively infected macrophages and was not detected in quiescently infected monocyte-derived macrophages (MDM) in which an HIV replication block was mapped to the late stages of the viral life cycle (A. V. Albright, R. M. Vos, and F. Gonzalez-Scarano, Virology 325:328-339, 2004). We demonstrate that the Anx2-Gag interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that Anx2 depletion is associated with a significant decline in the infectivity of released virions; this coincided with incomplete Gag processing and inefficient incorporation of CD63. Cumulatively, our data suggest that Anx2 is essential for the proper assembly of HIV in MDM.

Microarray analysis of activated mixed glial (microglia) and monocyte-derived macrophage gene expression.

Since macrophage activation can now be studied at a global level using modern microarray and proteomic analyses, discovery of novel macrophage activation genes is inevitable and important for understanding HIV-associated dementia (HAD). We isolated two different types of primary human macrophages: microglia and monocyte-derived macrophages (MDM) from brain tissue and whole blood, respectively. The microarray analysis of differentially regulated macrophage activation genes reported here supports our previous assertions that the mixed glia (MIX) cultured in starvation conditions (DMEM alone) are a non-activated, or "quiescent", tissue culture model for studying macrophage activation in the brain. Transcript levels from these quiescent cultures provided a background level of gene expression and allowed for the identification of upregulated macrophage activation genes in the MIX brain cultures upon treatment with an array of soluble activation factors: serum components, cytokines, and growth factors. We found that 914 genes in the MIX cultures and 734 genes in the MDM cultures had a greater than twofold increase in expression. We discovered 180 genes with expression that was increased more than twofold in both culture types. Microarray-specific statistical analyses were performed to complement fold change analysis: significance analysis of microarrays (SAM) and Partek Pro. In the MIX cultures, we detected over a 100-fold increase in IL-1beta and TIMP1 transcription; Caspase 9, S100A8 and 9, MMP12, IL-8, monocyte chemotactic protein 1 (MCP1), MRC-1, and IL-6 were also upregulated. Activation of starved MDM cultures resulted in fewer upregulated genes compared to MIX cultures. Genes upregulated in both MIX and MDM included CCL2 (MCP1), CCL7, CXCL5, TNFSF14, kinases, and phosphatases. These microarray data may provide leads for identifying previously unknown neurotoxins, disease biomarkers, and pathways responsible for the neuronal apoptosis observed in HAD and for the eventual identification of therapeutic targets and treatments.

Low-level HIV replication in mixed glial cultures is associated with alterations in the processing of p55(Gag).

We report a novel long-lived infection model in human mixed glial cultures (microglia) whereby cells harbor replication-competent HIV-1 for up to 2.5 months after infection; a model that potentially mimics latency within the central nervous system (CNS). Infection of mixed glial cultures in the presence of serum, cytokines, and growth factors (activating conditions) resulted in a robust productive infection of microglial cells as previously described for purified microglia. In contrast, similar mixed glial cells cultured in serum-free medium without cytokines or growth factors (mirroring a nonactivated CNS) supported HIV-1 entry, reverse transcription, integration, and transcription, yet released little or no infectious virus. We found instead that nonactivated mixed glial cells expressed almost 10-fold less Gag protein, but more importantly, analysis of the intracellular Gag products in quiescent cells showed an aberrant p55/p24 Gag processing phenotype that appeared to be due to the premature activity of the viral protease. These results suggest that the cellular environment in nonactivated microglia cells in these mixed glial cultures is not conducive to proper Gag processing and virus release. This long-lived infection model will be useful in identifying factors that are key for viral maturation in cells of the macrophage lineage.

Pathogenesis of human immunodeficiency virus-induced neurological disease.

Infection of the central nervous system by the type 1 human immunodeficiency virus (HIV-1) commonly results in a number of neurological impairments known, in their most severe form, as HIV-associated dementia (HAD). The persistence of HIV encephalitis (HIVE), the pathological correlate of HAD, in spite of highly active antiretroviral therapy (HAART) underscores the importance of continued research focused on the neurobiology of HIV. To elucidate direct and indirect mechanisms of HIV neuropathogenesis, current investigation is focused on neuroinvasion, HIV-1-mediated mechanisms of neuronal damage and apoptosis, and compartmentalized evolution of virus in the brain. The aim of this review is to provide a selective overview of the most recent research on the neurobiology of HIV, adding only a brief introduction regarding established principles.