Concordance between an FDA-approved companion diagnostic and an alternative assay kit for assessing homologous recombination deficiency in ovarian cancer.

OBJECTIVE: Authors evaluated the performance of a commercially available next-generation sequencing assay kit; this was based on genomic content from Illumina's TruSight™ Oncology 500 research assay that identifies BRCA variants and proprietary algorithms licensed from Myriad and, with additional genomic content, measures the homologous recombination deficiency (HRD) genomic instability score (GIS) in tumor tissue (TSO 500 HRD assay). METHODS: Data from the TSO 500 HRD assay were compared with data from the Myriad MyChoice®CDx PLUS assay (Myriad assay). Prevalence rates for overall HRD status and BRCA mutations (a deleterious or suspected deleterious BRCA1 or BRCA2 mutation or both) and assay agreement rates for HRD GIS and BRCA analysis were assessed in ovarian tumor samples. Pearson correlations of the continuous HRD GIS and analytic sensitivity and specificity were evaluated. RESULTS: The prevalence of overall HRD positivity was 51.2% (TSO 500 HRD assay) versus 49.2% (Myriad assay) and the prevalence of BRCA mutations was 27.6% (TSO 500 HRD assay) versus 25.5% (Myriad assay). After post-processing optimization, concordance of the HRD GIS was 0.980 in all samples and 0.976 in the non-BRCA mutation cohort; the area under the receiver operating characteristic curve was 0.995 and 0.992, respectively. CONCLUSIONS: Comparison between the Illumina and Myriad assays showed that overall HRD status, the individual components of BRCA analysis, and HRD GIS detection results were highly concordant (>93%), suggesting the TSO 500 HRD assay will approach the analytical accuracy of the FDA-approved Myriad assay.

Evaluation of potential biomarkers for lenvatinib plus pembrolizumab among patients with advanced endometrial cancer: results from Study 111/KEYNOTE-146.

BACKGROUND: Lenvatinib plus pembrolizumab demonstrated clinically meaningful benefit in patients with previously treated advanced endometrial carcinoma in Study 111/KEYNOTE-146 (NCT02501096). In these exploratory analyses from this study, we evaluated the associations between clinical outcomes and gene expression signature scores and descriptively summarized response in biomarker subpopulations defined by tumor mutational burden (TMB) and DNA variants for individual genes of interest. METHODS: Patients with histologically confirmed metastatic endometrial carcinoma received oral lenvatinib 20 mg once daily plus intravenous pembrolizumab 200 mg every 3 weeks for 35 cycles. Archived formalin-fixed paraffin-embedded tissue was obtained from all patients. T-cell-inflamed gene expression profile (TcellinfGEP) and 11 other gene signatures were evaluated by RNA sequencing. TMB, hotspot mutations in PIK3CA (oncogene), and deleterious mutations in PTEN and TP53 (tumor suppressor genes) were evaluated by whole-exome sequencing (WES). RESULTS: 93 and 79 patients were included in the RNA-sequencing-evaluable and WES-evaluable populations, respectively. No statistically significant associations were observed between any of the RNA-sequencing signature scores and objective response rate or progression-free survival. Area under the receiver operating characteristic curve values for response ranged from 0.39 to 0.54; all 95% CIs included 0.50. Responses were seen regardless of TMB (≥175 or <175 mutations/exome) and mutation status. There were no correlations between TcellinfGEP and TMB, TcellinfGEP and microvessel density (MVD), or MVD and TMB. CONCLUSIONS: This analysis demonstrated efficacy for lenvatinib plus pembrolizumab regardless of biomarker status. Results from this study do not support clinical utility of the evaluated biomarkers. Further investigation of biomarkers for this regimen is warranted. TRIAL REGISTRATION NUMBER: NCT02501096.

Biomarker Testing Journey Among Patients with Advanced Solid Tumors and Treatment Patterns by Homologous Recombination Repair Status: A Clinico-Genomic Database Study.

INTRODUCTION: Defects in the homologous recombination repair (HRR) pathway can include mutations in BRCA1 and BRCA2 (BRCAm) and other HRR genes (HRRm). These mutations are associated with a homologous recombination deficiency (HRD) phenotype. We evaluated testing journey and treatment patterns by BRCAm, HRRm, and HRD status in a real-world dataset. METHODS: Deidentified data for patients who had undergone comprehensive genomic profiling using FoundationOne®CDx were collected through December 31, 2020, from a real-world multi-tumor clinico-genomic database (CGDB) capturing data from clinics in the United States. Patients eligible for inclusion in this analysis had a confirmed diagnosis with advanced or metastatic disease between January 1, 2018, and December 31, 2019, for 1 of 15 solid tumor types. Objectives were to evaluate patient treatment patterns by BRCAm, HRRm, and HRD status and to describe the timing of when (throughout disease course) comprehensive genomic profiling was performed. RESULTS: Among 9457 patients included in the overall population with evaluable biomarker status, 7856 (83.1%) received ≥ 1 systemic therapy. Among the 7856 patients who received systemic therapy, 2324 (30.0%) underwent testing before first-line therapy, 4114 (52.4%) were tested after receiving first-line therapy and before receiving subsequent therapy (if any), 970 (12.3%) were tested after second-line therapy and before receiving subsequent therapy (if any), and 447 (5.7%) patients underwent testing after receiving third-line therapy. A higher proportion of patients with BRCAm, HRRm, or HRD-positive status were treated with poly(ADP-ribose) polymerase (PARP) inhibitors across all lines of therapy. There was no evidence of a meaningful difference in the proportion of patients who received other treatment (including chemotherapy and immunotherapy) by BRCAm, HRRm, or HRD status. CONCLUSION: The majority of patients from this real-world dataset underwent FoundationOne®CDx testing after initiation of first-line treatment. Testing appeared to influence treatment patterns, with a higher proportion of patients with BRCAm, HRRm, and HRD-positive disease receiving PARP inhibitors.

Real-world prevalence of homologous recombination repair mutations in advanced prostate cancer: an analysis of two clinico-genomic databases.

BACKGROUND: Homologous recombination repair mutation (HRRm) status may guide risk-stratification and treatment decisions, including polyadenosine diphosphate-ribose polymerase inhibitor use, in advanced prostate cancer. Although HRRm prevalence has been reported in single-institution studies or clinical trials, real-world HRRm prevalence in diverse populations is unknown. We describe HRRm in the clinical setting using two real-world clinicogenomic databases: the Flatiron Health and Foundation Medicine, Inc. Clinico-Genomic Database (CGDB), a national electronic health record-derived database, and the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE). METHODS: This cross-sectional analysis included 3757 individuals diagnosed with prostate cancer who had next generation sequencing (NGS) as standard of care. The CGDB included men with advanced/metastatic prostate cancer and genetic data included both germline and somatic pathogenic mutations. The GENIE analysis included men with prostate cancer whose received NGS as standard of care, but the data were filtered to include somatic mutations only. Due to key differences among databases, direct comparisons were not possible. Overall prevalence of HRRm was calculated and stratified by demographic and clinical characteristics. RESULTS: HRRm prevalence (combined germline and somatic) in CGDB (n = 487) was 24.6% (95% CI 20.9-28.7%), with no major differences across demographic and disease characteristic subgroups. HRRm prevalence (somatic) in GENIE (n = 3270) was 11.0% (95% CI 10.0-12.1%), which varied between 9.5% and 18.4% across treatment centers. CONCLUSIONS: Approximately one-quarter of patients with advanced/metastatic prostate cancer in the CGDB had germline and/or somatic HRRm, which is consistent with clinical trials such as the PROfound study that used a similar NGS platform and algorithm to define HRRm. In the GENIE database, HRRm prevalence varied by treatment center or NGS platform. More research is needed to understand real-world HRRm prevalence variations.

Association Between Homologous Recombination Repair Biomarkers and Survival in Patients With Solid Tumors.

PURPOSE: Mutations in BRCA1 and/or BRCA2 (BRCAm), other homologous recombination repair genes (HRRm), and homologous recombination deficiency (HRD) lead to an accumulation of genomic alterations that can drive tumorigenesis. The prognostic impact of these HRR pathway defects on overall survival (OS) in patients not receiving poly (ADP-ribose) polymerase inhibitors (PARPi) or immunotherapy is unclear. We evaluated the association of HRR biomarkers with OS in patients with advanced solid tumors receiving therapy excluding PARPi and immunotherapy. METHODS: Deidentified data were collected through December 31, 2020, from a real-world clinicogenomic database (CGDB) with data originating from approximately 280 cancer clinics in the United States. Patients age 18 years and older with an advanced/metastatic diagnosis between 2018 and 2019 for 1 of 15 solid tumors and available data in the CGDB were included. The primary analysis evaluated the association between HRR pathway biomarkers and OS, using start of second-line therapy as the index date (to reduce immortal time bias). RESULTS: A total of 9,457 patients had available data for BRCA/HRR and 5,792 for HRD status; 4,890 (51.7%) were women and mean (SD) age was 65.9 (11.5) years. For the primary analysis, adjusted hazard ratios for OS were BRCAm (n = 156) versus BRCA wild-type (wt; n = 3,131; 0.83 [95% CI, 0.60 to 1.17]); for HRRm (n = 467) versus HRRwt (n = 282; 0.95 [95% CI, 0.79 to 1.14]); and for HRD-positive (n = 447) versus -negative (n = 1,687; 1.22 [95% CI, 1.02 to 1.47]). Results were similar using start of first-line and start of third-line therapy as index dates. CONCLUSION: This large, real-world study found no association between OS and either BRCA or HRR status but identified a possible linkage between HRD positivity and shorter median OS in patients with advanced solid tumors who did not receive PARPi or immunotherapy.

Molecular determinants of clinical outcomes of pembrolizumab in recurrent ovarian cancer: Exploratory analysis of KEYNOTE-100.

OBJECTIVE: This prespecified exploratory analysis evaluated the association of gene expression signatures, tumor mutational burden (TMB), and multiplex immunohistochemistry (mIHC) tumor microenvironment-associated cell phenotypes with clinical outcomes of pembrolizumab in advanced recurrent ovarian cancer (ROC) from the phase II KEYNOTE-100 study. METHODS: Pembrolizumab-treated patients with evaluable RNA-sequencing (n = 317), whole exome sequencing (n = 293), or select mIHC (n = 125) data were evaluated. The association between outcomes (objective response rate [ORR], progression-free survival [PFS], and overall survival [OS]) and gene expression signatures (T-cell-inflamed gene expression profile [TcellinfGEP] and 10 non-TcellinfGEP signatures), TMB, and prespecified mIHC cell phenotype densities as continuous variables was evaluated using logistic (ORR) and Cox proportional hazards regression (PFS; OS). One-sided p-values were calculated at prespecified α = 0.05 for TcellinfGEP, TMB, and mIHC cell phenotypes and at α = 0.10 for non-TcellinfGEP signatures; all but TcellinfGEP and TMB were adjusted for multiplicity. RESULTS: No evidence of associations between ORR and key axes of gene expression was observed. Negative associations were observed between outcomes and TcellinfGEP-adjusted glycolysis (PFS, adjusted-p = 0.019; OS, adjusted-p = 0.085) and hypoxia (PFS, adjusted-p = 0.064) signatures. TMB as a continuous variable was not associated with outcomes (p > 0.05). Positive associations were observed between densities of myeloid cell phenotypes CD11c+ and CD11c+/MHCII-/CD163-/CD68- in the tumor compartment and ORR (adjusted-p = 0.025 and 0.013, respectively). CONCLUSIONS: This exploratory analysis in advanced ROC did not find evidence for associations between gene expression signatures and outcomes of pembrolizumab. mIHC analysis suggests CD11c+ and CD11c+/MHCII-/CD163-/CD68- phenotypes representing myeloid cell populations may be associated with improved outcomes with pembrolizumab in advanced ROC. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT02674061.

Association between gene expression signatures and clinical outcomes of pembrolizumab versus paclitaxel in advanced gastric cancer: exploratory analysis from the randomized, controlled, phase III KEYNOTE-061 trial.

BACKGROUND: In the randomized, controlled, phase III KEYNOTE-061 trial, second-line pembrolizumab did not significantly prolong overall survival (OS) versus paclitaxel in patients with PD-L1-positive (combined positive score ≥1) advanced gastric/gastroesophageal junction (G/GEJ) cancer but did elicit a longer duration of response and offered a favorable safety profile. This prespecified exploratory analysis was conducted to evaluate associations between tumor gene expression signatures and clinical outcomes in the phase III KEYNOTE-061 trial. METHODS: Using RNA sequencing data obtained from formalin-fixed, paraffin-embedded baseline tumor tissue samples, we evaluated the 18-gene T-cell-inflamed gene expression profile (TcellinfGEP) and 10 non-TcellinfGEP signatures (angiogenesis, glycolysis, granulocytic myeloid-derived suppressor cell (gMDSC), hypoxia, monocytic MDSC (mMDSC), MYC, proliferation, RAS, stroma/epithelial-to-mesenchymal transition/transforming growth factor-ß, WNT). The association between each signature on a continuous scale and outcomes was analyzed using logistic (objective response rate (ORR)) and Cox proportional hazards regression (progression-free survival (PFS) and OS). One-sided (pembrolizumab) and two-sided (paclitaxel) p values were calculated for TcellinfGEP (prespecified α=0.05) and the 10 non-TcellinfGEP signatures (multiplicity-adjusted; prespecified α=0.10). RESULTS: RNA sequencing data were available for 137 patients in each treatment group. TcellinfGEP was positively associated with ORR (p=0.041) and PFS (p=0.026) for pembrolizumab but not paclitaxel (p>0.05). The TcellinfGEP-adjusted mMDSC signature was negatively associated with ORR (p=0.077), PFS (p=0.057), and OS (p=0.033) for pembrolizumab, while the TcellinfGEP-adjusted glycolysis (p=0.018), MYC (p=0.057), and proliferation (p=0.002) signatures were negatively associated with OS for paclitaxel. CONCLUSIONS: This exploratory analysis of tumor TcellinfGEP showed associations with ORR and PFS for pembrolizumab but not for paclitaxel. TcellinfGEP-adjusted mMDSC signature was negatively associated with ORR, PFS, and OS for pembrolizumab but not paclitaxel. These data suggest myeloid-driven suppression may play a role in resistance to PD-1 inhibition in G/GEJ cancer and support a strategy of considering immunotherapy combinations which target this myeloid axis. TRIAL REGISTRATION NUMBER: NCT02370498.

Biomarker-directed, pembrolizumab-based combination therapy in non-small cell lung cancer: phase 2 KEYNOTE-495/KeyImPaCT trial interim results.

Although pembrolizumab confers clinical benefit in non-small cell lung cancer (NSCLC), only a subset of patients will respond due to a heterogenous tumor microenvironment. KEYNOTE-495/KeyImPaCT is an ongoing biomarker-directed, adaptively randomized phase 2 study investigating first-line pembrolizumab (200 mg every 3 weeks) + lenvatinib (20 mg daily), anti-CTLA-4 quavonlimab (25 mg every 6 weeks) or anti-LAG-3 favezelimab (200 mg or 800 mg every 3 weeks) in advanced NSCLC. Patients were categorized by T-cell-inflamed gene expression profile (TcellinfGEP) and tumor mutational burden (TMB) status and randomly assigned 1:1:1 to receive pembrolizumab + lenvatinib, pembrolizumab + quavonlimab or pembrolizumab + favezelimab. The primary outcome was investigator-assessed objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1 using pre-specified efficacy thresholds for each biomarker-defined subgroup (>5% (TcellinfGEPlowTMBnon-high (group I)), >20% (TcellinfGEPlowTMBhigh (group II) and TcellinfGEPnon-lowTMBnon-high (group III)) and >45% (TcellinfGEPnon-lowTMBhigh (group IV))). Secondary outcomes were progression-free survival, overall survival and safety. At data cutoff, ORR ranges were 0-12.0% in group I, 27.3-33.3% in group II, 13.6-40.9% in group III and 50.0-60.0% in group IV. ORR with pembrolizumab + lenvatinib in group III met the pre-specified efficacy threshold. The safety profile of each treatment arm was consistent with the known safety profile of each combination. These data demonstrate the feasibility of prospective TcellinfGEP and TMB assessment to study the clinical activity of first-line pembrolizumab-based combination therapies in advanced NSCLC. ClinicalTrials.gov registration: NCT03516981 .

Association Between Biomarkers and Clinical Outcomes of Pembrolizumab Monotherapy in Patients With Metastatic Triple-Negative Breast Cancer: KEYNOTE-086 Exploratory Analysis.

PURPOSE: In the two-cohort phase II KEYNOTE-086 study (ClinicalTrials.gov identifier: NCT02447003), first-line and second-line or later pembrolizumab monotherapy demonstrated antitumor activity in metastatic triple-negative breast cancer (mTNBC; N = 254). This exploratory analysis evaluates the association between prespecified molecular biomarkers and clinical outcomes. METHODS: Cohort A enrolled patients with disease progression after one or more systemic therapies for metastatic disease irrespective of PD-L1 status; Cohort B enrolled patients with previously untreated PD-L1-positive (combined positive score [CPS] ≥ 1) metastatic disease. The association between the following biomarkers as continuous variables and clinical outcomes (objective response rate [ORR], progression-free survival [PFS], and overall survival [OS]) was evaluated: PD-L1 CPS (immunohistochemistry), cluster of differentiation 8 (CD8; immunohistochemistry), stromal tumor-infiltrating lymphocyte (sTIL; hematoxylin and eosin staining), tumor mutational burden (TMB; whole-exome sequencing [WES]), homologous recombination deficiency-loss of heterozygosity, mutational signature 3 (WES), mutational signature 2 (apolipoprotein B mRNA editing catalytic polypeptide-like; WES), T-cell-inflamed gene expression profile (TcellinfGEP; RNA sequencing), and 10 non-TcellinfGEP signatures (RNA sequencing); Wald test P values were calculated, and significance was prespecified at α = 0.05. RESULTS: In the combined cohorts (A and B), PD-L1 (P = .040), CD8 (P < .001), sTILs (P = .012), TMB (P = .007), and TcellinfGEP (P = .011) were significantly associated with ORR; CD8 (P < .001), TMB (P = .034), Signature 3 (P = .009), and TcellinfGEP (P = .002) with PFS; and CD8 (P < .001), sTILs (P = .004), TMB (P = .025), and TcellinfGEP (P = .001) with OS. None of the non-TcellinfGEP signatures were associated with outcomes of pembrolizumab after adjusting for the TcellinfGEP. CONCLUSION: In this exploratory biomarker analysis from KEYNOTE-086, baseline tumor PD-L1, CD8, sTILs, TMB, and TcellinfGEP were associated with improved clinical outcomes of pembrolizumab and may help identify patients with mTNBC who are most likely to respond to pembrolizumab monotherapy.

Associations of Tissue Tumor Mutational Burden and Mutational Status With Clinical Outcomes With Pembrolizumab Plus Chemotherapy Versus Chemotherapy For Metastatic NSCLC.

Introduction: We evaluated tissue tumor mutational burden (tTMB) and mutations in STK11, KEAP1, and KRAS as biomarkers for outcomes with pembrolizumab plus platinum-based chemotherapy (pembrolizumab-combination) for NSCLC among patients in the phase 3 KEYNOTE-189 (ClinicalTrials.gov, NCT02578680; nonsquamous) and KEYNOTE-407 (ClinicalTrials.gov, NCT02775435; squamous) trials. Methods: This retrospective exploratory analysis evaluated prevalence of high tTMB and STK11, KEAP1, and KRAS mutations in patients enrolled in KEYNOTE-189 and KEYNOTE-407 and the relationship between these potential biomarkers and clinical outcomes. tTMB and STK11, KEAP1, and KRAS mutation status was assessed using whole-exome sequencing in patients with available tumor and matched normal DNA. The clinical utility of tTMB was assessed using a prespecified cutpoint of 175 mutations/exome. Results: Among patients with evaluable data from whole-exome sequencing for evaluation of tTMB (KEYNOTE-189, n = 293; KEYNOTE-407, n = 312) and matched normal DNA, no association was found between continuous tTMB score and overall survival (OS) or progression-free survival for pembrolizumab-combination (Wald test, one-sided p > 0.05) or placebo-combination (Wald test, two-sided p > 0.05) in patients with squamous or nonsquamous histology. Pembrolizumab-combination improved outcomes for patients with tTMB greater than or equal to 175 compared with tTMB less than 175 mutations/exome in KEYNOTE-189 (OS, hazard ratio = 0.64 [95% confidence interval (CI): 0.38‒1.07] and 0.64 [95% CI: 0.42‒0.97], respectively) and KEYNOTE-407 (OS, hazard ratio = 0.74 [95% CI: 0.50‒1.08 and 0.86 [95% CI: 0.57‒1.28], respectively) versus placebo-combination. Treatment outcomes were similar regardless of KEAP1, STK11, or KRAS mutation status. Conclusions: These findings support pembrolizumab-combination as first-line treatment in patients with metastatic NSCLC and do not suggest the utility of tTMB, STK11, KEAP1, or KRAS mutation status as a biomarker for this regimen.

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.

Baseline and post-treatment biomarkers of resistance to anti-PD-1 therapy in acral and mucosal melanoma: an observational study.

BACKGROUND: Immunotherapies targeting programmed cell death-1 (PD-1) and its ligands have improved clinical outcomes for advanced melanoma. However, many tumors exhibit primary resistance or acquire secondary resistance after an initial positive response. The mechanisms of resistance are not well understood, and no validated predictive biomarkers are available. This exploratory study aimed to characterize baseline differences and molecular changes arising during treatment in acral and mucosal melanomas that exhibited primary or secondary resistance to anti-PD-1 monotherapy. METHODS: This was an observational retrospective study of 124 patients who had been treated for metastatic acral or mucosal melanoma with anti-PD-1 monotherapy. Tumor samples were collected at baseline (all patients) and post-treatment (resistant tumors only) and were assayed by immunohistochemistry, whole-exome sequencing, and RNA sequencing. RESULTS: At baseline, more non-progressor than resistant tumors exhibited expression of PD-L1, immune cell infiltration, and high tumor mutational burden (TMB); baseline PD-L1 expression was also more common in secondary-resistant than in primary-resistant tumors as well as in late versus early secondary-resistant tumors. Non-progressor tumors also had higher median baseline expression of an 18-gene T cell-inflamed gene expression profile (TcellinfGEP). Among resistant tumors, the proportion of PD-L1-positive melanomas and the expression of the TcellinfGEP mRNA signature increased during treatment, while the expression of mRNA signatures related to WNT and INFA1 signaling decreased. There was evidence for greater changes from baseline in secondary-resistant versus primary-resistant tumors for some markers, including expression of RAS-related and WNT-related mRNA signatures and density of CD11c+ and FOXP3+ T cells. Greater changes in CD11c+ cell density were observed in early compared with late secondary-resistant tumors. CONCLUSIONS: Our findings suggest that TcellinfGEP and PD-L1 expression, TMB, immune cell infiltration, and RAS and WNT signaling warrant further investigation as potential mechanisms and/or biomarkers of anti-PD-1 therapy resistance in acral and mucosal melanomas. Confirmation of these findings in larger populations is needed.

Association of Tumor Mutational Burden with Efficacy of Pembrolizumab±Chemotherapy as First-Line Therapy for Gastric Cancer in the Phase III KEYNOTE-062 Study.

PURPOSE: This prespecified exploratory analysis evaluated the association between tumor mutational burden (TMB) status and outcomes of first-line pembrolizumab±chemotherapy versus chemotherapy in KEYNOTE-062. PATIENTS AND METHODS: In patients with advanced gastric cancer and evaluable TMB data, we evaluated the association between TMB (continuous variable; square root scale) assessed with FoundationOne CDx and clinical outcomes [objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)] using logistic (ORR) and Cox proportional hazards (PFS, OS) regression models. Clinical utility of TMB was assessed using the prespecified cutoff of 10 mut/Mb. RESULTS: TMB data were available for 306 of 763 patients (40.1%; pembrolizumab, 107; pembrolizumab+chemotherapy, 100; chemotherapy, 99). TMB was significantly associated with clinical outcomes in patients treated with pembrolizumab and pembrolizumab+chemotherapy (ORR, PFS, and OS; all P < 0.05) but not with chemotherapy (all P > 0.05). The overall prevalence of TMB ≥10 mut/Mb was 16% across treatment groups; 44% of patients who had TMB ≥10 mut/Mb had high microsatellite instability (MSI-H) tumors. Improved clinical outcomes (ORR, PFS, and OS) were observed in pembrolizumab-treated patients (pembrolizumab monotherapy and pembrolizumab+chemotherapy) with TMB ≥10 mut/Mb. When the analysis was limited to the non-MSI-H subgroup, both the positive association between clinical outcomes with pembrolizumab or pembrolizumab+chemotherapy and TMB as a continuous variable and the clinical utility of pembrolizumab (with or without chemotherapy) versus chemotherapy by TMB cutoff were attenuated. CONCLUSIONS: This exploratory analysis of KEYNOTE-062 suggests an association between TMB and clinical efficacy with first-line pembrolizumab-based therapy in patients with advanced gastric/gastroesophageal junction adenocarcinoma. However, after the exclusion of patients with MSI-H tumors, the clinical utility of TMB was attenuated.

Immune Biomarkers in Metastatic Castration-resistant Prostate Cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease in which molecular stratification is needed to improve clinical outcomes. The identification of predictive biomarkers can have a major impact on the care of these patients, but the availability of metastatic tissue samples for research in this setting is limited. OBJECTIVE: To study the prevalence of immune biomarkers of potential clinical utility to immunotherapy in mCRPC and to determine their association with overall survival (OS). DESIGN, SETTING, AND PARTICIPANTS: From 100 patients, mCRPC biopsies were assayed by whole exome sequencing, targeted next-generation sequencing, RNA sequencing, tumor mutational burden, T-cell-inflamed gene expression profile (TcellinfGEP) score (Nanostring), and immunohistochemistry for programmed cell death 1 ligand 1 (PD-L1), ataxia-telangiectasia mutated (ATM), phosphatase and tensin homolog (PTEN), SRY homology box 2 (SOX2), and the presence of neuroendocrine features. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The phi coefficient determined correlations between biomarkers of interest. OS was assessed using Kaplan-Meier curves and adjusted hazard ratios (aHRs) from Cox regression. RESULTS AND LIMITATIONS: PD-L1 and SOX2 protein expression was detected by immunohistochemistry (combined positive score ≥1 and >5% cells, respectively) in 24 (33%) and 27 (27%) mCRPC biopsies, respectively; 23 (26%) mCRPC biopsies had high TcellinfGEP scores (>-0.318). PD-L1 protein expression and TcellinfGEP scores were positively correlated (phi 0.63 [0.45; 0.76]). PD-L1 protein expression (aHR: 1.90 [1.05; 3.45]), high TcellinfGEP score (aHR: 1.86 [1.04; 3.31]), and SOX2 expression (aHR: 2.09 [1.20; 3.64]) were associated with worse OS. CONCLUSIONS: PD-L1, TcellinfGEP score, and SOX2 are prognostic of outcome from the mCRPC setting. If validated, predictive biomarker studies incorporating survival endpoints need to take these findings into consideration. PATIENT SUMMARY: This study presents an analysis of immune biomarkers in biopsies from patients with metastatic prostate cancer. We describe tumor alterations that predict prognosis that can impact future studies.

Putative Biomarkers of Clinical Benefit With Pembrolizumab in Advanced Urothelial Cancer: Results from the KEYNOTE-045 and KEYNOTE-052 Landmark Trials.

PURPOSE: In an exploratory analysis, we investigated the association between programmed death ligand 1 (PD-L1), tumor mutational burden (TMB), T-cell-inflamed gene expression profile (TcellinfGEP), and stromal signature with outcomes of pembrolizumab in urothelial carcinoma (UC). PATIENTS AND METHODS: Patients with advanced UC received first-line pembrolizumab 200 mg every 3 weeks in the single-arm phase II KEYNOTE-052 trial (NCT02335424) and salvage pembrolizumab 200 mg every 3 weeks or chemotherapy (paclitaxel/docetaxel/vinflunine) in the randomized phase III KEYNOTE-045 trial (NCT02256436). The association of each biomarker (continuous variable) with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) was evaluated using logistic regression (ORR) and Cox PH (PFS, OS), adjusted for ECOG PS; nominal P values were calculated without multiplicity adjustment (one-sided, pembrolizumab; two-sided, chemotherapy). Significance was prespecified at α = 0.05. RESULTS: In KEYNOTE-052, PD-L1, TMB, and TcellinfGEP were significantly associated with improved outcomes; stromal signature was significantly associated with worse outcomes. In KEYNOTE-045, although findings for TMB and TcellinfGEP with pembrolizumab were consistent with those of KEYNOTE-052, PD-L1 was not significantly associated with improved outcomes, nor was stromal signature associated with worse outcomes with pembrolizumab; chemotherapy was not associated with outcomes in a consistent manner for any of the biomarkers. Hazard ratio (HR) estimates at prespecified cutoffs showed an advantage for pembrolizumab versus chemotherapy regardless of PD-L1 or TMB, with a trend toward lower HRs in the combined positive score ≥10 and the TMB ≥175 mutation/exome subgroup. For TcellinfGEP, PFS and OS HRs were lower in the TcellinfGEP-nonlow subgroup regardless of treatment. CONCLUSIONS: Multiple biomarkers characterizing the tumor microenvironment may help predict response to pembrolizumab monotherapy in UC, and potential clinical utility of these biomarkers may be context-dependent.

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.

Evaluation of the TruSight Oncology 500 Assay for Routine Clinical Testing of Tumor Mutational Burden and Clinical Utility for Predicting Response to Pembrolizumab.

Pembrolizumab is approved for treating patients with unresectable or metastatic solid tumors with high tumor mutational burden (TMB), as assessed by the Food and Drug Administration-approved companion diagnostic FoundationOneCDx, after progression on prior treatment. To expand TMB assessment for enriching response to pembrolizumab, TMB measurement from TruSight Oncology 500 (TSO500) was evaluated in archival pan-tumor samples from 294 patients enrolled in eight pembrolizumab monotherapy studies. TSO500 is a panel-based next-generation sequencing assay with broad availability, quick turnaround time, and a standardized bioinformatics pipeline. TSO500 TMB was evaluated for correlation and concordance with two reference methods: FoundationOneCDx and whole-exome sequencing. The TSO500 cut-off for TMB-high was selected based on the receiver-operating characteristic curve analysis against each reference method's cut-off for TMB-high. Clinical utility of the selected TSO500 cut-off (10 mutations/Mb) was assessed by calculating the sensitivity, specificity, positive and negative predictive values, and objective response rate enrichment. There was high correlation and concordance of TSO500 TMB with both reference methods. TSO500 TMB was associated significantly with the best overall response, and the selected cut-off had comparable clinical utility with respective cut-offs for the reference methods in predicting response to pembrolizumab. As a commercial assay with global kit distribution complete with an off-the-shelf software package, TSO500 may provide additional access to immunotherapy for patients with tumors with TMB ≥10 mutations/Mb.

Hepatocellular carcinoma patients with high circulating cytotoxic T cells and intra-tumoral immune signature benefit from pembrolizumab: results from a single-arm phase 2 trial.

BACKGROUND: A limited number of studies have characterized genomic properties of hepatocellular carcinoma (HCC) patients in response to anti-PD-1 immunotherapy. METHODS: Herein, we performed comprehensive molecular characterization of immediate (D-42 to D-1) pre-treatment tumor biopsy specimens from 60 patients with sorafenib-failed HCC in a single-arm prospective phase II trial of pembrolizumab. Objective response rate was the primary efficacy endpoint. We used whole-exome sequencing, RNA sequencing, and correlative analysis. In addition, we performed single-cell RNA sequencing using peripheral blood mononuclear cells. RESULTS: The overall response rate of pembrolizumab in sorafenib-failed HCC patients was 10% ([6/60] 95% CI, 2.4-17.6). In a univariate analysis using clinicopathological features, female gender, PD-L1 positivity, and low neutrophil-to-lymphocyte ratio (NLR) were identified as contributing factors to pembrolizumab response. Somatic mutations in CTNNB1 and genomic amplifications in MET were found only in non-responders. Transcriptional profiles through RNA sequencing identified that pembrolizumab responders demonstrated T cell receptor (TCR) signaling activation with expressions of MHC genes, indicating increased levels of T cell cytotoxicity. In single-cell sequencing from 10 pre- and post-treatment peripheral blood mononuclear cells (PBMCs), patients who achieved a partial response or stable disease exhibited immunological shifts toward cytotoxic CD8+ T cells. Conversely, patients with progressive disease showed an increased number of both CD14+ and CD16+ monocytes and activation of neutrophil-associated pathways. CONCLUSIONS: Taken together, HCC patients with infiltration of cytotoxic T cells, along with increased active circulating CD8+ T cells during pembrolizumab treatment and down-regulation of neutrophil-associated markers, significantly benefited from pembrolizumab treatment. TRIAL REGISTRATION: NCT#03163992 (first posted: May 23, 2017).