Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

A Novel HRD Signature Is Predictive of FOLFIRINOX Benefit in Metastatic Pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) represents an aggressive disease with median overall survival (OS) of less than 1 year in the front-line setting. FOLFIRINOX and gemcitabine and paclitaxel (GP) are standard of care options for these patients; however, optimal selection of therapy is challenging. METHODS: Comprehensive genomic profiling was performed on 8358 PC patients. Outcomes were available for 1149 metastatic PC patients treated with 1L FOLFIRINOX or GP. A scar-based measure of HRD was called using a machine learning-based algorithm incorporating copy number and indel features. RESULTS: A scar-based HRD signature (HRDsig) was identified in 9% of patients. HRDsig significantly co-occurred with biallelic alterations in BRCA1/2, PALB2, BARD1, and RAD51C/D, but encompassed a larger population than that defined by BRCA1/BRCA2/PALB2 (9% vs. 6%). HRDsig was predictive of 1L FOLFIRNOX chemotherapy benefit with doubled OS relative to gemcitabine and paclitaxel (GP) (rwOS aHR 0.37 [0.22-0.62]), including 25% of the population with long-term (2 year+) survival in a real-world cohort of patients. Less benefit from FOLFIRINOX was observed in the HRDsig(-) population. Predictive value was seen in both the BRCA1/2/PALB2 mutant and wildtype populations, suggesting additional value to mutational profiling. CONCLUSION: A scar-based HRD biomarker was identified in a significant fraction of PC patients and is predictive of FOLFIRINOX benefit. Incorporating a biomarker like HRDsig could identify the right patients for platinum chemotherapy and potentially reduce FOLFIRINOX use by over 40%, minimizing toxicities with similar survival outcomes. Confirmatory studies should be performed.

Association of CD274 (PD-L1) Copy Number Changes with Immune Checkpoint Inhibitor Clinical Benefit in Non-Squamous Non-Small Cell Lung Cancer.

BACKGROUND: We sought to characterize response to immune checkpoint inhibitor (ICI) in non-squamous non-small cell lung cancer (NSCLC) across various CD274 copy number gain and loss thresholds and identify an optimal cutoff. MATERIALS AND METHODS: A de-identified nationwide (US) real-world clinico-genomic database was leveraged to study 621 non-squamous NSCLC patients treated with ICI. All patients received second-line ICI monotherapy and underwent comprehensive genomic profiling as part of routine clinical care. Overall survival (OS) from start of ICI, for CD274 copy number gain and loss cohorts across varying copy number thresholds, were assessed. RESULTS: Among the 621 patients, patients with a CD274 CN greater than or equal to specimen ploidy +2 (N = 29) had a significantly higher median (m) OS when compared with the rest of the cohort (N = 592; 16.1 [8.9-37.3] vs 8.6 [7.1-10.9] months, hazard ratio (HR) = 0.6 [0.4-1.0], P-value = .05). Patients with a CD274 copy number less than specimen ploidy (N = 299) trended toward a lower mOS when compared to the rest of the cohort (N = 322; 7.5 [5.9-11.3] vs 9.6 [7.9-12.8] months, HR = 0.9 [0.7-1.1], P-value = .3). CONCLUSION: This work shows that CD274 copy number gains at varying thresholds predict different response to ICI blockade in non-squamous NSCLC. Considering these data, prospective clinical trials should further validate these findings, specifically in the context of PD-L1 IHC test results.

Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study.

BACKGROUND: Tumour mutational burden (TMB) has been retrospectively correlated with response to immune checkpoint blockade. We prospectively explored the association of high tissue TMB (tTMB-high) with outcomes in ten tumour-type-specific cohorts from the phase 2 KEYNOTE-158 study, which assessed the anti-PD-1 monoclonal antibody pembrolizumab in patients with selected, previously treated, advanced solid tumours. METHODS: In the multi-cohort, open-label, non-randomised, phase 2 KEYNOTE-158 study, patients were enrolled from 81 academic facilities and community-based institutions across 21 countries in Africa, the Americas, Asia, and Europe. Eligible patients were aged 18 years or older, had a histologically or cytologically confirmed advanced (ie, unresectable or metastatic, or both) incurable solid tumour (eligible tumour types were anal, biliary, cervical, endometrial, mesothelioma, neuroendocrine, salivary, small-cell lung, thyroid, and vulvar), progression on or intolerance to one or more lines of standard therapy, had measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) assessed by independent central radiological review, Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of at least 3 months, adequate organ function, and a tumour sample for biomarker analysis. Participants were given pembrolizumab 200 mg intravenously every 3 weeks for up to 35 cycles. Tissue TMB (tTMB) was assessed in formalin-fixed paraffin-embedded tumour samples using the FoundationOne CDx assay (Foundation Medicine, Cambridge, MA, USA). The prespecified definition of tTMB-high status was at least 10 mutations per megabase. The primary endpoint was the proportion of patients with an objective response (complete or partial response) as per Response Evaluation Criteria in Solid Tumours (version 1.1) by independent central review. This prespecified analysis assessed the association between antitumour activity and tTMB in treated patients with evaluable tTMB data. Efficacy was assessed in all participants who received at least one dose of pembrolizumab, had evaluable tTMB data, and were enrolled at least 26 weeks before data cutoff (June 27, 2019), and safety was assessed in all participants who received at least one dose of pembrolizumab and had tTMB-high status. KEYNOTE-158 is registered at ClinicalTrials.gov, NCT02628067, and is ongoing. FINDINGS: Between Jan 15, 2016, and June 25, 2019, 1073 patients were enrolled. 1066 participants were treated as of data cutoff (June 27, 2019), of whom 805 (76%) were evaluable for TMB, and 105 (13%) of 805 had tTMB-high status and were assessed for safety. 1050 (98%) of 1066 patients enrolled by at least 26 weeks before data cutoff, of whom 790 (75%) were evaluable for TMB and included in efficacy analyses. 102 (13%) of these 790 patients had tTMB-high status (≥10 mutations per megabase), and 688 (87%) patients had non-tTMB-high status (<10 mutations per megabase). Median study follow-up was 37·1 months (IQR 35·0-38·3). Objective responses were observed in 30 (29%; 95% CI 21-39) of 102 patients in the tTMB-high group and 43 (6%; 5-8) of 688 in the non-tTMB-high group. 11 (10%) of 105 patients had treatment-related serious adverse events. 16 (15%) participants had a grade 3-5 treatment-related adverse event, of which colitis was the only such adverse event that occurred in more than one patient (n=2). One patient had fatal pneumonia that was assessed by the investigator to be treatment related. INTERPRETATION: tTMB-high status identifies a subgroup of patients who could have a robust tumour response to pembrolizumab monotherapy. tTMB could be a novel and useful predictive biomarker for response to pembrolizumab monotherapy in patients with previously treated recurrent or metastatic advanced solid tumours. FUNDING: Merck Sharp & Dohme Corp, a subsidiary of Merck & Co, Inc.

Tumor Mutational Burden as a Predictive Biomarker for Response to Immune Checkpoint Inhibitors: A Review of Current Evidence.

Treatment with immune checkpoint inhibitors (ICPIs) extends survival in a proportion of patients across multiple cancers. Tumor mutational burden (TMB)-the number of somatic mutations per DNA megabase (Mb)-has emerged as a proxy for neoantigen burden that is an independent biomarker associated with ICPI outcomes. Based on findings from recent studies, TMB can be reliably estimated using validated algorithms from next-generation sequencing assays that interrogate a sufficiently large subset of the exome as an alternative to whole-exome sequencing. Biological processes contributing to elevated TMB can result from exposure to cigarette smoke and ultraviolet radiation, from deleterious mutations in mismatch repair leading to microsatellite instability, or from mutations in the DNA repair machinery. A variety of clinical studies have shown that patients with higher TMB experience longer survival and greater response rates following treatment with ICPIs compared with those who have lower TMB levels; this includes a prospective randomized clinical trial that found a TMB threshold of ≥10 mutations per Mb to be predictive of longer progression-free survival in patients with non-small cell lung cancer. Multiple trials are underway to validate the predictive values of TMB across cancer types and in patients treated with other immunotherapies. Here we review the rationale, algorithm development methodology, and existing clinical data supporting the use of TMB as a predictive biomarker for treatment with ICPIs. We discuss emerging roles for TMB and its potential future value for stratifying patients according to their likelihood of ICPI treatment response. IMPLICATIONS FOR PRACTICE: Tumor mutational burden (TMB) is a newly established independent predictor of immune checkpoint inhibitor (ICPI) treatment outcome across multiple tumor types. Certain next-generation sequencing-based techniques allow TMB to be reliably estimated from a subset of the exome without the use of whole-exome sequencing, thus facilitating the adoption of TMB assessment in community oncology settings. Analyses of multiple clinical trials across several cancer types have demonstrated that TMB stratifies patients who are receiving ICPIs by response rate and survival. TMB, alongside other genomic biomarkers, may provide complementary information in selecting patients for ICPI-based therapies.

Comprehensive Genomic Profiling of Hodgkin Lymphoma Reveals Recurrently Mutated Genes and Increased Mutation Burden.

BACKGROUND: The genomic landscape of Hodgkin lymphoma (HL) has been difficult to characterize due to the paucity of neoplastic cells and an abundant microenvironment. Such characterization is needed in order to improve treatment strategies. MATERIALS AND METHODS: We performed comprehensive genomic profiling (CGP) using targeted next-generation sequencing on archival formalin-fixed paraffin embedded tumor samples from 63 patients to analyze the landscape of HL. RESULTS: CGP was successful for 49/63 archival specimens (78%), and revealed aberrations impacting genes including B2M, TP53, and XPO1 (E571). Of the 34 patients for whom total mutation burden (TMB; mutations/megabase [Mb]) was assessed, 5 (15%) had high TMB (≥20 mutations/Mb), 18 (53%) had intermediate TMB (6-19 mutations/Mb), and 11 (32%) had low TMB (≤5 mutations/Mb). We next tested 13 patients' plasma cell-free DNA with droplet digital polymerase chain reaction for the presence of XPO1 E571 mutation, which was confirmed in the plasma of 31% of patients. In three patients with serially collected plasma samples, XPO1 E571K allelic frequency changes corresponded with changes in tumor size on conventional radiographic imaging. CONCLUSION: The study demonstrates that comprehensive genomic profiling of archival Hodgkin lymphoma tumor samples is feasible and leads to the identification of genes that are recurrently mutated and that Hodgkin lymphoma has increased mutation burden in the majority of samples analyzed. Furthermore, tracking of XPO1 E571 mutant allele frequency in a subset of patients may also represent a potential disease-monitoring strategy and warrants further investigation. IMPLICATIONS FOR PRACTICE: This study provides the first evidence that comprehensive genomic profiling can be performed to map the genomic landscape of Hodgkin lymphoma and that a subpopulation of patients has mutations in TP53, B2M, XPO1, and other genes. It was found that 15% of patients have high mutation burden, which, in cancers such as melanoma, may indicate sensitivity to immune checkpoint inhibitors, and may thus be explored for Hodgkin lymphoma. Lastly, this work demonstrates that changes in the mutant allele frequency of XPO1 in serially collected plasma cell-free DNA samples correspond with treatment outcomes measured with conventional radiographic imaging.

A Novel gp41-Binding Adnectin with Potent Anti-HIV Activity Is Highly Synergistic when Linked to a CD4-Binding Adnectin.

The N17 region of gp41 in HIV-1 is the most conserved region in gp160. mRNA selection technologies were used to identify an adnectin that binds to this region and inhibits gp41-induced membrane fusion. Additional selection conditions were used to optimize the adnectin to greater potency (5.4 ± 2.6 nM) against HIV-1 and improved binding affinity for an N17-containing helical trimer (0.8 ± 0.4 nM). Resistance to this adnectin mapped to a single Glu-to-Arg change within the N17 coding region. The optimized adnectin (6200_A08) exhibited high potency and broad-spectrum activity against 123 envelope proteins and multiple clinical virus isolates, although certain envelope proteins did exhibit reduced susceptibility to 6200_A08 alone. The reduced potency could not be correlated with sequence changes in the target region and was thought to be the result of faster kinetics of fusion mediated by these envelope proteins. Optimized linkage of 6200_A08 with a previously characterized adnectin targeting CD4 produced a highly synergistic molecule, with the potency of the tandem molecule measured at 37 ± 1 pM. In addition, these tandem molecules now exhibited few potency differences against the same panel of envelope proteins with reduced susceptibility to 6200_A08 alone, providing evidence that they did not have intrinsic resistance to 6200_A08 and that coupling 6200_A08 with the anti-CD4 adnectin may provide a higher effective on rate for gp41 target engagement.IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1-infected patients is through the development of long-acting biologic inhibitors. This study describes the development and properties of an adnectin molecule that targets the most conserved region of the gp41 protein and inhibits HIV-1 with good potency. Moreover, when fused to a similar adnectin targeted to the human CD4 protein, the receptor for HIV-1, significant synergies in potency and efficacy are observed. These inhibitors are part of an effort to develop a larger biologic molecule that functions as a long-acting self-administered regimen for patients with HIV-1 infection.

Targeting HER2 in colorectal cancer: The landscape of amplification and short variant mutations in ERBB2 and ERBB3.

BACKGROUND: In contrast to lung cancer, few precision treatments are available for colorectal cancer (CRC). One rapidly emerging treatment target in CRC is ERBB2 (human epidermal growth factor receptor 2 [HER2]). Oncogenic alterations in HER2, or its dimerization partner HER3, can underlie sensitivity to HER2-targeted therapies. METHODS: In this study, 8887 CRC cases were evaluated by comprehensive genomic profiling for genomic alterations in 315 cancer-related genes, tumor mutational burden, and microsatellite instability. This cohort included both colonic (7599 cases; 85.5%) and rectal (1288 cases; 14.5%) adenocarcinomas. RESULTS: A total of 569 mCRCs were positive for ERBB2 (429 cases; 4.8%) and/or ERBB3 (148 cases; 1.7%) and featured ERBB amplification, short variant alterations, or a combination of the 2. High tumor mutational burden (≥20 mutations/Mb) was significantly more common in ERBB-mutated samples, and ERBB3-mutated CRCs were significantly more likely to have high microsatellite instability (P<.002). Alterations affecting KRAS (27.3%) were significantly underrepresented in ERBB2-amplified samples compared with wild-type CRC samples (51.8%), and ERBB2- or ERBB3-mutated samples (49.0% and 60.8%, respectively) (P<.01). Other significant differences in mutation frequency were observed for genes in the PI3K/MTOR and mismatch repair pathways. CONCLUSIONS: Although observed less often than in breast or upper gastrointestinal carcinomas, indications for which anti-HER2 therapies are approved, the percentage of CRC with ERBB genomic alterations is significant. Importantly, 32% of ERBB2-positive CRCs harbor short variant alterations that are undetectable by routine immunohistochemistry or fluorescence in situ hybridization testing. The success of anti-HER2 therapies in ongoing clinical trials is a promising development for patients with CRC. Cancer 2018;124:1358-73. © 2018 Foundation Medicine, Inc. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity.

A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

ALK Fusions in a Wide Variety of Tumor Types Respond to Anti-ALK Targeted Therapy.

BACKGROUND: Genomic fusions of the anaplastic lymphoma kinase gene (ALK) are a well-established therapy target in non-small cell lung cancer (NSCLC). From a survey of 114,200 clinical cases, we determined the prevalence of ALK rearrangements (rALK) in non-NSCLC tumors and report their responsiveness to therapies targeting ALK. MATERIALS AND METHODS: Comprehensive genomic profiling of 114,200 relapsed and metastatic malignancies, including both solid tumors and hematolymphoid cancers, was performed using a hybrid-capture, adaptor ligation-based next-generation sequencing assay. RESULTS: Of 114,200 clinical samples, 21,522 (18.8%) were NSCLC and 92,678 (81.2%) were other tumor types. Of the 876 (0.8%) cases with ALK fusions (fALK) or rALK, 675 (77.1%) were NSCLC and 201 (22.9%) were other tumor types. ALK fusions were significantly more frequent in NSCLC (3.1%) than non-NSCLC (0.2%; p < .0001). Patients with non-NSCLC tumors harboring fALK were significantly younger (p < .0001) and more often female (p < .0001) than patients with fALK-positive NSCLC. EML4 was more often the fusion partner in NSCLC (83.5%) versus non-NSCLC tumors (30.9%; p < .0001). CONCLUSION: ALK rearrangements can be identified in a wide variety of epithelial and mesenchymal malignancies beyond NSCLC. Anti-ALK therapies can be effective in non-NSCLC tumors driven by fALK, and further study of therapies targeting ALK in clinical trials involving a wider variety of cancer types appears warranted. IMPLICATIONS FOR PRACTICE: Rearrangements involving the ALK gene have been detected in dozens of cancer types using next-generation sequencing. Patients whose tumors harbor ALK rearrangements or fusions respond to treatment with crizotinib and alectinib, including tumors not normally associated with ALK mutations, such as non-Langerhans cell histiocytosis or renal cell carcinoma. Comprehensive genomic profiling using next-generation sequencing can detect targetable ALK fusions irrespective of tumor type or fusions partner.

Comprehensive Genomic Profiling of 282 Pediatric Low- and High-Grade Gliomas Reveals Genomic Drivers, Tumor Mutational Burden, and Hypermutation Signatures.

BACKGROUND: Pediatric brain tumors are the leading cause of death for children with cancer in the U.S. Incorporating next-generation sequencing data for both pediatric low-grade (pLGGs) and high-grade gliomas (pHGGs) can inform diagnostic, prognostic, and therapeutic decision-making. MATERIALS AND METHODS: We performed comprehensive genomic profiling on 282 pediatric gliomas (157 pHGGs, 125 pLGGs), sequencing 315 cancer-related genes and calculating the tumor mutational burden (TMB; mutations per megabase [Mb]). RESULTS: In pLGGs, we detected genomic alterations (GA) in 95.2% (119/125) of tumors. BRAF was most frequently altered (48%; 60/125), and FGFR1 missense (17.6%; 22/125), NF1 loss of function (8.8%; 11/125), and TP53 (5.6%; 7/125) mutations were also detected. Rearrangements were identified in 35% of pLGGs, including KIAA1549-BRAF, QKI-RAF1, FGFR3-TACC3, CEP85L-ROS1, and GOPC-ROS1 fusions. Among pHGGs, GA were identified in 96.8% (152/157). The genes most frequently mutated were TP53 (49%; 77/157), H3F3A (37.6%; 59/157), ATRX (24.2%; 38/157), NF1 (22.2%; 35/157), and PDGFRA (21.7%; 34/157). Interestingly, most H3F3A mutations (81.4%; 35/43) were the variant K28M. Midline tumor analysis revealed H3F3A mutations (40%; 40/100) consisted solely of the K28M variant. Pediatric high-grade gliomas harbored oncogenic EML4-ALK, DGKB-ETV1, ATG7-RAF1, and EWSR1-PATZ1 fusions. Six percent (9/157) of pHGGs were hypermutated (TMB >20 mutations per Mb; range 43-581 mutations per Mb), harboring mutations deleterious for DNA repair in MSH6, MSH2, MLH1, PMS2, POLE, and POLD1 genes (78% of cases). CONCLUSION: Comprehensive genomic profiling of pediatric gliomas provides objective data that promote diagnostic accuracy and enhance clinical decision-making. Additionally, TMB could be a biomarker to identify pediatric glioblastoma (GBM) patients who may benefit from immunotherapy. IMPLICATIONS FOR PRACTICE: By providing objective data to support diagnostic, prognostic, and therapeutic decision-making, comprehensive genomic profiling is necessary for advancing care for pediatric neuro-oncology patients. This article presents the largest cohort of pediatric low- and high-grade gliomas profiled by next-generation sequencing. Reportable alterations were detected in 95% of patients, including diagnostically relevant lesions as well as novel oncogenic fusions and mutations. Additionally, tumor mutational burden (TMB) is reported, which identifies a subpopulation of hypermutated glioblastomas that harbor deleterious mutations in DNA repair genes. This provides support for TMB as a potential biomarker to identify patients who may preferentially benefit from immune checkpoint inhibitors.

Advancing Evidence Generation for Circulating Tumor DNA: Lessons Learned from A Multi-Assay Study of Baseline Circulating Tumor DNA Levels across Cancer Types and Stages.

Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.

Evaluation of tissue- and plasma-derived tumor mutational burden (TMB) and genomic alterations of interest in CheckMate 848, a study of nivolumab combined with ipilimumab and nivolumab alone in patients with advanced or metastatic solid tumors with high TMB.

BACKGROUND: An accumulation of somatic mutations in tumors leads to increased neoantigen levels and antitumor immune response. Tumor mutational burden (TMB) reflects the rate of somatic mutations in the tumor genome, as determined from tumor tissue (tTMB) or blood (bTMB). While high tTMB is a biomarker of immune checkpoint inhibitor (ICI) treatment efficacy, few studies have explored the clinical utility of bTMB, a less invasive alternative for TMB assessment. Establishing the correlation between tTMB and bTMB would provide insight into whether bTMB is a potential substitute for tTMB. We explored the tumor genomes of patients enrolled in CheckMate 848 with measurable TMB. The correlation between tTMB and bTMB, and the factors affecting it, were evaluated. METHODS: In the phase 2 CheckMate 848 (NCT03668119) study, immuno-oncology-naïve patients with advanced, metastatic, or unresectable solid tumors and tTMB-high or bTMB-high (≥10 mut/Mb) were prospectively randomized 2:1 to receive nivolumab plus ipilimumab or nivolumab monotherapy. Tissue and plasma DNA sequencing was performed using the Foundation Medicine FoundationOne CDx and bTMB Clinical Trial Assays, respectively. tTMB was quantified from coding variants, insertions, and deletions, and bTMB from somatic base substitutions. Correlations between tTMB and bTMB were determined across samples and with respect to maximum somatic allele frequency (MSAF). Assay agreement and variant composition were also evaluated. RESULTS: A total of 1,438 and 1,720 unique tissue and blood samples, respectively, were obtained from 1,954 patients and included >100 screened disease ontologies, with 1,017 unique pairs of tTMB and bTMB measurements available for assessment. Median tTMB and bTMB were 3.8 and 3.5 mut/Mb, respectively. A significant correlation between tTMB and bTMB (r=0.48, p<0.0001) was observed across all sample pairs, which increased to r=0.54 (p<0.0001) for samples with MSAF≥1%. Assay concordance was highest for samples with MSAF≥10% across multiple disease ontologies and observed for both responders and non-responders to ICI therapy. The variants contributing to tTMB and bTMB were similar. CONCLUSIONS: We observed that tTMB and bTMB had a statistically significant correlation, particularly for samples with high MSAF, and that this correlation applied across disease ontologies. Further investigation into the clinical utility of bTMB is warranted.

A longitudinal circulating tumor DNA-based model associated with survival in metastatic non-small-cell lung cancer.

One of the great challenges in therapeutic oncology is determining who might achieve survival benefits from a particular therapy. Studies on longitudinal circulating tumor DNA (ctDNA) dynamics for the prediction of survival have generally been small or nonrandomized. We assessed ctDNA across 5 time points in 466 non-small-cell lung cancer (NSCLC) patients from the randomized phase 3 IMpower150 study comparing chemotherapy-immune checkpoint inhibitor (chemo-ICI) combinations and used machine learning to jointly model multiple ctDNA metrics to predict overall survival (OS). ctDNA assessments through cycle 3 day 1 of treatment enabled risk stratification of patients with stable disease (hazard ratio (HR) = 3.2 (2.0-5.3), P < 0.001; median 7.1 versus 22.3 months for high- versus low-intermediate risk) and with partial response (HR = 3.3 (1.7-6.4), P < 0.001; median 8.8 versus 28.6 months). The model also identified high-risk patients in an external validation cohort from the randomized phase 3 OAK study of ICI versus chemo in NSCLC (OS HR = 3.73 (1.83-7.60), P = 0.00012). Simulations of clinical trial scenarios employing our ctDNA model suggested that early ctDNA testing outperforms early radiographic imaging for predicting trial outcomes. Overall, measuring ctDNA dynamics during treatment can improve patient risk stratification and may allow early differentiation between competing therapies during clinical trials.

Pan-Cancer Analysis of Copy-Number Features Identifies Recurrent Signatures and a Homologous Recombination Deficiency Biomarker to Predict Poly (ADP-Ribose) Polymerase Inhibitor Response.

PURPOSE: Copy-number (CN) features reveal the molecular state of cancers and may have predictive and prognostic value in the treatment of cancer. We sought to apply published CN analysis methods to a large pan-cancer data set and characterize ubiquitous CN signatures across tumor types, including potential utility for treatment selection. METHODS: We analyzed the landscape of CN features in 260,333 pan-cancer samples. We examined the association of 10 signatures with genomic alterations and clinical characteristics and trained a machine learning classifier using CN and insertion and deletion features to detect homologous recombination deficiency signature (HRDsig) positivity. Clinical outcomes were assessed using a real-world clinicogenomic database (CGDB) of comprehensive genomic profiling linked to deidentified, electronic health record-derived clinical data. RESULTS: CN signatures were prevalent across cancer types and associated with diverse processes including focal tandem duplications, seismic amplifications, genome-wide loss of heterozygosity (gLOH), and HRD. Our novel HRDsig outperformed gLOH in predicting BRCAness and effectively distinguished biallelic BRCA and homologous recombination-repair wild-type (HRRwt) samples pan-tumor, demonstrating high sensitivity to detect biallelic BRCA in ovarian (93%) and other HRD-associated cancers (80%-87%). Pan-tumor prevalence of HRDsig was 6.4%. HRRwt cases represented a significant fraction of the HRDsig-positive cohort, likely reflecting a population with nongenomic mechanisms of HRD. In ovarian and prostate CGDBs, HRDsig identified more patients than gLOH and had predictive value for poly (ADP-ribose) polymerase inhibitor (PARPi) benefit. CONCLUSION: Tumor CN profiles are informative, revealing diverse processes active in cancer. We describe the landscape of 10 CN signatures in a large pan-cancer cohort, including two associated with HRD. We trained a machine learning-based HRDsig that robustly identified BRCAness and associated with biallelic BRCA pan-tumor, and was predictive of PARPi benefit in real-world ovarian and prostate data sets.

Deletions on 9p21 are associated with worse outcomes after anti-PD-1/PD-L1 monotherapy but not chemoimmunotherapy.

NCCN guidelines for first-line treatment of advanced non-squamous non-small-cell lung cancer (NSCLC) patients without targetable driver alterations includes either immunotherapy alone or in combination with chemotherapy. In this study, we investigated genomic predictors of survival after immunotherapy to guide this treatment decision. Cox proportional hazards regression was used to identify genomic correlates of survival in a cohort of EGFR/ALK-, non-squamous NSCLC patients treated with first-line pembrolizumab monotherapy (mono-IO) or pembrolizumab in combination with carboplatin/cisplatin and pemetrexed (chemo-IO) within a real-world clinico-genomic database. The effect of deletions on 9p21 was further evaluated in five additional tumor types. Among mono-IO treated non-squamous NSCLC patients, tumors with 9p21.3 gene deletions (CDKN2A, CDKN2B, MTAP) were associated with worse survival compared to the corresponding deletion-negative tumors (CDKN2A deletion HR = 1.8, P = 0.001). However, this association was not observed among chemo-IO treated patients (CDKN2A deletion HR = 1.1, P = 0.4). This finding remained after adjusting for clinical and genomic features including TMB and PD-L1. Deletions at 9p21.3 were not associated with differences in TMB, PD-L1, or tumor inflammation. Due to the high incidence of 9p21.3 deletions across tumor types, we performed a pan-cancer analysis and found CDKN2A deletion-positive tumors had worse survival following first-line immunotherapy treatment in multiple tumor types (HR = 1.4, P < 0.001). These results indicate deletions at 9p21.3 are a putative negative predictor of clinical benefit from first-line immune checkpoint inhibitors and may have utility in choosing between mono-IO vs chemo-IO regimens in NSCLC.

Blood-based tumor mutational burden as a biomarker for atezolizumab in non-small cell lung cancer: the phase 2 B-F1RST trial.

Tumor mutational burden (TMB) in circulating tumor DNA (ctDNA) has shown promise in predicting benefit from PD-L1/PD-1 inhibitors in retrospective studies. Aiming to assess blood TMB (bTMB) prospectively, we conducted B-F1RST ( NCT02848651 ), an open-label, phase 2 trial that evaluated bTMB as a predictive biomarker for first-line atezolizumab monotherapy in locally advanced or metastatic stage IIIB-IVB non-small cell lung cancer (n = 152). The co-primary endpoints were investigator-assessed objective response rate (ORR) per RECIST version 1.1 and investigator-assessed progression-free survival (PFS) between high and low bTMB subgroups at the pre-defined bTMB ≥ 16 (14.5 mutations per megabase) cutoff. Secondary endpoints included investigator-assessed PFS, overall survival (OS) and duration of response at various bTMB cutoffs, as well as safety. Investigator-assessed PFS in the bTMB ≥ 16 versus bTMB < 16 groups was not statistically significant. However, bTMB ≥ 16 was associated with higher ORR, and ORR improved as bTMB cutoffs increased. No new safety signals were seen. In exploratory analyses, patients with maximum somatic allele frequency (MSAF) < 1% had higher ORR than patients with MSAF ≥ 1%. However, further analysis showed that this effect was driven by better baseline prognostics rather than by MSAF itself. At 36.5-month follow-up, an exploratory analysis of OS found that bTMB ≥ 16 was associated with longer OS than bTMB < 16. Further study and assay optimization will be required to develop bTMB as a predictive, standalone biomarker of immunotherapy or for use in conjunction with other biomarkers.

Comprehensive pan-cancer genomic landscape of KRAS altered cancers and real-world outcomes in solid tumors.

Recent clinical development of KRAS inhibitors has heightened interest in the genomic landscape of KRAS-altered cancers. We performed a pan-cancer analysis of KRAS-altered samples from 426,706 adult patients with solid or hematologic malignancies using comprehensive genomic profiling; additional analyses included 62,369 liquid biopsy and 7241 pediatric samples. 23% of adult pan-cancer samples had KRAS alterations; 88% were mutations, most commonly G12D/G12V/G12C/G13D/G12R, and prevalence was similar in liquid biopsies. Co-alteration landscapes were largely similar across KRAS mutations but distinct from KRAS wild-type, though differences were observed in some tumor types for tumor mutational burden, PD-L1 expression, microsatellite instability, and other mutational signatures. Prognosis of KRAS-mutant versus other genomic cohorts of lung, pancreatic, and colorectal cancer were assessed using a real-world clinicogenomic database. As specific KRAS inhibitors and combination therapeutic strategies are being developed, genomic profiling to understand co-alterations and other biomarkers that may modulate response to targeted or immunotherapies will be imperative.

Atezolizumab versus chemotherapy in advanced or metastatic NSCLC with high blood-based tumor mutational burden: primary analysis of BFAST cohort C randomized phase 3 trial.

Tumor mutational burden (TMB) is being explored as a predictive biomarker for cancer immunotherapy outcomes in non-small cell lung cancer. BFAST (NCT03178552)-an open-label, global, multicohort trial-evaluated the safety and efficacy of first-line targeted therapies or immunotherapy in patients with unresectable Stage IIIB or IV advanced or metastatic non-small cell lung cancer who were selected for biomarker status using blood-based targeted next-generation sequencing. In the Phase 3 cohort C evaluating blood-based (b)TMB as a biomarker of atezolizumab efficacy, patients with bTMB of ≥10 (N = 471) were randomized 1:1 to receive atezolizumab or platinum-based chemotherapy per local standard of care. Cohort C did not meet its primary endpoint of investigator-assessed progression-free survival in the population with bTMB of ≥16 (hazard ratio, 0.77; 95% confidence interval: 0.59, 1.00; P = 0.053). Adverse events leading to treatment withdrawal occurred in 10% of patients in the atezolizumab arm and 20% in the chemotherapy arm. Adverse events of special interest occurred in 42% of patients in the atezolizumab arm and 26% in the chemotherapy arm. A prespecified exploratory analysis compared the bTMB clinical trial assay with the FoundationOne Liquid Companion Diagnostic assay and showed high concordance between assays. Additional exploration of bTMB to identify optimal cutoffs, confounding factors, assay improvements or cooperative biomarkers is warranted.