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Ankur R Parikh,1 Siraj M Ali,2 Alexa B Schrock,2 Lee A Albacker,2 Vincent A Miller,2 Phil J Stephens,2 Pamela Crilley,1 Maurie Markman1 1Eastern Regional Medical Center, Cancer Treatment Centers of America, Philadelphia, PA, USA; 2Foundation Medicine, Inc, Cambridge, MA, USA Abstract: In non-small-cell lung cancer (NSCLC) refractory to standard therapy and which lacks well-known oncogenic drivers, genomic profiling can still identify genomic alterations that may suggest potential sensitivity to targeted therapy. PTEN mutation in NSCLC may be sensitizing to analogs of rapamycin such as everolimus or temsirolimus, but more investigation is needed. We report the case of a patient with metastatic NSCLC harboring a PTEN mutation as well as high tumor mutational burden and PD-L1 positivity with a durable response to temsirolimus, but refractory to a checkpoint inhibitor. Even in the event of failure of treatment with checkpoint inhibitors in the background of a case with a higher tumor mutational burden and PD-L1 positivity, targeting specific genomic alterations may still result in patient benefit. Keywords: genomic profiling, temsirolimus, targeted therapy, immunotherapy

Microsatellite instability high (MSI-H) and mismatch repair deficient (dMMR) tumor status have been demonstrated to predict patient response to immunotherapies. We developed and validated a next-generation sequencing (NGS)-based companion diagnostic (CDx) to detect MSI-H solid tumors via a comprehensive genomic profiling (CGP) assay, FoundationOne®CDx (F1CDx). To determine MSI status, F1CDx calculates the fraction of unstable microsatellite loci across >2000 loci using a fraction-based (FB) analysis. Across solid tumor types, F1CDx demonstrated a high analytical concordance with both PCR (n = 264) and IHC (n = 279) with an overall percent agreement (OPA) of 97.7% and 97.8%, respectively. As part of a retrospective bridging clinical study from KEYNOTE-158 Cohort K and KEYNOTE-164, patients with MSI-H tumors as determined by F1CDx demonstrated an objective response rate (ORR) of 43.0% to pembrolizumab. In real-world cancer patients from a deidentified clinicogenomic database, F1CDx was at least equivalent in assessing clinical outcome following immunotherapy compared with MMR IHC. Demonstrated analytical and clinical performance of F1CDx led to the pan-tumor FDA approval in 2022 of F1CDx to identify MSI-H solid tumor patients for treatment with pembrolizumab. F1CDx is an accurate, reliable, and FDA-approved method for the identification of MSI-H tumors for treatment with pembrolizumab., (© 2024. The Author(s).)

The authors present a cohort of 661 young adult glioblastomas diagnosed using 2016 WHO World Health Organization Classification of Tumors of the Central Nervous System, utilizing comprehensive genomic profiling (CGP) to explore their genomic landscape and assess their relationship to currently defined disease entities. This analysis explored variants with evidence of pathogenic function, common copy number variants (CNVs), and several novel fusion events not described in literature. Tumor mutational burden (TMB) mutational signatures, anatomic location, and tumor recurrence are further explored. Using data collected from CGP, unsupervised machine-learning techniques were leveraged to identify 10 genomic classes in previously assigned young adult glioblastomas. The authors relate these molecular classes to current World Health Organization guidelines and reference current literature to give therapeutic and prognostic descriptions where possible., (© The Author(s) 2023. Published by Oxford University Press.)

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., Competing Interests: Competing interests: DF, DCP, EME, GRO, HT, JH, and LAA are employees of Foundation Medicine, Inc. and hold stock in Bristol Myers Squibb. GF, GG, and JP are employees of and hold stock in Bristol Myers Squibb. JB is an employee of and holds stock in Bristol Myers Squibb, and holds stock in Johnson and Johnson. NK holds stock in Bristol Myers Squibb. PD is a former employee of Bristol Myers Squibb., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

These analyses explore the impact of homologous recombination repair gene mutations, including BRCA1/2 mutations and homologous recombination deficiency (HRD), on the efficacy of the poly(ADP-ribose) polymerase (PARP) inhibitor talazoparib in the open-label, two-cohort, Phase 2 ABRAZO trial in germline BRCA1/2-mutation carriers. In the evaluable intent-to-treat population (N = 60), 58 (97%) patients harbor ≥1 BRCA1/2 mutation(s) in tumor sequencing, with 95% (53/56) concordance between germline and tumor mutations, and 85% (40/47) of evaluable patients have BRCA locus loss of heterozygosity indicating HRD. The most prevalent non-BRCA tumor mutations are TP53 in patients with BRCA1 mutations and PIK3CA in patients with BRCA2 mutations. BRCA1- or BRCA2-mutated tumors show comparable clinical benefit within cohorts. While low patient numbers preclude correlations between HRD and efficacy, germline BRCA1/2 mutation detection from tumor-only sequencing shows high sensitivity and non-BRCA genetic/genomic events do not appear to influence talazoparib sensitivity in the ABRAZO trial.ClinicalTrials.gov identifier: NCT02034916., (© 2023. Springer Nature Limited.)

Tumor cells need to activate a telomere maintenance mechanism, enabling limitless replication. The bulk of evidence supports that sarcomas predominantly use alternative lengthening of telomeres (ALT) mechanism, commonly associated with alterations in ATRX and DAXX. In our dataset, only 12.3% of sarcomas harbored alterations in these genes. Thus, we checked for the presence of other genomic determinants of high telomeric content in sarcomas. Our dataset consisted of 13555 sarcoma samples, sequenced as a part of routine clinical care on the FoundationOne®Heme platform. We observed a median telomeric content of 622.3 telomeric reads per GC-matched million reads (TRPM) across all samples. In agreement with previous studies, telomeric content was significantly higher in ATRX altered and POT1 altered sarcomas. We further observed that sarcomas with alterations in RAD51B or GID4 were enriched in samples with high telomeric content, specifically within uterus leiomyosarcoma for RAD51B and soft tissue sarcoma (not otherwise specified, nos) for GID4, Furthermore, RAD51B and POT1 alterations were mutually exclusive with ATRX and DAXX alterations, suggestive of functional redundancy. Our results propose a role played by RAD51B and GID4 in telomere elongation in sarcomas and open research opportunities for agents aimed at targeting this critical pathway in tumorigenesis., (© 2023. Springer Nature Limited and Centre of Excellence in Genomic Medicine Research, King Abdulaziz University.)

Amplification of HER2 can drive the proliferation of cancer cells, and several inhibitors of HER2 have been successfully developed. Recent advances in next-generation sequencing now reveal that HER2 is subject to mutation, with over 2,000 unique variants observed in human cancers. Several examples of oncogenic HER2 mutations have been described, and these primarily occur at allosteric sites outside the ATP-binding site. To identify the full spectrum of oncogenic HER2 driver mutations aside from a few well-studied mutations, we developed mutation-allostery-pharmacology (MAP), an in silico prediction algorithm based on machine learning. By applying this computational approach to 820 single-nucleotide variants, a list of 222 known and potential driver mutations was produced. Of these 222 mutations, 111 were screened by Ba/F3-retrovirus proliferation assays; 37 HER2 mutations were experimentally determined to be driver mutations, comprising 15 previously characterized and 22 newly identified oncogenic mutations. These oncogenic mutations mostly affected allosteric sites in the extracellular domain (ECD), transmembrane domain, and kinase domain of HER2, with only a single mutation in the HER2 orthosteric ATP site. Covalent homodimerization was established as a common mechanism of activation among HER2 ECD allosteric mutations, including the most prevalent HER2 mutation, S310F. Furthermore, HER2 allosteric mutants with enhanced covalent homodimerization were characterized by altered pharmacology that reduces the activity of existing anti-HER2 agents, including the mAb trastuzumab and the tyrosine kinase inhibitor lapatinib. Overall, the MAP-scoring and functional validation analyses provided new insights into the oncogenic activity and therapeutic targeting of HER2 mutations in cancer., Significance: This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers., (©2022 The Authors; Published by the American Association for Cancer Research.)

Treatment of non-small cell lung cancer is increasingly biomarker driven with multiple genomic alterations, including those in the epidermal growth factor receptor (EGFR) gene, that benefit from targeted therapies. We developed a set of algorithms to assess EGFR status and morphology using a real-world advanced lung adenocarcinoma cohort of 2099 patients with hematoxylin and eosin (H&E) images exhibiting high morphological diversity and low tumor content relative to public datasets. The best performing EGFR algorithm was attention-based and achieved an area under the curve (AUC) of 0.870, a negative predictive value (NPV) of 0.954 and a positive predictive value (PPV) of 0.410 in a validation cohort reflecting the 15% prevalence of EGFR mutations in lung adenocarcinoma. The attention model outperformed a heuristic-based model focused exclusively on tumor regions, and we show that although the attention model also extracts signal primarily from tumor morphology, it extracts additional signal from non-tumor tissue regions. Further analysis of high-attention regions by pathologists showed associations of predicted EGFR negativity with solid growth patterns and higher peritumoral immune presence. This algorithm highlights the potential of deep learning tools to provide instantaneous rule-out screening for biomarker alterations and may help prioritize the use of scarce tissue for biomarker testing., (© 2023. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press.)

Telomerase-mediated lengthening is a mechanism by which some cancer cells avoid senescence-mediated cell cycle arrest due to shortened telomeres. By reverse transcribing an RNA template, encoded by TERC, the enzyme telomerase synthesizes the elongation of telomeric DNA using the 3' end of the chromosome as a primer. TERC harbors a highly conserved template region consisting of 11 nucleotides spanning hg19 coordinates chr3:169482793-169482803. In human cell lines, when TERC was mutated to alter its template region, telomerase generated the predicted mutant telomeric repeats. However, it is unknown if this can occur in human clinical samples. Here, we report on the rare occurrence of two tumor samples where TERC template mutations were reflected in telomeric repeats., Competing Interests: RS, GMF, and LAA are employees of Foundation Medicine, Inc., a wholly owned subsidiary of Roche Holdings, Inc. and Roche Finance Ltd, and these employees have equity interest in an affiliate of these Roche entities.

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., (© 2022. The Author(s).)

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089)., Summary: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors., (©2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: PARP inhibitors (PARPi) have demonstrated efficacy in tumors with germline breast cancer susceptibility genes (gBRCA) 1 and 2 mutations, but further factors influencing response to PARPi are poorly understood., Experimental Design: Breast cancer tumor tissue from patients with gBRCA1/2 mutations from the phase III EMBRACA trial of the PARPi talazoparib versus chemotherapy was sequenced using FoundationOne CDx., Results: In the evaluable intent-to-treat population, 96.1% (296/308) had ≥1 tumor BRCA (tBRCA) mutation and there was strong concordance (95.3%) between tBRCA and gBRCA mutational status. Genetic/genomic characteristics including BRCA loss of heterozygosity (LOH; identified in 82.6% of evaluable patients), DNA damage response (DDR) gene mutational burden, and tumor homologous recombination deficiency [assessed by genomic LOH (gLOH)] demonstrated no association with talazoparib efficacy., Conclusions: Overall, BRCA LOH status, DDR gene mutational burden, and gLOH were not associated with talazoparib efficacy; however, these conclusions are qualified by population heterogeneity and low patient numbers in some subgroups. Further investigation in larger patient populations is warranted., (©2022 The Authors; Published by the American Association for Cancer Research.)

Cholangiocarcinoma (CCA) is a heterogeneous biliary tract cancer with a poor prognosis. Approximately 30% to 50% of patients harbor actionable alterations, including FGFR2 rearrangements. Pemigatinib, a potent, selective fibroblast growth factor receptor (FGFR) FGFR1-3 inhibitor, is approved for previously treated, unresectable, locally advanced or metastatic CCA harboring FGFR2 fusions/rearrangements, as detected by a US Food and Drug Administration-approved test. The next-generation sequencing (NGS)-based FoundationOneCDx (F1CDx) was US Food and Drug Administration approved for detecting FGFR2 fusions or rearrangements. The precision and reproducibility of F1CDx in detecting FGFR2 rearrangements in CCA were examined. Analytical concordance between F1CDx and an externally validated RNA-based NGS (evNGS) test was performed. Identification of FGFR2 rearrangements in the screening population from the pivotal FIGHT-202 study (NCT02924376) was compared with F1CDx. The reproducibility and repeatability of F1CDx were 90% to 100%. Adjusted positive, negative, and overall percentage agreements were 87.1%, 99.6%, and 98.3%, respectively, between F1CDx and evNGS. Compared with evNGS, F1CDx had a positive predictive value of 96.2% and a negative predictive value of 98.5%. The positive percentage agreement, negative percentage agreement, overall percentage agreement, positive predictive value, and negative predictive value were 100% for F1CDx versus the FIbroblast Growth factor receptor inhibitor in oncology and Hematology Trial-202 (FIGHT-202) clinical trial assay. Of 6802 CCA samples interrogated, 9.2% had FGFR2 rearrangements. Cell lines expressing diverse FGFR2 fusions were sensitive to pemigatinib. F1CDx demonstrated sensitivity, reproducibility, and high concordance with clinical utility in identifying patients with FGFR2 rearrangements who may benefit from pemigatinib treatment., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Cancer cells can proliferate indefinitely through telomere maintenance mechanisms. These mechanisms include telomerase-dependent elongation, mediated by TERT activation, and alternative lengthening of telomeres (ALT), linked to loss of ATRX or DAXX., Methods: We analyzed the telomeric content of 89,959 tumor samples within the Foundation Medicine dataset and investigated the genomic determinants of high telomeric content, linking them to clinical outcomes, when available., Results: Telomeric content varied widely by disease type with leiomyosarcoma having the highest and Merkel cell carcinoma having the lowest telomeric content. In agreement with previous studies, telomeric content was significantly higher in samples with alterations in TERC, ATRX, and DAXX. We further identified that amplifications in two genes, RAD21 and HGF, were enriched in samples with high telomeric content, which was confirmed using the PCAWG/ICGC dataset. We identified the minimal amplified region associated with high telomeric content for RAD21 (8q23.1-8q24.12), which excludes MYC, and for HGF (7q21.11). Our results demonstrated that RAD21 and HGF exerted an additive telomere lengthening effect on samples with existing alterations in canonical genes previously associated with telomere elongation. Furthermore, patients with breast cancer who harbor RAD21 alterations had poor median overall survival and trended towards higher levels of Ki-67 staining., Conclusions: This study highlights the importance of the role played by RAD21 (8q23.1-8q24.12) and HGF (7q21.11) in the lengthening of telomeres, supporting unlimited replication in tumors. These findings open avenues for work aimed at targeting this crucial pathway in tumorigenesis., (© 2022. The Author(s).)

Background: Tumor mutational burden (TMB) has been proposed as a predictive biomarker of response to immunotherapy. Efforts to standardize TMB scores for use in the clinic and to identify the factors that could impact TMB scores are of high importance. However, the biopsy collection site has not been assessed as a factor that may influence TMB scores., Methods: We examine a real-world cohort comprising 137,771 specimens across 47 tissues in 12 indications profiled by the FoundationOne assay (Foundation Medicine, Cambridge, MA) to assess the prevalence of biopsy sites for each indication and their TMB scores distribution., Results: We observe a wide variety of biopsy sites from which specimens are sent for genomic testing and show that TMB scores differ in a cancer- and tissue-specific manner. For example, brain or adrenal gland specimens from NSCLC patients show higher TMB scores than local lung specimens (mean difference 3.31 mut/Mb; p  < 0.01, 3.90 mut/Mb; p  < 0.01, respectively), whereas bone specimens show no difference., Conclusions: Our data shed light on the biopsied tissue as a driver of TMB measurement variability in clinical practice., Competing Interests: Competing interestsS.P.-C. and A.M.W. are employees of Bristol Myers Squibb. J.F.H., S.H.R. and L.A.A. are employees of Foundation Medicine., (© The Author(s) 2021.)

Background: Immune checkpoint inhibitors (ICIs) benefit patients with multiple cancer types, however, additional predictive biomarkers of response are needed. CD274 (programmed cell death ligand-1, PD-L1) gene rearrangements are positively associated with PD-L1 expression and may confer benefit to ICI, thus a pan-cancer characterization of these alterations is needed., Methods: We analyzed 283,050 patient samples across multiple tumor types that underwent comprehensive genomic profiling for activating CD274 rearrangements and other alterations. The DAKO 22C3 Tumor Proportion Scoring (TPS) method was used for PD-L1 immunohistochemistry (IHC) testing in a small subset with available data (n=55,423). A retrospective deidentified real-world clinico-genomic database (CGDB) was examined for ICI treatment outcomes. We also report a detailed case of CD274- rearranged metastatic rectal adenocarcinoma., Results: We identified 145 samples with functional rearrangements in CD274 . There were significant enrichments for PIK3CA , JAK2 , PDCD1LG2 , CREBBP , and PBRM1 co-mutations (ORs=2.1, 16.7, 17.8, 3.6, and 3.4, respectively, p<0.01). Genomic human papillomavirus (HPV)-16, Epstein-Barr virus, and mismatch repair genes also co-occurred (OR=6.2, 8.4, and 4.3, respectively, p<0.05). Median tumor mutational burden (TMB) was higher compared with CD274 wild-type samples (7.0 vs 3.5 mutations/Mb, p=1.7e-11), with disease-specific TMB enrichment in non-small cell lung, colorectal, unknown primary, and stomach cancers. PD-L1 IHC skewed toward positivity (N=39/43 samples with ≥1% positivity). Of eight patients from the CGDB, three remained on ICI treatment after 6 months. Separately, one patient with metastatic rectal adenocarcinoma experienced a pathologic complete response on chemoimmunotherapy., Conclusions: CD274 gene rearrangements are associated with increased PD-L1 IHC scores, higher TMB, and potential clinical benefit in ICI-treated patients with cancer., Competing Interests: Competing interests: ADK, KM, ZK, MM, JSR, LAA, RSPH, DIL, and JC are employees of Foundation Medicine, a wholly owned subsidiary of Roche Holdings and Roche Finance, and these employees have equity interest in an affiliate of these Roche entities., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The aim of the present study was to determine cell of origin (COO) from a platform using a DNA-based method, COO DNA classifier (COODC). A targeted exome-sequencing platform that applies the mutational profile of a sample was used to classify COO subtype. Two major mutational signatures associated with COO were identified: Catalogue of Somatic Mutations in Cancer (COSMIC) signature 23 enriched in activated B cell (ABC) and COSMIC signature 3, which suggested increased frequency in germinal center B cell (GCB). Differential mutation signatures linked oncogenesis to mutational processes during B-cell activation, confirming the putative origin of GCB and ABC subtypes. Integrating COO with comprehensive genomic profiling enabled identification of features associated with COO and demonstrated the feasibility of determining COO without RNA.

Background: The translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment., Case Presentation: Thirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0-4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases - including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis., Conclusions: CGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis., (Copyright © 2021. Published by Elsevier Inc.)

Background: Non-small cell lung cancer (NSCLC) patients bearing targetable oncogene alterations typically derive limited benefit from immune checkpoint blockade (ICB), which has been attributed to low tumor mutation burden (TMB) and/or PD-L1 levels. We investigated oncogene-specific differences in these markers and clinical outcome., Methods: Three cohorts of NSCLC patients with oncogene alterations (n=4189 total) were analyzed. Two clinical cohorts of advanced NSCLC patients treated with ICB monotherapy [MD Anderson (MDACC; n=172) and Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB; n=894 patients)] were analyzed for clinical outcome. The FMI biomarker cohort (n=4017) was used to assess the association of oncogene alterations with TMB and PD-L1 expression., Results: High PD-L1 expression (PD-L1 ≥50%) rate was 19%-20% in classic EGFR , EGFR exon 20 and HER2 -mutant tumors, and 34%-55% in tumors with ALK , BRAF V600E, ROS1 , RET , or MET alterations. Compared with KRAS- mutant tumors, BRAF non-V600E group had higher TMB (9.6 vs KRAS 7.8 mutations/Mb, p=0.003), while all other oncogene groups had lower TMB (p<0.001). In the two clinical cohorts treated with ICB, molecular groups with EGFR , HER2 , ALK , ROS1 , RET , or MET alterations had short progression-free survival (PFS; 1.8-3.7 months), while BRAF V600E group was associated with greater clinical benefit from ICB (CGDB cohort: PFS 9.8 months vs KRAS 3.7 months, HR 0.66, p=0.099; MDACC cohort: response rate 62% vs KRAS 24%; PFS 7.4 vs KRAS 2.8 months, HR 0.36, p=0.026). KRAS G12C and non-G12C subgroups had similar clinical benefit from ICB in both cohorts. In a multivariable analysis, BRAF V600E mutation (HR 0.58, p=0.041), PD-L1 expression (HR 0.57, p=0.022), and high TMB (HR 0.66, p<0.001) were associated with longer PFS., Conclusions: High TMB and PD-L1 expression are predictive for benefit from ICB treatment in oncogene-driven NSCLCs. NSCLC harboring BRAF mutations demonstrated superior benefit from ICB that may be attributed to higher TMB and higher PD-L1 expression in these tumors. Meanwhile EGFR and HER2 mutations and ALK , ROS1 , RET , and MET fusions define NSCLC subsets with minimal benefit from ICB despite high PD-L1 expression in NSCLC harboring oncogene fusions. These findings indicate a TMB/PD-L1-independent impact on sensitivity to ICB for certain oncogene alterations., Competing Interests: Competing interests: MVN is a consultant for Mirati and Merck/MSD, and reports funding to the institution from Mirati, Novartis, Pfizer, Ziopharm, AstraZeneca, and Checkmate; MM is an employee of Foundation Medicine, and owns stock in Roche; KS is an employee of Genentech, and owns stock in Roche; IB is an employee of Genentech, owns stock in Roche; VS is an employee of Roche, and owns stock in Roche; HX is an employee of Roche, and owns stock in Roche; HS is an employee of Genentech, and owns stock in Roche; XL is a Consultant for Eli Lilly, AstraZeneca, and EMD Serono; MEG is a former employee of Foundation Medicine; KM is an employee of Foundation Medicine, and owns stock in Roche; JPR reports licensing/royalties from Spectrum Pharmaceuticals; TC has received speaker’s fees from the Society for Immunotherapy of Cancer and Bristol-Myers Squibb, receives consulting fees from MedImmune and Bristol-Myers Squibb, and reports research funding to MD Anderson Cancer Center from Boehringer Ingelheim, MedImmune and Bristol-Myers Squibb; AT reports research grants from Eli Lilly, Millennium, Polaris, Genentech, Merck, Boehringer-Ingelheim, BMS, Ariad, Epizyme, Seattle Genetics, Takeda, and EMD Serono; advisory board member for BMS, Eli Lilly, Genentech, Roche, Novartis, Ariad, EMD Serono, Merck, Seattle Genetics, Astra-Zeneca, Boehringer-Ingelheim, Sellas Life Science, Takeda, Epizyme, and Huron; VP is an employee of Pfizer; DLG reports research grants from AstraZeneca, Jenssen R & D, Takeda, Ribun Therapeutics; and honoraria member of Senofi, AstraZeneca, Ribun Therapeutics; GS is an employee of Foundation Medicine, and owns stock in Roche; VAM is an employee of Foundation Medicine, and owns stock in Roche; BA is an employee of Foundation Medicine, and owns stock in Roche; GF is an employee of Foundation Medicine, and owns stock in Roche; LAA is an employee of Foundation Medicine, and owns stock in Roche; DS is an employee of Genentech, and owns stock in Roche; JZ reports research funding from Merck, Johnson and Johnson, and consultant fees from BMS, Johnson and Johnson, AstraZeneca, Geneplus, OrigMed, Innovent outside the submitted work; JVH reports research grants from NIH/NCI, American Cancer Society, Cancer Prevention & Research Institute of Texas, AACR Johnson & Johnson Lung Cancer, AZ, Spectrum, Checkmate Pharmaceuticals; Advisory Committees – AZ, BMS, GSK, Guardant Health, Kairos Venture Investments, BrightPath Biotherapeutics Hengrui Therapeutics, Eli Lilly, Spectrum, EMD Serono, Roche, Foundation One Medicine; Royalties & Licensing – Spectrum & Bio-Tree Systems, Inc., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Several studies have shown clinical outcomes data that support the use of CD274 (PD-L1 ) copy-number (CN) gains and/or losses as a biomarker for immune checkpoint inhibitor (ICPI). Here, we present the landscape of CD274 CN changes across a large cohort of solid tumor cases and correlate these with PD-L1 protein expression by immunohistochemistry., Methods: We analyzed all cases that underwent comprehensive genomic profiling (CGP) testing at Foundation Medicine between August 2014 and June 2020. CD274 CN changes were correlated with PD-L1 expression in tumor types where there were Food and Drug Administration approved companion diagnostic (CDx) claims and the CDx assay was used to assess PD-L1 expression., Results: In all, 244 584 samples representing 290 solid tumor types were included in the study. Overall, 17.6% (42 983/244 584) had CD274 CN gains (>specimen ploidy), 44.6% (108 970/244 584) were CD274 CN neutral, and 37.9% (92 631/244 584) had CD274 CN loss. Using different CN cut offs to define CD274 positivity resulted in different prevalence estimates: ploidy +1, 17.4% (42 636/244 584); ploidy +2, 6.2% (15 183/244 584); ploidy +3, 2.2% (5375/244 584); ploidy +4, 1.1% (2712/244 584); and ploidy +8, 0.2% (434/244 584). The prevalence of CN changes and CN positivity varied based on tumor type. CD274 CN gains were significantly associated with PD-L1 positivity in NSCLC, urothelial carcinoma, breast carcinoma, cervical carcinoma, esophagus squamous cell carcinoma (SCC) and head and neck SCC (ORs 3.3, 3.0, 2.0, 4.5. 3.8, 8.4, 1.4, respectively; p<0.05) and with microsatellite instability status in only clinically relevant tumor types (gastric adenocarcinoma, colorectal adenocarcinoma, uterine endometrial adenocarcinoma, esophageal adenocarcinoma and gastroesophageal junction adenocarcinoma (OR: 5.2, 1.9, 3.2, 3.7 and 6.5, respectively; p<0.05)). Conversely, CD274 CN changes were not significantly correlated with tumor mutational burden in almost all the tumor types., Conclusion: CD274 CN changes and PD-L1 expression were highly correlated in multiple tumor types. These prevalence data on CD274 CN changes across a large cohort of different solid tumors can be used to design future clinical studies to assess whether CD274 CN changes could be a potential biomarker for ICPI., Competing Interests: Competing interests: All authors of the manuscript are employees of Foundation Medicine, which is a wholly owned subsidiary of Roche and receives stock from Roche., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Molecular profiling of gliomas is vital to ensure diagnostic accuracy, inform prognosis, and identify clinical trial options for primary and recurrent tumors. This study aimed to determine the accuracy of reporting the whole arm 1p19q codeletion status from the FoundationOne platform., Methods: Testing was performed on glioma samples as part of clinical care and analyzed up to 395 cancer-associated genes (including IDH1/2 ). The whole arm 1p19q codeletion status was predicted from the same assay using a custom research-use only algorithm, which was validated using 463 glioma samples with available fluorescence in-situ hybridization (FISH) data. For 519 patients with available outcomes data, progression-free and overall survival were assessed based on whole arm 1p19q codeletion status derived from sequencing data., Results: Concordance between 1p19q status based on FISH and our algorithm was 96.7% (449/463) with a positive predictive value (PPV) of 100% and a positive percent agreement (PPA) of 91.0%. All discordant samples were positive for codeletion by FISH and harbored genomic alterations inconsistent with oligodendrogliomas. Median overall survival was 168 months for the IDH1/2 mutant, codeleted group, and 122 months for IDH1/2 mutant-only (hazard ratio (HR): 0.42; P < .05)., Conclusions: 1p19q codeletion status derived from FoundationOne testing is highly concordant with FISH results. Genomic profiling may be a reliable substitute for traditional FISH testing while also providing IDH1/2 status., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

We examined a large dataset of female metastatic breast cancers (MBCs) profiled with comprehensive genomic profiling (CGP) to identify the prevalence and distribution of immunotherapy responsiveness-associated biomarkers. DNA was extracted from 3831 consecutive MBCs: 1237 (ER pos /HER2 neg ), 1953 ER neg /HER2 amp , and 641 triple-negative breast cancer (TNBC). CGP was performed using the FoundationOne ® or FoundationOne ® CDx NGS assay. Tumor mutational burden (TMB) and microsatellite instability (MSI) were determined in a subset of cases. PD-L1 expression in immunocytes in a subset of cases was determined by immunohistochemistry using the companion diagnostic VENTANA PD-L1 SP142 Assay. The median age of the cohort was 54 years (range 20-89). Genomic alterations (GAs)/tumor were similar (range: 5.9-7.3). Markers of potential immune checkpoint inhibitor (ICPI) benefit included: CD274 (PD-L1) amplification (1%-3%), BRAF GA (1%-4%), TMB of ≥10 mutations/Mb (8%-12%), MSI-high (0.1%-0.4%), PBRM1 GA (1%), and positive PD-L1 staining of immunocytes ranging from 13% in ER pos /HER2 neg and 33% in ER neg /HER2 amp to 47% in the TNBC group. Potential markers of ICPI resistance included inactivating STK11 GA (1%-2%) and MDM2 amplification (3%-6%). MTOR pathway targets were common with lowest frequency in TNBC. ERBB2 short variant mutations were most frequent ER pos /HER2 neg and absent in TNBC. BRCA1/2 GA were least frequent in ER neg /HER2 amp . The demonstrations of clinical benefit of immunotherapy in MBC support the need for development and utilization of biomarkers to guide the use of ICPIs for these patients. In addition to guiding therapy selection, CGP shows potential to identify GA linked to response and resistance to ICPI in MBC., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Metastatic breast cancer is the leading cause of cancer death in women, but the genomics of metastasis in breast cancer are poorly studied., Methods: We explored a set of 11,616 breast tumors, including 5,034 metastases, which had undergone targeted sequencing during standard clinical care., Results: Besides the known hotspot mutations in ESR1, we observed a metastatic enrichment of previously unreported, lower-prevalence mutations in the ligand-binding domain, implying that these mutations may also be functional. Furthermore, individual ESR1 hotspots are significantly enriched in specific metastatic tissues and histologies, suggesting functional differences between these mutations. Other alterations enriched across all metastases include loss of function of the CDK4 regulator CDKN1B, and mutations in the transcription factor CTCF. Mutations enriched at specific metastatic sites generally reflect biology of the target tissue and may be adaptations to growth in the local environment. These include PTEN and ASXL1 alterations in brain metastases and NOTCH1 alterations in skin. We observed an enrichment of KRAS, KEAP1, STK11 and EGFR mutations in lung metastases. However, the patterns of other mutations in these tumors indicate that these are misdiagnosed lung primaries rather than breast metastases., Conclusions: An order-of-magnitude increase in samples relative to previous studies allowed us to detect novel genomic characteristics of metastatic cancer and to expand and clarify previous findings., Competing Interests: All authors were employees and may have held shares of Genentech/Roche or Foundation Medicine at the time this work was completed. Both companies are involved in the development of cancer drugs and clinical assays for metastatic breast cancer, and have multiple patents in related areas. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

A high tumour mutational burden (hypermutation) is observed in some gliomas 1-5 ; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Purpose: Amplifications of receptor tyrosine kinases (RTKS) are therapeutic targets in multiple tumor types (e.g. HER2 in breast cancer), and amplification of the chromosome 4 segment harboring the three RTKs KIT, PDGFRA, and KDR (4q12amp) may be similarly targetable. The presence of 4q12amp has been sporadically reported in small tumor specific series but a large-scale analysis is lacking. We assess the pan-cancer landscape of 4q12amp and provide early clinical support for the feasibility of targeting this amplicon., Experimental Design: Tumor specimens from 132,872 patients with advanced cancer were assayed with hybrid capture based comprehensive genomic profiling which assays 186-315 genes for all classes of genomic alterations, including amplifications. Baseline demographic data were abstracted, and presence of 4q12amp was defined as 6 or more copies of KIT/KDR/PDGFRA. Concurrent alterations and treatment outcomes with matched therapies were explored in a subset of cases., Results: Overall 0.65% of cases harbored 4q12amp at a median copy number of 10 (range 6-344). Among cancers with >100 cases in this series, glioblastomas, angiosarcomas, and osteosarcomas were enriched for 4q12amp at 4.7%, 4.8%, and 6.4%, respectively (all p < 0.001), giving an overall sarcoma (n = 6,885) incidence of 1.9%. Among 99 pulmonary adenocarcinoma cases harboring 4q12amp, 50 (50%) lacked any other known driver of NSLCC. Four index cases plus a previously reported case on treatment with empirical TKIs monotherapy had stable disease on average exceeding 20 months., Conclusion: We define 4q12amp as a significant event across the pan-cancer landscape, comparable to known pan-cancer targets such as NTRK and microsatellite instability, with notable enrichment in several cancers such as osteosarcoma where standard treatment is limited. The responses to available TKIs observed in index cases strongly suggest 4q12amp is a druggable oncogenic target across cancers that warrants a focused drug development strategy., Implications for Practice: Coamplification of the receptor tyrosine kinases (rtks) KIT/KDR/PDGFRA (4q12amp) is present broadly across cancers (0.65%), with enrichment in osteosarcoma and gliomas. Evidence for this amplicon having an oncogenic role is the mutual exclusivity of 4q12amp to other known drivers in 50% of pulmonary adenocarcinoma cases. Furthermore, preliminary clinical evidence for driver status comes from four index cases of patients empirically treated with commercially available tyrosine kinase inhibitors with activity against KIT/KDR/PDGFRA who had stable disease for 20 months on average. The sum of these lines of evidence suggests further clinical and preclinical investigation of 4q12amp is warranted as the possible basis for a pan-cancer drug development strategy., (© 2019 The Authors. The Oncologist published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

We characterized the landscape and drug sensitivity of ERBB2 (HER2) mutations in cancers. In 11 datasets (n = 211,726), ERBB2 mutational hotspots varied across 25 tumor types. Common HER2 mutants yielded differential sensitivities to eleven EGFR/HER2 tyrosine kinase inhibitors (TKIs) in vitro, and molecular dynamics simulations revealed that mutants with a reduced drug-binding pocket volume were associated with decreased affinity for larger TKIs. Overall, poziotinib was the most potent HER2 mutant-selective TKI tested. Phase II clinical testing in ERBB2 exon 20-mutant non-small cell lung cancer resulted in a confirmed objective response rate of 42% in the first 12 evaluable patients. In pre-clinical models, poziotinib upregulated HER2 cell-surface expression and potentiated the activity of T-DM1, resulting in complete tumor regression with combination treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: Merkel cell carcinoma (MCC) is a rare, aggressive cutaneous malignancy, which has demonstrated sensitivity to immune checkpoint inhibitor therapy. Here, we perform the largest genomics study in MCC to date to characterize the molecular landscape and evaluate for clinical and molecular correlates to immune checkpoint inhibitor response., Experimental Design: Comprehensive molecular profiling was performed on 317 tumors from patients with MCC, including the evaluation of oncogenic mutations, tumor mutational burden (TMB), mutational signatures, and the Merkel cell polyomavirus (MCPyV). For a subset of 57 patients, a retrospective analysis was conducted to evaluate for clinical and molecular correlates to immune checkpoint inhibitor response and disease survival., Results: Genomic analyses revealed a bimodal distribution in TMB, with 2 molecularly distinct subgroups. Ninety-four percent ( n = 110) of TMB-high specimens exhibited an ultraviolet light (UV) mutational signature. MCPyV genomic DNA sequences were not identified in any TMB-high cases (0/117), but were in 63% (110/175) of TMB-low cases. For 36 evaluable patients treated with checkpoint inhibitors, the overall response rate was 44% and response correlated with survival at time of review (100% vs. 20%, P < 0.001). Response rate was 50% in TMB-high/UV-driven and 41% in TMB-low/MCPyV-positive tumors ( P = 0.63). Response rate was significantly correlated with line of therapy: 75% in first-line, 39% in second-line, and 18% in third-line or beyond ( P = 0.0066). PD-1, but not PD-L1, expression was associated with immunotherapy response (77% vs. 21%, P = 0.00598, for PD-1 positive and negative, respectively)., Conclusions: We provide a comprehensive genomic landscape of MCC and demonstrate clinicogenomic associates of immunotherapy response., (©2019 American Association for Cancer Research.)

Background: PD-L1 expression and tumor mutational burden (TMB) have emerged as important biomarkers of response to immune checkpoint inhibitor (ICI) therapy. These biomarkers have each succeeded and failed in predicting responders for different cancer types. We sought to describe the PD-L1 expression landscape across the spectrum of ICI-responsive human cancers, and to determine the relationship between PD-L1 expression, TMB, and response rates to ICIs., Methods: We assessed 9887 clinical samples for PD-L1 expression and TMB., Results: PD-L1 expression and TMB are not significantly correlated within most cancer subtypes, and they show only a marginal association at the tumor sample level (Pearson's correlation 0.084). Across distinct tumor types, PD-L1 expression and TMB have nonoverlapping effects on the response rate to PD-1/PD-L1 inhibitors and can broadly be used to categorize the immunologic subtypes of cancer., Conclusion: Our results indicate that PD-L1 expression and TMB may each inform the use of ICIs, point to different mechanisms by which PD-L1 expression regulates ICI responsiveness, and identify new opportunities for therapeutic development., Funding: Funding was provided by Foundation Medicine Inc., the Johns Hopkins Bloomberg-Kimmel Institute for Cancer Immunotherapy, the Viragh Foundation, the National Cancer Institute Specialized Program of Research Excellence (SPORE) in Gastrointestinal Cancers (P50 CA062924), the NIH Center Core Grant (P30 CA006973), the Norman & Ruth Rales Foundation, and the Conquer Cancer Foundation.

After sequential treatment with first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), EGFR-mutant non-small cell lung cancers frequently harbor multiple resistance mutations in exon 20 of EGFR including T790M, mediating resistance to first-generation TKIs, and at codons 792, 796, or 797 mediating resistance to third-generation TKIs. However, whether these resistance mutations are in cis or trans has therapeutic implications for patients. We analyzed a cohort of 29 patients with NSCLC harboring EGFR mutations at codons 792, 796, or 797 to establish the configuration of these mutations. We performed hybrid capture-based, next-generation sequencing on formalin-fixed paraffin-embedded biopsy tissue or liquid biopsy. 27 samples had both a T790M mutation and a mutation at codons 792, 796, or 797. In all of these cases, the mutations were found in the cis configuration; the trans configuration was not observed. Two patients' samples harbored a mutation at codon 797 but no T790M mutation. In these two cases, longitudinal analysis showed earlier biopsies harbored EGFR T790M, which was undetectable following osimertinib treatment. Treatment of one these patients with both first- and third-generation EGFR TKIs resulted in a mixed response. Here we describe multiple configurations of EGFR T790M and third-generation TKI resistance mutations at codons 792, 796, and 797. These mutations are most commonly found in cis, which confers resistance to all current EGFR TKIs. We also describe two patients that exhibited T790M loss with acquisition of a mutation at codon 797. In addition, one of these patients, with an EGFR C797S in a lung biopsy was subsequently found to have EGFR C797N in a later biopsy of pleural fluid, highlighting the dynamic multiclonal nature of advanced NSCLC., Competing Interests: We have read the journal's policy and the authors of this manuscript have the following competing interests: All authors affiliated with Foundation Medicine were employees and owned FMI stock (MEG, MM, LY, JS, JG, MK, VAM, PJS, GMF, SA, LAA). JPG is an External Speaker and consultant for Foundation Medicine Inc. This affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. All other authors have no other financial conflicts to disclose.

Rearrangements involving the neurotrophic receptor kinase genes (NTRK1, NTRK2, and NTRK3; hereafter referred to as TRK) produce oncogenic fusions in a wide variety of cancers in adults and children. Although TRK fusions occur in fewer than 1% of all solid tumors, inhibition of TRK results in profound therapeutic responses, resulting in Breakthrough Therapy FDA approval of the TRK inhibitor larotrectinib for adult and pediatric patients with solid tumors, regardless of histology. In contrast to solid tumors, the frequency of TRK fusions and the clinical effects of targeting TRK in hematologic malignancies are unknown. Here, through an evaluation for TRK fusions across more than 7,000 patients with hematologic malignancies, we identified TRK fusions in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), histiocytosis, multiple myeloma, and dendritic cell neoplasms. Although TRK fusions occurred in only 0.1% of patients (8 of 7,311 patients), they conferred responsiveness to TRK inhibition in vitro and in vivo in a patient-derived xenograft and a corresponding AML patient with ETV6-NTRK2 fusion. These data identify that despite their individual rarity, collectively, TRK fusions are present in a wide variety of hematologic malignancies and predict clinically significant therapeutic responses to TRK inhibition.

Immune evasion is a well-recognized hallmark of cancer and recent studies with immunotherapy agents have suggested that tumors with increased numbers of neoantigens elicit greater immune responses. We hypothesized that the immune system presents a common selective pressure on high mutation burden tumors and therefore immune evasion mutations would be enriched in high mutation burden tumors. The JAK family of kinases is required for the signaling of a host of immune modulators in tumor, stromal, and immune cells. Therefore, we analyzed alterations in this family for the hypothesized signature of an immune evasion mutation. Here, we searched a database of 61,704 unique solid tumors for alterations in the JAK family kinases (JAK1/2/3, TYK2). We used The Cancer Genome Atlas and Cancer Cell Line Encyclopedia data to confirm and extend our findings by analyzing gene expression patterns. Recurrent frameshift mutations in JAK1 were associated with high mutation burden and microsatellite instability. These mutations occurred in multiple tumor types including endometrial, colorectal, stomach, and prostate carcinomas. Analyzing gene expression signatures in endometrial and stomach adenocarcinomas revealed that tumors with a JAK1 frameshift exhibited reduced expression of interferon response signatures and multiple anti-tumor immune signatures. Importantly, endometrial cancer cell lines exhibited similar gene expression changes that were expected to be tumor cell intrinsic (e.g. interferon response) but not those expected to be tumor cell extrinsic (e.g. NK cells). From these data, we derive two primary conclusions: 1) JAK1 frameshifts are loss of function alterations that represent a potential pan-cancer adaptation to immune responses against tumors with microsatellite instability; 2) The mechanism by which JAK1 loss of function contributes to tumor immune evasion is likely associated with loss of the JAK1-mediated interferon response.

Genomic profiling is widely predicted to become a standard of care in clinical oncology, but more effective data sharing to accelerate progress in precision medicine will be required. Here, we describe cancer-associated genomic profiles from 18,004 unique adult cancers. The dataset was composed of 162 tumor subtypes including multiple rare and uncommon tumors. Comparison of alteration frequencies to The Cancer Genome Atlas identified some differences and suggested an enrichment of treatment-refractory samples in breast and lung cancer cohorts. To illustrate novelty within the dataset, we surveyed the genomic landscape of rare diseases and identified an increased frequency of NOTCH1 alterations in adenoid cystic carcinomas compared with previous studies. Analysis of tumor suppressor gene patterns revealed disease specificity for certain genes but broad inactivation of others. We identified multiple potentially druggable, novel and known kinase fusions in diseases beyond those in which they are currently recognized. Analysis of variants of unknown significance identified an enrichment of SMAD4 alterations in colon cancer and other rare alterations predicted to have functional impact. Analysis of established, clinically relevant alterations highlighted the spectrum of molecular changes for which testing is currently recommended, as well as opportunities for expansion of indications for use of approved targeted therapies. Overall, this dataset presents a new resource with which to investigate rare alterations and diseases, validate clinical relevance, and identify novel therapeutic targets. Cancer Res; 77(9); 2464-75. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Chromosome instability (CIN) is deleterious to normal cells because of the burden of aneuploidy. However, most human solid tumors have an abnormal karyotype implying that gain and loss of chromosomes by cancer cells confers a selective advantage. CIN can be induced in the mouse by inactivating the spindle assembly checkpoint. This is lethal in the germline but we show here that adult T cells and hepatocytes can survive conditional inactivation of the Mad2l1 SAC gene and resulting CIN. This causes rapid onset of acute lymphoblastic leukemia (T-ALL) and progressive development of hepatocellular carcinoma (HCC), both lethal diseases. The resulting DNA copy number variation and patterns of chromosome loss and gain are tumor-type specific, suggesting differential selective pressures on the two tumor cell types.

Background: The integration of genomics with immunotherapy has potential value for cancer vaccine development. Given the clinical successes of immune checkpoint modulators, interest in cancer vaccines as therapeutic options has been revived. Current data suggest that each tumor contains a unique set of mutations (mutanome), thus requiring the creation of individualized cancer vaccines. However, rigorous analysis of non-individualized cancer immunotherapy approaches across multiple cancer types and in the context of known driver alterations has yet to be reported. We therefore set out to determine the feasibility of a generalizable cancer vaccine strategy based on targeting multiple neoantigens in an HLA-A/B subtype-directed manner., Methods: A cancer gene-focused, hybrid capture-based genomic analysis was performed on 63,220 unique tumors. Neoantigens were predicted using a combined peptide processing and MHC-I binding prediction tool (IEDB) for all recurrent (>10 tumors) missense alterations and non-frameshift indels for the two most common HLA-A/B subtypes in North American/European populations., Results: Despite being overwhelmingly unique overall, many mutanomes (~45%) contain at least one mutation from a set of ten mutations chosen to maximize the number of unique tumors. This held true for tumors driven by KRAS G12C (n = 1799), PIK3CA E545K (n = 1713), or EGFR L858R (n = 478) alterations, which define distinct sample subsets. We therefore hypothesized that sets of carefully selected mutations/neoantigens may allow the development of broadly applicable semi-universal cancer vaccines. To test the feasibility of such an approach, antigen processing and MHC-I binding prediction was applied for HLA subtypes A*01:01/B*08:01 and A*02:01/B*44:02. In tumors with a specific HLA type, 0.7 and 2.5% harbored at least one of a set of ten neoantigens predicted to bind to each subtype, respectively. In comparison, KRAS G12C-driven tumors produced similar results (0.8 and 2.6% for each HLA subtype, respectively), indicating that neoantigen targets still remain highly diverse even within the context of major driver mutations., Conclusions: This "best case scenario" analysis of a large tumor set across multiple cancer types and in the context of driver alterations reveals that semi-universal, HLA-specific cancer vaccine strategies will be relevant to only a small subset of the general population. Similar analysis of whole exome/genome sequencing, although not currently feasible at scale in a clinical setting, will likely uncover further diversity.

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is a saprophytic fungus that is ubiquitous in the environment and is commonly associated with allergic sensitization and severe asthma in humans. Although A. fumigatus is recognized by multiple microbial pattern-recognition receptors, we found that an A. fumigatus-derived glycosphingolipid, asperamide B, directly activates invariant natural killer T (iNKT) cells in vitro in a CD1d-restricted, MyD88-independent and dectin-1-independent fashion. Moreover, asperamide B, when loaded onto CD1d, directly stained, and was sufficient to activate, human and mouse iNKT cells. In vivo, asperamide B rapidly induced airway hyperreactivity, which is a cardinal feature of asthma, by activating pulmonary iNKT cells in an interleukin-33 (IL-33)-ST2-dependent fashion. Asperamide B is thus the first fungal glycolipid found to directly activate iNKT cells. These results extend the range of microorganisms that can be directly detected by iNKT cells to the kingdom of fungi and may explain how A. fumigatus can induce severe chronic respiratory diseases in humans.

Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80(+) lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Blockade of TIM-4 inhibited the phagocytosis of antigen-specific T cells by TIM-4 expressing lymph node medullary macrophages, resulting in an increase in the number of antigen-specific T cells and the abrogation of respiratory tolerance. Moreover, specific depletion of medullary macrophages inhibited the induction of respiratory tolerance, highlighting the key role of TIM-4 and medullary macrophages in tolerance. Therefore, TIM-4-mediated clearance of antigen specific T cells represents an important previously unrecognized mechanism regulating respiratory tolerance.

Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4-CD8-, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2(-/-) mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-gamma production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2(-/-) non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell-mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1-PD-L1 interactions in mediating tissue tolerance.