Background: Genomic fusions are potent oncogenic drivers across cancer types and many are targetable. We demonstrate the clinical performance of DNA-based comprehensive genomic profiling (CGP) for detecting targetable fusions. Materials and Methods: We analyzed targetable fusion genes in >450 000 tissue specimens profiled using DNA CGP (FoundationOne CDx, FoundationOne). Using a de-identified nationwide (US-based) non-small cell lung cancer (NSCLC) clinico-genomic database, we assessed outcomes in patients with nonsquamous NSCLC (NonSqNSCLC) who received matched therapy based on a fusion identified using DNA CGP. Lastly, we modeled the added value of RNA CGP for fusion detection in NonSqNSCLC. Results: We observed a broad diversity of fusion partners detected with DNA CGP in conjunction with targetable fusion genes (ALK, BRAF, FGFR2, FGFR3, NTRK1/2/3, RET, and ROS1). In NonSqNSCLC with oncogenic ALK, NTRK, RET, and ROS1 fusions detected by DNA CGP, patients treated with a matched tyrosine kinase inhibitor had better real-world progression-free survival than those receiving alternative treatment regimens and benefit was observed regardless of the results of orthogonal fusion testing. An estimated 1.3% of patients with NonSqNSCLC were predicted to have an oncogenic driver fusion identified by RNA, but not DNA CGP, according to a model that accounts for multiple real-world factors. Conclusion: A well-designed DNA CGP assay is capable of robust fusion detection and these fusion calls are reliable for informing clinical decision-making. While DNA CGP detects most driver fusions, the clinical impact of fusion detection is substantial for individual patients and exhaustive efforts, inclusive of additional RNA-based testing, should be considered when an oncogenic driver is not clearly identified.

Supplementary Figure S1. Reports cell cycle analysis after 4 days of drug treatment.

Background Mutations in the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), encoded by the PIK3CA gene, cause dysregulation of the PI3K pathway in 35–40% of patients with HR+/HER2– breast cancer. Preclinically, cancer cells harboring double or multiple PIK3CA mutations (mut) elicit hyperactivation of the PI3K pathway leading to enhanced sensitivity to p110α inhibitors. Methods To understand the role of multiple PIK3CA mut in predicting response to p110α inhibition, we estimated the clonality of multiple PIK3CA mut in circulating tumor DNA (ctDNA) from patients with HR+/HER2– metastatic breast cancer enrolled to a prospectively registered clinical trial of fulvestrant ± taselisib, and analyzed the subgroups against co-altered genes, pathways, and outcomes. Results ctDNA samples with clonal multiple PIK3CA mut had fewer co-alterations in receptor tyrosine kinase (RTK) or non- PIK3CA PI3K pathway genes compared to samples with subclonal multiple PIK3CA mut indicating a strong reliance on the PI3K pathway. This was validated in an independent cohort of breast cancer tumor specimens that underwent comprehensive genomic profiling. Furthermore, patients whose ctDNA harbored clonal multiple PIK3CA mut exhibited a significantly higher response rate and longer progression-free survival vs subclonal multiple PIK3CA mut. Conclusions Our study establishes clonal multiple PIK3CA mut as an important molecular determinant of response to p110α inhibition and provides rationale for further clinical investigation of p110α inhibitors alone or with rationally-selected therapies in breast cancer and potentially other solid tumor types.

Precision oncology is a rapidly evolving field. Reliable tools are required for comprehensive genomic profiling (CGP) in clinical and research settings. NGS provides capabilities to detect multiple alterations, but complex workflows may be difficult to implement. Roche Diagnostics and Foundation Medicine have developed the AVENIO Tumor Tissue CGP Kit, a streamlined and easy to implement Research-Use-Only CGP assay to enable translational research labs. The AVENIO Tumor Tissue CGP Kit profiles 324 genes for cancer alterations, tumor mutation burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH)). The kit contains reagents to create libraries. A software provides access to Foundation Medicine's bioinformatics pipeline. Five laboratories tested 160 samples, consisting of cell line and formalin fixed paraffin embedded (FFPE) tumor specimens. To assess reproducibility, 144 samples were evaluated across operators, lots, and instruments. To evaluate diagnostic sensitivity and specificity, 314 FFPE samples were tested and positive and negative predictive agreements determined against an orthogonal NGS method. Across five labs, pass rate was 98.1%. Sequencing quality metrics were high, with most samples reaching 1500x-2500x median coverage. Sites had 99.7% agreement for short variants. In the 144 sample reproducibility study, samples were highly uniform across variables, achieving >90% on-target rate. In the 314 sample concordance study, pass rate was 99.06%, with most samples achieving 2000x-2500x median coverage. Out of 314 samples, PPA to an orthogonal NGS-based method were: 98.3% short variants, 90.5% rearrangements, 94.8% copy number, 100% MSI, 100% TMB, and 96.8% gLOH. NPA reached 99.97% across short variants. Detected alterations included EGFR T7090M, BRAF V600E, KRAS 12/13 mutations, and EML4-ALK fusions. CGP tests are being utilized in precision oncology, but NGS workflows and bioinformatics pipelines can be difficult to implement. The AVENIO CGP Tumor Tissue Kit is highly reproducible across sites and operators, achieving high sequencing quality metrics and diagnostic sensitivity.

PURPOSE To examine the overlap of homologous recombination deficiency (HRD) and microsatellite instability high (MSI-H) status, and to dissect driver versus bystander status of BRCA1/2 mutations ( BRCAm) in this context. METHODS A pan-cancer comprehensive genomic profiling cohort (n = 213,199) was examined for overlap between BRCAm and MSI-H status. BRCA1/2 variant zygosity was examined and correlated with MSI-H status, tumor mutational burden, and genome-wide loss of heterozygosity (gLOH). Clinical histories of two patients with prostate cancer with co-occurring BRCAm and MSI-H are described. RESULTS HRD and MSI-H phenotypes were generally mutually exclusive events ( P < .001). BRCAm that co-occurred together with high tumor mutational burden or MSI-H were predominantly monoallelic bystander alterations. In breast, ovarian, and pancreatic cancers, very few BRCAm occurred in the context of MSI-H; however, in prostate cancer, 12.8% of BRCA1 and 3.4% of BRCA2 alterations co-occurred with MSI-H. In these BRCA-associated cancers, co-occurring BRCAm were generally monoallelic and were not associated with elevated gLOH. Two patients with prostate cancer with co-occurring BRCAm and MSI-H showed resistance to poly (ADP-ribose) polymerase inhibition but sensitivity to subsequent anti–programmed cell death protein 1 therapy. CONCLUSION MSI-H status and HRD are generally mutually exclusive phenomena across cancer types, but may rarely co-occur, especially in prostate cancer. Although MSI-H samples had a higher BRCAm prevalence relative to microsatellite-stable tumors, these BRCA1/2 mutations were generally monoallelic and were not associated with elevated gLOH. Our findings suggest that most BRCAm coexisting with microsatellite instability are likely bystander events that may not result in sensitivity to poly (ADP-ribose) polymerase inhibitors.

There are more than 70 distinct sarcomas, and this diversity complicates the development of precision-based therapeutics for these cancers. Prospective comprehensive genomic profiling could overcome this challenge by providing insight into sarcomas’ molecular drivers. Through targeted panel sequencing of 7494 sarcomas representing 44 histologies, we identify highly recurrent and type-specific alterations that aid in diagnosis and treatment decisions. Sequencing could lead to refinement or reassignment of 10.5% of diagnoses. Nearly one-third of patients (31.7%) harbor potentially actionable alterations, including a significant proportion (2.6%) with kinase gene rearrangements; 3.9% have a tumor mutational burden ≥10 mut/Mb. We describe low frequencies of microsatellite instability (<0.3%) and a high degree of genome-wide loss of heterozygosity (15%) across sarcomas, which are not readily explained by homologous recombination deficiency (observed in 2.5% of cases). In a clinically annotated subset of 118 patients, we validate actionable genetic events as therapeutic targets. Collectively, our findings reveal the genetic landscape of human sarcomas, which may inform future development of therapeutics and improve clinical outcomes for patients with these rare cancers.

Based on the approvals of crizotinib and entrectinib by the Food and Drug Administration for the treatment of ROS1 positive nonsmall cell lung cancer (NSCLC), we sought to examine the mutational profile of a variety of solid tumors (excluding sarcomas) with ROS1 fusions that underwent comprehensive genomic profiling. A review of our database was performed to extract all nonsarcoma patients with ROS1 fusions that were discovered by the hybrid capture‐based DNA only sequencing assays. We examined the coalterations representing potentially targetable biomarkers, resistance alterations and other alterations in these cases. In addition, we examined the histologic characteristics and protein expression with immunohistochemistry (IHC). From a series of clinically advanced nonsarcoma solid tumors, 356 unique cases with ROS1 fusions included 275 (77.2%) NSCLC and 81 (22.8%) non‐NSCLC. Ten novel ROS1 fusions were discovered. Importantly, the NSCLC ROS1 fusion pos tumors had a higher PD‐L1 IHC expression positivity when compared to the NSCLC ROS1 fusion neg population ( P = .012, Chi‐squared). The frequency of known and likely anti‐ ROS1 targeted therapy resistance genomic alterations in NSCLC was 7.3% (20/275) and in non‐NSCLC was 4.9% (4/81). Overall, the coalteration profile of ROS1 fusion pos NSCLC and non‐NSCLC was similar with only three genes altered significantly more frequently in non‐NSCLC vs NSCLC: TERT , PTEN , APC . In our study, we characterized a large cohort of ROS1 fusion pos NSCLC and non‐NSCLC solid tumors and discovered 10 novel ROS1 fusions.

Background: Adult primary retroperitoneal sarcomas (RPSs) are a group of heterogeneous tumors with different histological subtypes. Comprehensive genomic profiling (CGP) analyses have recently provided significant insights into the biology of sarcomas by identifying genomic alterations (GAs) which could benefit from targeted therapies. Methods: RPS were evaluated by CGP using next-generation sequencing of up to 406 cancer-related genes. Tumor mutational burden (TMB) was determined on 0.83 to 1.14 mut/Mb of sequenced DNA. Finally, PD-L1 expression was determined. Results: Overall, 296 cases of primary RPS were analyzed. Liposarcoma (LPS) subtype had more GA/tumor than leiomyosarcoma (LMS) subtypes, with follicular dendritic cell sarcomas harboring the highest and synovial sarcomas the lowest. TP53 and Rb1 alterations were the highest in LMS, and CDK4/6 and MDM2 in LPS. However, both the TMB and targetable GA rates were low across subtypes. PD-L1 immunostaining was low positive in 21% and high positive in 5% of patients, respectively. Conclusions: CGP analysis revealed that potentially actionable genomic targets were rare in our cohort of RPS. Moreover, RPSs seem less likely to respond to immune checkpoint inhibitors based on putative biomarkers status. Nevertheless, genomic stratification according to histological subtypes led to description of GAs that can inform future clinical trials design.

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.

Genomic studies performed in cancer patients and tumor-derived cell lines have identified a high frequency of alterations in components of the mammalian switch/sucrose non-fermentable (mSWI/SNF or BAF) chromatin remodeling complex, including its core catalytic subunit, SMARCA4. Cells exhibiting loss of SMARCA4 rely on its paralog, SMARCA2, making SMARCA2 an attractive therapeutic target. Here we report the genomic profiling of solid tumors from 131,668 cancer patients, identifying 9434 patients with one or more SMARCA4 gene alterations. Homozygous SMARCA4 mutations were highly prevalent in certain tumor types, notably non-small cell lung cancer (NSCLC), and associated with reduced survival. The large sample size revealed previously uncharacterized hotspot missense mutations within the SMARCA4 helicase domain. Functional characterization of these mutations demonstrated markedly reduced remodeling activity. Surprisingly, a few SMARCA4 missense variants partially or fully rescued paralog dependency, underscoring that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations who may benefit from SMARCA2-targeted therapy.

Abstract Genomic studies performed in cancer patients have identified a high frequency of alterations in the core catalytic subunit of the mammalian switch/sucrose non-fermentable (mSWI/SNF) chromatin remodeling complex, SMARCA4. Cells with inactivating SMARCA4 mutations rely on its paralog, SMARCA2, making it an attractive therapeutic target. Here we report the results of genomic profiling of 131,668 cases from patients with solid tumors and identified 8,588 cases with one or more SMARCA4 alterations. We observed a high prevalence of homozygous SMARCA4 truncating and non-truncating mutations in certain tumor types, notably non-small cell lung cancer (NSCLC) and cancers of unknown primary. We found that homozygous SMARCA4 mutations in NSCLC were mutually exclusive with other mutations in mSWI/SNF and oncogenic drivers, including KRAS and EGFR. A retrospective study of NSCLC patients who were treated in the Flatiron Health network (>265 oncology practices in the U.S.) and underwent FoundationOne® or FoundationOne CDx® tumor sequencing as part of routine care revealed that real-world patients with homozygous truncating SMARCA4 mutations had significantly reduced overall survival. The large sample size of our cohort also elucidated novel hotspot missense mutations within the helicase domain of SMARCA4. Functional modeling of these mutations using biochemical and cell-based (ATAC-seq) assays demonstrated that all evaluated mutations have significantly reduced nucleosome remodeling activity upon in vitro reconstitution in SMARCA4-deficient cells. We also addressed whether SMARCA4 missense mutations could rescue the cell growth defects and loss in chromatin accessibility induced upon SMARCA2 loss. Interestingly, we observe that a few missense mutants were able to either partially or fully rescue growth, suggesting hypomorphic activity under the selective pressure of SMARCA2 loss. Overall, these studies highlight NSCLC patients with homozygous truncating SMARCA4 mutations as a novel population with an unmet need who may benefit from SMARCA2-targeted therapy and underscore that careful selection criteria must be employed to identify patients with inactivating, homozygous SMARCA4 missense mutations. Citation Format: Tharu M. Fernando, Robert Piskol, Russell Bainer, Ethan Sokol, Sally Trabucco, Qing Zhang, Huong Trinh, Sophia Maund, Marc Kschonsak, Subhra Chaudhuri, Tom Januario, Zora Modrusan, Robert L. Yauch. Characterization and therapeutic implications of SMARCA4 mutations in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5869.

Abstract Massive tumor sequencing allows us to progress beyond cataloging driver mutations, to discovery of the intricate patterns of genomic alterations that are required to enable a successful tumor. We used patterns of mutation co-occurrence to map activation of the ras pathway in over 16,000 lung adenocarcinomas. This revealed a network of 40 highly mutually exclusive mutations in 19 genes, 18 of which are known ras pathway genes (the RTKs EGFR, ERBB2, MET, ALK, RET, ROS1, KIT, FGFR2; NRAS and KRAS, BRAF and CRAF, MEK1/2, CBL, NF1, SOS1, and RIT1), indicating that each mutation is a strong pathway driver and is redundant with other pathway mutations. Together, these mutations cover 71% of all lung adenocarcinomas and include several previously uncharacterized mutations. Analysis of the remaining tumors uncovers additional patterns of ras pathway activation. Some have additional mutations in ras pathway genes that have the opposite pattern—they are co-occurrent with each other, either in an additive manner where several mutations are enriched with each other, such as rare raf and ras family mutations, or synergistic, where a particular pair of mutations is highly co-occurrent, often in the same gene. These pairs are exclusive of other pathway mutations, suggesting that they are also strong pathway activators. We also see partially exclusive mutations, including amplifications of several genes and loss-of-function RAF family mutations that are likely to be weak activators of the ras pathway in additional tumors. Finally, this approach also identifies recurrent mutations that are independent of other pathway mutations, giving clinical evidence that their function in human tumors is distinct from the common function of other ras pathway mutations. We validated these patterns of exclusivity, additivity, and synergy in 80,000 genomic profiles from 400 other tumor types, and identified additional exclusive ras pathway alterations in several other tissues. Across all cancers, 30% of all tumors had exclusive mutations indicating that they were ras pathway driven. The individual mutations varied greatly between tissues, suggesting that there exist extensive tissue-specific enhancers and inhibitors of each oncogenic mutation, which may be of great relevance to drug and biomarker development. This initial foray into the future of massive tumor genomic datasets shows that we can transform genomic data from individual mutations into patterns of pathway activation, and distinguish pathway-driving mutations from other hotspots, providing a better understanding of the oncogenic strategies used by each human tumor. The degree of mutational exclusivity seen can predict the degree of oncogene addiction, and patterns of co-mutation can suggest strategies for combination therapies, as well as allow reverse translation to enable better matching of preclinical and basic research models to oncogenic strategies that are most relevant to human tumors. Citation Format: Gerard Manning, Nick Lounsbury, Ryan Hartmaier, Sally Trabucco, Ethan Sokol. Dissecting the playbook of cancer: Genomic analysis of 100,000 human tumors reveals elaborate patterns of activation of the RTK-RAS-MAPK pathway [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA30.

Introduction: Follicular lymphoma (FL) is a slow growing lymphatic cancer characterized by translocations in and overexpression of BCL-2, which inhibits apoptosis. This pathogenesis may generate neoantigens (NAs) that are recognizable by T-cells as part of a patients' immune surveillance and are unique to specific FL mutations. Tumor mutation burden (TMB), and NA prevalence and prognostic nature, have previously been characterized for diffuse large B-cell lymphoma (DLBCL), identifying that TMB correlated with NA burden (NAB). While TMB did not correlate with outcomes, the presence of a NA, especially BCL2 NA, correlated with outcomes in de novo DLBCL. In addition, the majority of patients were predicted to have ≥1 NA (Paulson, EHA 2019). However, to date, the prevalence of NAs in FL, and their association with clinical outcomes, have not been characterized. The aim of our study was to characterize NA prevalence and evaluate the prognostic value of NA biomarkers, assessed by a targeted, comprehensive genomic profiling (CGP) platform, on progression-free survival (PFS) for patients with de novo FL. Methods: CGP data on 465 genes were available from patients with FL who provided biopsy samples at screening for the Phase III PRIMA trial (NCT00140582; intent-to-treat [ITT] population =1018; patients received rituximab [R] maintenance vs observation after response to initial first-line treatment with R-CVP [cyclophosphamide, vincristine, prednisone], R-CHOP [cyclophosphamide, doxorubicin, vincristine, prednisone] or R-FCM [fludarabine, cyclophosphamide, mitoxantrone]). CGP was used to calculate TMB, HLA type (OptiType) and NA prediction (NetMHCpan). The prevalence of NAs at time of screening was analyzed by the number and proportion of patients. The prognostic value of NAs were evaluated against the Cox Proportional Hazards null model for PFS including terms for treatment, country and response to induction treatment. The Akaike Information Criterion (AIC), a likelihood ratio test (LRT) p-value vs the null when including an additional NA term and its associated hazard ratio (95% CI), were calculated. A visualization of the genetic mutational landscape was generated using dimension reduction methods. Results: In total, 247 of the 1202 enrolled patients were assayed with CGP. Baseline characteristics and survival in the biomarker evaluable population (BEP) were consistent with the ITT population. We calculated a median TMB of 6.7 mutations per megabase for the PRIMA cohorts with a median of two predicted NAs per patient. The majority of patients (83%) were predicted to have ≥1 NA. TMB moderately correlated with NAB (0.42 pearson coefficient), Figure A. TMB was not associated with outcomes (hazard ratio [HR] 95% CI: 0.98 [0.94-1.02]) and similarly the presence of a NA was not associated with PFS (HR [95% CI]: 0.90 [0.56-1.44]). The most prevalent predicted NAs (n [patients]; %) were in BCL2 (54; 21.9%), CREBBP (49; 19.8%), EZH2 (14; 5.7%), KMT2D (13; 5.3%), and CARD11 (10; 4.0%). In a pooled analysis of patients from both arms, only presence of EZH2 NA improved the AIC of the null model (AIC=1135.6) to 1134.7 (LRT p=0.09 better than 0.1), Table. The HR (95% CI) of EZH2 NA in this model was 0.46 (0.17-1.25), indicating better survival for the EZH2 NA-negative group. Within each treatment group, HR (95% CI) for EZH2 was 0.27 (0.07-1.13) in the observation group and 1.05 (0.25-4.44) in the R group, Figure B. Inspecting the tSNE mutations by gene, a neighborhood pattern could be seen between EZH2 and BCL2 connected by KMT2D and flanked by CARD11 and CREBBP, Figure C. Conclusions: We observed that similarly to de novo DLBCL, TMB and NAB were also correlated in patients with de novo FL. While NAB was associated with clinical outcomes in de novo DLBCL, we did not observe these associations in FL. In patients with de novo FL,who discontinued R after initial treatment response, the absence of EZH2 predicted neoantigens were associated with PFS. This is consistent with results on EZH2 mutation status reported by Huet, et al.(Blood Cancer J 2017). A caveat to this hypothesis generating analysis is the small number of PFS events in the EZH2 neoantigen group. These insights may inform future personalized strategies in FL. Disclosures Henneges: Genentech (via Syneos Health): Current Employment; University of Wurzburg: Ended employment in the past 24 months. Jin:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Foundation Medicine Inc: Current Employment. Venstrom:Foundation Medicine, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Trabucco:F. Hoffmann-La Roche, Bristol-Myers Squibb Co., BioNTech: Current equity holder in publicly-traded company; Bristol-Myers Squibb Co: Current equity holder in publicly-traded company; BioNTech: Current equity holder in publicly-traded company; Loxo Oncology: Divested equity in a private or publicly-traded company in the past 24 months; Foundation Medicine, Inc.: Current Employment; Patent pending with Foundation Medicine and Genentech: Patents & Royalties: Patent pending. Nielsen:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Penuel:Genentech, Inc./ F. Hoffmann-La Roche: Current Employment; F. Hoffmann-La Roche: Current equity holder in publicly-traded company. Salles:Abbvie, Amgen, Celgene, Gilead, Janssen, Kite, Morphosys, Novartis, Roche, Takeda: Other: Participation to educational events; Abbvie, Autolus, BMS/Celgene, Debiopharm, Genmab, Kite/Gilead, Epizyme, Janssen, Karyopharm, Morphosys, Novartis, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees; Abbvie, Amgen, Celgene, Gilead, Janssen, Kite, Morphosys, Novartis, F. Hoffmann-La Roche, Takeda: Honoraria; Abbvie, Autolus, BMS/Celgene, Debiopharm, Genmab, Kite/Gilead, Epizyme, Janssen, Karyopharm, Morphosys, Novartis, F. Hoffmann-La Roche, Takeda: Consultancy. Paulson:Genentech, Inc.: Current Employment; F. Hoffmann-La Roche: Current equity holder in private company, Current equity holder in publicly-traded company.

Abstract Comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) provides an opportunity to noninvasively monitor a patient’s tumor burden via liquid biopsy. Liquid biopsies can assess a patient’s mutational landscape through time and provide information on treatment response and relapse. It has become increasingly common to have paired genomic data analysis between liquid and tissue biopsies from the same patient. However, the impact of clinical, temporal, and biologic factors on percentage of positive agreement (PPA) of variant detection between these biopsies is unclear. For our study, we leveraged two databases containing CGP data from paired liquid and tissue biopsy specimens tested by Foundation Medicine (FMI): the FMI database (>1700 paired samples) and the Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB). The CGDB is a subset of the FMI database linked with the Flatiron Health nationwide de-identified EHR-derived database, which includes demographic, treatment, and clinical outcomes information (>500 paired samples). We report pan-solid tumor and per-indication prevalence and PPA for variants commonly assayed in both liquid and tissue tests. We found that PPA depended on the tumor DNA concentration in plasma and tissue biopsies, as well as the amount of time between tests. We also investigated the effect of treatments administered in the time between liquid and tissue tests on the detected variants and observed some well-described resistance alterations at a higher frequency in liquid samples, including a higher prevalence of ESR1 point mutations in breast cancer, more EGFR T790M alterations in lung cancer, and frequent KRAS Q61H alterations in colorectal cancer. Liquid biopsies can also contain DNA shed from multiple metastatic sites. We observed evidence of this in the form of polyclonal resistance alterations, which may also account for differences in PPA over time. Our findings indicate that while PPA is generally high between samples, it may be influenced by factors such as intervening therapies, resistance, therapy efficacy, and polyclonality of liquid samples. Citation Format: Zoe June F. Assaf, Smruthy Sivakumar, Dexter X. Jin, Sophia L. Maund, Svetlana Lyalina, Guneet Walia, Ethan S. Sokol, Sally E. Trabucco. Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A17.

Abstract Background: Genomic instability refers to the tendency to accumulate genomic alterations. MSI-High (MSI-H) tumors are characterized by a mismatch repair deficiency triggering hypermutation, whereas HRD tumors display genomic lesions that result in genomic loss of heterozygosity (gLOH). These mechanistic differences have critical implications for treatment strategies, where immune checkpoint blockade is often efficacious in MSI-H tumors and PARP inhibition is linked to response in HRD tumors. We investigated the patterns of co-occurrence or mutual exclusivity between these two mechanisms of genomic instability. Methods: Comprehensive genomic profiling was performed on >130K tumors to coding exons and select introns of up to 465 genes. MSI was determined on up to 114 loci and high gLOH (≥14%) was used as a surrogate of HRD (PMID: 27908594). Results: In this cohort, HRD was most prevalent in triple-negative breast and fallopian tube cancers and absent in cancers such as skin neuroendocrine cancers. HRD was strongly associated with biallelic loss of homologous recombination (HR) pathway genes and not monoallelic loss of these genes. High MSI was most frequently found in endometrial and small intestine cancers, and absent in cancers such as fallopian tube cancers and GIST. MSI-H tumors and HRD tumors were mutually exclusive (6.5% of MSI-H tumors were HRD vs 25.7% of tumors that were not MSI-H; OR = 5.0; p<1e-108). Conclusions: We show that high MSI and HRD events are mutually exclusive across tumor types, suggesting that co-occurrence of these genomic instability mechanisms may be evolutionarily disadvantageous. High gLOH is associated with biallelic loss of HR pathway genes, supporting its use as a surrogate for HRD. The genomic differences between HRD and MSI-H may underlie differential susceptibility to targeted therapeutics. Gene% Biallelic loss in gLOH-H% Biallelic loss in gLOH-LowBiallelic loss p-value% Monoallelic loss in gLOH-H% Monoallelic loss in gLOH-LowMonoallelic loss p-valueATM2.45%1.54%1.24E-251.15%1.83%1.12E-17BARD10.25%0.03%5.15E-290.25%0.28%0.40BRCA15.15%0.23%<1.00E-1000.49%0.57%0.10BRCA25.32%0.76%<1.00E-1000.74%1.11%4.62E-09BRIP10.36%0.12%1.97E-160.41%0.49%0.07CDK120.49%0.31%3.95E-060.30%0.52%1.31E-07CHEK20.65%0.44%8.20E-060.54%0.86%1.92E-09FANCA0.51%0.25%1.36E-120.38%0.47%0.04FANCC0.22%0.07%2.03E-100.11%0.26%1.64E-07FANCG0.10%0.03%1.08E-050.08%0.12%0.04RAD51B0.39%0.15%5.36E-150.04%0.09%0.01RAD51C0.32%0.05%2.66E-320.10%0.12%0.41RAD51D0.18%0.02%2.52E-230.08%0.11%0.20 Citation Format: Dexter X. Jin, Ethan S. Sokol, Sally E. Trabucco, Garrett M. Frampton, Luciana Molinero. Microsatellite instability (MSI) and homologous recombination deficiency (HRD) are mutually exclusive mechanisms of genomic instability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3538.

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.

11541 Background: We performed CGP on 315 cases of RS to discover targetable genomic alterations (GA) and their potential impact on potential targeted and immunotherapy (IO) selection. Methods: FFPE tissues from 315 clinically advanced RS tissues underwent hybrid-capture based CGP (DNA and RNA). Tumor mutational burden (TMB) was determined on up to 1.2 Mbp of sequenced DNA, and tumor cell PD-L1 expression was determined by IHC (Dako 22C3) (low = 1-49%; High = >50% tumor cell staining). Results: 155 liposarcomas (LPS), 74 leiomyosarcomas (LMS), 46 pleomorphic sarcomas (PLS), 7 solitary fibrous tumors (SFT), 6 malignant peripheral nerve sheath tumors (MPNST), 5 synovial sarcomas (SS) and 5 dendritic follicular cell sarcomas (DFCS) were studied; 17 cases were excluded. Median age was 59. There were 5.1 GA/tumor, none were MSI-high, median TMB was low at 2.4; PD-L1 IHC staining was low-positive in 21% and high-positive in 5%. MPNST patients were younger (median 28 years vs. 59 years). LPS was more frequent in men and LMS in women. LPS had more GA/tumor than LMS, with DFCS having the highest and SS the lowest. TP53 and RB1 GA were the highest in LMS and CDK4/6 and MDM2 GA the highest in LPS. Molecular targets in mTOR pathway were most frequently altered. Targetable gene fusions in ALK, ROS1 and NTRK1-3 were rare. Non-targetable fusions in HMGA2 (LPS and some PLS), STAT6 (SFT) and SS18 (SS) were also identified. Conclusions: RS in our cohort are predominantly composed of LPS, LMS and PLS and we identified a small proportion with “actionable” genomic targets on CGP, albeit in association with uncertain mTOR pathway inhibitor benefit and uncommon targetable kinase fusions. Our analysis suggests that a small subset of RS may respond to immunotherapy based on putative biomarker expression. [Table: see text]

Introduction: B cell lymphoma/leukemias (BCL) are a diverse set of malignancies. The genomic landscape of many BCL subtypes have been described. However, genomic ancestry has rarely been investigated. We applied SNP-based genomic ancestry prediction to comprehensive genomic profiling (CGP) data to identify significant enrichment of ancestry by subtype. We also explored enrichment of genomic alterations (GAs) by ancestry. Methods: During routine clinical care, 2834 unique patient (pt) samples of BCLs underwent CGP for 406 DNA genes and 265 RNA genes to detect all classes of GAs on the FoundationOne® Heme platform. This dataset was enriched for relapsed/refractory pts as they are more likely to have genomic testing as part of clinical care (referral bias). Each pt was assigned an ancestry of American (AMR), African (AFR), East Asian (EAS), European (EUR), or South Asian (SAS) using a SNP-based machine learning methodology (J. Newberg et al., AACR 2019). AMR was defined using a mix of Hispanic and Latin American populations. Enrichment analyses were performed using Fisher's exact test with FDR correction. Results: We compared the ancestry composition of each BCL subtype to the overall ancestry composition of the rest of the sample set (Fig 1A). Pts of AFR ancestry were overrepresented in plasmablastic lymphoma (PBL) (OR=7.2, P<0.05); EAS pts were overrepresented in Burkitt lymphoma (BL) (OR=4.99, P<0.05); and AMR pts were overrepresented in acute B-cell lymphoblastic leukemia/lymphoma (B-ALL) (OR=3.2, P<0.001). AMR SNPs have been associated with increased risk of B-ALL and worse prognosis (PMID: 21297632). We also investigated GAs enriched in specific ancestries. B-ALL AMR pts were enriched for GAs in IL7R, IGH, CRLF2, JAK2, and IKZF1 compared to other ancestries in B-ALL (Fig 1B). These genes were associated with the high-risk Philadelphia chromosome-like ALL (Ph-like ALL) molecular subtype of B-ALL (PMID: 30181314). We found 33% of all B-ALL contained GAs consistent with Ph-like ALL (PMID: 30181314). While we noted enrichment of AMR pts in the overall B-ALL cohort, we identified additional enrichment in the Ph-like B-ALL cohort with 47% of Ph-like B-ALL pts being of AMR ancestry (OR=1.85, p<0.001). AMR pts accounted for almost half the Ph-ALL pts in this cohort; however, even in B-ALL pts without Ph-like genomic features, 32% were of AMR ancestry suggesting this enrichment is not simply due to increased CGP testing in Ph-ALL. In diffuse large B cell lymphoma (DLBCL), we found pts to be primarily of EUR ancestry, however we identified ancestry bias in GAs (Fig 1C). CD79B alterations were enriched in DLBCL pts of SAS ancestry, although not significant after FDR correction, consistent with previous reports of increased Activate B-Cell (ABC) cell of origin (COO) subtype and BTK signaling in pts from South East Asia (PMID: 31189540). CDKN2A, also frequently altered in ABC COO subtype, trended towards enrichment in EAS ancestry. CUX1, a tumor suppressor involved in PI3K signaling, was strongly enriched in AFR pts in both DLBCL and B-ALL. One CUX1 insertion variant (G870_G871insSGG) was particularly common in AFR pts with BCL (7/9 AFR, 2/9 AMR), which has been reported previously in Myelodysplastic syndromes (PMID: 24030381). CUX1 alterations have been reported to be associated with increased PI3K signaling suggesting in part PI3K inhibitor trials should proactively include pts of AFR ancestry (PMID: 24316979). Finally, EZH2 alterations were slightly enriched in AMR DLBCL pts, but showed no ancestry bias in follicular lymphoma (FL) pts, of interest given the recent EZH2 inhibitor approval in FL. Conclusions: This study described multiple important genomic differences in BCL using genomic ancestry rather than patient-reported descriptive variables. Enrichment of AMR ancestry was observed in B-ALL overall, and in Ph-like B-ALL. In addition, enrichment of CUX1 alterations was observed in both DLBCL and B-ALL of AFR ancestry. While precision medicine holds the promise to advance cancer care, many acknowledge the potential to unintentionally deepen existing health disparities (PMID: 31112478). Further analysis of ancestry informative markers in BCL, including enrichment of ancestry markers in specific cancer subtypes and ancestry-associated enrichment of specific GAs, may lead to insights into cancer biology and contribute to ongoing cancer health disparities research. Disclosures Moore: Foundation Medicine, Inc: Current Employment; Roche Holdings: Current equity holder in publicly-traded company. Evens:Abbvie: Consultancy, Honoraria; Mylteni: Consultancy, Honoraria; Research To Practice: Honoraria, Speakers Bureau; MorphoSys: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. Newberg:Roche Holdings: Current equity holder in publicly-traded company; Foundation Medicine, Inc: Current Employment. Severson:Foundation Medicine, Inc: Current Employment; Roche Holdings: Current equity holder in publicly-traded company. Mills:Foundation Medicine, Inc: Current Employment; Abbvie: Current equity holder in publicly-traded company; Roche Holdings: Current equity holder in publicly-traded company; Abbott: Current equity holder in publicly-traded company; Merck: Current equity holder in publicly-traded company. Vergilio:Roche Holdings: Current equity holder in publicly-traded company; Foundation Medicine, Inc: Current Employment. Trabucco:F. Hoffmann-La Roche, Bristol-Myers Squibb Co., BioNTech: Current equity holder in publicly-traded company; Patent pending with Foundation Medicine and Genentech: Patents & Royalties: Patent pending; Foundation Medicine, Inc.: Current Employment; Bristol-Myers Squibb Co: Current equity holder in publicly-traded company; BioNTech: Current equity holder in publicly-traded company; Loxo Oncology: Divested equity in a private or publicly-traded company in the past 24 months. Ganesan:M2GEN: Research Funding; Foundation Medicine, Inc: Consultancy; Inspirata: Consultancy; Merck: Consultancy, Current Employment, Current equity holder in publicly-traded company; Silagene: Consultancy; Foghorn Therapeutics: Consultancy; Roche: Consultancy; Novartis: Consultancy.