Recurrent Somatic Copy Number Alterations Research Articles

Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)

Abstract Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression. We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents. These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects. To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma. Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6941.

Recurrent Somatic Copy Number Alterations and Their Association with Oncogene Expression Levels in High-Grade Ovarian Serous Carcinoma.

Somatic copy number alterations (SCNAs) are frequently observed in high-grade ovarian serous carcinoma (HGOSC). However, their impact on gene expression levels has not been systematically assessed. In this study, we explored the relationship between recurrent SCNA and gene expression using The Cancer Genome Atlas Pan Cancer dataset (OSC, TCGA, PanCancer Atlas) to identify cancer-related genes in HGOSC. We then investigated any association between highly correlated cancer genes and clinicopathological parameters, including age of diagnosis, disease stage, overall survival (OS), and progression-free survival (PFS). A total of 772 genes with recurrent SCNAs were observed. SCNA and mRNA expression levels were highly correlated for 274 genes; 24 genes were classified as a Tier 1 gene in the Cancer Gene Census in the Catalogue of Somatic Mutations in Cancer (CGC-COSMIC). Of these, 11 Tier 1 genes had highly correlated SCNA and mRNA expression levels: TBL1XR1, PIK3CA, UBR5, EIF3E, RAD21, EXT1, RECQL4, KRAS, PRKACA, BRD4, and TPM4. There was no association between gene amplification and disease stage or PFS. EIF3E, RAD21, and EXT1 were more frequently amplified in younger patients, specifically those under the age of 55 years. Patients with tumors carrying PRKACA, BRD4, or TPM4 amplification were associated with a significantly shorter OS. RECQL4 amplification was more frequent in younger patients, and tumors with this amplification were associated with a significantly better OS.

DNA cytosine deamination is associated with recurrent Somatic Copy Number Alterations in stomach adenocarcinoma.

Stomach Adenocarcinoma (STAD) is a leading cause of death worldwide. Somatic Copy Number Alterations (SCNAs), which result in Homologous recombination (HR) deficiency in double-strand break repair, are associated with the progression of STAD. However, the landscape of frequent breakpoints of SCNAs (hotspots) and their functional impacts remain poorly understood. In this study, we aimed to explore the frequency and impact of these hotspots in 332 STAD patients and 1,043 cancer cells using data from the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE). We studied the rates of DSB (Double-Strand Breaks) loci in STAD patients by employing the Non-Homogeneous Poisson Distribution (λ), based on which we identified 145 DSB-hotspots with genes affected. We further verified DNA cytosine deamination as a critical process underlying the burden of DSB in STAD. Finally, we illustrated the clinical impact of the significant biological processes. Our findings highlighted the relationship between DNA cytosine deamination and SCNA in cancer was associated with recurrent Somatic Copy Number Alterations in STAD.

Molecular profiling of EBV associated diffuse large B-cell lymphoma

Epstein-Barr virus (EBV) associated diffuse large B-cell lymphoma (DLBCL) represents a rare aggressive B-cell lymphoma subtype characterized by an adverse clinical outcome. EBV infection of lymphoma cells has been associated with different lymphoma subtypes while the precise role of EBV in lymphomagenesis and specific molecular characteristics of these lymphomas remain elusive. To further unravel the biology of EBV associated DLBCL, we present a comprehensive molecular analysis of overall 60 primary EBV positive (EBV+) DLBCLs using targeted sequencing of cancer candidate genes (CCGs) and genome-wide determination of recurrent somatic copy number alterations (SCNAs) in 46 cases, respectively. Applying the LymphGen classifier 2.0, we found that less than 20% of primary EBV + DLBCLs correspond to one of the established molecular DLBCL subtypes underscoring the unique biology of this entity. We have identified recurrent mutations activating the oncogenic JAK-STAT and NOTCH pathways as well as frequent amplifications of 9p24.1 contributing to immune escape by PD-L1 overexpression. Our findings enable further functional preclinical and clinical studies exploring the therapeutic potential of targeting these aberrations in patients with EBV + DLBCL to improve outcome.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

P053: Recurrent somatic copy number alterations and their association with gene expression levels on cancer genes in ovarian serous cystadenocarcinoma

P053: Recurrent somatic copy number alterations and their association with gene expression levels on cancer genes in ovarian serous cystadenocarcinoma

Comprehensive Somatic Copy Number Analysis Using Aqueous Humor Liquid Biopsy for Retinoblastoma

Simple SummaryAqueous humor (AH) liquid biopsy is an enriched source of cell-free circulating tumor-derived DNA for retinoblastoma (RB). The use of this AH liquid biopsy allows for genomic analysis of eyes in the absence of tumor tissue. Development of this platform was critical because direct tumor biopsy is prohibited in RB due to risk of extraocular tumor spread. In this retrospective study, we provide comprehensive, whole-genome analysis of the somatic copy number alterations (SCNAs) in 68 eyes of 64 RB patients. We show that the prevalence of specific SCNAs differ between eyes that required immediate enucleation (surgical removal) and eyes that were attempted to be saved but subsequently failed treatment, requiring secondary enucleation. Increases in chromosomal instability, or higher number of broad genomic alterations, predict higher risk clinical and biomarker features in these eyes. Prospective analyses are needed to further determine the clinical relevance and application of these findings.Aqueous humor (AH) liquid biopsy has been established as a surrogate tumor biopsy for retinoblastoma (RB). Previous AH studies have focused on highly recurrent RB somatic copy number alterations (SCNAs) including gain of 1q, 2p, 6p, and loss of 13q and 16q. In this retrospective study, we provide a comprehensive, whole-genome analysis of RB SCNAs and evaluate associated clinical features for 68 eyes of 64 RB patients from whom AH was obtained between December 2014 and October 2020. Shallow whole-genome sequencing of AH cell-free DNA was performed to assess for SCNAs. The prevalence of specific non-highly recurrent SCNAs, such as 20q gain and 8p loss, differed between primarily and secondarily enucleated eyes. Increases in chromosomal instability predict more advanced seeding morphology (p = 0.015); later age of diagnosis (p < 0.0001); greater odds of an endophytic tumor growth pattern (without retinal detachment; p = 0.047); tumor heights >10 mm (p = 0.09); and containing 6p gain, a biomarker of poor ocular prognosis (p = 0.004). The AH liquid biopsy platform is a high-yield method of whole-genome RB SCNA analysis, and SCNAs are associated with numerous clinical findings in RB eyes. Prospective analyses are encouraged to further elucidate the clinical relevance of specific SCNAs in RB.

Abstract PD13-01: Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases

Abstract BACKGROUND: Brain metastatic disease occurs in 10-30% of metastatic breast cancer cases. The incidence of brain metastases is increasing, yet overall survival remains &amp;lt; 2 years. Treatment of brain metastases is limited with current clinical practice centered on radiation, chemotherapy and surgery. Although these treatments may prolong survival in the short term, targeting oncogenic alterations in brain metastases may deliver a more sustained clinical benefit. In this multicenter study, we comprehensively characterized DNA and RNA alterations that aberrantly drive specific oncogenic pathway activity pertinent to breast cancer brain metastases (BCBM). METHODS: RNA sequencing was performed on a cohort of patient-matched primary and resected brain metastatic tumours (45 patients; N=90 samples). Whole exome DNA sequencing (WXS) was performed for 18/45 patients (54 trios consisting of primary tumor, brain metastasis and matched normal tissue). An independent brain metastatic WXS cohort (N=21 patients) (PMID: 26410082) was also analysed resulting in a total of 39 patient samples. Recurrent somatic copy number alterations (SCNA), somatic single nucleotide variants (SNVs) and mutational signatures were identified from WXS data. Expressed gene fusions were detected computationally from RNA-Seq (N=45 cases) RESULTS: Of the 45 BCBM patients, median age at diagnosis was 51 years [25, 67], median overall survival 57 months (range 18-255) with median brain metastases free survival 34 months (range 5-216).Clinical molecular subtype of the primary tumour included 13 ER+/HER2- (29%), 16 HER2+ (35.5%) and 16 TNBC (35.5%). Regions of significant recurrent amplifications and deletions in BCBM (N=39 patients) were identified in 4q12, 10q11.21, 8p11.23, 8q23.3 and 17q12 (FDR &amp;lt; 0.10). Recurrent expressed gene fusions identified in known cancer driver genes were associated with chromatin modification, MAPK and HER signaling pathways. Mutational signature analysis of SNVs identified signatures associated with ageing, mismatch repair and homologous recombination deficiency (HRD) mutational processes. The relative contribution of the HRD signature was significantly increased in brain metastases compared to matched primary tumour (p &amp;lt; 0.05). Concordantly increased HRD in the brain metastatic transcriptome was confirmed in these patients by gene set variation analysis (GSVA) of homologous recombination pathway genes from KEGG database in RNA-Seq data. Moreover, in the extended BCBM RNA-Seq (N=45 patients) GSVA pathway scores were elevated in brain metastases relative to primary tumours, validating the functional significance of altered DNA repair defects in brain metastases CONCLUSIONS: Here, we report recurrent genetic drivers, supported by altered functional transcriptome, unique to brain metastasis that may have clinical implications for prognosis and treatment choice. Specifically, targeting defects in the homologous recombination repair mechanism may represent new therapeutic strategies and management opportunities for breast cancer brain metastases patients. Citation Format: Nicola S Cosgrove, Damir Varešlija, Stephen Keelan, Simon Furney, Jodi M Carter, Steven N Hart, Siddhartha Yadav, Arnold DK Hill, Steffi Oesterreich, Adrian V Lee, Fergus J Couch, Leonie Young. Homologous recombination deficiency represents a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-01.

Genomic and Transcriptomic Profiling Reveals Distinct Subsets Associated with Outcomes in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is a rare subtype of aggressive non-Hodgkin's lymphoma in which the genomic factors determining the clinical behavior are not fully understood. The genetic heterogeneity in MCL motivates us to characterize the genetic landscape, to define pattern of clonal evolution, and to determine molecular subgroups. Methods We performed whole-exome sequencing on 152 DNA samples derived from 134 MCL patients, which includes 123 untreated and 11 relapsed patients, and 16 with longitudinal samples. 95 patients received high dose cytarabine-based immunochemotherapy from BDH-MCL01 clinical trial (NCT02858804). 48 samples have matched RNA sequencing data for which 42 from untreated and 6 from relapsed patients. We used GATK, MutSig2CV, and GISTIC2.0 to identify driver genetic lesions, applied MutationalPatterns to define mutational signatures, and utilized ABSOLUTE and PhylogicNDT to determine pattern of clonal evolution. We applied NNMF consensus clustering to identify subgroups and evaluated association between genomic features and clinical outcomes. Results The median non-silent mutational burden was 29 per sample (range 8-72). 34 recurrently mutated genes were identified, containing previously reported driver mutations (TP53, ATM, CCND1, KMT2D, NSD2, SMARCA4, ARID1A, NOTCH1, NOTCH2, BIRC3, TRAF2, UBR5) and novel mutations (SP140, SVEP1, LRP1B, LRP2, PCDH10). 7 copy number gain and 13 loss regions were detected as recurrent somatic copy number alterations (frequency&amp;gt;10%, q&amp;lt;0.1) (FigA). In multivariable Cox models of PFS and OS, TP53 mutation/del(17p13), SP140 mutation/del(2q36), and mutations in NOTCH1, PCDH10, and del(9p) showed prognostic value independent of MIPI risk and IGHV mutation status. We defined the clonal status of genetic lesions and pattern of clonal evolution. Del(11q22) and del(9p) tend to be clonal while mutations in NSD2, LRP1B, CTNNA2 were more likely to be subclonal (q&amp;lt;0.05) (Fig B). Clonality analysis further enabled inference of temporal relationships between pairs of events. We further determined clonal evolution pattern by measuring the dynamic changes of fraction of cancer cells harboring each genetic lesion. 11 of 16 (69%) longitudinal samples had extreme clonal evolution (CCF change &amp;gt; 0.5), 4 with modest evolution (0.2 ≤ CCF change ≤ 0.5), 1 without evolution (CCF change &amp;lt; 0.2). Patients with extreme evolution had inferior survival than with those with modest and no evolution (median survival from first sampling was 47.5 months vs not reached, p=0.041, second sampling was 17.1 months vs not reached, p=0.023). We classified MCL into four subsets based on genetic lesions, each with distinct gene expression profile and clinical behavior (Fig C, D). Cluster 1 (C1) and cluster 2-4 (C2-4) were associated with indolent and aggressive types of MCL. Consistent with different cellular origins for two types of MCL, C1 and C2-4 were enriched for gene expression signatures of memory and CCR6 negative light zone B cells, respectively. C1 featured mutated IGHV, CCND1 mutation, amp(11q13) and active BCR signaling. C2 was enriched with del (11q22), del (1p21), ATM mutation and had upregulation of genes involved in the NF-kB and DNA repair pathways while C3 was characterized by mutations in SP140, NOTCH1 and NSD2 and downregulation of gene expression in the NF-kB, BCR signaling, MYC and inflammatory pathways. Interestingly, C4 had the highest incidence of blastoid or pleomorphic MCL (23.7%, p=0.016) and harbored del(17p), del(13q), del(9p) and mutations in TP53 and TRAF2. This cluster carried enrichment of MYC pathway activation and hyperproliferation signatures. These unique gene expression signatures indicated that coordinate genomic factors captured biologic heterogeneity. Importantly, patients in these four clusters had distinct outcomes with median PFS of not reached for C1, 41.2 months for C2, 30.7 months for C3, and 16.1 months for C4 (log rank, p&amp;lt;0.001). The differences of OS and PFS remained significant when only considered 95 patients with high dose cytarabine-based immunochemotherapy. Conclusion Our study provides a portrait of the MCL genetic landscape, uncovers pattern of clonal evolution in MCL, classifies patients with genetic features, links the cluster with gene expression and clinical outcome. The outcome-associated genetic signatures will guide the development of therapies in patients with the greatest need. Figure Disclosures No relevant conflicts of interest to declare.

KAT6A amplifications are associated with shorter progression-free survival and overall survival in patients with endometrial serous carcinoma.

Somatic copy number alterations (CNA) are common in endometrial serous carcinoma (ESC). We used the Tumor Cancer Genome Atlas Pan Cancer dataset (TCGA Pan Can) to explore the impact of somatic CNA and gene expression levels (mRNA) of cancer-related genes in ESC. Results were correlated with clinico-pathologic parameters such as age of onset, disease stage, progression-free survival (PFS) and overall survival (OS) (n = 108). 1,449 genes with recurrent somatic CNA were identified, observed in 10% or more tumor samples. Somatic CNA and mRNA expression levels were highly correlated (r> = 0.6) for 383 genes. Among these, 45 genes were classified in the Tier 1 category of Cancer Genome Census-Catalogue of Somatic Mutations in Cancer. Eighteen of 45 Tier 1 genes had highly correlated somatic CNA and mRNA expression levels including ARNT, PIK3CA, TBLXR1, ASXL1, EIF4A2, HOOK3, IKBKB, KAT6A, TCEA1, KAT6B, ERBB2, BRD4, KEAP1, PRKACA, DNM2, SMARCA4, AKT2, SS18L1. Our results are in agreement with previously reported somatic CNA for ERBB2, BRD4 and PIK3C in ESC. In addition, AKT2 (p = 0.002) and KAT6A (p = 0.015) amplifications were more frequent in tumor samples from younger patients (<60), and CEBPA (p = 0.028) and MYC (p = 0.023) amplifications were more common with advanced (stage III and IV) disease stage. Patients with tumors carrying KAT6A and MYC amplifications had shorter PFS and OS. The hazard ratio (HR) of KAT6A was 2.82 [95 CI 1.12–7.07] for PFS and 3.87 [95 CI 1.28–11.68] for OS. The HR of MYC was 2.25 [95 CI 1.05–4.81] and 2.62[95 CI 1.07–6.41] for PFS and OS, respectively.

Abstract A70: Aqueous humor is superior to blood as a liquid biopsy for retinoblastoma

Abstract Purpose: To evaluate whether blood demonstrates similar potential as aqueous humor (AH) to be used as a liquid biopsy for retinoblastoma (RB) and whether tumor-derived cell-free DNA (cfDNA) can be isolated as effectively from the blood as AH. Methods: AH was extracted via clear corneal limbal paracentesis from RB eyes at diagnosis or during intravitreal injection of chemotherapy. Matched peripheral venous blood samples were drawn. Shallow whole-genome sequencing was performed to assess for cell-free tumor DNA fractions and highly recurrent somatic copy number alterations (SCNAs) in the blood and AH samples. Results: Seven samples of AH taken at diagnosis and 13 samples at the time of intravitreal injection were compared to matched blood samples. The presence of any detectable SCNA in the AH was 11/20 and 0/20 in the blood (p=&amp;lt;0.001). The median size distribution of cfDNA molecules in the AH was 157.5 bp versus 181.5 bp in the blood (p=&amp;lt;0.001). Conclusions: The AH appears to be superior to the blood as a source of cell-free tumor DNA for retinoblastoma, thus a better target for development as a liquid biopsy for this cancer. Citation Format: Liya Xu, Jesse L Berry, Ashley Polski, Rima Jubran, Peter Kuhn, Jonathan W. Kim, James Hicks. Aqueous humor is superior to blood as a liquid biopsy for retinoblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A70.

Abstract A45: Targeted drug therapies for osteosarcoma

Abstract Osteosarcoma (OS) is a highly aggressive cancer that has had no new treatments options in over 30 years. OS is a heterogeneous disease that is characterized by widespread and recurrent somatic copy-number alterations (SCNAs) with relatively few recurrent point mutations. Our laboratory has recently published that SCNAs contain key oncogenic drivers that can be used to identify patient-specific candidates for targeted therapies (Sayles, Breese, et al., Cancer Discovery Jan. 2019). Using patient-derived tumor xenografts (PDX), we demonstrated that targeting of patient-specific oncogenes within SCNAs leads to significant decrease in tumor burden. However, no single-agent therapy was able to regress tumors completely, suggesting that combination therapies would be required for disease management. In order to assess the applicability of our PDX models to the patient tumor and the stability of the oncogenes within SCNAs, we performed whole-genome sequencing (WGS). We observed that SCNAs are highly stable across samples from the same patient in addition to multiple PDX passages and PDX-derived cell lines, highlighting the equivalence between the PDX models and their derived cell lines to the human disease. This allows us to use the PDX cell lines as a surrogate for the identification of combination drug therapies that may be of benefit in OS. Currently, we have generated 6 PDX cell lines that encompass the most common SCNAs observed in patients, including MYC, CCNE1, and CDK4 gain and alterations in the PI3K/PTEN pathway. We have performed a single-agent drug screen with 38 agents and have identified several efficacious compounds, including HDAC inhibitors and cell cycle and Wee1 inhibitors. We also observed a differential response between PDX cell lines to various chemotherapeutics, including gemcitabine, paclitaxel, and SN-38. We are currently testing combination drug therapies in vitro and will validate using our PDX xenografts in vivo. While these experiments are still ongoing, we can report that MYC-driven OS PDX xenografts show a striking resensitization to cisplatin after AURKB inhibitor (barasertib) pretreatment. We have 2 aggressive PDX models with high MYC amplification generated from metastatic lesions. These PDX models are resistant to cisplatin, which is part of the standard of care. We treated these PDX with barasertib and then with cisplatin for two cycles. This resulted in a decrease in tumor volume for the combination therapy compared to vehicle or either single agent alone. Additionally, we observed retention of the platinum adduct 24 hrs after cisplatin dosing only when tumors were pretreated with barasertib. Further work is needed to assess the mechanism of this synergy and whether it can be of use in other SCNA-driven OS and to identify other possible combination therapies that could be of importance in this disease. Citation Format: Leanne C. Sayles, Marcus R. Breese, Henry Martell, Alex G. Lee, Stanley Leung, Avanthi T. Shah, E. Alejandro Sweet-Cordero. Targeted drug therapies for osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A45.

Abstract B066: Breast and prostate cancers harbor common somatic copy number alterations that consistently differ by race and are associated with survival

Abstract African Americans (AAs) tend to have more aggressive diseases and worse outcomes for many cancer types compared to European Americans (EAs). Large structural changes on chromosomes, such as somatic copy number alterations (SCNAs), are commonly present in many cancers and associated with clinical outcomes. While studies focusing on single tumor types have identified racial differences in SCNA frequencies and their possible association with cancer outcomes, pan-cancer race-differentiated SCNAs and their clinical implications have not been fully explored. Focusing on two of the most common hormonally driven cancers, breast cancer in women and prostate cancer in men, and using data from The Cancer Genome Atlas (TCGA), we first identified recurrent SCNAs using GISTIC2 in AAs and EAs separately within each cancer. For each GISTIC2 identified SCNA region, we calculated the magnitude deviation from the expected null value (i.e. copy number (CN) = 2) using the area under the log2(CN) (cnAUC) for each tumor. Modeling cnAUC as the outcome variable, we identified SCNAs that differed between AAs and EAs in breast cancers (n=58 SCNAs; permutation p&amp;lt;10-4) and prostate cancers (n=78 SCNAs; permutation p=0.006). Six of nine race-differentiated SCNAs common to breast and prostate cancers had consistent magnitude differences by race across both tumor types, and all six were of higher magnitude in AAs. These six race-differentiated SCNAs were found at chromosomes 5q11.2-q14.1, 5q15-q21.1, 8q21.11-q21.13, 8q21.3-q24.3, 11q22.3, and 13q12.3-q21.3 with the chromosome 8q regions being the only amplifications. Unsupervised hierarchical clustering of the SCNAs in these six race-differentiated regions identified three patient groups in each tumor type with significant survival differences. Of particular importance, within triple negative breast cancers, these SCNA patient groups showed the most significant difference in progression-free survival (p=0.019). While there was a higher frequency of AAs among the SCNA-defined patient groups with worse survival, no significant racial difference in survival was observed within each SCNA-defined patient group. This observation supports the idea that racial disparities in breast and prostate cancer outcomes might be in part due to racial differences in tumor biology that drive structural chromosomal changes shared across these common tumor types. Our ability to derive SCNA defined patient groups associated with survival based on race-differentiated genomic aberrations demonstrates both their clinical relevance and the need to understand the biologic mechanisms that give rise to them. Citation Format: Yalei Chen, Sudha M Sadasivan, Ruicong She, Indrani Datta, Kanika Taneja, Chitale Dhananjay, Nilesh Gupta, Melissa B Davis, Lisa A Newman, Craig G Rogers, Pamela L Paris, Jia Li, Benjamin A Rybicki, Albert M Levin. Breast and prostate cancers harbor common somatic copy number alterations that consistently differ by race and are associated with survival [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B066.

Validation of the Genetically-Defined DLBCL Subtypes and Generation of a Parsimonious Probabilistic Classifier

Diffuse large B-cell lymphoma (DLBCL) is a clinically and molecularly heterogeneous disease with recognized transcriptional subtypes associated with normal cells of origin, activated B-cell (ABC) and germinal center B-cell (GCB) tumors. Emerging data suggested that additional heterogeneity existed, prompting us to comprehensively characterize genomic signatures of 304 newly diagnosed DLBCLs from patients treated with state-of-the-art therapy. We integrated recurrent mutations, somatic copy number alterations (SCNAs) and structural variants (SVs) and identified 5 genetically distinct DLBCL clusters (C1- C5 DLBCLs; Chapuy, Stewart, Dunford, et al. Nat Med 2018). Specifically, we identified two genetically distinct ABC subtypes, including favorable-risk C1 DLBCLs with features of extrafollicular origin and alterations also seen in transformed marginal zone lymphomas (NOTCH2 and NF-κB pathway member mutations and BCL6 SVs). Unfavorable-risk C5 ABC DLBCLs harbored frequent 18q/BCL2 copy gain and co-occurring CD79B and MYD88L265P mutations. We also identified two genetically distinct GCB subtypes, including unfavorable-risk C3 DLBCLs with frequent BCL2 SVs, mutations in chromatin-modifying enzymes (CREBBP, MLL2, EZH2) and BCR/PI3K signaling pathway members (including inactivating PTEN mutations and copy loss). Favorable-risk C4 GCB DLBCLs had frequent mutations in core and linker histones and signaling intermediates (SGK1, BRAF and STAT3). Additionally, we identified an ABC/GCB-independent subtype, C2 DLBCLs, characterized by frequent bi-allelic TP53 inactivation, 9p21.23/CDKN2A copy loss and associated genomic instability reflected in recurrent SCNAs, increased genome doublings and a distinct outcome following induction therapy. A next step in utilizing the characterized genetic substructure was to confirm it in an independent series and develop a molecular classifier that allows prospective identification of C1-C5 DLBCLs. To this end, we accessed whole exome sequencing, copy number and SV data from a recent cohort of newly diagnosed DLBCLs (39 tumor-normal pairs, 462 tumor-only samples; Schmitz et al. NEJM 2018). All samples were re-analyzed using our mutational and SCNA pipelines and our newly generated tumor-only algorithm (Chapuy, Stewart, Dunford, et al. Nat Med 2018) to avoid batch effects and harmonize the datasets. SVs were used as reported. Purity and ploidy were inferred using ABSOLUTE and samples with missing data or low purity were removed. For the combined cohort (579 samples), we assessed our previously characterized 158 genetic drivers (Chapuy, Stewart, Dunford, et al. Nat Med 2018) and confirmed equal distribution of their marginal frequencies (R=0.88, p=1.5e-51), excluding batch effects. Next, we applied non-negative matrix factorization (NNF) consensus clustering to the combined dataset (158 genetic drivers vs. 579 tumors) and confirmed the C1-C5 DLBCL genetic clusters. Notably, tumors from both series contributed at comparable frequencies to the respective C1-C5 DLBCLs. We also noted an enrichment of the alternative genetic labels from Schmitz et al. in 3 of our C1-C5 DLBCL subtypes (B2N in C1 DLBCLs, p&amp;lt;0.0001; EZB in C3 DLBCLs, p&amp;lt;0.0001; MCD in C5 DLBCLs, p&amp;lt;0.0001). These data confirmed the identity of the C1-C5 DLBCL clusters in an independent cohort. Next, we developed a molecular classifier that prospectively identified C1-C5 DLBCLs using a minimum number of easy-to-measure features. The NMF-defined classes of the combined cohort were used as gold-standard training and validation datasets. We tested different models for classification and selected an artificial neural network approach which provides accurate classification of individual samples and well-calibrated confidence metrics. To minimize potential overtraining, we developed a reduced input feature set of the 22 most discriminating features, constructed confidence metrics for each sample and trained an ensemble of Feed-Forward Neural Networks via 10-fold cross validation. With this approach, our classifier had 84% accuracy for the total set and 94% accuracy for the high-confidence samples (70% of all samples). The newly developed parsimonious classifier will allow prospective identification of the independently confirmed C1-C5 DLBCL subtypes in newly diagnosed patients, a necessity for clinical application. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents &amp; Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Shipp:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck &amp; Co.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

409P - Integrated genomic and transcriptomic analysis revealed mutagenic patterns of dedifferentiated liposarcoma and leiomyosarcoma in Chinese patients

BackgroundL-type sarcoma (liposarcoma + leiomyosarcoma) is the most common subtype of soft tissue sarcoma (STS). Leiomyosarcoma (LMS) and dedifferentiated LPS (DDLPS) are more aggressive with worse outcomes than well-differentiated sarcoma. Efforts have thus been made to decipher the difference of oncogenesis between DDLPS and LMS, and assist in the choice of treatment. MethodsWES was conducted on 32 STS (20 LMS and 12 DDLPS) and matched peripheral blood. Somatic SNVs and indels were identified by GATK tools. Recurrent somatic copy-number alterations (SCNA) were called using Control-FREEC and GISTIC2.0. We further conducted RNA-Seq on 8 DDLPS samples and 8 LMS samples, and use STAR-Fusion to reveal the distinct chromosome rearrangement patterns of LMS and DDLPS samples. ResultsWe identified top 20 highly variable genes between LMS and DDLPS. 55% LMS (n = 11) carry deletions or mutations of TP53. All DDLPS carry wildtype TP53, but chromosome 12q14–15 region which encodes MDM2, CDK4 and HMGA2 are highly amplified in all DDLPS samples, suggesting distinct mechanisms of TP53 pathway inactivation. Hippo pathway is reported to be involved in resistance to cytotoxic drugs and modulate tumour immunogenicity. In our study, 70% LMS (n = 14) and 58% DDLPS (n = 7) carry at least one Hippo pathway alterations, top variable genes in LMS include DCHS1, LLGL1, AJUBA et al. Among 8 LMS and 8 DDLPS who underwent RNA-Seq, we identified 4 fusion transcripts in 3 LMS and 36 fusion transcripts in 8 DDLPS (P = 0.007). Fusion transcripts involving chromosome 12 (Chr12) are only identified in DDLPS, including 10 interchromosomal and 12 intrachromosomal rearrangements. MDM2 and RAB3IP are the most common fusion partners. There was also significant correlation between MDM2/CDK4 co-amplification and Chr12 rearrangement (P < 0.001). All DDLPS with MDM2/CDK4 co-amplification showed elevated MDM2 and CDK4 co-expression. ConclusionsLMS and DDPLS carry distinct mutagenic patterns, ranging from SNVs to gross genomic instability. Chromosome rearrangement of DDLPS, particularly in Chr12, result in both gene amplification and fusion events, and these neoantigens produced by gene fusion may open up alternative avenues for immunotherapy. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Cytolytic activity correlates with the mutational burden and deregulated expression of immune checkpoints in colorectal cancer

BackgroundMicrosatellite unstable colorectal cancers (MSI+ CRCs) expressing PD-L1, respond to anti-PD-1 or anti-PD-L1 checkpoint blockade, whereas microsatellite-stable tumors do not respond the same. Our aim was to examine how the immune landscape relates to different aspects of the CRC’s biology, including neoepitope burden.MethodsWe used TCGA data to stratify patients based on a cytolytic T-cell activity expression index and correlated immune cytolytic activity (CYT) with mutational, structural, and neoepitope features of each tumor sample. The expression of several immune checkpoints was verified in an independent cohort of 72 CRC patients, relative to their MSI status, using immunohistochemistry and RT-qPCR.ResultsCRC exhibits a range of intertumoral cytolytic T-cell activity, with lower cytolytic levels in the tumor, compared to the normal tissue. We separated CRC patients into CYT-high and CYT-low subgroups. High cytolytic activity correlated with increased mutational load in colon tumors, the count of MHC-I/−II classically defined and alternatively defined neoepitopes, high microsatellite instability and deregulated expression of several inhibitory immune checkpoints (VISTA, TIGIT, PD-1, IDO1, CTLA-4, and PD-L1, among others). Many immune checkpoint molecules (IDO1, LAG3, TIGIT, VISTA, PD-1, PD-L1 and CTLA-4) expressed significantly higher in MSI+ CRCs compared to MSS tumors. The expression of Treg markers was also significantly higher in CYT-high tumors. Both individual and simultaneous high levels of CTLA-4 and PD-L1 had a positive effect on the patients’ overall survival. On the reverse, simultaneous low expression of both genes led to a significant shift towards negative effect. Assessed globally, CYT-low CRCs contained more recurrent somatic copy number alterations. PD-L1 protein was absent in most samples in the independent cohort and stained lowly in 33% of MSI CRCs. PD-L1+ CRCs stained moderately for CD8 and weakly for FOXP3. CYT-high colon tumors had higher TIL load, whereas CYT-high rectum tumors had higher TAN load compared to their CYT-low counterparts.ConclusionsOverall, we highlight the link between different genetic events and the immune microenvironment in CRC, taking into consideration the status of microsatellite instability. Our data provide further evidence that MSI+ and CYT-high tumors are better candidates for combinatorial checkpoint inhibition.

Abstract 2880: Targeted drug therapies for osteosarcoma

Abstract Osteosarcoma (OS) is a highly aggressive cancer that has had no new treatments options in over 30 years. OS is a heterogeneous disease which is characterized by widespread and recurrent somatic copy-number alterations (SCNAs) with relatively few recurrent point mutations. We have previously reported that SCNAs contain key oncogenic drivers that can be used to identify patient-specific candidates for targeted therapies (Sayles and Breese, et al. Cancer Discovery 2018). Using patient-derived tumor xenografts (PDX), we demonstrated that targeting of patient-specific oncogenes within SCNAs leads to significant decrease in tumor burden. However, no single agent therapy was able to regress tumors completely suggesting that combination therapies would be required for disease management. In order to assess the applicability of OS PDX models to the patient tumor and the stability of the oncogenes within SCNAs, we preformed whole genome sequencing (WGS). We observed that SCNAs are highly stable across samples from the same patient in addition to multiple PDX passages and PDX derived cells lines, highlighting the equivalence between the PDX models and their derived cell lines to the human disease. This allows us to use the PDX cell lines as a surrogate for the identification of combination drug therapies that may be of benefit in OS. Currently, we have generated six PDX cell lines that encompass the most common SCNAs observed in patients including MYC, CCNE1 and CDK4 gain and alterations in the PI3K/PTEN pathway. We have performed single agent drug screens in these cell lines and have identified heterogeneous responses as would be anticipated by the genomic diversity observed in OS. We are currently testing combination drug therapies and will validate using the PDX xenografts in vivo. We have found that MYC driven OS PDX cell lines and PDX xenografts show a striking re-sensitization to cisplatin after AURKB inhibitor (barasertib) pretreatment. We have two PDX and derived cell lines that were generated from highly aggressive metastatic disease. Both of these PDX have high MYC amplification and were resistant to cisplatin, which is part of the standard of care. We pretreated the PDX cell lines for 24hrs with barasertib then added cisplatin and saw a synergistic effect in vitro. Further studies in the PDX model demonstrated a decrease in tumor volume for the combination therapy compared to vehicle or either single agent alone. Additionally, we observed retention of the platinum adduct 24 hrs after cisplatin dosing only when tumors were pretreated with barasertib. Further work is needed to assess the mechanism of this synergy and whether it can be of use in other SCNA driven OS and to identify other possible combination therapies that could be of importance in this disease. Citation Format: Leanne C. Sayles, Amanda Koehne, Kieren Marini, Alex G. Lee, Stanley G. Leung, Avanthi T. Shah, Marcus R. Breese, E. Alejandro Sweet-Cordero. Targeted drug therapies for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2880.

Elucidating the transcriptional program of feline injection-site sarcoma using a cross-species mRNA-sequencing approach

BackgroundFeline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans.MethodsUsing high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios.ResultsWe found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro.Conclusions(1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS.

Genome-Informed Targeted Therapy for Osteosarcoma.

Osteosarcoma is a highly aggressive cancer for which treatment has remained essentially unchanged for more than 30 years. Osteosarcoma is characterized by widespread and recurrent somatic copy-number alterations (SCNA) and structural rearrangements. In contrast, few recurrent point mutations in protein-coding genes have been identified, suggesting that genes within SCNAs are key oncogenic drivers in this disease. SCNAs and structural rearrangements are highly heterogeneous across osteosarcoma cases, suggesting the need for a genome-informed approach to targeted therapy. To identify patient-specific candidate drivers, we used a simple heuristic based on degree and rank order of copy-number amplification (identified by whole-genome sequencing) and changes in gene expression as identified by RNA sequencing. Using patient-derived tumor xenografts, we demonstrate that targeting of patient-specific SCNAs leads to significant decrease in tumor burden, providing a road map for genome-informed treatment of osteosarcoma. SIGNIFICANCE: Osteosarcoma is treated with a chemotherapy regimen established 30 years ago. Although osteosarcoma is genomically complex, we hypothesized that tumor-specific dependencies could be identified within SCNAs. Using patient-derived tumor xenografts, we found a high degree of response for "genome-matched" therapies, demonstrating the utility of a targeted genome-informed approach.This article is highlighted in the In This Issue feature, p. 1.

Comprehensive Genomic Analysis of Primary Mediastinal B-Cell Lymphoma

Primary mediastinal large B-cell lymphomas (PMBL) typically occur in young women who present with localized, large mediastinal masses. These tumors share certain clinical, pathomorphological and transcriptional features with classical Hodgkin lymphoma (cHL). To date, PMBL genetic analyses focused on limited sets of genes and recurrent somatic copy number alterations (SCNAs). Previously, we identified frequent 9p24.1/PD-L1/PD-L2 copy gains and increased expression of the PD-1 ligands as a genetically-defined immune escape mechanism in PMBL. The demonstrated efficacy of PD-1 blockade in relapsed/refractory PMBL led to recent FDA approval and underscored the importance of characterizing targetable genetic vulnerabilities in this disease.For these reasons, we obtained diagnostic biopsy specimens from 37 patients with PMBL (median age 34; female 70%) and performed whole exome sequencing (WES) with an expanded bait set to capture structural variants (SVs). Somatic alterations (mutations, SCNAs and SVs) were determined using established analytical pipelines including our algorithm for evaluating tumors without paired normal samples. Genes more frequently mutated than by chance, Candidate Cancer Genes (CCGs), were identified with MutSig2CV and recurrent SCNAs were defined with GISTIC2.0. SVs were characterized with a recently described 4-algorithm pipeline (Nature Medicine, 2018;24(5):679-690).First, we identified 15 CCGs (q-value <0.1) including genes with known roles in PMBL, such as IL4R and TNFAIP3 and mutational drivers in additional B-cell lymphomas (B2M, GNA13, STAT6, IKZF3, XPO1, TP53, PAX5) and other cancers (TP53, ZNF217 and XPO1). Overlaying the predicted protein changes onto available 3D protein structures highlighted the likely biological functions of specific alterations, such as mutational clustering in the STAT6 DNA-binding domain.We next analyzed the PMBL mutational signatures and identified 3 cases as hypermutators with MSI signatures, including 2 with MLH1 frameshift mutations and 1 with a nonsense PMS2 mutation. Despite the young age of the PMBL patient cohort, the majority of remaining mutations were caused by spontaneous deamination at CpGs, a genetic signature usually associated with aging. The next most prevalent mutational signatures were APOBEC and, infrequently, AID. We observed a higher median mutational density in PMBL (7.56 mutations/MB), compared to diffuse large B-cell lymphoma (DLBCL) and most solid cancers, providing a potential basis for increased neoantigen production and responsiveness to PD-1 blockade.Next, we identified 18 recurrent SCNAs, including 10 copy gains (2 focal and 8 arm level) and 8 copy losses (7 focal and 1 arm level). Copy gains of 9p24.1/PD-L1/PD-L2 were detected in 70% of cases. SVs were defined at base-pair resolution and included infrequent (2/37) tandem duplications of both PD-1 ligands and inactivating CTIIA SVs (deletions and inversions) in 10% (4/37) of cases.Although PMBL had a higher mutational density than DLBCL, the PMBL alterations involved a smaller number of median genetic drivers (9 [PMBL] vs 17 [DLBCL], respectively). Combined analyses of recurrent CCGs, SCNAs and SVs revealed that certain candidate driver genes were perturbed by multiple mechanisms. Examples include: TNFAIP3 (59% overall, 41% mutations, 24% copy loss, 6% biallelic); and B2M (51% overall, 30% mutations, 27% copy loss, 6% biallelic).Concurrent analyses of the 3 types of genetic alterations also revealed multiple bases of perturbing specific signaling pathways. In this PMBL series, 73% (27/33) of tumors exhibited one or more alterations of JAK/STAT pathway components: IL4R mutations (32%), JAK2 (9p24.1 focal copy gain [70%]) and STAT6 mutations (43%). Additionally, 59% of PMBLs had alterations of antigen presentation pathway components including B2M copy loss or mutations, copy loss of 6q21.33 (which includes the HLA class I/II loci) and SVs of CTIIA. These findings provide a genetic framework for analyzing the precise mechanism of action of PD-1 blockade in PMBL.Taken together, these findings underscore the importance of a comprehensive genomic analysis in PMBL and define additional candidate treatment targets and pathogenetic mechanisms in this disease.____BC, CS and AD contributed equally. GG and MAS contributed equally. DisclosuresRodig:Merck: Research Funding; KITE: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding. Shipp:Merck: Research Funding; AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.