Mutations In ATR Research Articles

Human Myeloma Cell Lines Mutation Landscape: Toward a Rationale Use of Cell Lines to Assess Drug Efficacy Prediction in Myeloma

Human myeloma cell lines (HMCLs) are widely used for the assessment of (new) drug efficacy in vitro or in vivo in mouse models. HMCLs have been mainly derived from patients at ultimate relapse and frequently from extramedullary disease, peripheral blood (secondary plasma cell leukemia), pleural effusion or ascites. Of HMCLs derived from patients at diagnosis most were from primary plasma cell leukemia. While HMCLs have been extensively characterized for 14q32 translocation and gene expression profile, the global mutation profile was never performed in a large collection of HMCLs. We thus performed a whole exon sequencing (WES) in 33 independent HMCLs displaying either recurrent 14q32 translocations (involving CCND1 or CCND3 [n=11], c-MAF or MAFB [n=8] and NSD2 gene [n=9]), an unknown non-recurrent translocation (n=3) or none translocation (n=2). We identified variants (missense, non-sense, essential splice, indels) and analyzed the copy number variation (CNV) to report deletion and amplification.DNA was captured for exons and enriched with Sureselect target enrichment system (Agilent) then sequenced on HiSeq 2500. Fastq reads were aligned on hg19/GRCh37 reference genome, variants were called using GaTK. Single Nucleotide Polymorphisms (SNPs) were filtered out by removing over-represented variants (>15%), of the remaining variants, only non-neutral variants were kept, as defined by SnpEff algorithm. Copy Number Variations (CNV) data were calculated from read-depth using XHMM.HMCLs display a median of 1603 mutations per sample impacting a median of 1345 genes per sample, MM1S and NAN7 harboring the highest and the lowest burden of mutations with 4725 and 1298 individual mutations, respectively. HMCLs harbored the same most frequent mutations found in primary myeloma cells that were KRAS, NRAS, BRAF, TP53, FAM46C and DIS3 . K/NRAS and TP53 were the most mutated genes across the collection (16 and 23 HMCLs, respectively). Presence of mutations was analyzed according to the heterogeneity in translocation and in the growth factor dependency (IGF1 versus IL6-dependent), to the mutant status of several genes (TP53, NRAS, KRAS ...) and to the cell response to several targeted and untargeted drugs. In order to provide a comprehensive landscape of abnormalities for all HMCLs (variants, CNV and Gene Expression Profiling [GEP], e.g. Figure 1), we also investigated the major pathways involved in growth and resistance, which are p53, DNA repair, epigenetic regulation, NFkB, ERK/MEK, AKT/mTOR, cell cycle and cell death pathways. For instance, the Multiple Factorial Analysis (MFA) of variants and GEP for p53 pathway is shown in Figure 2: HMCLs are segregated mostly according to TP53 or ATM status, mutations being mostly exclusive between the 2 genes. Contrary to TP53 mutations, ATM and ATR mutations are rare events (n=9 and 7, respectively), both occurring mostly in TP53 wild-type HMCLs. This global analysis underlines that, below TP53 status, quite all HMCLs display an abnormal p53 pathway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synthetic lethality of LP-184, a next generation acylfulvene, in ex vivo PDX models with homologous recombination defects.

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed < 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

ATR and BRCA2 Simultaneous Mutation in a ccRCC With Sarcomatoid Differentiation and Extensive Metastases: A Case Report

The genomic landscape and driver-gene mutations differ significantly among diverse histological subtypes of clear cell renal cell carcinoma (ccRCC) due to the intratumoral heterogeneity. Frequent mutations in canonical DNA damage response genes, such as BRCA1/2 or ATR serine/threonine kinase (ATR) haven't been reported even in large-scale genomic profiling of ccRCC researches. Herein, we reported a rare ccRCC harboring ATR and BRCA2 simultaneous mutation with complicated morphologies and extensive metastases. Our case indicates that the deleterious alteration of DNA damage response genes, increasing CD8+ TILs, high PD1/PD-L1 expression and high TMB might contribute to this patient's tumor metastasis and aggressive biological behavior.

Abstract 6474: FAT1 and CASP8 co-mutations are associated with poor prognosis in HPV negative head and neck squamous cell carcinoma

Abstract INTRODUCTION: FAT1 alterations with somatic mutations or deletions are observed in head and neck squamous cell carcinomas (HNSCC). These alterations lead to loss of function of FAT1. FAT1 loss of function was known to be associated with aberrant Wnt activation, cell migration and invasion. In this study, we present analysis and outcomes of co-mutations seen in FAT1-mutation positive HNSCC. METHODS: TCGA data were analyzed for mutation, gene expression data and overall survival. We examined mutation data in 415 HPV negative samples and 72 HPV positive samples. The Fisher's Exact test was used to determine if differences in mutation frequency existed between the different cohorts. Mantel-Cox method was used for overall survival (OS) comparison between the cohorts. RESULTS: FAT1 somatic mutation was the third most commonly mutated gene in HPV-negative HNSCC while it was not common in HPV-positive HNSCC. FAT1 mutation was seen in 25.8% of HPV negative HNSCC (107/415), while 4.2% (3/72) HPV positive HNSCC had FAT1 mutation. We then analyzed the frequency of co-mutations in FAT1-mutated HPV-negative HNSCC. CDKN2A, NOTCH1, CASP8, FBXW7, ZFHX4, HRAS, DCC, ATR mutations were more common in FAT1-mutated cancers. CASP8 mutation was seen in 24.30% (26 out of 107 samples) of FAT1-mutations positive HPV negative HNSCC, while it was less common in FAT1-mutation negative HNSCC (9.08% 28/308) (P=0.002). The patients with FAT1 and CASP8 co-mutation were associated with shorter overall survival when compared with patients with FAT1-mutation (19.8 months vs 52.3 months P=0.02). FAT1 only or CASP8 only mutation without co-mutation did not affect survival. To determine the mutation impact of the FAT1 and CASP8 mutations in HNSCC, differentially expressed genes between mutated and non-mutated genes were analyzed in HNSCC. CASP8 mutation was the top gene among the genes with somatic mutation in HNSCC and associated with gene expression changes in more than 6000 genes. FAT1 was ranked 11th resulting in differential gene expression in more than 1000 genes. DISCUSSION: FAT1 somatic mutations are commonly seen in HPV negative HNSCC. We have demonstrated for the first time that CASP8 and FAT1 co-mutations are associated with poor prognosis with decreased overall survival in HPV negative HNSCC. Both CASP8 and FAT1 mutations are among the top genes with mutation impact on differential gene expression in HNSCC suggesting significance of these mutations in HNSCC. Citation Format: Emrullah Yilmaz, David Lee, Andrew Cowan, Gregory Gan, Michelle Ozbun, Yan Guo. FAT1 and CASP8 co-mutations are associated with poor prognosis in HPV negative head and neck squamous cell carcinoma [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 6474.

Mutational profiling of lung adenocarcinoma in China detected by next-generation sequencing.

NSCLC is the most common type of lung cancers. The purpose of this study is to screen cancer-related mutations in early LUAD in China through NGS technology, determine their correlation with clinical characteristics and provide basis for treatment decisions. In this study, we performed a 583 gene panel to detect the mutational spectrum of the tumors which were collected from 98 LUAD patients. The sequencing data and clinical characteristics were analyzed. Mutations were identified in 94.9% of patients. EGFR had the highest mutation frequency which was detected in 66% of the patients and was significantly associated with female gender and non-smoking history. Other genes with high mutation frequency were TP53 (37%), ERBB2 (24%), BCOR (22%), ZFHX3 (19%), BTG1 (17%), ATR (16%), WWTR1 (15%), etc. TP53 mutations were significantly associated with medium and low differentiation of tumors; BCOR and BLM mutations with gender; WWTR1 mutations with age; and ATR mutations with visceral pleura invasion were observed. 61% of the patients harbored at less one actionable alteration associated with FDA-recognized or investigational drugs. Multiple mutations in LUAD patients in this study have not previously been reported in NSCLC. Moreover, mutations in driver genes including EGFR, TP53, BCOR, BLM, WWTR1, and ATR were significantly related to clinical features. The panel used in this study is an effective approach for molecular analysis and can be applied in personalized treatment decision-making and drug development.

Hsa_circ_0007841: A Novel Potential Biomarker and Drug Resistance for Multiple Myeloma.

Purpose: Circular RNA (circRNA) is a key regulatory factor in the development and progression of human tumors. However, the working mechanism and clinical significance of most circRNAs remain unknown in human cancers, including multiple myeloma (MM).Patients and Methods: This study employs high-throughput circRNA microarray with bioinformatics to identify differentially expressed circRNAs in patients with MM. The hsa_circ_0007841 expressions were observed in the MM tissues of 86 patients. Drug-resistant cell lines and pathological features were also detected. In addition, the relationship between hsa_circ_0007841 expressions in the MM tissues and the pathological features of patients with MM were evaluated and role of hsa_circ_0007841 as a potential biomarker and therapeutic target was assessed.Results: The results show that in the MM cell lines and drug-resistant cell lines, hsa_circ_0007841 expression was significantly upregulated, which was closely associated with disease prognosis. Specifically, hsa_circ_0007841 upregulation was correlated with chromosomal aberrations such as gain 1q21, t (4:14) and mutations in ATR and IRF4 genes. This finding was corroborated in large samples. Finally, bioinformatics analysis showed that eight differentially expressed miRNAs and 10 candidate mRNAs interacted with hsa_circ_0007841, shedding some new light on the basic functional research.Conclusion: This study may be the first to report that hsa_circ_0007841 is significantly upregulated in MM. It also suggests that hsa_circ_0007841 may be a novel biomarker for MM and its involvement in the progression of MM.

314P - Next-generation DNA sequencing (NGS) results for tumours from phase II ABRAZO study of talazoparib after platinum or cytotoxic non-platinum regimens in patients (pts) with advanced breast cancer (ABC) and germline BRCA1/2 (gBRCA) mutations

314P - Next-generation DNA sequencing (NGS) results for tumours from phase II ABRAZO study of talazoparib after platinum or cytotoxic non-platinum regimens in patients (pts) with advanced breast cancer (ABC) and germline BRCA1/2 (gBRCA) mutations

Association of LRP1B Mutation With Tumor Mutation Burden and Outcomes in Melanoma and Non-small Cell Lung Cancer Patients Treated With Immune Check-Point Blockades.

Background: Tumor mutation burden (TMB) have been served as the most prevalent biomarkers to predict immunotherapy response. LRP1B (low-density lipoprotein receptor-related protein 1B) is frequently mutated in melanoma, non-small cell lung cancer (NSCLC) and other tumors; however, its association with TMB and survival in patients with immunotherapy remains unknown.Methods: We curated somatic mutation data and clinicopathologic information from 332 melanoma immunotherapy samples for discovery and 113 NSCLC samples for further corroboration. Bayesian variants non-negative matrix factorization was used to extract tumor mutational signatures. Multivariate Cox and logistic regression models were applied to adjust confounding factors. The CIBERSORT and GSEA algorithm were separately used to infer leukocyte relative abundance and significantly enriched pathways.Results: Patients with LRP1B mutation were identified to be associated with prolonged survival in both immunotherapy cohort. Higher tumor mutation burden was found in LRP1B mutated patients, and the association remained significant after controlling for age, gender, stage, mutations in TP53 and ATR, and mutational signatures. Immune response and cell cycle regulation circuits were among the top enriched pathways in samples with LRP1B mutations.Conclusion: Our studies suggested sequencing even a single, frequently mutated gene may provide insight into genome-wide mutational burden, and may serve as a biomarker to predict immune response.

DNA damage repair (DDR) gene alterations as a predictive biomarker for response to platinum-containing chemotherapy in advanced biliary tract cancer (BTC).

4078 Background: Although many studies using whole-exome sequencing or targeted sequencing have reported the molecular profile of BTC, its clinical implications remains unclear. In this study, we assessed a predictive role of DDR gene mutations in advanced BTC patients treated with platinum-containing regimen. Methods: Eighty-eight patients with pathologically-confirmed BTC who received first-line gemcitabine-cisplatin combination (n = 69) or fluoropyrimidine-oxaliplatin combination (n = 19) were included in this analysis. Targeted exome sequencing was performed using Foundation Medicine T7 assay or in-house OncoPanel AMC. Germline or somatic mutations in ATM, ATR, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, FAM175A, GEN1, MLH1, MSH2, MSH6, MRE11A, NBN, PALB2, PMS2, RAD50, RAD51, RAD51C, RAD51D, and XRCC2 were classified as DDR gene mutations. Data regarding baseline characteristics and treatment outcomes were retrospectively obtained from medical records. Results: The median age was 62 years (range, 25-78), with male comprising 64.8% (n = 57). By primary tumor site, 21 patients with GBC (23.9%), 44 with ICC (50.0%) and 23 with ECC (26.1%) were included. Most patients received palliative chemotherapy for their initially metastatic (50.0%) or recurred (44.3%) disease; the rest 5.7% had locally advanced disease. The median PFS and OS of overall patients were 7.1 and 16.1 months, respectively with median follow-up duration of 20.2 months. DDR gene mutations were found in 63.5% of patients. BRCA2 (18.2%) was most frequently mutated, followed by ATM (13.6%), and ATR (8.0%). DDR gene mutations were significantly associated with prolonged PFS (presence vs. absence; median, 6.9 vs. 5.7 months; P = 0.013) and OS (median, 21.0 vs. 13.3 months, P = 0.009). The impact of DDR gene mutations remained significant in multivariate analyses for PFS that included other prognostic factors (hazard ratio, 0.51; P = 0.009), but not for OS. Conclusions: The presence of DDR gene mutations might be a promising predictive biomarker for response to platinum-based chemotherapies in advanced BTC. Future investigation using novel agents targeting DDR gene alteration in BTC are warranted.

Combined Targeted Resequencing of Cytosine DNA Methylation and Mutations of DNA Repair Genes with Potential Use for Poly(ADP-Ribose) Polymerase 1 Inhibitor Sensitivity Testing

Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.

Abstract PR08: Longitudinal sampling of ctDNA reveals actionable mutations to optimize treatment of patients with high-grade serous ovarian cancer

Abstract Tumor cells leak their DNA into the blood stream, which allows us to use plasma sample from a patient to measure biomarker levels. In particular, circulating tumor DNA (ctDNA) sampling together with next-generation sequencing technologies provide a rapid and noninvasive test for quantifying tumor response to a treatment as well as finding potentially actionable mutations. The main objective of our study was to use ctDNA to establish a therapeutic window for targeting potentially actionable mutations to optimize the treatment of high-grade serous ovarian cancer (HGSOC) patients. We employed a targeted sequencing panel of 508 clinically annotated cancer genes to screen for actionable copy-number variations (CNV) and single-nucleotide variants (SNV) in 56 plasma and 20 fresh tumor samples from 13 patients with stage IIB-IVB HGSOC treated at the Turku University Hospital, Finland. All patients were surgically debulked and had received standard first-line carboplatin and paclitaxel chemotherapy. We analyzed longitudinal samples (from 3-6 time points per patient) from 11 patients during primary therapy, and from two patients with multiple (two and three samples per patient) relapsed disease. DNA isolated from tumor tissue and plasma was analyzed for genetic alterations by targeted deep-sequencing. White blood cell DNA was used as germline controls. Pathogenic germline mutations in 14 known ovarian cancer susceptibility genes were evaluated with CADD annotation framework and ClinVar database. Minimum requirements for passed somatic SNVs were CADD phred score 10, sequencing depth 100 (ctDNA) or 30 reads (tumor and blood), variant read count 4 or frequency 0.001. Variants detected in blood with > 2 reads were excluded. Additionally, a variant that passed filtering in at least one sample of a patient was included, regardless of threshold, if present in any other specimen. Thresholds of < 0.7 and > 4 copies were used as functionally relevant losses and gains, respectively. Clinically relevant germline pathogenic mutations in BRCA2, ATR, APC, and RB1 genes were identified from blood samples of five (38%) patients. The preoperative samples were also characterized by the highest somatic mutation burden, with a median total variant allele frequency (VAF) of 0.16 (range 0.02-0.77). Importantly, clinically relevant CNVs were detected in preoperative ctDNA of 6 (46%) patients, in potentially targetable driver genes such as ERBB2, PIK3CA, and CDK12. In six patients undergoing neoadjuvant chemotherapy (NACT), we noted a significant decrease in total ctDNA mutation burden during treatment, consistent with a drop in CA-125 levels. Interestingly, the levels of CNV counts increased in ctDNA during NACT in two patients, which associated with improved response to chemotherapy. During adjuvant treatment after debulking surgery, potentially actionable mutations were detected in all patients, with median total VAF of 0.03 (range 0.006-0.43). Importantly, in patients with no macroscopic residual disease, we detected a median of 2.5 (range 1-21) mutations in adjuvant treatment samples, indicating a molecularly persistent disease. Further, a subset of these alterations in, e.g., FANCA and PRCKB persisted during adjuvant treatment, revealing a potential therapeutic window for targeted treatments. Finally, two patients with progressive disease were characterized by predominantly either ctDNA gains or losses. Further, potentially actionable mutations in, e.g., TNXRD1, which sensitizes to AKT inhibitors, could be identified and tracked during disease progression. Taken together, longitudinal analysis of a panel-based ctDNA targeted sequencing can reliably detect very low VAF alterations, which can be used to identify and track actionable genomic alterations. Thus, ctDNA analysis can open a therapeutic window to target molecular residual disease in patients with newly diagnosed and advanced HGSOC. This abstract is also being presented as Poster B06. Citation Format: Kaiyang Zhang, Liina Salminen, Jaana Oikkonen, Kaisa Huhtinen, Johanna Hynninen, Seija Grénman, Sakari Hietanen, Rainer Lehtonen, Anniina Färkkilä, Sampsa Hautaniemi. Longitudinal sampling of ctDNA reveals actionable mutations to optimize treatment of patients with high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR08.

ATR is required to complete meiotic recombination in mice

Precise execution of recombination during meiosis is essential for forming chromosomally-balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these remain poorly understood, particularly in mammals. Here we report that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated resected DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

Abstract 5380: Whole-exome sequencing of Brazilian non-small cell lung cancer

Abstract Background: Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer with low survival rates. NSCLC patients harboring EGFR mutations are benefited by targeted therapy with tyrosine kinase inhibitors. The mutational profiling and the knowledge about recurrent mutations in Brazilian NSCLC is limited. Therefore, the detection of the mutation landscape of Brazilian NSCLC tumors is crucial for tailored therapy. Aim: To evaluate the mutational profiling of NSCLC of a Brazilian series. Methods: We analyzed a series of 40 NSCLC patients from Barretos Cancer Hospital. Clinical staging and exposure data were collected from all patients. Exome sequencing was performed in paired tumor/blood tissue DNA using the Illumina HiSeq2500™ System. Germline variations were excluded and the Genome Analysis Toolkit and VarScan were used for variants annotation and mutation detection. Results: Overall, 29 (72.5%) of cases were adenocarcinomas and 11 (27.5%) were squamous cell carcinoma (SCC) and the series was enriched by initial stages cases (I, n=16; II, n=12; III, n=6; IV, n=6). Regarding tobacco exposure, 33 patients were current smokers or former smokers and 7 patients were never smokers. In accordance, most of the cases presented the mutation signature 4, related to tobacco exposure, which is characterized by C>A substitutions and signature 29, related to tobacco chewing habit, which is characterized by C>A and C>T substitutions. All SCC patients were current or former smokers. TP53 gene was the most recurrently mutated gene observed in 48% (n=14) of the adenocarcinomas and 100% (n=11) of SCC cases (p=0,003). In adenocarcinomas, besides of TP53 gene, recurrent mutations were also found in KRAS (28%; n=8), STK11(21%; n=6), USP36 and APOB (14%; n=4/each), POLQ and CHD5 (10% n=3/each), EGFR and ANO3 (7%; n=2/each) and PDGFRA, BRAF, ERBB2, ERBB4, NRAS, RB1, CD22, CTNNA2 and ATR mutations were identified in only one adenocarcinoma case (3%). In SCC, besides of TP53 gene, recurrent mutations were also found in RB1, CD22, CTNNA2 and CHD7 (27%; n=3/each), ATR and APOB (18%; n=2/each), KRAS, PDGFRA, ERBB2, ERBB4, POLQ, CHD5 and ANO3 were identified in only one SCC case (9%). EGFR, STK11, BRAF, NRAS and USP36 mutations were exclusively found in adenocarcinomas and CHD7 mutations were exclusively found in SCC. Conclusions: The mutational landscape of lung adenocarcinoma and SCC are clearly different. A noteworthy number of pathogenic mutations was identified in this initial stages-enriched series. Our results may contribute to improve the knowledge about the molecular pathogenesis of NSCLC to better guide the clinical management with targeted therapies. Citation Format: Leticia Ferro Leal, Adriane Feijo Evangelista, Pedro R. de Marchi, Ysadhora Christiane Camargo Rodrigues, Eduardo Caetano Albino da Silva, Rui Manuel Reis. Whole-exome sequencing of Brazilian non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5380.

Genetic heterogeneity and actionable mutations in HER2-positive primary breast cancers and their brain metastases.

Brain metastases constitute a challenge in the management of patients with HER2-positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR, homozygous deletion in CDKN2A and amplification in KRAS. Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53. Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor.

The Cell Cycle Checkpoint Regulator ATR Is Required for Internal Aluminum Toxicity-Mediated Root Growth Inhibition in Arabidopsis.

Aluminum (Al) can target multiple sites of root cells for toxicity, including the cell wall, the plasma membrane and symplastic components. Previous work revealed that the cell cycle checkpoint regulator (ATR) Ataxia Telangiectasia-mutated and Rad3-related is required for Al toxicity-induced root growth inhibition in als3 and that the symplastic component DNA is an important target site of Al for the toxicity. However, whether monitoring DNA integrity through ATR-regulated pathway is required for Al-induced root growth inhibition in other Al-sensitive mutants remains unknown. In this study, we demonstrated that the atr mutation could also rescue the Al hypersensitivity and Al-induced cell cycle arrest in star1, which supports the hypothesis that ALS3 and STAR1 function together to be involved in the detoxification of Al in Arabidopsis. However, mutation of ATR could not rescue the Al-sensitive phenotype of almt1 or stop1, both of which are defective in external detoxification mechanisms of Al. We further showed that the Al hypersensitivity and Al-induced quiescent center (QC) differentiation in als1 could also be rescued by the atr mutation. Therefore, our results suggest that ATR-regulated pathway is involved in the modulation of internal Al toxicity-mediated root growth inhibition in Arabidopsis.

Abstract IA8: The genetics and genomics of African esophageal cancer

Abstract Esophageal squamous cell carcinoma (ESCC) is common in many Black populations of sub-Saharan Africa, with high incidence regions in Eastern and Southern Africa. Clinical presentation in Africa is late, and treatment is mainly palliative with a very poor prognosis. Various environmental risk factors have been identified, but the possible contribution of inherited genetic variants to disease risk is an important question which is unresolved. Also, limited data are available on the somatic mutations which are driving tumor development. This presentation will review current knowledge on the role of germline genetic variants and somatic mutations in the development of African ESCC. Genetic association studies of African ESCC have been limited to the analysis of small numbers of single nucleotide polymorphisms (SNPs) in candidate genes, and carried out only in the Black and Mixed Ancestry populations of the Western Cape of South Africa. Several positive associations have been reported, particularly in the Mixed Ancestry population, but none have achieved genome-wide levels of significance or been replicated in independent studies. We are currently testing SNPs which have been associated with ESCC in genome-wide association studies (GWAS) from Asian and European populations for association with ESCC in the South African population. Black cases with a histologically confirmed diagnosis of ESCC and matching population controls were recruited, after informed consent and institutional ethical approval, from the Western Cape and Gauteng provinces of South Africa. SNPs were genotyped either by individual TaqMan assays (Applied Biosystems) or in a multiplex MassARRAY (Agena Bioscience) and genotypes were tested for association. Our initial studies found no significant evidence of association at previously reported GWAS loci ATP1B2, CASP8, c20orf54, HEATR3, PDE4D, PLCE1, PTPN2, RUNX1, SMG6, ST6GAL1, TMEM173 and UNC5L loci. However, evidence for association was observed for the SNP rs2239815 at XBP1 on chromosome 22q12 (OR =1.41, P = 0.00087), and the SNPs rs4822983 (OR = 1.31, P = 0.0013) and rs1033667 (OR = 1.29, P = 0.0025) in CHEK2. Genotyping of a larger SNP panel in an expanded collection of cases and controls is in progress to extend these findings. Detection of somatic mutations in ESCC tumour tissue has in the past been limited to sequencing of candidate genes and copy number studies, with TP53 mutations being detected in a minority of South African and Kenyan patients. We have carried out pilot whole exome sequencing in 10 matched blood/tumour pairs from South African ESCC patients and detected mutations in TP53, ATR, GNAS, MAGI2, FBXW7, FLT3, NFE2L2, TET2 and ZNF750. Follow up sequencing of the TP53 gene detected missense or truncating mutations in 18/26 (69%) of tumours. A recent sequencing study of 59 Malawian ESCC patients (Liu et al) observed recurrent mutations in TP53, CDKN2A, NFE2L2, CHEK2, NOTCH1, FAT1 and FBXW7, and transcriptomic profiles were used to divide ESCCs into 3 subtypes. Priorities for the future should include well powered GWAS in high risk populations from several African countries, with collaborative studies to compare genetic risk factors across populations and meta-analysis to provide power to detect risk loci with moderate effects. Whole-exome or whole genome sequencing of substantial panels of blood/tumour pairs are needed to establish the major drivers of tumorigenesis in African ESCC and to derive mutational signatures which may provide insight into causal environmental factors. Citation Format: Christopher G. Mathew. The genetics and genomics of African esophageal cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA8.

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment

Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased Tcell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.

STAG2 Mutations Are an Independent Prognostic Factor in Patients with Myelodysplastic Syndromes

▪INTRODUCTION: STAG2is a component of the cohesin complex involved in the cohesion of sister chromatids, transcriptional regulation and DNA repair. Recent sequencing studies have identified cohesion mutations in myeloid malignancies with data suggesting a potential adverse impact on survival.METHODS: We conducted whole exome sequencing of 114 previously untreated patients with MDS or CMML. Exome capture hybrid was performed using Agilent SureSelect All Exon V4. Sequencing was performed with Illumina HiSeq 2000 and aligned to the hg19 human genome reference. Common virtual normal in house pool was used for somatic variant calling. Clinical and demographic data was obtained from clinical records. Generalized linear models were used to study the association of response (OR = overall and CR = complete) and potential risk factors. Response was defined following 2006 IWG criteria. The Kaplan-Meier produce limit methods were used to estimate the median overall survival (OS) and leukemia-free survival (LFS).RESULTS: A total of 10 (9%) patients carried high-confidence STAG2 mutations. Patient characteristics are shown in Table 1. Patients with STAG2 mutations tended to have lower Hgb (8.6 vs 10.5 g/dL, p=0.003) and higher median number of driver mutations (4 vs 2, p<0.001). Two patients (20%) had MDS-MLD and the remaining 8 (80%) had MDS-EB, therefore, STAG2 mutation was associated with excess blasts (rho=0.314, p=0.001). There were no cases of CMML with STAG2 mutation. Seven (70%) patients had Low/Int-1 IPSS category and where therefore lower-risk by IPSS and 3 (30%) had Int-2/High IPSS and were therefore higher-risk by IPSS. A total of 65 (57%) patients in the study cohort, 6 (67%) of patients with STAG2 mutation, were treated with hypomethylating agents. STAG2 mutations correlated with normal karyotype (rho=0.190, p=0.04), RUNX1 (rho=0.232, p=0.013), SRSF2 (rho=0.192, p=0.04), ATR (rho=0.303, p=0.001) and NRAS (rho=0.279, p=0.003) mutations. The mean variant allele frequency of STAG2 mutations was 0.35 (range 0.11-072). Seven appeared as clonal driver mutations and 3 as subclonal based on clonal heterogeneity testing. Presence of STAG2 mutation did not significantly impact OR (OR=0.45, 95%CI: 0.08-2.65, p=0.376) or CR (OR=0.32, 95% CI: 0.04-2.97, p=0.318) to therapy with hypomethylating agents. Median follow up of the cohort was 21.6 months (range 1-102 months). Presence of STAG2 mutations was associated with decreased OS (HR=2.45, 95% CI: 1.04-5.80, p=0.041) (Figure 1A). Within the lower-risk IPSS group, STAG2 mutations were associated with an even greater impact on OS (HR = 3.32, 95%CI 1.12-9.82, p=0.030). By multivariate analysis including other prognostic variables such as cytogenetics, Hbg <8g/dL, platelets <50x109/L, ANC <0.8 x109/L or blast >10%, STAG2 mutation retained its independent adverse impact on OS (HR 2.69, 1.03-6.99, p=0.042). STAG2 mutations did not predict for shorter LFS (HR = 2.30, 95% CI 0.51-10.27, p=0.263) (Figure 1B).CONCLUSION: STAG2 mutations can be found in close to 10% patients with MDS. As previously reported, they tend to appear in patients with MDS-EB and correlate with survival outcomes. In our study, presence of STAG2 mutation was independently associated with adverse overall survival in patients with MDS, particularly in those classified as lower-risk by IPSS, but did not predict for shorter LFS. Contrary to previous reported data, STAG2 mutations did not predict for higher responses to azacitidine or decitabine. [Display omitted] [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Ravandi:Seattle Genetics: Consultancy, Honoraria, Research Funding; BMS: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. DiNardo:Novartis: Other: advisory board, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Agios: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding. Daver:BMS: Research Funding; Karyopharm: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Sunesis: Consultancy, Research Funding; Ariad: Research Funding; Kiromic: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Kantarjian:Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Regional and temporal heterogeneity of epithelial ovarian cancer tumor biopsies: implications for therapeutic strategies.

Stage III/IV epithelial ovarian cancer (EOC) is a systemic disease. The clonal relationship among different tumor lesions at diagnosis (spatial heterogeneity) and how tumor clonal architecture evolves over time (temporal heterogeneity) have not yet been defined. Such knowledge would help to develop new target-based strategies, as biomarkers which can adjudge the success of therapeutic intervention should be independent of spatial and temporal heterogeneity.The work described in this paper addresses spatial and temporal heterogeneity in a cohort of 71 tumor biopsies using targeted NGS technology. These samples were taken from twelve high grade serous (HGS) and seven non HSG-EOC, both at the time of primary surgery when the tumor was naïve to chemotherapy and after chemotherapy.Matched tumor lesions growing in the ovary or at other anatomical sites show very different mutational landscapes with branched tumor evolution. Mutations in ATM, ATR, TGFB3, VCAM1 and COL3A1 genes were shared across all lesions. BRCA1 and BRCA2 genes were frequently mutated in synchronous lesions of non HGS-EOC. Relapsed disease seems to originate from resistant clones originally present at the time of primary surgery rather than from resistance acquired de novo during platinum based therapy. Overall the work suggests that EOC continues to evolve. More detailed mapping of genetic lesions is necessary to improve therapeutic strategies.