ATM Alterations Research Articles

Germline mutations in MSH2 and ATM gene in patients with GIST (gastrointestinal stromal tumor) and second epitelial tumors.

e23520 Background: In adult GISTs are frequently sporadic, while rarely GISTs are linked to Carney Triad and Carney-Stratakis Syndrome and NF1. GISTs with second primary tumors are reported in 4-33% of patients in literature and genetic counseling is suggested to explore an underlying germline mutations pathway. Methods: In our Academic Hospital Centre (EURACAN member) in Florence, Italy, we are following patients with GIST and multiple primary tumors with genetic counseling (72 GISTs with second tumors/185 patients with GIST) and germline analysis of the following genetic panel is performed as clinically indicated: BRCA1, BRCA2, MUTYH, MLH1, MSH2, MSH6, CDH1, ATM, TP53, PTEN, CHECK2, PALB2, BARD1, BRIP1, BLM, RAD51C, RAD51D, XRCC2, PMS2, MRE11A, RAD50, NBN, FAM175A, EPKAM, TSK1, MEN1 by sequencing analysis with Illumina MiSeq by kit multiplicom BRCA Hereditary cancer Mastr plus, and bioinformatic analysis by software SOPHIADDM (Sophia genetics) for point genetic alterations of BRCA1 NM_007294.3, BRCA2 NM_000059.3, MUTYH NM_000249, MSH2 NM_000251, MSH6 NM_000179, CDH1 NM_00444360, ATM NM_000051, TP53 NM_000546, PTEN NM_000314, CHEK2 NM_001005735, PALB2 NM_024675, BARD1 NM_000465, BRIP1 NM_032043, BLM NM_000057, RAD51C NM_002876, RAD51D NM_001142571, XRCC2 NM_005431, PMS2 NM_000535, MRE11A NM_005590, RAD50 NM_006732, NBN NM_002485, FAM175A NM_139076, EPCAM NM_002354, STK1 NM_000455, MEN1 NM_000244 and MLPA (Multiplex Ligation-dependent Probe Amplification) test analysis for patients with kit P087-BRCA1,P045-BRCA2(CHEK2, P248-MLH1-MSH2, P003-MLH1/MSH2, P072-MSH6-MUTYH (MRC-Holland). Results: In 3 patients germline mutations have been observed: 1 patient showed the c.1192dupG, p.(Ala398Glyfs*19) pathogenic mutation in exon 7 of MSH2 gene, confirmed by Sanger Sequencing, 1 patient showed c.565-?_1130+?del mutation consisting in heterozygous 3-4-5-6 exons deletion of MSH2 gene, confirmed by MLPA analysis, and in 1 patient the following ATM alteration has been identified in heterozygosis: ATM c.5319+2T > C, p.(?). In the 2 patients with Lynch syndrome with colon adenocarcinoma (MSI-H), synchronous GISTs (1 patient quadruple WT and 1 patient kit ex 11 mutated ) were diagnosed; in the patient with ATM mutation, the diagnosis of GIST (kit ex 11 mutated) occurred after prostate adenocarcinoma and before colon adenocarcinoma (MSI-H). Conclusions: Our analysis suggests that GIST diagnosis could be tumor-related to multiple hereditary tumor syndromes as Lynch Syndrome and Ataxia-Teleangectasia syndrome, the latter being linked in eterozygosis to tumor susceptibility to breast in female. This report represents a high value in terms of genetic counseling for relatives and in terms of therapeutic implications for the patients.

Circulating tumor DNA-based genomic profiling of small bowel adenocarcinoma.

3523 Background: Small bowel adenocarcinoma (SBA) is a rare malignancy, with lower incidence, later stage at diagnosis, and worse overall survival compared to other intestinal cancers, such as colorectal cancer (CRC). Since the majority of small bowel tumors are not accessible to endoscopic biopsy, comprehensive genomic profiling using circulating tumor DNA (ctDNA) may enable non-invasive detection of targetable genomic alterations (GA) in SBA patients. In this study, we characterize the ctDNA GA landscape in SBA. Methods: Analysis of 299 ctDNA samples prospectively collected from 265 SBA patients between 2017 to 2020 was performed using a 73 gene next generation sequencing panel (Guardant360). A subset of patients underwent longitudinal analysis of changes in GA associated with systemic therapy. Results: Of the 265 patients, 160 (60.3%) were male; the median age was 66 (range: 21-93 years). The most common GA identified in SBA patients included TP53 [58%], KRAS [44%], and APC [40%]. MSI was detected in 3.4% of SBA patients. When stratified by primary tumor location, APC, KRAS, TP53, PIK3CA, and ARID1A were the most common GA identified in both duodenal and jejunal adenocarcinomas. ERBB2, BRCA2 and CDK6 alterations were enriched in duodenal adenocarcinoma, while NOTCH and BRAF alterations were enriched in jejunal adenocarcinoma. The most common currently-targetable GA identified were ATM [18%], PIK3CA [17%], EGFR [15%], CDK4/6 [11%], BRAF [10%], and ERBB2 [10%]. Unique differences in GA between SBA and CRC were identified: i) the majority of ERBB2 alterations are mutations (89%) in the extracellular domain and kinase domain, not amplifications (11%); ii) the majority of BRAF alterations are non V600E mutations (69%) and amplifications (28%); iii) there is a significantly lower rate of APC mutations (40%). Alterations in DNA damage response pathway proteins, including ATM and BRCA 1/2, were identified in 30% of SBA patients. ATM alterations were more common in patients ³65 years old. The most common mutations predicted to be related to clonal hematopoiesis of indeterminate potential were TP53, KRAS and GNAS. Longitudinal ctDNA analysis in 4 SBA patients revealed loss of mutations associated with therapeutic response (TP53 R342*, MAPK3 R189Q) and acquired mutations associated with therapeutic resistance (NF1 R1968*, MET S170N, RAF1 L613V). Conclusions: This study represents the first large-scale blood-based ctDNA genomic profiling of SBA. SBA represents a unique molecular entity with differences in frequency and types of GA compared to CRC. Variations in GA were noted based on anatomic origin within the small intestine. Longitudinal ctDNA monitoring revealed novel GA associated with therapeutic resistance. Identification of multiple targetable GA may facilitate clinical decision making and improve patient outcomes in SBA, especially when a tissue biopsy is not feasible or sufficient for comprehensive genomic profiling.

Frequency of homologous recombination repair alterations in ovarian cancer in Chinese population.

e17531 Background: Ovarian Cancer (OC) is the most lethal cancer of all gynecological malignancies.Circulating tumor DNA (ctDNA) has received substantial attention in recent years resulting from the non-invasive, safe and effective method with considerable potential for clinical diagnosis and treatment management in patients with OC. Here, we assessed the mutational feature in homologous recombination repair (HRR) using ctDNA in OC. Methods: Plasma ctDNA was isolated from blood of patients and then was analyzed by AcornMed Biotechnology NGS-based assay for 808 genes panel for genomic alterations. The somatic and germline pathogenic mutations were identified in 12 HRR genes (ATM, BRCA1, BRCA2, BRIP1, CHEK1, CHEK2, FANCA, PALB2, RAD51B, RAD51C, RAD51D, RAD54L). Results: At our institution, 85 patients underwent NGS analysis of ovarian cancer specimens. The median age was 57 (range from 26 to 83). Twenty-six patients(42.34%) harbored a mutation in at least 1 of the HRR genes in their tumor. The most commonly altered HRR gene was BRCA1 (18.25%), followed by BRCA2 (8.76%), ATM (5.84%), RAD51D(3.65%), CHEK2 (2.92%), FANCA(2.19%) and RAD51C (0.73%). To determine the difference of mutation landscape in HRR between Chinese and western populations, we compared prevalence and spectrum in cases between our cohort and the cohort of Heeke et al 1(Table). Prevalence of HRR were different from the Western cases(42.34% vs 20%, P＜0.05). Of note, BRCA1, BRCA2, ATM and CHEK2 alterations rate was higher in our cohort, and BRIP1 and PALB2 were only detected in the western cohort. Conclusions: CtDNA can characterize the mutational feature of HRR in OC. Around 42.3% of patients with OC harbour germline or somatic HRR mutations. The expanded use of PARP inhibitors in HRR deficient tumours using a signature of HRR by ctDNA in clinical practice requires validation.[Table: see text]

Downregulation of ATM and BRCA1 Predicts Poor Outcome in Head and Neck Cancer: Implications for ATM-Targeted Therapy.

ATM and BRCA1 are DNA repair genes that play a central role in homologous recombination repair. Alterations of ATM and BRCA1 gene expression are found in cancers, some of which are correlated with treatment response and patient outcome. However, the role of ATM and BRCA1 gene expression in head and neck cancer (HNC) is not well characterized. Here, we examined the prognostic role of ATM and BRCA1 expression in two HNC cohorts with and without betel quid (BQ) exposure. The results showed that the expression of ATM and BRCA1 was downregulated in BQ-associated HNC, as the BQ ingredient arecoline could suppress the expression of both genes. Low expression of either ATM or BRCA1 was correlated with poor overall survival (OS) and was an independent prognostic factor in multivariate analysis (ATM HR: 1.895, p = 0.041; BRCA1 HR: 2.163, p = 0.040). The combination of ATM and BRCA1 expression states further improved on the prediction of OS (HR: 4.195, p = 0.001, both low vs. both high expression). Transcriptomic analysis showed that inhibition of ATM kinase by KU55933 induced apoptosis signaling and potentiated cisplatin-induced cytotoxicity. These data unveil poor prognosis in the HNC patient subgroup with low expression of ATM and BRCA1 and support the notion of ATM-targeted therapy.

Interpreting Sequence Variation in PDAC-Predisposing Genes Using a Multi-Tier Annotation Approach Performed at the Gene, Patient, and Cohort Level.

We investigated germline variation in pancreatic ductal adenocarcinoma (PDAC) predisposition genes in 535 patients, using a custom-built panel and a new complementary bioinformatic approach. Our panel assessed genes belonging to DNA repair, cell cycle checkpoints, migration, and preneoplastic pancreatic conditions. Our bioinformatics approach integrated annotations of variants by using data derived from both germline and somatic references. This integrated approach with expanded evidence enabled us to consider patterns even among private mutations, supporting a functional role for certain alleles, which we believe enhances individualized medicine beyond classic gene-centric approaches. Concurrent evaluation of three levels of evidence, at the gene, sample, and cohort level, has not been previously done. Overall, we identified in PDAC patient germline samples, 12% with mutations previously observed in pancreatic cancers, 23% with mutations previously discovered by sequencing other human tumors, and 46% with mutations with germline associations to cancer. Non-polymorphic protein-coding pathogenic variants were found in 18.4% of patient samples. Moreover, among patients with metastatic PDAC, 16% carried at least one pathogenic variant, and this subgroup was found to have an improved overall survival (22.0 months versus 9.8; p=0.008) despite a higher pre-treatment CA19-9 level (p=0.02). Genetic alterations in DNA damage repair genes were associated with longer overall survival among patients who underwent resection surgery (92 months vs. 46; p=0.06). ATM alterations were associated with more frequent metastatic stage (p = 0.04) while patients with BRCA1 or BRCA2 alterations had improved overall survival (79 months vs. 39; p=0.05). We found that mutations in genes associated with chronic pancreatitis were more common in non-white patients (p<0.001) and associated with longer overall survival (52 months vs. 26; p=0.004), indicating the need for greater study of the relationship among these factors. More than 90% of patients were found to have variants of uncertain significance, which is higher than previously reported. Furthermore, we generated 3D models for selected mutant proteins, which suggested distinct mechanisms underlying their dysfunction, likely caused by genetic alterations. Notably, this type of information is not predictable from sequence alone, underscoring the value of structural bioinformatics to improve genomic interpretation. In conclusion, the variation in PDAC predisposition genes appears to be more extensive than anticipated. This information adds to the growing body of literature on the genomic landscape of PDAC and brings us closer to a more widespread use of precision medicine for this challenging disease.

Evaluation of Gene Expression Alterations of ATM, TP53, and POLM in Gastric Biopsy Specimens of Patients with Gastritis and its Association with Helicobacter Pylori Infection

Evaluation of Gene Expression Alterations of ATM, TP53, and POLM in Gastric Biopsy Specimens of Patients with Gastritis and its Association with Helicobacter Pylori Infection

Olaparib efficacy in patients with metastatic castration-resistant prostate cancer (mCRPC) carrying circulating tumor (ct) DNA alterations in BRCA1, BRCA2 or ATM: Results from the PROfound study.

27 Background: ctDNA testing offers additional opportunities for homologous recombination repair (HRR) gene alteration determination in patients who are not able to access tumor tissue testing. Alteration testing in ctDNA, for BRCA1, BRCA2 and ATM alterations was performed retrospectively in the PROfound study (phase 3 trial of olaparib versus physician’s choice of abiraterone or enzalutamide in men with HRR gene-mutated mCRPC [NCT02987543]). Methods: ctDNA samples were sequenced at FMI, using the FoundationOne Liquid CDx assay for alterations in BRCA1, BRCA2 (BRCA) and ATM, using plasma samples collected during screening in PROfound. Only patients who consented and provided plasma samples from Cohort A (BRCA/ ATM alteration positive by tissue testing) were tested for ctDNA alterations. Radiographic progression-free survival (rPFS) assessed by blinded independent central review (BICR) in patients positive for alterations in ctDNA was analysed via stratified log-rank test. Additional efficacy endpoints (Objective Response Rate [ORR], Overall Survival[OS]) were also assessed. Results: In total, 181/245 (73.9%) Cohort A patients consented and provided a plasma sample for ctDNA testing, of which 139/181 (76.8%) had a ctDNA result reported (either mutation positive or mutation negative) and 42 patient samples failed testing due to insufficient DNA yield or a technical failure of the test.BRCA/ ATM alterations were identified in111/139 (79.9%) patients and 28/139 patients did not report a BRCA/ATM mutation either due to lack of ctDNA shedding from the tumour or ctDNA levels below the sensitivity of the assay. Patients who carried BRCA/ ATM ctDNA alterations had comparable demography, baseline characteristics and proportions of BRCA1, BRCA2 and ATM mutated patients to the overall Cohort A patients. rPFS by BICR results, in patients with BRCA/ ATM alterations in ctDNA, are presented in the Table. Key secondary efficacy endpoints (ORR, OS) will also be reported for ctDNA alteration positive patients. Conclusions: ctDNA testing in patients with mCRPC is feasible and most, but not all patients have concordant results. Patients who were positive for alterations in BRCA1, BRCA2 or ATM showed a consistent rPFS improvement (hazard ratio [HR] 0.33 [0.21, 0.53]) when compared with the ITT population identified by tumor tissue testing (HR 0.34 [0.25, 0.47]). This suggests that using ctDNA to identify patients carrying BRCA1, BRCA2 or ATM alterations who will benefit from olaparib may be beneficial. Clinical trial information: NCT02987543. [Table: see text]

Exploratory gene-by-gene analysis of olaparib in patients (pts) with metastatic castration-resistant prostate cancer (mCRPC): PROfound.

126 Background: The Phase 3 PROfound trial (NCT02987543) met its primary endpoint and key secondary endpoints, including improved overall survival (OS) for olaparib in men with mCRPC with alterations in BRCA1, BRCA2, or ATM (Cohort A). We report gene-by-gene analysis of olaparib antitumor activity among the 15 prespecified homologous recombination repair (HRR) genes. Methods: Pts were randomized to olaparib (300 mg bid; n=256) or physician’s choice of enzalutamide or abiraterone (control; n=131). Exploratory analyses in pts with alterations in BRCA1 and/or BRCA2 (BRCA, regardless of co-occurring alterations with other HRR genes) or in single genes were conducted. Results: Evidence of olaparib antitumor activity was observed in subgroups with &gt;10 pts (table). Data for pts with alterations in only BRCA1, BRCA2, PPP2R2A, RAD51B, RAD54L, PALB2, BRIP1, CHEK1, BARD1, and RAD51D will be reported (no FANCL or RAD51C enrolled). Conclusions: Small subgroups limit interpretation for some genes. Olaparib antitumor activity is greatest in pts with BRCA alterations, with a spectrum of clinical sensitivity to olaparib as defined by rPFS and OS across the broader population with alterations in other HRR genes. Clinical trial information: NCT02987543. [Table: see text]

Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia.

BackgroundSeveral genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high‐risk alterations such as del(11q)/ATM‐mutations and del(17p)/TP53‐mutations have not been established.MethodsWe integrated next‐generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response.ResultsTargeted sequencing analysis of the co‐occurrence of high‐risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co‐occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9‐edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition.ConclusionsOur work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

Correlation between comprehensive genomic profiling (CGP) utilizing tissue-based testing (T-CGP) and cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

422 Background: Early systemic dissemination is a salient feature of PDAC and the ability to reliably detect as well as monitor cfDNA based alterations may have predictive and/or prognostic value. However, a clear correlation between the clonality of the primary tumor and micro-metastatic disease has not been established in PDAC. We examined the correlation between T-CGP and cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of PDAC pts who had both T-CGP (October 2010 – March 2020) and cfDNA based CGP (May 2016 – March 2020), with an average of 645 days between tests (Range: 21 – 2101 days) For T-CGP, exonic regions and select intronic regions of cancer related genes were sequenced to a high uniform depth (&gt;500X median coverage) using FoundationOne Heme (Exons: 315 genes; Select Introns: 28 genes) and FoundationOne CDx (Exons: 310 genes; Select Introns: 34 genes). cfDNA based CGP, was performed using either FoundationOne Liquid (Exons: 70 genes; Select Introns: 7 genes) or Foundation-ACT (Exons: 59 genes; Select Introns: 6 genes) platform. Results: Eighty-one pts were identified with both T-CGP and cfDNA based CGP - median age was 61 years, 59.3% (48/81) were male and 66.2% (49/74) had reported metastatic disease. The median tumor DNA content available for T-CGP analysis was 112.3 ng. The median cfDNA concentration was 1.7 ng/µL, and the number of reported ct-DNA based somatic alterations correlated significantly with the concentration of ct-DNA (Spearman’s R = 0.19, p &lt; 10-8). 75/81 (92.6%) and 41/81 (50.6%) of pts had at least one pathogenic somatic alteration detected by T-CGP and cfDNA based CGP, respectively. KRAS, TP53 and CDKN2A were the most frequently altered genes in both T-CGP and cfDNA based CGP (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [ BRCA2, ATM, and CHEK2 alterations] were detected in 7.4% (6/81) of T-CGP and 7.4% (6/81) cfDNA based CGP, respectively. Two BRAF fusions were detected from samples tested with T-CGP, however these were not detected with the cfDNA assay. Conversely, an ALK-EML4 fusion was detected using both platforms. Conclusions: The concordance between T-CGP and cfDNA based CGP in HR-DDR gene alterations may be indicative of the germline status of some of these alterations; nevertheless, it suggests the predictive utility of cfDNA based CGP in PDAC. The differential prevalence of other gene alterations between the two modalities is likely reflective of the cfDNA yield available for CGP and to a lesser extent, clonal evolution. cfDNA based CGP holds promise as a complimentary predictive tool to T-CGP. [Table: see text]

Comprehensive genomic profiling (CGP) utilizing cell-free DNA (cfDNA) in patients (pts) with pancreatic ductal adenocarcinoma (PDAC).

421 Background: PDAC has a propensity for early systemic dissemination and most pts with primary tumors radiographically confined to the pancreas harbor micro-metastases. cfDNA based CGP offers a non-invasive mechanism to identify actionable genomic targets in PDAC and account for serial clonal evolution in response to therapeutic selection pressure. We interrogated the Foundation Medicine database to characterize cfDNA based CGP in pts with PDAC. Methods: We performed a retrospective analysis of pts with PDAC who underwent cfDNA based CGP between May 2016 and February 2020. cfDNA based CGP was done utilizing either the FoundationOne Liquid (F1-Liquid) or the Foundation-ACT (F-ACT) testing platform. Samples were interrogated for exonic and select intronic regions of cancer-related genes for both F1-Liquid (Exons: 70 genes; Select Introns: 7 genes) and F-ACT (Exons: 59 genes; Select Introns: 6 genes). Variant zygosity and somatic/germline status (SGZ) for short variant mutations was computationally predicted without matched normal tissue using an investigational method. A Gaussian Mixture Model (GMM) was used to identify latent molecular classes in samples harboring somatic, pathogenic variants (n=613); the Bayesian Information Criteria (BIC) was used for model selection. Results: We identified 1,009 pts with cfDNA based CGP - median age was 65, 55% were male, and median cfDNA concentration was 24 ng/µL. cfDNA yield had an influence on the detection of somatic alterations (p &lt;0.001). 613/1009 (60.8%) of pts had at least one somatic alteration detected on cfDNA based CGP. TP53 (53.7%), KRAS (40.3%) and CDKN2A (6.5%) were the most frequently altered genes (Table1). Homologous Recombination DNA Damage Repair (HR-DDR) gene alterations [somatic and/or germline BRCA2, ATM, and CHEK2 alterations], were detected in 12.3% (124/1009) pts – 11.4% (46/403) in KRAS mutated pts and 12.9% (78/606) in KRAS wildtype (WT) pts. Fusions were detected only in KRAS WT pts (1.5%, 6/403). Pts with cfDNA based CGP could be classified into 4 latent classes – class 1 ( KRAS - 100%, TP53 - 100% and CDKN2A – 33.3%), class 2 ( KRAS - 65.7%), class 3 ( KRAS – 68.2%, CDKN2A/B – 7.7% ) and class 4 ( KRAS – 38.5%, TP53 - 26.7%). Conclusions: The putative role of cfDNA based CGP in monitoring therapeutic response and clonal evolution in pts with PDAC may be predicated on cfDNA yield. In clinical scenarios where pre-treatment tissue availability is limited, cfDNA based CGP may be have utility in leveraging the predictive value of somatic and/or germline HR-DDR gene alterations. The prognostic and/or predictive relevance of the four latent classes needs validation in clinically annotated data sets. [Table: see text]

Panel gene profiling of small bowel adenocarcinoma: Results from the NADEGE prospective cohort.

Small bowel adenocarcinoma (SBA) is a rare tumour. Large genomic analyses with prognostic assessments are lacking. The NADEGE cohort has enrolled 347 patients with all stage SBA from 2009 to 2012. Next-generation sequencing investigates the presence of 740 hotspot somatic mutations in a panel of 46 genes involved in carcinogenesis. The mismatch repair (MMR) status was assessed by immunochemistry. We have collected 196 tumour samples and 125 had conclusive results for mutation analysis. The number of mutations was 0 in 9.6% of tumours, only 1 in 32.0%, 2 in 26.4% and ≥3 in 32.0%. Overall, at least one genomic alteration was observed in 90.4% of tumour. The most frequent genomic alteration was in KRAS (44.0%), TP53 (38.4%), PIK3CA (20.0%), APC (18.4%), SMAD4 (14.4%) and ERBB2 (7.2%) genes. KRAS mutations were more frequent in synchronous metastatic tumours than in localised tumours (72.7% vs 38.2%, P = .003). There was no significant difference in the mutation rates according to primary location for the most frequently altered gene. ATM, FGFR3 and FGFR1 gene alterations were associated with Lynch syndrome and IDH1 mutations with Crohn disease. dMMR tumours were associated with younger age, localised tumours, less KRAS but more SMARCB1 mutations. No genomic alteration was associated with overall survival. There is a trend for better survival in patient with dMMR tumours. In conclusion, there is a different genomic alteration profile in SBA according to predisposing diseases. No association between genomic alterations and prognoses was observed except for a trend of better prognoses associated with dMMR.

PTEN, ATM, IDH1 mutations and MAPK pathway activation as modulators of PFS and OS in patients treated by first line EGFR TKI, an ancillary study of the French Cooperative Thoracic Intergroup (IFCT) Biomarkers France project

ObjectivesTumor mutation screening is standard of care for patients with stage IV NSCLC. Since a couple of years, widespread NGS approaches used in routine diagnostics to detect driver mutations such as EGFR, KRAS, BRAF or MET allows the identification of other alterations that could modulated the intensity or duration of response to targeted therapies. The prevalence of co-occurring alterations that could affect response or prognosis as not been largely analyzed in clinical settings and large cohorts of patients. Thanks to the IFCT program “Biomarkers France“, a collection of samples and data at a nation-wide level was available to test the impact of co-mutations on first line EGFR TKI in patients with EGFR mutated cancers. Materials and methodsTargeted NGS was assessed on available (n = 208) samples using the Ion AmpliSeq™ Cancer Hotspot Panel v2 to screen for mutations in 50 different cancer genes. ResultsThis study showed that PTEN inactivating mutations, ATM alterations, IDH1 mutations and complex EGFR mutations were predictors of short PFS in patients with a stage 4 lung adenocarcinoma receiving first line EGFR TKI and that PTEN, ATM, IDH1 and KRAS mutations as well as alterations in the MAPK pathway were related to shorter OS. ConclusionThese findings may lead to new treatment options in patients with unfavorable genotypes to optimize first line responses.

Advanced Prostate Cancer with ATM Loss: PARP and ATR Inhibitors

BackgroundDeleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. ObjectiveTo characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. Design, setting, and participantsWe studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. Outcome measurements and statistical analysisATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. Results and limitationsOverall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. ConclusionsATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. Patient summaryOf aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR.

Clinical and Biological Impact of TP53 Alterations in Del(11q) Chronic Lymphocytic Leukemia

Large-scale next-generation sequencing (NGS) studies have suggested common patterns of co-occurrence or mutual exclusivity between genetic alterations in chronic lymphocytic leukemia (CLL). However, little is known about how most of these alterations cooperate to drive CLL pathogenesis, as well as the impact of these concurrencies in clinical outcome. In this regard, we investigated the clinical and biological impact of the co-occurrence of high-risk lesions such as del(11q)/ATM mutation and del(17p)/TP53 mutation by integrating NGS and CRISPR/Cas9 approaches. To address these questions, we first analyzed the mutational profile of 271 CLLs (17.3% del(11q); 10.7% del(17p)). The most frequently mutated genes were NOTCH1 (20%), TP53 (14%), SF3B1 (11%) and ATM (10%). Within del(11q), 32% showed TP53 alterations (53% biallelic; 47% monoallelic). Interestingly, patients harboring combined del(11q) and TP53 alterations by either mutation or deletion (del(11q) TP53ALT) exhibited significantly shorter overall survival (OS) than del(11q) CLLs without TP53 alterations (del(11q) TP53WT) and those TP53 altered without del(11q) (no del(11q) TP53ALT) (median 17 vs. 88, 36 months; P=0.0004, P=0.02). Conversely, we observed a significant lack of ATM mutations in CLLs with biallelic TP53 alterations (P=0.002) and a mutual exclusivity between biallelic TP53 and biallelic ATM losses (P=0.03)(Fig 1A). Based on the NGS results, we next used the CRISPR/Cas9 system to model monoallelic and biallelic ATM and TP53 loss in vitro. We generated isogenic HG3-Cas9 CLL-derived cell lines harboring monoallelic del(11q) (targeting 11q22.1/11q23.3 regions) and further loss-of-function mutations in ATM and/or TP53 to mimic all the possible combinations observed in our CLL cohort. By proliferation assays, we noted that the introduction of TP53 mutations increased the proliferation rates in both HG3WT and HG3-del(11q) cells. In contrast, the introduction of an ATM truncating mutation on the remaining allele of the HG3-del(11q) TP53MUT clone, suppressed this proliferative advantage, with growth rates comparable to those of HG3-del(11q). Accordingly, DNA content analysis by propidium iodide revealed that cells harboring biallelic ATM and TP53 loss also showed mitotic and cell cycle defects. To further evaluate the implications of these alterations in the clonal dynamics of CLL in vivo, we performed fluorescence-based clonal competition experiments by injecting these edited cell lines intravenously into NGS mice. First, we observed that HG3-TP53MUT cells outgrew HG3WT cells in spleen of xenotransplanted mice 14 days after injection (P&amp;lt;0.001). In a second experiment, HG3-del(11q), HG3-del(11q) TP53MUT and HG3-del(11q) ATMMUTTP53MUT cells were injected. Strikingly, HG3-del(11q) TP53MUT cells were able to outcompete HG3-del(11q) cells in spleen and bone marrow (P&amp;lt;0.001; P&amp;lt;0.001). By contrast, HG3-del(11q) ATMMUTTP53MUT cells failed to engraft neither in spleen nor bone marrow, being outcompeted by the other injected cells (P&amp;lt;0.001), providing biological insights on the mutual exclusivity of these genetic events in CLL (Fig 1B). We next assessed whether these cell models could predict responses of these combined abnormalities to BTK and PI3K inhibitors. We observed that HG3-del(11q) TP53MUT and HG3-TP53MUT cells showed partial response to ibrutinib and idelalisib, although the IC50 values were still higher than the ones observed in HG3WT clones, especially with idelalisib (27.4 and 20.6 vs. 1.8 uM, respectively). Nonetheless, we found that HG3-del(11q) TP53MUT cells were highly sensitive to novel preclinical drugs that have been shown to be effective in TP53 deficient cells such ATR inhibitors, with an IC50 value comparable to HG3WT cells (mean IC50 0.55 vs. 0.67 uM) (Fig 1C). In summary, we show that mutations in TP53 can appear in a subset of monoallelic del(11q) CLL cases, conferring a synergistic clonal advantage in vivo, and therefore a dismal clinical impact on the OS of this CLL subgroup. In addition, the biological basis of mutual exclusivity of biallelic ATM and TP53 alterations in CLL was assessed, underscoring the importance of the number of alleles affected by these alterations, and establishing novel pre-clinical models for the study of the biology and therapeutic response of concurrent genetic abnormalities in the disease. Funding: PI18/01500 FI19/00191 CD19/00222 *MQA CPC equal contr Disclosures No relevant conflicts of interest to declare.

Alterations of DNA damage response genes correlate with response and overall survival in anti-PD-1/PD-L1-treated advanced urothelial cancer.

DNA damage response (DDR) gene alterations in cancer are associated with a higher tumor mutational burden (TMB) and may impact clinical outcomes of urothelial cancer (UC). Here, we explore the prognostic role of DDR alterations in advanced UC treated with anti‐PD‐1/PD‐L1 agents. The study included 53 patients who had FoundationOne genomic sequencing and received anti‐PD‐1/PD‐L1 therapy. Fisher exact test and trend test were used to assess differences in objective response rate (ORR). Overall survival (OS) was measured from the time of initial UC diagnosis and Cox proportional hazard regression analysis was performed to calculate hazard ratio (HR) and 95% confidence interval (CI). The cohort had a median age of 66 with 64% receiving platinum‐based chemotherapy. DDR alterations (including ATM) were associated with a non‐significantly higher ORR to PD‐1/PD‐L1 blockade (41% vs. 21%, p = 0.136). Patients with DDR alterations (excluding ATM) had non‐significantly longer OS, likely due to a small sample size (HR = 0.53, 95% CI 0.20–1.38, p = 0.19). ATM alterations were associated with a non‐significantly higher ORR (40% vs. 29%, p = 0.6), but also with significantly shorter OS (HR = 5.7, 95% CI 1.65–19.74, p = 0.006). Patients with ≥ 3 DDR alterations (including ATM) had substantially higher TMB (p = 0.01) and higher ORR (80%) with PD‐1/PD‐L1 blockade versus 24% ORR in patients with <3 DDR alterations. In summary, DDR alterations were associated with non‐significantly higher ORR and longer OS for patients with advanced UC receiving anti‐PD‐1/PD‐L1 agents. ATM alterations were associated with shorter OS.

Genomic and epigenomic insights into the origin, pathogenesis, and clinical behavior of mantle cell lymphoma subtypes

AbstractMantle cell lymphoma (MCL) is a mature B-cell neoplasm initially driven by CCND1 rearrangement with 2 molecular subtypes, conventional MCL (cMCL) and leukemic non-nodal MCL (nnMCL), that differ in their clinicobiological behavior. To identify the genetic and epigenetic alterations determining this diversity, we used whole-genome (n = 61) and exome (n = 21) sequencing (74% cMCL, 26% nnMCL) combined with transcriptome and DNA methylation profiles in the context of 5 MCL reference epigenomes. We identified that open and active chromatin at the major translocation cluster locus might facilitate the t(11;14)(q13;32), which modifies the 3-dimensional structure of the involved regions. This translocation is mainly acquired in precursor B cells mediated by recombination-activating genes in both MCL subtypes, whereas in 8% of cases the translocation occurs in mature B cells mediated by activation-induced cytidine deaminase. We identified novel recurrent MCL drivers, including CDKN1B, SAMHD1, BCOR, SYNE1, HNRNPH1, SMARCB1, and DAZAP1. Complex structural alterations emerge as a relevant early oncogenic mechanism in MCL, targeting key driver genes. Breakage-fusion-bridge cycles and translocations activated oncogenes (BMI1, MIR17HG, TERT, MYC, and MYCN), generating gene amplifications and remodeling regulatory regions. cMCL carried significant higher numbers of structural variants, copy number alterations, and driver changes than nnMCL, with exclusive alterations of ATM in cMCL, whereas TP53 and TERT alterations were slightly enriched in nnMCL. Several drivers had prognostic impact, but only TP53 and MYC aberrations added value independently of genomic complexity. An increasing genomic complexity, together with the presence of breakage-fusion-bridge cycles and high DNA methylation changes related to the proliferative cell history, defines patients with different clinical evolution.

Exploiting defects in homologous recombination repair for metastatic, castration-resistant prostate cancer

ABSTRACT The PROfound trial highlights that there is a benefit in testing genes involved in homologous recombination (HR) and forms the rationale for testing in all patients with metastatic, castration-resistant prostate cancer (mCRPC). This trial also demostrates that olaparib improves progression free survival (PFS), objective response rate (ORR), and time to pain progression in patients who harbor alterations in BRCA1, BRCA2, and ATM. These are groundbreaking findings – this is the first trial that demonstrates the efficacy of olaparib versus standard therapy in a genomically-selected patient population with metastatic prostate cancer. Although this trial does not demonstrate improvements in overall survival (OS), we believe that this may be an underestimation based on trial-design. Future studies of olaparib are likely to yield further promising results.

Abstract 3536: Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss

Abstract Background: ATM, a protein kinase involved in double-strand DNA break repair, is mutated in a significant fraction of prostate cancers, and targeted therapies such as ATR inhibitors appear promising for ATM-deficient tumors. However, few ATM-deficient primary prostate cancers have been clinically and genomically characterized. Design: We validated an automated and dichotomously-scored clinical-grade immunohistochemistry (IHC) assay to detect ATM protein loss using prostate cancer cell lines and 52 very high-grade prostate tumors (Gleason score 5+4=9 or 5+5=10) with known ATM genomic status. We examined the frequency of ATM protein loss among 29 tumors with known pathogenic germline ATM mutations or variants of unknown significance (VUS). Finally, we screened more than 800 additional primary prostate carcinomas using tissue microarrays (TMA) for ATM protein loss. Targeted somatic genomic sequencing using HRD Plus (Myriad Genetics) or UW-OncoPlex was conducted in a subset of immunostained tumors in each cohort. Results: ATM protein loss by IHC was found in 13% (7/52) of very high-grade (primary Gleason pattern 5) tumors with somatic genomic sequencing data. Of 7 cases with ATM protein loss, 4 had a deleterious ATM mutation and 3 had apparent single copy ATM genomic loss. Of the remaining 45 cases without ATM protein loss, none had ATM genomic alterations. In the cohort of tumors with pathogenic germline ATM mutations, 74% (17/23) had ATM protein loss, but only 45% (9/20) with evaluable sequencing had a second pathogenic alteration in ATM detected (all of these had protein loss). Of those with germline mutations and ATM protein loss, the majority (76% or 13/17) had an apparent clonal alteration with homogeneous protein loss in all tumor cells sampled. In contrast, among 6 cases with germline ATM VUS, only one had protein loss. On TMA analysis, ATM loss was present in 3% (25/824) of evaluable tumors overall, and was significantly more common in tumors from Grade Group 5 (12/130; 9.2%) compared to all other Grade Groups (13/694; 1.9%) (P&amp;lt;0.0001). Of tumors found by TMA screening with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 58% (7/12) with heterogeneous ATM protein loss had detectable pathogenic alterations in ATM. Of these, 82% (9/11) were inferred to have somatic ATM variants. Conclusions: Validated ATM IHC is a sensitive assay for detecting underlying ATM alterations and may be useful to evaluate VUS in ATM. However, not all tumors with ATM protein loss have detectable genomic alterations. ATM protein loss is likely an early clonal event in tumors with germline mutations and is significantly enriched in high-grade prostate cancers. Citation Format: Harsimar Kaur, Jessica Hicks, Colin Pritchard, Angelo De Marzo, William Isaacs, Jerry Lanchbury, Kirsten Timms, Emmanuel Antonarakis, Tamara L. Lotan. Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss [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 3536.

Abstract 3541: Biallelic alterations in BRCA2, ATM, or CDK12 in metastatic prostate cancer via circulating tumor DNA

Abstract Background: Genomic instability is a hallmark of metastatic prostate cancer. Defects in homologous recombination repair genes such BRCA2 are associated with response to platinum-based chemotherapy and poly ADP ribose polymerase (PARP) inhibitors. However, monoallelic DNA damage repair (DDR) mutations are unlikely to result in therapeutic vulnerability. Here, we used clinically-practical liquid biopsies to distinguish between monoallelic and biallelic DDR defects. Our secondary objective was to characterize the downstream genomic alterations enabled by each class of underlying DDR defect. Methods: We performed targeted sequencing of 1615 cell-free DNA samples from 882 patients with mPCa using a custom prostate cancer panel. Samples with deleterious BRCA2 mutation(s) were subjected to deep sequencing of tumor suppressor introns to identify structural rearrangements. Hypermutated mismatch-repair defective tumors were excluded from analysis. Results: 101 patients had at least one deleterious germline or somatic mutation in one of 19 assessed DDR genes in the targeted panel. BRCA2, ATM, and CDK12 were most commonly affected, collectively mutated in 16.0% of patients with sufficient circulating tumor DNA (ctDNA) content to be assessed for both germline and somatic mutations (n = 630). In 345 patients with &amp;gt;20% ctDNA content, eight somatic biallellic BRCA2 deletions were identified. In patients with &amp;gt;1 deleterious mutation in BRCA2, ATM, and CDK12, disruption of the second allele (via mutation, deletion, or copy-neutral loss of heterozygosity) was detected in 97%, 85%, and 91% of cases, respectively. Patients with a mutation in these genes had fewer TP53 mutations than DDR-intact controls (p&amp;lt;0.05). However, BRCA2-defective patients instead harbored frequent compound deletions and/or rearrangements that disrupted tumor-suppressor genes (e.g. TP53, PTEN, RB1). In patients with CDK12 mutations, tumor-suppressor loss was rare but copy number gains of MDM2, CCND1, CDK4/6 were common. ATM-loss was not associated with evidence of genomic instability. Conclusions: In metastatic prostate cancer, deleterious mutations in BRCA2, ATM or CDK12, as detected in ctDNA, are frequently accompanied by alteration to the second allele. This means that detection of one deleterious mutation in these genes may be a surrogate for biallelic loss. In BRCA2 and CDK12-mutant genomes, tumor suppressor genes can be disrupted via structural rearrangements. ATM-mutant genomes show scant evidence for TP53 disruption. Citation Format: Evan Warner, Steven Yip, Matti Annala, Andrew Murtha, Cameron Herberts, Kim Chi, Alexander Wyatt. Biallelic alterations in BRCA2, ATM, or CDK12 in metastatic prostate cancer via circulating tumor DNA [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 3541.