DNA Damage Repair Pathway Genes Research Articles

Impact of DNA damage repair alterations on real-world response to therapy in metastatic colorectal cancer.

e15557 Background: Alterations in the DNA damage repair (DDR) pathway genes contribute to tumor development. These alterations can be exploited by cytotoxic chemotherapy regimens that induce DNA damage. Platinum chemotherapy agents have been shown to be particularly effective in breast and ovarian cancer patients with alterations in the DDR pathway. However, the clinical impact of DDR alterations in metastatic colorectal cancer (mCRC) is still poorly understood. In this study, we utilize a large national database to evaluate clinical outcomes in patients with mCRC and alterations in DDR pathway genes. Methods: We reviewed 5602 patients that received standard fluorouracil based first-line chemotherapy regimens for mCRC using the nationwide Flatiron Health EHR-derived de-identified database. Chemotherapy regimens were categorized as a platinum-based doublet (FOLFOX, CAPOX), irinotecan-based doublets (FOLFIRI), or triplet (FOLFOXIRI). Overall survival (OS) was summarized within groups via Kaplan-Meier survival estimates. OS was compared between groups in the context of univariable and multivariable Cox proportional hazards models controlling for age, gender, DDR status, stage at diagnosis, ECOG, RAS/RAF status, MMR status, albumin and CEA at diagnosis. OS was defined as the time in months between date of first line therapy initiation to death. Results: DDR gene alterations were indentified in 35.7% (2001) patients.The most frequent mutations were in ATM (13.3%) and BRCA2 (8.3%). There was no significant difference in OS based on DDR mutation status. Within DDR altered patients, there was no difference in OS between those who received a platinum-based doublet, irinotecan-based doublet or triplet first-line therapy. Time to next treatment or death (TTNTD) was significantly improved with an irinotecan-containing doublet regimen and triplet regimen compared to a platinum-based doublet in a multivariable model (HR 0.79, p = 0.001 and HR 0.68, p = 0.002, respectively). There was no difference in OS in DDR-mutated patients that received an irinotecan-based regimen in the first line followed by an oxaliplatin-based regimen in the second line or the reverse sequence. When evaluating the impact of commonly co-mutated genes on survival we observed a worse OS in patients with a KRAS mutation in addition to a DDR mutation (HR 1.19, p = 0.045). An APC mutation was associated with an improved OS in the entire cohort, regardless of DDR status (HR 0.76, p < 0.001). Conclusions: In this real-world population, approximately one third of patients with mCRC harbored an alteration in a DDR pathway gene. In these patients, TTNTD was greater with irinotecan-containing first line therapies (doublet or triplet). However, OS was similar regardless of which first-line chemotherapy was used.

Characterization of DNA damage repair (DDR) alterations and the tumor immune microenvironment (TIME) in advanced non-small cell lung cancer (NSCLC).

8603 Background: Exploiting the DDR pathway with synthetic lethality agents such as PARP inhibitors has been clinically validated in several cancers. In NSCLC, novel agents leveraging defects in the DDR pathway are being studied alone and in combination with checkpoint inhibitors. To expand on combination approaches and inform biomarker selected strategies, we analyzed DDR alteration prevalence and impact on the TIME in metastatic NSCLC. Methods: We retrospectively analyzed de-identified next-generation sequencing data from patients (pts) with metastatic NSCLC in the Tempus Database. Tumors were sequenced with the Tempus xT DNA and xR RNA assays. DDR pathway genes were defined as previously described (PMID 29617664). Pts were stratified based on the presence (DDRmt) or absence (DDRwt) of a pathogenic somatic alteration or copy number deletion in a DDR pathway gene. The associations of DDR alteration with immune cell infiltration predicted from gene expression patterns, PD-L1 from IHC, TMB (TMB-H, >10 mut/Mb), and microsatellite instability (MSI) were examined. Wilcoxon rank-sum and Pearson’s Chi-squared/Fisher's exact tests were used to assess statistical significance (p<0.05, q<0.05 for false discovery rate correction for multiple testing). Results: Across 14,127 samples, 5,255 (37%) were DDRmt and 8,872 (63%) were DDRwt. The median age at diagnosis was 67 (IQR 60-74). Current/former smokers (83%; n=10,445) had a higher prevalence of DDRmt (86% vs. 82%; p<0.001). The most prevalent DDRmt was SMARCA4 (18%), followed by PTEN (13%), BRCA2 (11%), ATM (9.0%), ERCC3 (6.6%), PMS2 (6.0%), and MUTYH (5.1%). TMB-H was more common (18% [n=931] vs. 11% [n=1001]; p<0.001) and median TMB was higher (5.4 vs. 4.6; p<0.001) in DDRmt pts compared to DDRwt. MSI-H was more prevalent in the DDRmt group (1.1% [n=57] vs. <0.1% [n=7]; p<0.001), while PD-L1 positivity was similar in DDRmt and DDRwt pts (55% [n=1918] vs. 58% [n=3314]; p=0.071). Compared to DDRwt, pts with DDRmt exhibited modest changes in immune cell infiltration patterns (Table). Conclusions: In this large, real-world cohort of pts with metastatic NSCLC, DDRmt was associated with distinct TIME patterns including high TMB, MSI-H, and modest changes in immune cell infiltrates. These findings warrant further studies to understand how DDR pathway alterations mediate changes in the TIME to inform novel combinatorial immunotherapeutic approaches in NSCLC.[Table: see text]

Association Between Polymorphisms in DNA Damage Repair Pathway Genes and Female Breast Cancer Risk.

Breast cancer risk have been discussed to be associated with polymorphisms in genes as well as abnormal DNA damage repair function. This study aims to assess the relationship between genes single nucleotide polymorphisms (SNPs) related to DNA damage repair and female breast cancer risk in Chinese population. A case-control study containing 400 patients and 400 healthy controls was conducted. Genotype was identified using the sequence MassARRAY method and expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) in tumor tissues was analyzed by immunohistochemistry assay. The results revealed that ATR rs13091637 decreased breast cancer risk influenced by ER, PR (CT/TT vs. CC: adjusted odds ratio [OR] = 1.54, 95% confidence interval [CI]: 1.04-2.27, p = 0.032; CT/TT vs. CC: adjusted OR = 1.63, 95%CI: 1.14-2.35, p = 0.008) expression. Stratified analysis revealed that PALB2 rs16940342 increased breast cancer risk in response to menstrual status (AG/GG vs. AA: adjusted OR = 1.72, 95%CI: 1.13-2.62, p = 0.011) and age of menarche (AG/GG vs. AA: adjusted OR = 1.54, 95%CI: 1.03-2.31, p = 0.037), whereas ATM rs611646 and Ku70 rs132793 were associated with reduced breast cancer risk influenced by menarche (GA/AA vs. GG: adjusted OR = 0.50, 95%CI: 0.30-0.95, p = 0.033). In a summary, PALB2 rs16940342, ATR rs13091637, ATM rs611646, and Ku70 rs132793 were associated with breast cancer risk.

Correlations between pathogenic variants in DNA repair genes and anticancer treatment efficacy in stage IV non-small cell lung cancer: A large real-world cohort and review of the literature.

Mutations in genes involved in DNA damage repair (DDR), a hallmark of cancer, are associated with increased cancer cell sensitivity to certain therapies. This study sought to evaluate the association of DDR pathogenic variants with treatment efficacy in patients with advanced non-small cell lung cancer (NSCLC). A retrospective cohort of consecutive patients with advanced NSCLC attending a tertiary medical center who underwent next-generation sequencing in 01/2015-8/2020 were clustered according to DDR gene status and compared for overall response rate (ORR), progression-free survival (PFS) (patients receiving systemic therapy), local PFS (patients receiving definitive radiotherapy), and overall survival (OS) using log-rank and Cox regression analyses. Of 225 patients with a clear tumor status, 42 had a pathogenic/likely pathogenic DDR variant (pDDR), and 183 had no DDR variant (wtDDR). Overall survival was similar in the two groups (24.2 vs. 23.1 months, p = 0.63). The pDDR group had a higher median local PFS after radiotherapy (median 45 months vs. 9.9 months, respectively; p = 0.044), a higher ORR (88.9% vs. 36.2%, p = 0.04), and a longer median PFS (not reached vs. 6.0 months, p = 0.01) in patients treated with immune checkpoint blockade. There was no difference in ORR, median PFS, and median OS in patients treated with platinum-based chemotherapy. Our retrospective data suggest that in patients with stage 4 NSCLC, pathogenic variants in DDR pathway genes may be associated with higher efficacy of radiotherapy and immune checkpoint inhibitors (ICIs). This should be further explored prospectively.

MCL-319 LP-284 - A Highly Potent Small Molecule Targeting Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL). Approximately 40%-50% of MCL patients carry inactivating mutations in the gene ataxia telangiectasia mutated (ATM), a core component of the cell's DNA damage response system. MCL cells with mutations in ATM are known to have increased chromosomal imbalance, copy number loss, and hypersensitivity to DNA-damaging agents. LP-184, a small-molecule DNA-damaging agent, is known to be synthetically lethal in tumors with DNA-repair deficiencies, including tumors with ATM mutations. However, DNA damage by LP-184 is dependent on activation by the oxidoreductase prostaglandin reductase 1 (PTGR1), which is expressed at low levels in many hematological cancers. We synthesized LP-284, an enantiomer of LP-184, which retains synthetic lethality in cancers with impaired DNA damage repair pathway genes (e.g., ERCC1, ERCC2, ERCC3, ERCC6), but LP-284's activity is independent of PTGR1 expression. We demonstrated that LP-284 has enhanced tumoricidal activity in multiple hematological cancer cell lines using a B-cell NHL panel. The IC50 values for all 6 MCL cell lines treated with LP-284 were among the lowest for all hematological cell lines tested with LP-284. These MCL cell lines included cell lines resistant to ibrutinib, zanubrutinib, venetoclax, and bortezomib. Additionally, among the 6 MCL cell lines tested, the 3 cell lines with mutations in the nucleotide excision repair (NER) or homologous recombination (HR) DNA damage repair pathways had lower LP-284 IC50 values than the 3 cell lines without NER or HR mutations. Because ATM orchestrates the activities of the NER and HR pathways, MCL patients with ATM deficiencies are likely to have better responses to LP-284 treatment. Collectively, these data indicate that LP-284 is a promising preclinical DNA-damaging agent that possesses nanomolar-range anti-tumor activities in multiple and diverse MCL cell lines, with the potential to treat MCL patients who are resistant to other therapies.

Characterization of prostate cancer adrenal metastases: dependence upon androgen receptor signaling and steroid hormones

Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression. Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo. Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice. PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases. This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).

Pan-Cancer analysis of the mutation frequency of genes in DDR pathway in Chinese population.

e15109 Background: The DNA damage repair (DDR) pathway is of vital importance to maintain human genomic stability. And the variation of DDR pathway related genes is closely related to tumorigenesis, tumor progression and therapeutic response. In order to comprehensively study the mutation status of DDR pathway related genes in human cancers, we evaluated the genomic features of Chinese pan-cancer patients and investigated the mutation frequency of genes in DDR pathway. Methods: A total of 5018 patients consisting of more than 22 cancer types were enrolled in this study. Utilizing target next-generation sequencing (NGS) platform, genomic profiling of 599 cancer-related genes was performed on tumor tissue or blood samples. And many DDR pathway related genes are included and detected mutations such as TP53, BRCA2, BRCA1, ATM, BLM, FANCA, POLE and so on. Results: We observed 91.23% (4578/5018) of cases harbored at least one alteration of the DDR pathway genes. The mutation frequency ranges from 71.88% to 100.00% among the 22 cancer types. Among them the DDR gene mutation frequency of lymphoma, esophagus and esophagogastric junction cancer and endometrial cancer are all 100%, and mutation frequency of bowel cancer, hepatobiliary cancer, melanoma, breast cancer, esophageal cancer, gastric cancer and central nervous system cancer are also more than 90% respectively. The highest mutation rate gene is a well-known tumor suppressor gene TP53, which mutated in 2650 patients and the mutation rate is 52.81%. And it primarily mutated in esophageal cancer, with a mutation rate of 81.43%. Besides the mutation rate of TP53 in gastric cancer, bowel cancer, breast cancer, ovarian cancer, pancreatic cancer, bladder cancer and hepatobiliary cancer are all more than 50%. Followed by is BRCA2 gene, which is also a tumor suppressor gene and the mutation rate is 16.04%. Next is ATM, BLM and FANCA, the mutation rate of which is 11.68%, 11.12% and 8.97% respectively. Conclusions: We find a unique genomic feature of DDR pathway in the comprehensively analysis of the mutation profile of the Chinese pan-cancer patients, which may provide potential therapeutic targets in the future.

Abstract PD3-02: Linking proliferative signal to DNA-damage signal in a tumor cell: A contextual synergy between the PI3K pathway and the DDR pathway in TNBC

Abstract The phosphoinositide 3-kinase (PI3K) pathway signals not only for cell survival and proliferation in tumor cells but also controls DNA damage repair (DDR) and maintains HR (homologous recombination) steady-state the translational significance of which has been reported [Juvekar A. et al., 2012]. In breast cancers (BC), alterations of the PI3K pathway genes are both subtype-specific as well as contextual, and alterations of DDR pathway genes are one such contextual event occurring with the upregulation of the PI3K pathway. We hypothesized that there is contextual cooperation between the cellular signals of the PI3K and DDR pathways. We sought to identify the co-alterations of the PI3K and DDR pathway genes in our triple-negative BC (TNBC) patient cohort to (A) interrogate the contextuality of such alterations and (B) understand the mechanism of the contextual cooperation using an algorithmically chosen combination of pathway targeted drugs. We analyzed the somatic alterations profile of genes (FoundationOne, USA) in 264 BC patients (Avera Cancer Institute, SD, USA). We experimentally validated the cooperation between the pathways using alteration-guided targeted inhibitors; isoform-specific PI3K and PARP inhibitor(s) in the TNBC model, to demonstrate the efficacy of a combination of a node-specific targeted inhibitor(s) of the two pathways. The predominant type of mutation of genes in the PI3K pathway in our patients with TNBC was found in PTEN (Y68C, Y180*, loss, loss exons 1-5, and deletion exon1) and PIK3CA (amplification, E545K, H1047R). The other common mutation found was in TP53 (>80%) and somatic BRCA1/2 (~15%) genes. The interaction between the two pathways was evaluated, applying STRING10 to test the association at the highest 0.900 confidence views. The highest level of efficiency of drugs is demonstrated following the treatment of p110beta inhibitor (AZD 6482) in combination with PARP inhibitor (Olaparib/Talazoparib) plus DNA damaging agent (carboplatin) in BRCA-incompetent PTEN-null TNBC cells (SUM149) as compared to the efficiency of a combination of a PI3K pathway targeted inhibitor (of p110alpha) with a PARP inhibitor in BRCA-competent PTEN WT, but PIK3CA altered TNBC cells (BT20). The context-dependent synergy between the PI3K and DDR pathways in the TNBC model is comprehended at the levels of (1) load of DNA damage, (2) PARP activity, (3) of BRAC1/2 competency or HRD scores, and (4) mode of upregulation of PI3K pathway. We present an algorithm for a rational combination of the PI3K and the DDR pathway targeted drugs in TNBC. Citation Format: Nandini Dey, Jennifer Carlson Aske, Pradip De. Linking proliferative signal to DNA-damage signal in a tumor cell: A contextual synergy between the PI3K pathway and the DDR pathway in TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-02.

Genetic Analysis Reveals the Prognostic Significance of the DNA Mismatch Repair Gene MSH2 in Advanced Prostate Cancer.

DNA damage repair is frequently dysregulated in advanced prostate cancer and has been linked to cancer susceptibility and survival outcomes. The aim of this study is to assess the influence of genetic variants in DNA damage repair pathways on the prognosis of prostate cancer. Specifically, 167 single nucleotide polymorphisms (SNPs) in 18 DNA damage repair pathway genes were assessed for association with cancer-specific survival (CSS), overall survival (OS), and progression-free survival (PFS) in a cohort of 630 patients with advanced prostate cancer receiving androgen deprivation therapy. Univariate analysis identified four SNPs associated with CSS, four with OS, and two with PFS. However, only MSH2 rs1400633 C > G showed a significant association upon multivariate analysis and multiple testing adjustments (hazard ratio = 0.75, 95% confidence interval = 0.63-0.90, p = 0.002). Furthermore, rs1400633 risk allele C increased MSH2 expression in the prostate and other tissues, which correlated with more aggressive prostate cancer characteristics. A meta-analysis of 31 gene expression datasets revealed significantly higher MSH2 expression in prostate cancer than in normal tissues (p < 0.001), and this high expression was associated with a poor prognosis of prostate cancer (p = 0.002). In summary, we identified MSH2 rs1400633 as an independent prognostic biomarker for prostate cancer survival, and the association of MSH2 with cancer progression lends relevance to our findings.

Comprehensive Next-Generation Sequencing Reveals Novel Predictive Biomarkers of Recurrence and Thoracic Toxicity Risks After Chemoradiation Therapy in Limited Stage Small Cell Lung Cancer

Although definitive chemoradiation therapy (dCRT) remains the most effective treatment modality in limited stage small cell lung cancer (SCLC), some patients progress quickly or develop serious radiation-induced thoracic toxicity (RITT). Molecular correlates of response to dCRT remain to be explored.Genomic profiling was performed retrospectively on 231 patients with limited-stage SCLC treated with dCRT between 2015 and 2019 using a customized panel covering cancer and radiation therapy response-related genes. Exploratory associations of progression-free survival, overall survival, and RITT with clinical features, tumor genetics, genomic and molecular pathway alterations, and single nucleotide polymorphisms were conducted.In addition to the common SCLC genes, such as TP53, RB1, and NOTCH1/2, potentially actionable mutations in EGFR, KRAS, and BRCA1/2 were among the top alterations in the cohort. At the single-gene level, CDK4 and GATA6 alterations were independent predictors of poor survival by multivariate analysis. At the genomic level, high tumor mutational burden was strongly associated with favorable survival outcome. Pathway-level analysis showed that activating mutations in the MAPK/ERK pathway genes, particularly those in EGFR/ERBB2, correlated with poor survival. Combined analysis enabled optimized risk stratification of post-dCRT survival. On the other hand, our study also confirmed that single nucleotide polymorphisms in MTHFR, CYP2B6, NQO1, and LIG4 were risk alleles of high-grade RITT. Remarkably, somatic loss-of-function mutations in the DNA damage repair pathway genes were associated with increased risk of high-grade RITT, particularly pneumonitis, which likely reflect a complex interplay between the tumor and its immune microenvironment.Taken together, by examining the mutational landscape of a large cohort of limited-stage SCLC, we identified novel molecular predictors of survival and RITT. Our findings also implicate several key molecular pathways, including the MAPK/ERK and DNA damage repair pathways, in the regulation of dCRT response.

Abstract 2206: Investigation of DNA damage repair mutated characteristics and correlation with immunogenic biomarker in Chinese head and neck cancer patients

Abstract Background: DNA damage repair (DDR) pathway have been reported in several cancer, and known as a predictive biomarker of platinum-based chemotherapy, PARP inhibitor therapy, radiotherapy, and immunotherapy. However, the predictive value and mutated characteristics of DDR in patients with head and neck cancer (HNC) is still uncertain. The purpose of this study is to assess the frequency of DDR mutations, and the correlation between DDR mutations and immunogenic biomarker such as tumor mutation burden (TMB), microsatellite instability (MSI) and PD-L1 expression in Chinese HNC patients. Methods: Genomic profiling of DNA was performed through a next-generation sequencing (NGS) (panel on 381/733-gene) on tissue from Chinese patients with head and neck cancer between January 23, 2017 and April 10 2020. TMB was defined as total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was determined by immunohistochemistry staining using Dako 22C3. Germline or somatic mutations of 35 DDR genes were classified as DDR gene mutations. Result: Genetic mutation of 319 HNC patients were analyzed using NGS, of which 32 (10.03%) harbored alterations in DDR genes. The most frequently mutated DDR genes were BRCA2 (3.23%), followed by FANCA (2.02%), BRCA1 (1.61%), RAD51 (0.81%), RAD50 (0.81%), PMS2 (0.81%), and PLAB2 (0.81%). The frequency of KMT2C, CD274, FBXW7, APC, NOTCH1, PDCD1LG2, RB1, LRP1B, and JAK2 mutations was higher in DDR mutations (DDRmut) than in DDR wild-type (DDRwt) patients. PD-L1 status analysis was performed in 265 HNC patients, including 24 DDRmut patients and 241 DDRwt patients. But there was no significant difference on PD-L1 expression between DDRmut t and DDRwt (P=0.93). 310 HNC patients (including 31 DDRmut patients and 279 DDRwt patients) could be evaluated for TMB, the results showed that DDRmut (10.13Muts/Mb) was associated with higher TMB than DDRwt (5.65Muts/Mb) (P = 0.0031). Of the 308 HNC patients (including 31 DDRmut patients and 277 DDRwt), only 3 (0.97%) were diagnosed as MSI-H, including 2 (6.45%) patients with DDRmut and 1 (0.36%) patient with DDRwt. Conclusions: The DDR pathway genes alteration characteristics was presented in Chinese HNC patients, and was associated with a higher TMB level. These findings provide a theoretical basis for future investigation of clinical therapy in DDR mutated Chinese HNC patients. Citation Format: Yong Chen, Yan Wang, Yating Zheng, Wenzhuan Xie, Mengli Huang. Investigation of DNA damage repair mutated characteristics and correlation with immunogenic biomarker in Chinese head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2206.

Analysis of DNA damage repair pathway genes as a predictive biomarker for HAIC in hepatocellular carcinoma.

e16159 Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide.Hepatic arterial infusion chemotherapy (HAIC) with oxaliplatin, fluorouracil, and leucovorin (FOLFOX) is one of the effective treatment for advanced HCC patients. DNA damage repaired (DDR) pathway has been widely researched in many cancers recent years, which was a promising predictive biomarker for the response to platinum-based chemotherapy and Immunotherapy. But the DDR characteristics of different subset pathways in HCC, and its correlation with HAIC efficacy were unknown. Methods: Whole exome sequencing (WES) data of 366 HCC patients was obtained from the Cancer Genome Altas (TCGA). Next generation sequencing (NGS, panel on 381/733-gene) was performed on FFPE tumor samples from 2092 Chinese HCC patients (Chinese cohort). Germ-line or somatic mutations of 32 DDR pathway genes (including Mismatch Repair and Homologous Recombination) were classified as DDR gene mutations. TMB was defined as total number of somatic non-synonymous mutations in coding region. Whole exome sequencing (WES) data and clinical data of 21 HCC advanced patients treated with HAIC were obtained to survival analysis. Results: In total, 59.84% (219/366) of HCC patients in TCGA harboring DDR mutation and 67.38% (1316/2092) in Chinese cohort. In Chinese HCC cohort, the mean TMB level of DDR mutant group was significant higher than wild-type group (mean TMB, mutation vs wild-type = 8.12 vs 6.42 Muts/Mb, P &lt; 0.0001). The top three mutation frequency DDR genes were BRCA2 (17%), ATM (13%) and BARD1 (12%), respectively. The highest mutation frequency DDR subset pathways were Fanconi anemia (FA, 44.14%, 862/2092), homologous recombination repair (HRR, 38.71%, 756/2092) and check point factors (CHP, 17.31%, 338/2092) come in second and third, respectively. Next we compared the TMB level between different DDR subset pathways, the mutant of nucleotide excision repair (NER) pathway harbored the highest medium TMB level (7.26 Muts/Mb). The survival analysis was performed on HCC patients treated with HAIC with FOLFOX. There was no difference in clinical baseline information between DDR mutation group (n = 6) and wild-type group (n = 15). The progression-free survival (PFS) of DDR mutation group were significantly longer than wild-type group (median PFS, mutation vs. wild-type = 8.9 vs. 4.5 months; HR 0.34[95% CI 0.13-0.85]; P = 0.0349), and an extending trend on overall survival (OS) without significant difference (median OS, 15.7 vs 8.9 months; HR 0.37[95% CI 0.13-1.00]; P = 0.0921). Conclusions: The DDR pathways was associated with higher TMB level. Preliminary data from clinical cohorts suggested better treatment outcomes of HAIC with FOLFOX in DDR mutation HCC patients.

Molecular Profiling of Exceptional Responders to Cancer Therapy

The vast majority of metastatic cancers cannot be cured. Palliative treatment may relieve disease symptoms by stopping or slowing cancer growth and may prolong patients' lives, but almost all patients will inevitably develop disease progression after initial response. However, for reasons that are not fully understood, a very few patients will have extraordinary durable responses to standard anticancer treatments. We analyzed exceptional responders treated at Fox Chase Cancer Center between September 2009 and November 2017. An exceptional response was defined as a complete response lasting more than 1 year or a partial response or stable disease for more than 2 years. Tumor samples were analyzed using an Ambry Genetics test kit with a 142-gene panel. Messenger RNA expression was evaluated using NanoString's nCounter PanCancer Pathways Panel and Immune Profiling Panel and compared with matched controls for gender, age, and cancer type. Twenty-six exceptional responders with metastatic bladder, kidney, breast, lung, ovarian, uterine, and colon cancers were enrolled. Mutations were identified in 45 genes. The most common mutation was an EPHA5 nonsynonymous mutation detected in 87.5% of patients. Mutations in DNA damage repair pathway genes were also frequent, suggesting increased genome instability. We also found varying expression of 73 genes in the Pathways panel and 85 genes in the Immune Profiling panel, many of them responsible for improvement in tumor recognition and antitumor immune response. The genomic instability detected in our exceptional responders, plus treatment with DNA damage compounds combined with favorable anticancer immunity, may have contributed to exceptional responses to standard anticancer therapies in the patients studied. With recent advances in the treatment of cancer, there is increased emphasis on the importance of identifying molecular markers to predict treatment outcomes, thereby allowing precision oncology. In this study, it was hypothesized that there is a "specific biologic signature" in the biology of the cancer in long-term survivors that allows sensitivity to systemic therapy and durability of response. Results showed that DNA damage repair pathway alterations, combined with favorable anticancer immunity, may have contributed to exceptional responses. It is very likely that an in-depth examination of outlier responses will become a standard component of drug development in the future.

Enhanced response of radioresistant carcinoma cell line to heterogeneous dose distribution of grid; the role of high-dose bystander effect

Purpose The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect. Materials and methods Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using ‘medium transfer’ and ‘cell-to-cell contact’ strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed. Results Various parameters (ɑ/β, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes (Xrcc6 and H2afx) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed. Conclusions Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.

Germline Contributions to Clonal Hematopoiesis in Solid Cancer Patients

Clonal hematopoiesis (CH), defined by expansion of clones in the hematopoietic system, has been linked to internal (aging) and external (smoking and oncologic therapy) factors in patients with advanced solid tumors. The effects of inherited variants, however, have yielded inconclusive results in few studies so far. While comparison of CH prevalence between monozygotic and dizygotic pairs failed to detect genetic predisposition to CH, others have shown associations with germline variants near TERT and TET2, and a potential link between pathogenic CHEK2 germline variants and CH has been proposed. Here, utilizing prospective tumor-blood paired sequencing data from a large institutional clinical cohort, we link inherited pathogenic variants in cancer predisposition genes to CH and show interactions between germline and somatic mutations in DNA damage repair (DDR) genes. We analyzed sequencing data from 32,748 cancer patients who have undergone MSK-IMPACT testing using paired tumor and blood samples to study the associations between CH and rare pathogenic and likely-pathogenic (P/LP) germline variants. P/LP germline mutations were identified in the blood using an in-house developed random forest algorithm, trained using known P/LP mutations. Following joint anonymization of P/LP germline mutations and CH variants, linear regression models by gene were used to test for associations, accounting for known CH confounders. In our cohort, 9,697 patients (29.6%) harbored at least one CH mutation, while P/LP germline variants were detected in 1,793 of these CH patients. P/LP germline variants in CHEK2 (OR=1.29, p=4.49x10-2) were found to be significantly associated with CH in a pan-cancer analysis. Interestingly, germline P/LP and CH co-mutations in CHEK2 were very rare; we identified only one patient with CH and germline mutations in CHEK2 out of 161 patients with germline CHEK2 mutations, compared to 304 patients with CH CHEK2 mutation when CHEK2 is wild-type in the germline. In contrast, ATM and TP53 showed increased number of patients with co-mutations in P/LP germline and CH mutations in the same gene, with 14 out of 84 patients with germline mutations in ATM compared to 254 when not mutated (OR=7.05, p=2.08x10-7) , and 3 out of 16 compared to 387 for TP53 (OR=10.94, p=5.44x10-3). Further, we found mutual exclusivity between CHEK2 P/LP germline variants and CH mutations in PPM1D (OR=0.18, p=1.79x10-2). This relationship was replicated by comparing CHEK2 germline mutations and somatic PPM1D mutations in the solid tumor samples within the same cohort and same pattern of mutual exclusivity also persisted in comparisons of other DDR pathway genes (i.e. ATM vs PPM1D and TP53 vs PPM1D). When studying specific cancer types, a significant association between CHEK2 P/LP germline variants and CH in breast cancer patients was also identified (OR=2.33, p=2.64x10-3). Furthermore, trends between germline mutations in ATM and CH rates in breast cancer (OR=1.84, p=9.82x10-2) and lung adenocarcinoma (OR=2.22, p=8.91x10-2) patients were observed. Our results support the already reported link between rare pathogenic germline mutations in CHEK2 with CH in cancer patients, with stronger association among breast cancer patients (Comen E. et al, 2019). Moreover, this study suggests a close relationship between inherited variants and CH mutations within the DDR genes in solid tumor patients and points out to interesting mutual exclusivity patterns between these same genes. Intriguingly, hematopoietic stem cells have been largely related to proficient DDR systems in order to regulate HSC maintenance and tissue homeostasis in the hematopoietic system. All in all, associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities cancer patients harboring these germline mutations. Disclosures Bolton: GRAIL: Research Funding. Papaemmanuil:Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Illumina: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Kyowa Hakko Kirin: Consultancy, Honoraria; MSKCC: Patents &amp; Royalties. Levine:Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Astellas: Consultancy; Morphosys: Consultancy; Novartis: Consultancy; Amgen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Berger:Illumina: Research Funding; Grail: Research Funding; Roche: Consultancy. Zehir:Illumina: Honoraria; Memorial Sloan Kettering Cancer Center: Current Employment.

High-Dose Melphalan Significantly Increases Mutational Burden in Multiple Myeloma Cells at Relapse: Results from a Randomized Study in Multiple Myeloma

High-Dose Melphalan Significantly Increases Mutational Burden in Multiple Myeloma Cells at Relapse: Results from a Randomized Study in Multiple Myeloma

A Comprehensive Analysis of Alterations in DNA Damage Repair Pathways Reveals a Potential Way to Enhance the Radio-Sensitivity of Esophageal Squamous Cell Cancer.

Esophageal squamous cell cancer (ESCC) is a common malignancy with a poor 5-year overall survival in China. Altered DNA damage repair (DDR) pathways are associated with a predisposition to cancer and contribute to therapeutic response and resistance in cancers. However, alterations of DDR pathway genes in ESCC are still largely unknown. In this study, we employed genome sequencing data of 192 samples, comparative genomic hybridization data of 123 cases, and gene expression microarray data of 119 patients to firstly perform a comprehensive analysis of the gene alterations of 7 DDR pathways in ESCC. Gene mutations and copy number variations (CNVs) were observed in all 7 DDR pathways, and especially, CNVs were the dominant alteration types. Compared with other pathways, two DNA double-strand break (DSB) repair pathways homologous recombination (HR) and non-homologous end joining (NHEJ), carried significant gene mutations and CNVs especially gene amplifications. Most genes including RAD54B, NBS1, RAD51B, and PRKDC were significantly amplified and over-expressed in ESCC. Amplification and high expression of DSB repair pathway genes were associated with poorer overall survival. Gene set variation analysis further showed that DSB repair pathways were up-regulated in ESCC. Besides, we firstly demonstrated that combination of mirin and NU7441, two inhibitors for HR and NHEJ respectively, with ionizing radiation treatment significantly enhanced DSBs, reduced clonogenic cell survival, inhibited cell proliferation, and promoted cell apoptosis in ESCC cells with DSB pathway gene amplification. These findings suggest that DSB repair pathways were significantly altered in ESCC and inhibiting DSB repair pathways might enhance the radio-sensitivity of ESCC with DSB repair up-regulation.

Beyond BRCA1 and BRCA2: Deleterious Variants in DNA Repair Pathway Genes in Italian Families with Breast/Ovarian and Pancreatic Cancers.

The 5–10% of breast/ovarian cancers (BC and OC) are inherited, and germline pathogenic (P) variants in DNA damage repair (DDR) genes BRCA1 and BRCA2 explain only 10–20% of these cases. Currently, new DDR genes have been related to BC/OC and to pancreatic (PC) cancers, but the prevalence of P variants remains to be explored. The purpose of this study was to investigate the spectrum and the prevalence of pathogenic variants in DDR pathway genes other than BRCA1/2 and to correlate the genotype with the clinical phenotype. A cohort of 113 non-BRCA patients was analyzed by next-generation sequencing using a multigene panel of the 25 DDR pathways genes related to BC, OC, and PC. We found 43 unique variants in 18 of 25 analyzed genes, 14 classified as P/likely pathogenic (LP) and 28 as variants of uncertain significance (VUS). Deleterious variants were identified in 14% of index cases, whereas a VUS was identified in 20% of the probands. We observed a high incidence of deleterious variants in the CHEK2 gene, and a new pathogenic variant was detected in the RECQL gene. These results supported the clinical utility of multigene panel to increase the detection of P/LP carriers and to identify new actionable pathogenic gene variants useful for preventive and therapeutic approaches.

A Patient-centric Approach to Pancreatic Cancer Research: the Pancreatic Cancer Action Network Experience

Abstract The Pancreatic Cancer Action Network (PanCAN) set the goal of doubling survival from pancreatic cancer by 2020 almost a decade ago. During this time, several initiatives were put in place to directly assist patients today and to support research to help the patients of tomorrow. Through the Patient Central call center, pancreatic cancer patients receive critical information to assist with their care, search for clinical trials for which they are eligible, participate in a Patient Registry, connect with patients or caregivers with similar experiences, or enroll in the Know Your Tumor (KYT) molecular profiling service. Now that more than 1000 patients have received KYT reports, the outcomes indicate that patients that receive therapies targeted to tumor-specific molecular alterations experience significant improvements in progression-free survival and overall survival compared to control groups. Subset analysis also reveals benefits for individuals with defects in DNA damage repair pathway genes who are treated with a platinum-containing regimen. PanCAN's experience with the challenges and benefits of implementing a nation-wide patient support and molecular profiling effort, including cost-effectiveness analysis and examples of extraordinary responses, will be presented. Further advances in improving patient outcomes are anticipated to come from the Precision Promise adaptive clinical trial platform and an Early Detection Initiative, both of which are in the planning stages. The 5-year relative survival rate for pancreatic cancer in the USA has climbed from 6% to 9% over the past decade, building a foundation upon which further, substantive improvements in survival can be realized in future years.

Exome sequencing of oral leukoplakia and oral squamous cell carcinoma implicates DNA damage repair gene defects in malignant transformation

ObjectivesTo map the genomic pathways of patients with oral leukoplakia (OLK) which transformed to cancer (progressive) and those which did not (non-progressive), and to compare their exomic profiles. Materials and methodsWhole exome sequencing was performed on 42 sequential samples from five progressive and eight non-progressive patients. Association of genomic variant frequencies with progression or lesion severity were analysed by non-parametric tests (Kruskal-Wallis and Mann-Whitney-Wilcoxon) and multivariate sparse partial least squares discriminant analysis (sPLS-DA). Enrichment analysis was used to characterise the effect of mutations upon biological pathways. Confirmatory studies used qPCR and immunohistochemistry. ResultsUsing sPLS-DA, the variant frequency of a small number of genes could be used to classify the samples based on lesion severity or progressive status. Enrichment analysis showed that DNA damage repair gene related pathways were highly impacted in lesions which progressed to cancer. Multivariate analysis of a set of 148 DNA damage repair genes could be used to classify progressive lesions using mutation frequency. BRCA1, BRCA2 and other double strand break (DSB) repair Fanconi anaemia (FA)/BRCA pathway genes were prominent contributors to this classification. ConclusionPatients with progressive and non-progressive OLK can be differentiated using the frequency of exomic variants, particularly in DNA damage repair pathway genes. To our knowledge, this is the first report of FA/BRCA (DSB) pathway involvement in malignant transformation of OLK to oral squamous cell carcinoma (OSCC).