Damage Repair Pathways Research Articles

Comparative analysis of PD-L1 expression and molecular alterations in primary versus metastatic lung adenocarcinoma: a real-world study in China.

Programmed death-ligand 1 (PD-L1) is the only Food and Drug Administration-approved biomarker for monitoring response to immune checkpoint inhibitor (ICI) therapy in patients with lung adenocarcinoma. Understanding the nuances of molecular phenotypes, clinical attributes, and PD-L1 expression levels in primary and metastatic lung adenocarcinoma may help predict response to therapy and assist in the clinical management of lung adenocarcinoma. A total of 235 primary and metastatic lesion specimens from patients with non-small cell lung cancer (NSCLC) an institution in Shandong, China were analyzed. PD-L1 expression was assessed by immunohistochemistry using the 22C3 antibody, and the molecular phenotype was determined by next-generation sequencing of 450 genes. The molecular phenotypes of the primary and metastatic lesions were compared. Elevated PD-L1 expression was significantly associated with advanced and metastatic disease (P = 0.001). The distribution of PD-L1 expression varied based on the anatomical location, showing a higher frequency of elevated PD-L1 expression in distal metastases than in the primary tumor. Metastatic lesions exhibited a higher proportion of carcinogenic pathway gene alterations and a greater number of DNA damage-repair pathway gene alterations than the primary lesions. Notably, CDKN2A copy number deletions were more prevalent in metastatic lesions than in primary lesions. Clinical data stemming from research conducted at the Memorial Sloan Kettering Cancer Center revealed an association between the absence of CDKN2A expression and a poorer prognosis in stage I lung adenocarcinoma. Samples of metastatic tumors exhibited a higher proportion of elevated PD-L1 expression, a greater number of pathway alterations, and a higher occurrence of CDKN2A copy number deletions than primary samples. This highlights the importance of reinforcing the clinical management and follow-up of patients with CDKN2A deficiency, particularly within the subset of stage I lung adenocarcinoma.

Clinicopathological and molecular analysis of microsatellite instability in prostate cancer: a multi-institutional study in China.

Microsatellite instability (MSI), or mismatch repair-deficiency (dMMR), is rare in prostate cancers (PCas). The histological and molecular features of PCas with MSI/dMMR are incompletely described. Thus, we sought to identify the characteristics of PCas with MSI/dMMR. We analyzed 1,141 primary treatment-naive PCas by MMR-related protein immunohistochemistry (MLH1, PMS2, MSH2, and MSH6). We identified eight cases exhibiting MSI/dMMR (0.7%, 8/1141). Of these, six tumors had both MSH2 and MSH6 protein loss, one had both MLH1 and PMS2 protein loss, and one had only MSH6 loss. Histologically, MSI/dMMR-PCas frequently demonstrated high histological grade (Grade Group 4 or 5), ductal/intraductal histology (6/8 cases), pleomorphic giant-cell features (4/8 cases), and conspicuous tumor lymphocytic infiltration (8/8 cases). Polymerase chain reaction-based analysis of seven MSI/dMMR tumors revealed two MSI-H tumors with loss of both MSH2 and MSH6 proteins. Subsequently, the seven cases underwent next-generation sequencing (NGS) analysis with a highly validated targeted panel; four were MSI. All cases had a high tumor mutation burden (median: 45.3 mutations/Mb). Overall, the MSI/dMMR-PCas showed a high frequency of DNA damage-repair pathway gene changes, including five with pathogenic somatic or germline MMR gene mutations. Activating mutations in the MAPK pathway, PI3K pathway, and WNT/β-catenin pathway were common. TMPRSS2::ERG rearrangement was identified in one case (1/7, 14.3%). Several pathological features are associated with MSI/dMMR in PCas. Identification of these features may help to select patients for genetic screening. As MSI/dMMR-PCas are enriched for actionable mutations, patients should be offered NGS to guide standard-of-care treatment.

Abstract 308: Low molecular weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer

Abstract Background: Low molecular weight cyclin E (LMW-E) are oncogenic forms of cyclin E that are post translationally generated from the full-length cyclin E1 (FL-cycE). LMW-E is detected in breast cancer cells and tumor tissues, but not in normal mammary epithelial cells or adjacent normal tissues. Unlike FL-cycE, LMW-E drives mammary epithelial cell transformation in human cells and spontaneous mammary tumor formation in transgenic mouse models, but the oncogenic mechanisms of LMW-E and its unique function(s) independent of FL-cycE are not fully understood. It is currently assumed that LMW-E drives the tumorigenic process by promoting G1/S cell cycle transition and accelerating mitotic exit. Biochemical features such as longer protein half-life, higher affinity to its kinase partner CDK2, and resistance to endogenous CDK inhibitors such as p21 and p27 all promote the tumorigenic ability of LMW-E. Clinical studies in breast cancer reveal that overexpression of LMW-E predicts recurrence and poor survival in breast cancer patients independent of molecular subtype, Ki67 status, nodal status, or tumor grade, suggesting LMW-E may drive breast cancer development independent of its role in cell proliferation. In the current study, we tested the hypothesis that LMW-E promotes genomic instability by deregulating DNA replication and damage repair. Results: We generated immortalized pre-cancerous human mammary epithelial cells (hMECs) to express doxycycline inducible LMW-E or FL-cycE in CCNE1 knock-out background. We found that FL-cycE overexpression led to DNA replication stress and DNA damage accumulation, resulting in reduced cell viability. LMW-E overexpression, on the other hand, promoted cell survival under replication stress, resulting in persistent genomic instability. RNA-sequencing results showed LMW-E but not FL-cycE overexpression enhanced DNA replication and damage repair pathways. Molecularly, LMW-E interacted with and facilitated pre-replication complex assembly. LMW-E also mediated DNA repair by upregulating RAD51 and C17orf53, showing a dominant repairing effect over DNA damage induced by FL-cycE. Moreover, targeting the replication stress response pathway ATR-CHK1-RAD51 with small molecule inhibitors significantly decreased viability of LMW-E overexpressing hMECs and breast cancer cells. Lastly, we showed that positive LMW-E status was associated with genomic instability in tumors from a cohort of 725 patients diagnosed with early-stage breast cancer, further supporting our hypothesis that LMW-E promotes genomic instability to fuel breast cancer development. Conclusions: Collectively, our findings delineated a novel role for LMW-E in breast tumorigenesis mediated by replication stress tolerance and genomic instability, providing novel therapeutic strategies for LMW-E overexpressing breast cancers. Citation Format: Mi Li, Spiridon Tsavachidis, Fuchenchu Wang, Tuyen Bui, Tuyen D. Nguyen, Linjie Luo, Asha S. Multani, Melissa L. Bondy, Kelly K. Hunt, Khandan Keyomarsi. Low molecular weight cyclin E deregulates DNA replication and damage repair to promote genomic instability in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 308.

The DDR-related gene signature with cell cycle checkpoint function predicts prognosis, immune activity, and chemoradiotherapy response in lung adenocarcinoma

BackgroundAs a DNA surveillance mechanism, cell cycle checkpoint has recently been discovered to be closely associated with lung adenocarcinoma (LUAD) prognosis. It is also an essential link in the process of DNA damage repair (DDR) that confers resistance to radiotherapy. Whether genes that have both functions play a more crucial role in LUAD prognosis remains unclear.MethodsIn this study, DDR-related genes with cell cycle checkpoint function (DCGs) were selected to investigate their effects on the prognosis of LUAD. The TCGA-LUAD cohort and two GEO external validation cohorts (GSE31210 and GSE42171) were performed to construct a prognosis model based on the least absolute shrinkage and selection operator (LASSO) regression. Patients were divided into high-risk and low-risk groups based on the model. Subsequently, the multivariate COX regression was used to construct a prognostic nomogram. The ssGSEA, CIBERSORT algorithm, TIMER tool, CMap database, and IC50 of chemotherapeutic agents were used to analyze immune activity and responsiveness to chemoradiotherapy.Results4 DCGs were selected as prognostic signatures, and patients in the high-risk group had a lower overall survival (OS). The lower infiltration levels of immune cells and the higher expression levels of immune checkpoints appeared in the high-risk group. The damage repair pathways were upregulated, and chemotherapeutic agent sensitivity was poor in the high-risk group.ConclusionsThe 4-DCGs signature prognosis model we constructed could predict the survival rate, immune activity, and chemoradiotherapy responsiveness of LUAD patients.

Adherence to genetic testing guidelines in pancreatic cancer and impact on treatment.

e16238 Background: NCCN June 2019 Guidelines recommend that clinicians consider germline (GL) testing for patients(pts) diagnosed with pancreatic cancer (PDAC) and consider molecular analysis of tumors in pts with metastatic disease. Up to 25% of advanced PDAC pts have GL or somatic mutations in the DNA damage repair (DDR) pathway which are predictive of response to platinum chemotherapy (plat) and maintenance therapy with olaparib. Referral for genetic testing (GT) at our institution is currently made at provider discretion. We aimed to determine rates of GT, factors associated with GT, and outcomes of those tested. Methods: Frequency of GT testing was examined in pts with non-endocrine PDAC and >2 visits between 6/2019 and 6/2021 at Mount Sinai Health System. Clinicopathological variables and treatment outcomes were also recorded. Results: 149 pts were included. 71 pts (48%) were male. 25 pts (17%) had a personal history of cancer and 8 (5.3%) with history of DDR related cancer (prostate, breast, ovarian). 66 pts (44%) had GL testing: 42 (28%) at diagnosis with the remainder later in treatment. The rate of GL testing increased every year: 33% (2019), 44% (2020), 61% (2021). There was no statistically significant association between decision to do GL testing and age, ECOG, race, personal or family history (FH) of cancer. A FH of DDR related cancer was positively associated with GL testing (47% vs 27% p=0.039). 8 pts (12% of pts tested) had pathological gMut: BRCA1 (1), BRCA2 (1), ATM (2), PALB2 (2) , NTHL1 (1), both CHEK2 and APC (1). 6 pts (75%) had GL testing at diagnosis. One g BRCA2 pt had a plat response. Neither g BRCA pt received a PARP inhibitor. 24 pts in total had a response to plat, 16 (67%) of whom had GL testing. 94 pts (63%) had molecular tumor testing (including 65% of pts with metastases). Pathogenic mut in DDR genes were identified in 9 pts: ARID1A (2), BRCA2 (3), ATM (2), PALB2 (2), CHEK2 (1). Four of these pts had corresponding gMuts. One pt with somatic ATM mut had g BRCA1 mut. Two pts with BRCA2 somatic mut did not have GL testing. Conclusions: GT based on provider discretion resulted in suboptimal rates of GL testing. However, since inception of national guidelines, testing rates nearly doubled. Rates of GL DDR mutations were lower than previously reported, with varying response to plat in this cohort. Future studies are needed to understand barriers to GT and improve implementation.[Table: see text]

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.

Predictive value of ectopic HORMAD1 tumor expression for high-dose platinum-based chemotherapy benefit in patients with high-risk HER2-negative breast cancer.

541 Background: The meiotic DNA break regulator HORMAD1 is aberrantly expressed in many cancers and is associated with increased genomic instability. The susceptibility of HORMAD1 expressing tumors to agents targeting DNA damage repair (DDR) pathways is poorly understood since clinical data within the context of a randomized clinical trial (RCT) is lacking. Here, we retrospectively studied HORMAD1 expression as a putative predictive biomarker in an RCT for benefit of adjuvant high-dose platinum-based chemotherapy (HDCT) with autologous stem cell support in patients with high-risk HER2-negative early breast cancer (BC). Methods: Patients with stage III BC participated in an RCT comparing HDCT to conventional chemotherapy (CDCT; Rodenhuis et all, NEJM, 2003; Steenbruggen et all, JAMA Oncol, 2020). We studied the subgroup with HER2-negative BC for whom tumor BRCA1-like classification was previously determined using a validated DNA comparative genomic hybridization algorithm (Vollebergh et all, BCR, 2014). Tumor HORMAD1 expression was determined on FFPE samples using RNAscope, an RNA in situ hybridization method, and classified as negative (no expression) or positive (any expression detected). Results: For 195/246 (79.3%) HER2-negative patients treated according to protocol, HORMAD1 RNAscope status was available; dropout was due to absence or insufficient quality of tumor specimens. HORMAD1 positivity was enriched in triple-negative breast cancer (TNBC) (23/47; 48.9%). Furthermore, in all HER2-negative BCs, HORMAD1 positivity (45/195; 23.1%) was associated with age ≤40 years, histological grade III, <10 positive lymph nodes, breast-conserving surgery, BRCA1-like profile, and tumor-infiltrating lymphocytes (TILs) >10%. Such association, although not significant, was also observed within TNBC. During a median follow-up of 20.3 years, 124 (63.6%) recurrences and 115 deaths (59.0%) occurred. The prognostic effect of HORMAD1 positivity on overall survival (OS) varied with follow-up time and was borderline significant at 10 years and significant thereafter (10-year: adjusted (adj.) HR 0.47, 95% CI 0.21-1.04; 15-year: adj. HR 0.25, 95% CI 0.07-0.91). Benefit on RFS from HDCT over CDCT was stronger in patients with HORMAD1-positive tumors (adj. HR 0.18, 95% CI 0.06-0.54) than in patients with HORMAD1-negative tumors (adj. HR 0.69, 95% CI 0.46-1.02) (P-interaction = 0.02). Similar results were observed for OS. Conclusions: In this retrospective sub study of 195 patients with high-risk HER2-negative BC participating in an RCT, tumor HORMAD1 expression is predictive for benefit of high-dose platinum-based chemotherapy. Our observations are consistent with the prior observations that HORMAD1 expression is associated with genomic instability and impaired DDR pathways. Further research is warranted to validate our findings. Clinical trial information: NCT03087409.

Clinical Impact of Immune Checkpoint Inhibitor (ICI) Response, DNA Damage Repair (DDR) Gene Mutations and Immune-Cell Infiltration in Metastatic Melanoma Subtypes.

Molecular and histopathological analysis of melanoma subtypes has revealed distinct epidemiological, genetic, and clinical features. However, immunotherapy for advanced metastatic melanoma patients does not differ based on subtype. Response to immune checkpoint inhibitors (ICI) has been shown to vary, therefore, predictive biomarkers are needed in the design of precision treatments. Targeted sequencing and histopathological analysis (CD8 and CD20 immunohistochemistry) were performed on subtypes of metastatic melanoma (cutaneous melanoma (CM, n = 10); head and neck melanoma (HNM, n = 7); uveal melanoma (UM, n = 4); acral lentiginous melanoma (AM, n = 1) and mucosal melanoma (MM, n = 1) treated with ICI). Progression-free survival (PFS) was significantly associated with high CD8 expression (p = 0.025) and mutations in DNA damage repair (DDR) pathway genes (p = 0.012) in all subtypes but not with CD20 expression. Our study identified that immune cell infiltration and DDR gene mutations may have an impact in response to ICI treatment in metastatic melanoma but differs among subtypes. Therefore, a comprehensive understanding of the immune infiltration cells’ role and DDR gene mutations in metastatic melanoma may identify prognostic biomarkers.

TET1 mutations as a predictive biomarker for immune checkpoint inhibitors in colon adenocarcinoma

BackgroundThe ten-eleven translocation 1 (TET1), which is essential for active DNA demethylation, plays a multifaceted role in the pathogenesis of colorectal cancer. The study has demonstrated the association of TET1 mutations with a high response to immune checkpoint inhibitors (ICIs) in diverse cancers. However, the relationship between TET1 mutations and the response to ICIs in colon cancer is still lacking.MethodsThe prognosis, predictive markers, immune characteristics, mutation number of DNA damage repair (DDR) pathways, pathway enrichment, and drug sensitivity conditions were all compared between TET1-mutated and wild-type patients with colon adenocarcinoma (COAD).ResultsThe overall survival of patients with TET1 mutations in the ICI-treated cohort was significantly longer than those without (p = 0.0059). Compared with the wild-type patients, TET1-mutated patients had higher tumor mutational burden and neoantigen load, enhanced abundance of tumor-infiltrating immune cells, increased expression of immune-related genes, and mutation number of DDR pathways. Additionally, the patients with TET1 mutations were found to be more sensitive to lapatinib and 5-fluorouracil.ConclusionThese findings suggest that TET1 mutations may serve as a potential biomarker for the response to ICIs in COAD patients.

Clinico-genomic characterization of patients with metastatic urothelial carcinoma in real-world practice and development of a novel bladder immune prognostic index (BIPI).

548 Background: Real-world data (RWD) linking clinical outcomes with comprehensive genomic profiling (CGP) may enable identification of biomarkers to guide treatment selection and stratification in future trials. The primary objective was to characterize patients with metastatic urothelial carcinoma (mUC) included in a clinic-genomic database (CGDB), comprised of the electronic health record-derived Flatiron Health database with linked FoundationOne CGP results. As secondary objective, a novel Bladder Immune Prognostic Index (BIPI) was developed. Methods: A retrospective exploratory analysis was performed of de-identified RWD, retrieved from the CGDB. Data from mUC patients starting first-line single-agent immune checkpoint inhibitors (ICIs) and an unmatched group treated with front-line platinum-based chemotherapy (CHT) between Jan 1, 2011, and Sept 30, 2019, were analyzed and correlated with overall survival (OS). Known driver alterations, tumor mutational burden (TMB), and PD-L1 expression were described. A BIPI predicting outcome with ICIs was developed using a Cox-LASSO model and validated externally in a phase II trial (NCT02951767). Results: Of the 1021 patients with mUC identified in CGDB, 118 ICI-treated and 268 CHT-treated patients were included. Median follow-up duration was 9.4 and 14.5 months, respectively. Median OS was 5.4 months (95%CI, 3.3–9.2) with ICIs and 8.2 months (95%CI, 6.8–10.0) with CHT. In ICI-treated patients, low albumin and metastatic disease at initial presentation were associated with worse OS [HR (95%CI) 2.15 (1.18–3.90), p =.012; 2.58 (1.30–5.10), p =.007, respectively] whereas surgery for organ-confined disease and high TMB (≥10 mut/Mb) were associated with improved OS (HR (95%CI) 0.56 (0.36–0.88), p =.012; 0.58 [0.35–0.95]; p=.03), respectively. In CHT-treated patients, those with high APOBEC had worse OS (HR 1.43 [95% CI, 1.06–1.94]; p=.02). Neither PD-L1 (HR 0.96 [0.37-2.46]; p =.93), FGFR3 mutations (HR 0.98 [0.65-1.47]; p =.92) nor DNA damage-repair pathway alterations (HR 1.06 [0.73-1.52]; p =.77) were associated with OS. A novel BIPI for ICI-treated patients combining clinical and genomic variables (non-metastatic at initial diagnosis, normal albumin level, previous surgery for organ-confined disease, high TMB) was developed. Patients were categorized in 3 groups (low, intermediate, high risk) which correlated with OS. Median OS (95%CI) for low, intermediate and high risk was 11.7 (8.9−17.7), 4.1 (2.5–NE) and 2.4 months (1.0–4.0), (p <.001). Same results were observed in the validation cohort from an independent phase II immunotherapy trial in mUC (p <.001). Conclusions: This is the first time RWD including CGP were used to develop and validate a novel BIPI in mUC. This prognostic index may help patient selection in everyday practice and inform future trial design.

REV1 promotes lung tumorigenesis by activating the Rad18/SERTAD2 axis

REV1 is the central member of the family of TLS polymerases, which participate in various DNA damage repair and tolerance pathways and play a significant role in maintaining genomic stability. However, the role of REV1 in tumors is rarely reported. In this study, we found that the expression of REV1 was significantly upregulated in lung cancer tissues compared with matched adjacent tissues and was associated with poor prognosis. Functional experiments demonstrated that REV1 silencing decreased the growth and proliferation capacity of lung cancer cells. Mechanistically, REV1 upregulated the expression of SERTAD2 in a Rad18-dependent manner, thereby promoting lung carcinogenesis. A novel REV1 inhibitor, JH-RE-06, suppressed lung tumorigenesis in vivo and in vitro and was shown to be safe and well tolerated. Our study confirmed that REV1 is a potential diagnostic marker and therapeutic target for lung cancer and that JH-RE-06 may be a safe and efficient therapeutic agent for NSCLC.

Whole-Genome Sequencing Reveals Comprehensive Genomic Profiles of Radiation-Induced Sarcomas

<h3>Purpose/Objective(s)</h3> Radiation-induced sarcoma (RIS) is a rare secondary malignancy resulting from ionizing radiation-related treatment after a long latency period. Despite the unfavorable clinical outcomes, the genomic footprints of ionizing radiation in RIS development remain largely unknown. Hence, this study aimed to characterize RIS genomes and the genomic alterations in them. <h3>Materials/Methods</h3> We performed whole-genome sequencing (WGS) of 11 cases of RIS matched with normal genomes to identify somatic alterations potentially associated with RIS development. <h3>Results</h3> The mutation abundance of RIS genomes including one hypermutated genome was variable. Cancer-related genes might show different types of genomic alterations. For instance, NF1, NF2, NOTCH1, NOTCH2, PIK3CA, RB1, and TP53 showed singleton somatic mutations; MYC, CDKN2A, RB1, and NF1 showed recurrent copy number alterations; and NF2, ARID1B, and RAD51B showed recurrent structural variations (SVs). The effects of non-homologous end joining on short insertions-deletions and SVs were substantial in RIS genomes, compared with in spontaneous osteosarcoma genomes, representing the genomic hallmark of RIS genomes. In addition, frequent chromothripsis and predisposing germline variants in DNA damage-repair pathways were identified. <h3>Conclusion</h3> Taken together, WGS-scale characterization of RIS genomes may pave the way for advanced diagnostic and therapeutic strategies for RIS.

Genomic alterations associated with mutational signatures, DNA damage repair and chromatin remodeling pathways in cervical carcinoma

Despite recent advances in the prevention of cervical cancer, the disease remains a leading cause of cancer-related deaths in women worldwide. By applying the GISTIC2.0 and/or the MutSig2CV algorithms on 430 whole-exome-sequenced cervical carcinomas, we identified previously unreported significantly mutated genes (SMGs) (including MSN, GPX1, SPRED3, FAS, and KRT8), amplifications (including NFIA, GNL1, TGIF1, and WDR87) and deletions (including MIR562, PVRL1, and NTM). Subset analyses of 327 squamous cell carcinomas and 86 non-squamous cell carcinomas revealed previously unreported SMGs in BAP1 and IL28A, respectively. Distinctive copy number alterations related to tumors predominantly enriched for *CpG- and Tp*C mutations were observed. CD274, GRB2, KRAS, and EGFR were uniquely significantly amplified within the Tp*C-enriched tumors. A high frequency of aberrations within DNA damage repair and chromatin remodeling genes were detected. Facilitated by the large sample size derived from combining multiple datasets, this study reveals potential targets and prognostic markers for cervical cancer.

Histone deacetylase 6 acts upstream of DNA damage response activation to support the survival of glioblastoma cells

DNA repair promotes the progression and recurrence of glioblastoma (GBM). However, there remain no effective therapies for targeting the DNA damage response and repair (DDR) pathway in the clinical setting. Thus, we aimed to conduct a comprehensive analysis of DDR genes in GBM specimens to understand the molecular mechanisms underlying treatment resistance. Herein, transcriptomic analysis of 177 well-defined DDR genes was performed with normal and GBM specimens (n = 137) from The Cancer Genome Atlas and further integrated with the expression profiling of histone deacetylase 6 (HDAC6) inhibition in temozolomide (TMZ)-resistant GBM cells and patient-derived tumor cells. The effects of HDAC6 inhibition on DDR signaling were examined both in vitro and intracranial mouse models. We found that the expression of DDR genes, involved in repair pathways for DNA double-strand breaks, was upregulated in highly malignant primary and recurrent brain tumors, and their expression was related to abnormal clinical features. However, a potent HDAC6 inhibitor, MPT0B291, attenuated the expression of these genes, including RAD51 and CHEK1, and was more effective in blocking homologous recombination repair in GBM cells. Interestingly, it resulted in lower cytotoxicity in primary glial cells than other HDAC6 inhibitors. MPT0B291 reduced the growth of both TMZ-sensitive and TMZ-resistant tumor cells and prolonged survival in mouse models of GBM. We verified that HDAC6 regulated DDR genes by affecting Sp1 expression, which abolished MPT0B291-induced DNA damage. Our findings uncover a regulatory network among HDAC6, Sp1, and DDR genes for drug resistance and survival of GBM cells. Furthermore, MPT0B291 may serve as a potential lead compound for GBM therapy.

Repair pathways for radiation DNA damage under normoxic and hypoxic conditions: Assessment with a panel of repair-deficient human TK6 cells.

Various types of DNA lesions are produced when cells are exposed to ionizing radiation (IR). The type and yield of IR-induced DNA damage is influenced by the oxygen concentration. Thus, different DNA repair mechanisms may be involved in the response of normoxic and hypoxic cells to irradiation with IR. However, differences between the repair mechanisms of IR-induced DNA damage under normoxic versus hypoxic conditions have not been clarified. Elucidating the relative contribution of individual repair factors to cell survival would give insight into the repair mechanisms operating in irradiated normoxic and hypoxic cells. In the present study, we used a panel of repair-deficient human TK6 cell lines that covered seven repair pathways. Cells were irradiated with X-rays under normoxic and hypoxic conditions, and the sensitivities of each mutant relative to the wild-type (i.e. relative sensitivity) were determined for normoxic and hypoxic conditions. The sensitivity of cells varied depending on the type of repair defects. However, for each repair mutant, the relative sensitivity under normoxic conditions was comparable to that under hypoxic conditions. This result indicates that the relative contribution of individual repair pathways to cell survival is comparable in normoxic and hypoxic cells, although the spectrum of IR-induced DNA damage in hypoxic cells differs from that of normoxic cells.

P53 Activation in Genetic Disorders: Different Routes to the Same Destination.

The tumor suppressor p53 is critical for preventing neoplastic transformation and tumor progression. Inappropriate activation of p53, however, has been observed in a number of human inherited disorders that most often affect development of the brain, craniofacial region, limb skeleton, and hematopoietic system. Genes related to these developmental disorders are essentially involved in transcriptional regulation/chromatin remodeling, rRNA metabolism, DNA damage-repair pathways, telomere maintenance, and centrosome biogenesis. Perturbation of these activities or cellular processes may result in p53 accumulation in cell cultures, animal models, and perhaps humans as well. Mouse models of several p53 activation-associated disorders essentially recapitulate human traits, and inactivation of p53 in these models can alleviate disorder-related phenotypes. In the present review, we focus on how dysfunction of the aforementioned biological processes causes developmental defects via excessive p53 activation. Notably, several disease-related genes exert a pleiotropic effect on those cellular processes, which may modulate the magnitude of p53 activation and establish or disrupt regulatory loops. Finally, we discuss potential therapeutic strategies for genetic disorders associated with p53 misactivation.

Abstract 2068: Radiotherapy is associated with a deletion signature that contributes to poor cancer patient outcomes

Abstract Diffuse gliomas are highly aggressive brain tumors that invariably relapse despite treatment with chemo- and radiotherapy. Treatment with alkylating chemotherapy can drive tumors to develop a hypermutator phenotype. In contrast, the genomic effects of radiation therapy (RT) remain unknown. We analyzed the mutational spectra following treatment with ionizing radiation in sequencing data from 190 paired primary-recurrent gliomas from the Glioma Longitudinal Analysis (GLASS) dataset and 2116 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified a significant increase in the burden of small deletions following radiation therapy that was independent of other factors and was significantly associated with the clinically applied RT-dosage in Gy (P = 1e-02, multivariable log-linear regression). These novel deletions demonstrated distinct characteristics when compared to pre-existing deletions present prior to RT-treatment and deletions in RT-untreated tumors. Radiation therapy-acquired deletions were characterized by a larger deletion size (GLASS and metastatic cohort, P = 1.2e-02 and P = 8e-11, respectively; Mann-Whitney U test), an increased distance to repetitive DNA elements (P &amp;lt; 2.2e-16, Kolmogorov-Smirnov test) and a reduction in microhomology at breakpoints (P = 3.2e-02, paired Wilcoxon signed-rank test). These observations suggested that canonical non-homologous end joining (c-NHEJ) was the preferred pathway for DNA double strand break repair of RT-induced DNA damage. Furthermore, radiotherapy resulted in frequent chromosomal deletions and significantly increased frequencies of CDKN2A homozygous deletions. Finally, a high burden of RT-associated deletions was associated with worse clinical outcomes (GLASS and metastatic cohort, P &amp;lt; 1e-04 and P = 2.6e-02, respectively; Wald test). Our results collectively suggest that effective repair of RT-induced DNA damage is detrimental to patient survival and that inhibiting c-NHEJ may be a viable strategy for improving the cancer-killing effect of radiotherapy. Taken together, the identified genomic scars as a result of radiation therapy reflect a more aggressive tumor with increased levels of resistance to follow up treatments. Citation Format: Emre Kocakavuk, Kevin J. Anderson, Kevin C. Johnson, Frederick S. Varn, Samirkumar B. Amin, Erik P. Sulman, Floris P. Barthel, Roel G. Verhaak. Radiotherapy is associated with a deletion signature that contributes to poor cancer patient outcomes [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 2068.

Abstract 2050: Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma

Abstract Background: Previous study indicated that &amp;gt;80% of osteosarcomas (OSs) have genomic instability characteristics similar to BRCA1/2-deficient cancers. Such BRCA-like traits could manifest a deficiency in the DNA damage repair (DDR) pathway. Patients with alterations in DDR pathway was reported sensitive to platinum-based chemotherapy and PARP inhibitors. In order to seek more precision therapeutic opportunities, we explored the molecular profile of Chinese OS patients to identify whether the analogous phenomena exists as reported. Material and Methods: 44 archival Chinese OS patients from 3D Medicines and a public dataset including 35 OS patients (download from cBioPortal), were collected and analyzed next-generation sequencing (NGS) results on tumor tissues for gene mutations, copy number alterations (CNAs) and fusions in 33 cancer-associated DDR genes. Simultaneously, 44 Chinese OS patients' tumor analysis generated homologous recombination deficiency (HRD) scores (defined as the unweighted sum of LOH, TAI and LST scores) through the 733 gene panel sequencing. Results: Among 44 Chinese OS patients, 18 (40.9%) of OS had DDR pathway gene somatic alterations, and TP53 (31.8%) were the most frequently mapped gene. Other DDR somatic alterations were identified in BRCA2 (6.8%), BARD1 (4.5%), BRCA1 (2.3%), PMS2 (2.3%), FANCA (2.3%) and CHEK1 (2.3%). It was worth mention that all gene alterations were copy number reduction except TP53. Collectively, the frequencies of HRR and mismatch repair (MMR) alterations were 15.9% and 2.3%, respectively. HRD score were available in 18 patients, the median was 40.5 (range, 0-81). As to public dataset, 6 (17.1%) patients harbored somatic alterations in DDR genes. The variations were BRCA2 (5.7%), ATR (2.9%), BARD1 (2.9%), FANCC (2.9%), MLH1 (2.9%), MSH6 (2.9%) and PMS2 (2.9%). The HRR mutations frequency was 11% and MMR was 8.6%. Conclusions: This is the first large-scale comprehensive genomic instability characteristics analysis for Chinese patients with OS. Chinese patients and public dataset patients covers many common features. The DDR gene alteration frequency and HRD score are higher in OS patients, the association of variations in DDR genes and HRD score could be considered substitutable in predicting platinum-based therapy and PARP inhibitor response in clinical practice. Citation Format: Yonggang Wang, Hongtao Li, Jing Xia, Shiqing Chen, Yuezong Bai. Comprehensive genomic instability characteristics analysis in Chinese patients with osteosarcoma [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 2050.

DNA repair- and nucleotide metabolism-related genes exhibit differential CHG methylation patterns in natural and synthetic polyploids (Brassica napus L.)

Polyploidization plays a crucial role in the evolution of angiosperm species. Almost all newly formed polyploids encounter genetic or epigenetic instabilities. However, the molecular mechanisms contributing to genomic instability in synthetic polyploids have not been clearly elucidated. Here, we performed a comprehensive transcriptomic and methylomic analysis of natural and synthetic polyploid rapeseeds (Brassica napus). Our results showed that the CHG methylation levels of synthetic rapeseed in different genomic contexts (genes, transposon regions, and repeat regions) were significantly lower than those of natural rapeseed. The total number and length of CHG-DMRs between natural and synthetic polyploids were much greater than those of CG-DMRs and CHH-DMRs, and the genes overlapping with these CHG-DMRs were significantly enriched in DNA damage repair and nucleotide metabolism pathways. These results indicated that CHG methylation may be more sensitive than CG and CHH methylation in regulating the stability of the polyploid genome of B. napus. In addition, many genes involved in DNA damage repair, nucleotide metabolism, and cell cycle control were significantly differentially expressed between natural and synthetic rapeseeds. Our results highlight that the genes related to DNA repair and nucleotide metabolism display differential CHG methylation patterns between natural and synthetic polyploids and reveal the potential connection between the genomic instability of polyploid plants with DNA methylation defects and dysregulation of the DNA repair system. In addition, it was found that the maintenance of CHG methylation in B. napus might be partially regulated by MET1. Our study provides novel insights into the establishment and evolution of polyploid plants and offers a potential idea for improving the genomic stability of newly formed Brassica polyploids.

Abstract 2040: Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma patients

Abstract Background: DNA damage repair (DDR) pathway have attracted much attention because they modulate cancer risk, progression and therapeutic response. DDR deficiency sensitize the tumor to poly ADP-ribose polymerase inhibitors (PARPi). Also alterations in DDR genes are associated with higher tumor mutation burden (TMB) and better response to immune checkpoint inhibitors (ICI). Here we conduct a large-scale genomic analysis to clarify the landscape of DDR gene mutations in hepatocellular carcinoma and the correlation between individual gene mutation and tumor mutation burden. Methods: Tumor samples from 1427 patients with advanced hepatocellular carcinoma were assayed through next-generation sequencing with 381 cancer-related genes including 30 predefined DDR genes: MRE11A, RAD50, BARD1, BLM, BRCA1, BRCA2, BRIP1, PALB2, MLH1, MSH2, MSH6, PMS2, POLD1, ATM, ATR, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCG, FANCF, FANCL, RAD51, CUL3, ERCC1, POLE, MUTYH, and PRKDC. TMB (mutations per megabase [mut/Mb]) was calculated based on nonsilent somatic mutations in coding regions. Wilcoxon test was used for the comparisons and statistical significance was set at p = 0.05. Results: Somatic DDR gene alterations were found in 18.8% of the HCC patients. The most common mutational types were copy number variation (30.54%), frameshift (21.89%), and nonsense (20%). The most frequently altered DDR genes are ATM (2.6%) and BRCA2 (2.2%), followed by FANCA (0.9%), PALB2 (0.8%), and MLH1 (0.7%). As to different functional pathways of DDR, the gene alteration frequency from high to low is HRR (6.1%), DS (3.2%), FA (2.5%), MMR (2.2%), BER (0.6%), NHEJ (0.6%), and NER (0.3%). The TMB of patients carrying somatic DDR gene alterations was significantly higher than those without somatic DDR gene alterations (7.26 vs 6.45 mut/MB, p&amp;lt;0.001). As to individual gene, alterations in RAD50, MLH1, MSH2, CHEK2, and FANCE were significantly correlated with higher tumor mutation burden while alterations in other DDR genes were not. Conclusion: This is the largest study to clarify the landscape of DDR gene mutations in hepatocellular carcinoma. This study confirms that mutations in DDR genes are relatively common and are correlated with higher tumor mutation burden. This also indicates not all DDR genes are equal since mutations of certain individual genes were associated with higher TMB whereas others were not; further studies are needed to confirm this. Citation Format: Mengmei Yang, Wenzhuan Xie, Mengli Huang, Xinhua Zhu. Analysis of DNA damage repair gene alterations and their association with tumor mutation burden in 1427 hepatocellular carcinoma 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 2040.