Homologous Recombination DNA Repair Defects Research Articles

Monoterpene indole alkaloids with anticancer activity from Tabernaemontana species

AbstractIndole alkaloids, predominantly found in plants, are a large group of natural product-derived compounds characterized by a remarkable chemical diversity associated with significant biological properties. Among these, terpenoid indole alkaloids are the largest group of secondary metabolites. Tabernaemontana species (Apocynaceae) are widely distributed in tropical and subtropical regions of the world and used in traditional medicine to treat a variety of illnesses, including tumours. These species biosynthesize large quantities of structurally complex monoterpene indole and bisindole alkaloids. Given the compelling biological properties of indole alkaloids, the phytochemical study of Tabernaemontana species has been widely addressed to identify potential anticancer compounds. Several indole alkaloids have shown antiproliferative effect due to different mechanisms, namely by inducing apoptosis or arresting cell cycle, in diverse cancer cell lines, including multidrug-resistant phenotypes. This review primarily aims to underscore the anticancer activity of indole and bisindole alkaloids isolated from several Tabernaemontana species. Moreover, our recent contributions to the field are also highlighted, focusing on the study of Tabernaemontana elegans. The anticancer evaluation, namely the reversion of P-glycoprotein-mediated multidrug resistance, of two sets of monoterpene indole alkaloid derivatives, obtained by modification of some functional groups of two major monoterpene indole alkaloids, is reviewed, as well as the results obtained for a derivative that targeted homologous recombination DNA repair defects.

Whole-genome sequencing based assessment of HER2 focal amplification for precision oncology of breast cancers.

609 Background: Focal oncogene amplification confers a growth advantage to tumor cells and drives cancer evolution. Herein, we aimed to explore focally amplified HER2 in breast carcinomas. Methods: We performed whole genome sequencing (WGS) and whole-transcriptome sequencing (WTS) in 787 breast cancer samples. HER2 focal amplification was defined as having a segment length less than 3Mbp and ≥4 higher copy number than the surrounding region. Results: Most of the patients were premenopausal women (n = 574, 72.9%) with a median age of 37 (range 21-76) years. We identified 57 (58.8%) and 27 (5.0%) samples with HER2 focal amplification in HER2-positive (n = 97) and even HER2-negative (n = 536) by immunohistochemistry (IHC) and in-situ hybridization (ISH) according to ASCO guideline for breast cancer, respectively. Regardless of HER2-positivity, HER2 focal amplification was significantly associated with higher HER2 RNA expression. Among 28 patients who received six cycles of docetaxel/carboplatin/trastuzumab/pertuzumab neoadjuvant therapy followed by curative surgery, 21 patients had HER2 focal amplification and 14 (66.7%) achieved pathologic complete remission (pCR). Intriguingly, all patients without HER2 focal amplification (n = 7) failed to achieve pCR. HER2 focal amplification showed enrichment of TP53 mutations (OR 0.245, q < 0.001), but was mutually exclusive with GATA3 mutations (OR 2.82, q = 0.055). The mutational spectrums suggested that the activity in APOBEC family of cytidine deaminases were responsible for the somatic single-nucleotide variants of breast cancer with HER2 focal amplification. Compared to samples without HER2 focal amplification, samples with HER2 focal amplification were less likely to have defective homologous recombination DNA repair pathway. HER focal amplifications were closely associated with various types of structural variations. Translocation partners of HER2 were widespread across the entire genome with notable exceptions on chromosome 2, and most of them were close to super-enhancers implying enhancer hijacking as one of mechanisms of HER2 overexpression. Breast cancers with or without HER2 focal amplification showed distinct RNA expression patterns. Notably, activity of genes involved in doxorubicin-resistance and AKT1 signaling were significantly associated with HER2 focal amplification. Conclusions: Our analysis demonstrates that WGS enables better classification of the HER2 amplification status in breast cancer than IHC with ISH, suggesting WGS as a sensitive tool for screening breast cancers for anti-HER2-targeted therapies.

Targeted therapy of advanced parathyroid carcinoma guided by genomic and transcriptomic profiling.

Parathyroid carcinoma (PC) is an ultra-rare malignancy with a high risk of recurrence after surgery. Tumour-directed systemic treatments for PC are not established. We used whole-genome and RNA sequencing in four patients with advanced PC to identify molecular alterations that could guide clinical management. In two cases, the genomic and transcriptomic profiles provided targets for experimental therapies that resulted in biochemical response and prolonged disease stabilization: (a) immune checkpoint inhibition with pembrolizumab based on high tumour mutational burden and a single-base substitution signature associated with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) overactivation; (b) multi-receptor tyrosine kinase inhibition with lenvatinib due to overexpression of FGFR1 (Fibroblast Growth Factor Receptor 1) and RET (Ret Proto-Oncogene) and, (c)later in the course of the disease, PARP (Poly(ADP-Ribose) Polymerase) inhibition with olaparib prompted by signs of defective homologous recombination DNA repair. In addition, our data provided new insights into the molecular landscape of PC with respect to the genome-wide footprints of specific mutational processes and pathogenic germline alterations. These data underscore the potential of comprehensive molecular analyses to improve care for patients with ultra-rare cancers based on insight into disease biology.

NR4A3 Expression Is Consistently Absent in Acinic Cell Carcinomas of the Breast: A Potential Nosologic Shift

Acinic cell carcinoma (AciCC) is a tumor that is recognized in both the breast and salivary glands. Recently, the recurrent genomic rearrangement, t(4;9)(q13;q31) was identified in salivary AciCC that results in constitutive upregulation of the nuclear transcription factor NR4A3, which can be detected by immunohistochemistry. In this study, we sought to evaluate NR4A3 expression in breast AciCC using immunohistochemistry. Strong and diffuse nuclear staining was considered a positive result. Sixteen AciCCs were studied, including 8 pure AciCCs and 8 AciCCs admixed with other types (invasive carcinoma of no special type in 5 cases and metaplastic carcinoma in 3 cases). All 16 AciCCs showed negative results for NR4A3 expression. Four cases with available material were evaluated for rearrangements of the NR4A3 gene by fluorescence in situ hybridization and no rearrangements were observed. Whole-genome sequencing of 1 AciCC revealed a TP53 splice-site mutation, high levels of genomic instability, and genomic features of homologous recombination DNA repair defects; a structural variant analysis of this case did not reveal the presence of a t(4;9) rearrangement. We conclude that breast AciCCs consistently lack NR4A3 rearrangement or overexpression, unlike most of the salivary AciCCs, and that consistent with previous results, breast AciCCs are associated with genomic alterations more similar to those seen in triple-negative breast carcinomas than salivary gland AciCCs. These results suggest that unlike other salivary gland–like tumors that occur in the breast, the molecular underpinnings of the salivary gland and breast AciCCs are different and that the salivary gland and breast AciCCs likely represent distinct entities.

KRAS Mutation Variants and Co-occurring PI3K Pathway Alterations Impact Survival for Patients with Pancreatic Ductal Adenocarcinomas.

KRAS variant alleles may have differential biological properties which impact prognosis and therapeutic options in pancreatic ductal adenocarcinomas (PDA). We retrospectively identified patients with advanced PDA who received first-line therapy and underwent blood and/or tumor genomic sequencing at the University of Washington between 2013 and 2020. We examined the incidence of KRAS mutation variants with and without co-occurring PI3K or other genomic alterations and evaluated the association of these mutations with clinicopathological characteristics and survival using a Cox proportional hazards model. One hundred twenty-six patients had genomic sequencing data; KRAS mutations were identified in 111 PDA and included the following variants: G12D (43)/G12V (35)/G12R (23)/other (10). PI3K pathway mutations (26% vs. 8%) and homologous recombination DNA repair (HRR) defects (35% vs. 12.5%) were more common among KRAS G12R vs. non-G12R mutated cancers. Patients with KRAS G12R vs. non-G12R cancers had significantly longer overall survival (OS) (HR 0.55) and progression-free survival (PFS) (HR 0.58), adjusted for HRR pathway co-mutations among other covariates. Within the KRAS G12R group, co-occurring PI3K pathway mutations were associated with numerically shorter OS (HR 1.58), while no effect was observed on PFS. Patients with PDA harboring KRAS G12R vs. non-G12R mutations have longer survival, but this advantage was offset by co-occurring PI3K alterations. The KRAS/PI3K genomic profile could inform therapeutic vulnerabilities in patients with PDA.

Next generation sequencing identifies potential PARP inhibitor sensitive mutations

Abstract Introduction/Objective Poly ADP-ribose polymerase (PARP) inhibitors are a novel and important drug class targeting homologous recombination DNA repair defects (HRD) and have been approved for use in breast, pancreatic and ovarian cancers. Originally targeted for loss of function mutations in BRCA1 and BRCA2, many other genes are involved in the HRD pathway; Rimar et al detailed 19 DNA repair genes associated with homologous recombination and PARP inhibitor sensitivity. Our 214 gene NGS panel, Iowa Cancer Mutation Profile, includes BRCA1, BRCA2 and 15 other HRD pathway genes. We reviewed cases from the prior 12 months to determine the frequency of HRD7 pathway gene variants in various tumor types with potential PARP inhibitor sensitivity. Methods/Case Report Iowa Cancer Mutation Profile NGS test results from June 4, 2020 through May 7, 2021 were reviewed for variants involving ATM, ATR, BAP1, BLM, BRCA1, BRCA2, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCD2, MRE11A, NBN, PALB2, RAD51c and RAD51d, categorized as pathogenic, likely pathogenic or of unknown significance and had the tumor type identified. Additional chart review for PARP inhibitor therapy was performed in cases of breast, pancreatic, and ovarian cancer. Results (if a Case Study enter NA) A total of 599 cases were reviewed with 234 found to have variants in genes with possible PARP inhibitor sensitivity. Of these 2% (n=8) and 11% (n=43) of variants were categorized as pathogenic or likely pathogenic while most (n=334) were categorized as variants of unknown significance. The pathogenic and likely pathogenic variants included mutations in ATM (n=13), BRCA2 (n=12), BAP1 (n=8), FANCA (n=5), BRCA1 (n=4), NBM (n=3), FANCD2 (n=2), ATR (n=1), CDK12 (n=1), FANCC (n=1), and RAD51d (n=1), and frameshift (n=19) and nonsense (n=19) alterations were most common. Non-small cell lung cancer was the most frequent tumor type identified (n=78). At this time no PARP inhibitors were identified for use in cases of breast (n=9), pancreatic (n=3) and ovarian cancer (n=33). Conclusion Review of our institutional results for mutations in HRD pathway genes identified possible PARP inhibitor sensitivity in 8% (46 of 599) of cases during the period of review in a wide variety of tumor types. Our results suggest that variants with possible sensitivity to PARP inhibitor therapy are frequently identified from many tumor types and should be a component of solid tumor mutation profiling.

Molecular characterization of high-grade serous ovarian cancers occurring in younger and older women

ObjectivesTo determine if the mutational landscapes and genomic features of homologous recombination DNA repair defects (HRD) vary between younger and older patients with high-grade serous ovarian cancer (HGSOC). MethodsYounger and older women were defined as bottom and top age quartiles, respectively. HGSOCs from 15 younger (median 49 years, range 35–53) and 15 older women (median 72 years, range 70–87) were subjected to whole-exome sequencing (WES). For validation, HGSOC WES data were obtained from The Cancer Genome Atlas (TCGA), including 38 younger (median 45 years, range 34–50) and 30 older women (median 74 years, range 68–84). Mutational profiles, BRCA1/2 status, genomic HRD features, and for TCGA cases RNA-sequencing-based HRD transcriptomic signatures were assessed. ResultsIn the institutional cohort, pathogenic germline BRCA1/2 mutations were more frequent in younger (5/15) than older women (0/15, p = 0.042). No somatic BRCA1/2 mutations were identified. HGSOCs from older patients preferentially displayed aging-related mutational signatures and, in contrast to younger patients, harbored CCNE1 amplifications (3/15, 20%). In the TCGA cohort, pathogenic germline BRCA1 (younger 8/38, older 0/30, p = 0.007) but not BRCA2 mutations (young 3/38, older 4/30, p = 0.691) were more frequent in younger patients. Again, no somatic BRCA1/2 mutations were identified. HGSOCs from younger women more frequently displayed genomic features of HRD (all, p < 0.05), a significant HRD gene-signature enrichment, but less frequently CCNE1 amplification (p = 0.05). Immunoreactive CLOVAR subtypes were more common in HGSOCs from younger women, and proliferative subtypes in HGSOCs from older women (p = 0.041). ConclusionsHGSOC patients diagnosed at an older age less frequently harbor pathogenic BRCA1 germline mutations and genomic features of HRD than younger women. Individualized treatment options, particularly pertaining to use of PARP inhibitors, in older women may be warranted.

Whole-exome analysis of metaplastic breast carcinomas with extensive osseous differentiation.

Metaplastic breast carcinoma (MBC) is a rare type of triple-negative breast cancer that shows vast histological and genetic heterogeneity. Osseous differentiation can be found in different subtypes of MBC. Whether MBCs with osseous differentiation are underpinned by specific genetic alterations has yet to be defined. The aim of this study was to investigate the repertoire of somatic mutations and copy number alterations (CNAs) in three MBCs with extensive osseous differentiation. Tumour and normal DNA samples from three MBCs with extensive osseous differentiation were subjected to whole-exome sequencing. Somatic mutations, CNAs and mutational signatures were determined by use of a validated bioinformatics pipeline. Our analyses revealed clonal TP53 hotspot mutations associated with loss of heterozygosity of the wild-type allele coupled with mutations affecting genes related to the Wnt and/or the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathways in all cases analysed. All cases showed a dominant mutational signature 1, with two cases showing a secondary signature 3 in addition to other features of homologous recombination DNA repair defects. The oncostatin M receptor gene, which plays a role in mesenchymal differentiation and bone formation, was found to be mutated in two MBCs with extensive osseous differentiation and in none of 35 previously published 35 MBCs. Our findings suggest that MBCs with osseous differentiation have somatic mutations similar to those of other forms of MBC.

Functional Categorization of BRCA1 Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

Because BRCA1 is a high-risk breast/ovarian cancer susceptibility gene, BRCA1 sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic BRCA1 variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, BRCA1 VUS may be categorized based on their functional impact on this pathway. Two hundred thirty-eight BRCA1 VUS-comprising most BRCA1 VUS known in the Netherlands and Belgium-were tested for their ability to complement Brca1-deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic BRCA1 variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families. All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants. We have functionally categorized 238 BRCA1 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.

Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer

Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.

Abstract OT2-03-02: A pilot trial of hyperthermia in combination with olaparib in breast cancer patients with chest wall recurrences

Abstract Background: Chest wall recurrences represent a significant source of morbidity for breast cancer patients causing symptoms including bleeding, ulceration, infection and pain. Approximately 5 to 10% of breast cancer patients undergoing a mastectomy will have a chest wall or nodal recurrence within ten years after surgery. Loco-regional recurrence isolated to the chest wall occurs in 50 to 70% of these patients and only one third of these patients having synchronous metastases. After radiation, patients with unresectable isolated loco-regional breast cancer have very few treatment options. Hyperthermia, temperature elevation to 40° to 45°C for 60 minutes, is a potent radiotherapy and chemotherapy sensitizer without significant toxicity. Additionally, superficial hyperthermia can improve the complete response and local control in breast cancer patients with chest wall disease. Chest wall hyperthermia induces degradation of the BRCA2 protein, thereby leading to defective homologous recombination DNA repair, and sensitization to PARP-1 inhibitors. Krawczyk and colleagues (2017) showed that mild hyperthermia innately sensitizes homologous recombination proficient tumor cells to treatment with a PARP-1 inhibitor in pre-clinical models. Olaparib, is an oral targeted therapy that is an inhibitor of the poly ADP ribose polymerase (PARP) and is currently approved for treatment for patients with metastatic ovarian cancer and metastatic breast cancer with BRCA1 or BRCA2 mutation. Our hypothesis is that the combination of olaparib and chest wall hyperthermia will be a safe and tolerable combination for breast cancer patients with chest wall metastases. Trial Design: In this novel dose-escalation pilot trial, breast cancer patients with chest wall recurrences will be treated with olaparib at the appropriate dose level, concurrently with chest wall hyperthermia involving heating the skin to 43 ° Celsius for 1 hour. We plan to treat patients first with olaparib given twice a day in tablet form at the appropriate dose level for one week and then add hyperthermia twice a week for 3 weeks, for a total duration of 4 weeks of treatment. Eligibility criteria: Female breast cancer patients regardless of ER/PR/HER2 status who have chest wall recurrences greater than 2 cm are eligible for this study. Patients are eligible after any line of therapy and if they have wild-type germ-line BRCA1 or BRCA2 status. Specific Aims: We will evaluate the safety and determine the maximum tolerated dose of the combination of olaparib and chest wall hyperthermia. We will assess the local progression free survival, one-year progression free survival, overall response and quality of life scores for these patients. We plan to obtain skin biopsies to evaluate biomarkers of homologous recombination deficiency including BRCA1, BRCA2, RAD51 as well as γH2AX as an indicator of DNA damage. Statistical Methods: A 3+3 dose-escalation design will be used to determine the MTD of olaparib in combination with hyperthermia. We will initially enroll 3 subjects at each dose level. If one of the three experiences that level of toxicity, we will accrue 3 more subjects at that dose. If at any time there are two or more dose-limiting toxicities (in the 3-6 subjects) on a given dose, we will terminate accrual to the trial. No patient will be treated at a higher dose until the 3 or 6 patients have completed their 30 day toxicity evaluation period at the current dose. The survival endpoints of PFS and local PFS will be analyzed using log-rank test with Kaplan-Meier curves, as well as Cox proportional hazard models. Present Accrual and target accrual: Target accrual is 12 patients in the next two years. This study opened in May of 2019 at Thomas Jefferson University in Philadelphia, PA with full support from AstraZeneca. Clinical trial information: NCT03955640 Citation Format: Saveri Bhattacharya, Matthew Schiewer, Rita C. Murphy, Pramila Rani Anne, Nicole Simone, Voichita Bar Ad, Maysa Abu Khalaf, Daniel P. Silver, Rebecca Jaslow, Frederick M. Fellin, Allison M. Zibelli, Ana Maria Lopez, Adam Berger, Theodore N. Tsangaris, Melissa A. Lazar, Alliric I. Willis, Paul Stauffer, Mark D. Hurwitz. A pilot trial of hyperthermia in combination with olaparib in breast cancer patients with chest wall recurrences [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-03-02.

Endometrial Cancers in BRCA1 or BRCA2 Germline Mutation Carriers: Assessment of Homologous Recombination DNA Repair Defects.

Endometrial cancer (EC) is not considered a component of the hereditary breast and ovarian cancer syndrome but can arise in patients with germline BRCA1/2 (gBRCA1/2) mutations. Biallelic BRCA1/2 alterations are associated with genomic features of homologous recombination DNA repair deficiency (HRD) in cancer. We sought to determine if ECs in gBRCA1/2 mutation carriers harbor biallelic alterations and/or features of HRD. Of 769 patients with EC who underwent germline panel testing, 10 pathogenic gBRCA1/2 mutation carriers were identified, and their tumor- and normal-derived DNA was subjected to massively parallel sequencing targeting at least 410 cancer-related genes. Three gBRCA1/2-associated ECs were identified in 232 ECs subjected to whole-exome sequencing by The Cancer Genome Atlas. Somatic mutations, copy number alterations, loss of heterozygosity, microsatellite instability (MSI), and genomic HRD features were assessed. Of the 13 patients included who had EC, eight harbored pathogenic gBRCA1 mutations and five harbored gBRCA2 mutations. Eight (100%) and two (40%) ECs harbored biallelic BRCA1 and BRCA2 alterations through loss of heterozygosity of the wild-type allele. All ECs harbored somatic TP53 mutations. One monoallelic/sporadic gBRCA2-associated EC had MLH1 promoter methylation and was MSI high. High large-scale state transition scores, a genomic feature of HRD, were found only in ECs with bi- but not monoallelic BRCA1/2 alterations. The Signature Multivariate Analysis HRD signature Sig3 was enriched in biallelic gBRCA1/2 ECs, and the three ECs from The Cancer Genome Atlas with BRCA1 biallelic alterations subjected to whole-exome sequencing displayed a dominant HRD-related mutational signature 3. A subset of gBRCA1/2-associated ECs harbor biallelic BRCA1/2 alterations and genomic features of HRD, which may benefit from homologous recombination-directed treatment regimens. ECs in BRCA2 mutation carriers might be sporadic and even MSI high, and may potentially benefit from immune-checkpoint inhibition.

Homologous recombination DNA repair defects in PALB2-associated breast cancers

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.

CDK12 controls G1/S progression by regulating RNAPII processivity at core DNA replication genes.

CDK12 is a kinase associated with elongating RNA polymerase II (RNAPII) and is frequently mutated in cancer. CDK12 depletion reduces the expression of homologous recombination (HR) DNA repair genes, but comprehensive insight into its target genes and cellular processes is lacking. We use a chemical genetic approach to inhibit analog‐sensitive CDK12, and find that CDK12 kinase activity is required for transcription of core DNA replication genes and thus for G1/S progression. RNA‐seq and ChIP‐seq reveal that CDK12 inhibition triggers an RNAPII processivity defect characterized by a loss of mapped reads from 3′ends of predominantly long, poly(A)‐signal‐rich genes. CDK12 inhibition does not globally reduce levels of RNAPII‐Ser2 phosphorylation. However, individual CDK12‐dependent genes show a shift of P‐Ser2 peaks into the gene body approximately to the positions where RNAPII occupancy and transcription were lost. Thus, CDK12 catalytic activity represents a novel link between regulation of transcription and cell cycle progression. We propose that DNA replication and HR DNA repair defects as a consequence of CDK12 inactivation underlie the genome instability phenotype observed in many cancers.

Abstract 2723: Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Abstract Chordomas are rare tumors of the axial skeleton and skull base with few therapeutic options and no clinically validated molecular drug targets. The value of comprehensive genomic analyses for guiding medical therapy of patients with advanced-stage chordoma is unknown. We performed whole-exome and genome sequencing of tumor and matched germline control samples from 11 patients with locally advanced or metastatic chordoma within the MASTER program, a prospective clinical sequencing program of the German Cancer Consortium. All patients were pretreated and had progressive disease prior to molecular analysis. Genomic profiling showed that advanced chordomas are frequently characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair. First, DNA copy number profiles showed high numbers of structural variants greater than 10 million base pairs in size in the majority of cases. Second, all patients harbored somatic aberrations of at least 2 genes known to be involved in HR, and 10/11 cases harbored somatic alterations in 3 or more HR pathway genes. For example, 8 patients showed heterozygous BRCA2 deletions, which were associated with heterozygous deletions of ERCC6 in 6 patients and RAD54L in 7 patients, as well as PTEN alterations (heterozygous deletion, heterozygous mutation and deletion of the wildtype allele or loss of heterozygosity). Other recurrently altered HR genes included ATR, CHEK2, FANCC, FANCD2, FANCG, RAD18, RAD51B, and XRCC3. Third, pathogenic germline alterations were detected in 3 patients. A heterozygous BRCA2 frameshift mutation (p.T3085fs*26; ACMG Class 5), a heterozygous NBN frameshift mutation (p.K219Nfs*16; ACMG Class 5), and a heterozygous CHEK2 missense mutation (p.R145W; ACMG Class 4) were accompanied by somatic deletion of the respective wildtype alleles. Fourth, a mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and coincided with genomic instability. The high prevalence of an HR deficiency “footprint” in chordoma patients prompted us to explore the clinical efficacy of the poly(ADP-ribose) polymerase(PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells. Olaparib treatment of a patient whose tumor showed a prominent exposure to an HR deficiency-associated mutational signature, a high degree of genomic instability, and 13 heterozygous HR gene alterations halted tumor growth for 10 months. Whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain, providing novel insight into the mechanisms of PARP inhibitor resistance. In summary, our study has uncovered a key biological feature of advanced chordoma that represents an immediately actionable therapeutic target and provides a rationale for genomics-guided clinical trials of PARP inhibition in this intractable tumor entity. Citation Format: Stefan Gröschel, Daniel Hübschmann, Francesco Raimondi, Peter Horak, Gregor Warsow, Martina Fröhlich, Barbara Klink, Laura Gieldon, Barbara Hutter, Kortine Kleinhenz, David Bonekamp, Oliver Marschal, Priya Chudasama, Jagoda Mika, Marie Groth, Sebastian Uhrig, Stephen Krämer, Christoph Heining, Christoph Heilig, Daniela Richter, Eva Reisinger, Katrin Pfütze, Roland Eils, Stephan Wolf, Christof von Kalle, Christian Brandts, Claudia Scholl, Wilko Weichert, Stephan Richter, Sebastian Bauer, Roland Penzel, Evelin Schröck, Albrecht Stenzinger, Richard Schlenk, Benedikt Brors, Robert Russell, Hanno Glimm, Matthias Schlesner, Stefan Fröhling. Defective homologous recombination DNA repair as therapeutic target in advanced chordoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2723.

Abstract 1686: Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors

Abstract Most adult gastrointestinal stromal tumors (GIST) are driven by activating KIT or PDGFRA mutations. The remaining 10-15% of cases, often referred to as wildtype (WT) GIST, display either alterations of the succinate dehydrogenase complex (SDH) or RAS pathway mutations. To gain additional insight into the biology of GIST, we performed whole-exome or genome and RNA sequencing in 38 GIST patients (WT, n=15; KIT-mutant, n=21; PDGFRA-mutant, n=2) enrolled in a prospective molecular stratification trial of NCT Heidelberg/Dresden and the German Cancer Consortium (DKTK) designed for younger adults with advanced-stage cancer across histologies and patients with rare tumors (NCT/DKTK MASTER). Of the 15 patients with WT GIST, 3 had pathogenic germline mutations in NF1 and 9 harbored SDH alterations (germline, n=5; somatic, n=4). In the 3 patients with quadruple-negative GIST - defined by the absence of KIT, PDGFRA, SDH, or RAS pathway alterations - we detected novel gene fusions affecting RET, FGFR2, and FGF4, respectively. To delineate biologically relevant subgroups of GIST based on RNA sequencing data from the entire cohort (n=34), we used 3 different clustering methods and 4 different measures of stability and consistency. Despite the underlying clinical and molecular heterogeneity, we identified 3 distinct transcriptional subgroups that were characterized by (i) SDH deficiency, (ii) recurrent somatic RB1 alterations and mutational signatures associated with defective homologous recombination DNA repair, and (iii) elevated PDGFRA expression, respectively. Furthermore, we used random forest analysis to identifiy genes that are significantly (p&amp;lt;0.005) differentially expressed between the 3 subgroups. Interestingly, quadruple-negative cases did not form a separate cluster or clustered within a specific subgroup. Collectively, our data illustrate the molecular heterogeneity of advanced-stage GIST and support comprehensive molecular profiling approaches to capture the entire spectrum of clinically actionable genetic alterations, such as diverse fusion genes affecting kinase signaling pathways in quadruple-negative cases or pathogenic germline mutations in patients with inconspicuous family histories. The finding of two separate transcriptional clusters among patients with SDH-proficient GIST may be reflective of distinct regulatory pathways whose molecular underpinnings and clinical actionability warrant further study. Citation Format: Peter Horak, Matea Hajnic, Laura Gieldon, Mario Hlevnjak, Susan Richter, Barbara Hutter, Johanna Falkenhorst, Sebastian Uhrig, Gregor Warsow, Nagarajan Paramasivam, Stefan Gröschel, Barbara Klink, Simon Kreutzfeldt, Christoph Heining, Christoph E. Heilig, Martina Fröhlich, Stephan Richter, Christian Brandts, Wilko Weichert, Philipp Jost, Olaf Neumann, Marc Zapatka, Albrecht Stenzinger, Alexander Marx, Benedikt Brors, Evelin Schröck, Sebastian Bauer, Peter Hohenberger, Hanno Glimm, Claudia Scholl, Stefan Fröhling. Comprehensive genomic and transcriptomic profiling of gastrointestinal stromal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1686.

Homologous recombination DNA repair defects in copy-number high serous-like endometrial cancers

Objective: Endometrial cancers (ECs) of copy-number high (serous-like) molecular subtype are characterized by high levels of copy-number alterations and recurrent TP53 mutations akin to high-grade serous ovarian (HGSOCs) and basal-like breast cancers, which are sensitive to platinum agents and/or PARP inhibitors. We aimed to assess whether copy-number high (serous-like) ECs show defects in homologous recombination DNA repair (HRD) akin to HGSOCs and basal-like breast cancers.

Defective homologous recombination DNA repair as therapeutic target in advanced chordoma

Chordomas are rare bone tumors with few therapeutic options. Here we show, using whole-exome and genome sequencing within a precision oncology program, that advanced chordomas (n = 11) may be characterized by genomic patterns indicative of defective homologous recombination (HR) DNA repair and alterations affecting HR-related genes, including, for example, deletions and pathogenic germline variants of BRCA2, NBN, and CHEK2. A mutational signature associated with HR deficiency was significantly enriched in 72.7% of samples and co-occurred with genomic instability. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib, which is preferentially toxic to HR-incompetent cells, led to prolonged clinical benefit in a patient with refractory chordoma, and whole-genome analysis at progression revealed a PARP1 p.T910A mutation predicted to disrupt the autoinhibitory PARP1 helical domain. These findings uncover a therapeutic opportunity in chordoma that warrants further exploration, and provide insight into the mechanisms underlying PARP inhibitor resistance.

Abstract P2-01-02: Whole exome sequencing analysis of the progression from ductal carcinoma in situ to invasive ductal carcinoma

Abstract Introduction: Ductal carcinoma in situ (DCIS) is a bona fide non-obligate precursor of invasive carcinoma. Single cell sequencing studies have revealed intra-lesion genetic heterogeneity in DCIS and shown that progression to invasive ductal carcinoma (IDC) may occur through different mechanisms, including the selection of a subpopulation of tumor cells, acquisition of new genetic alterations or multi-clonal invasion. Here, we sought to investigate the genetic heterogeneity of DCIS, and to document further the clonal selection process accompanying progression to IDC. Materials and methods: Synchronous DCIS (n=16) and IDC (n=15) samples from 14 patients were microdissected separately, and DNA samples of tumor and matched normal tissues were subjected to whole-exome sequencing (WES; n=27) or massively parallel targeted sequencing of all coding regions of ≥410 cancer-related genes (n=4). Somatic genetic alterations and mutational signatures were identified using state-of-the-art bioinformatics algorithms. PyClone was employed to define the clonal architecture of each DCIS and IDC and infer the clonal shifts accompanying progression from DCIS to IDC. Results: DCIS were found to harbor recurrent somatic mutations affecting PIK3CA (50%), GATA3 (44%), TP53 (38%), CBFB (19%), PTEN (13%), and AKT1 (13%), which are genes known to be significantly mutated in invasive breast cancers. Despite the genomic similarities between matched DCIS and IDCs, NOTCH2 and MYC were found to be amplified solely in the IDC component of two cases, and PPM1D amplification was restricted to the DCIS component of another case. The mutational signature ascribed to aging (i.e. signature 1) was the predominant mutational signature in the DCIS and IDCs analyzed. PyClone analysis revealed that all synchronous DCIS and IDC studied here were clonally related and confirmed the previous observation that DCIS displays intra-lesion genetic heterogeneity. Evidence of clonal selection in the progression from DCIS to IDC was observed in three cases, whereby a minor DCIS subclone likely constituted the substrate for the development of IDC. In one of these cases, from a patient with a BRCA1 germline pathogenic mutation, we observed a shift from the mutational signature associated with defective homologous recombination DNA repair (i.e. signature 3) to the APOBEC-related mutational signatures (i.e. signatures 2 and 13) in the progression from DCIS to IDC. Conclusion: Intra-lesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC. Our findings corroborate the notion that DCIS is a direct non-obligate precursor of IDC, and that clonal selection in the progression of DCIS to IDC may be present in a subset of cases, but is unlikely to constitute the most frequent mechanism of progression. Citation Format: Lee JY, Bi R, Pareja F, Geyer FC, Brown D, Wen HY, Norton L, Hicks J, Weigelt B, Reis-Filho JS. Whole exome sequencing analysis of the progression from ductal carcinoma in situ to invasive ductal carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-02.

Abstract P4-04-01: The landscape of somatic genetic alterations in breast cancers from CHEK2 germline mutation carriers

Abstract Introduction: Checkpoint kinase 2 (CHEK2) is a tumor suppressor gene, which regulates cell cycle in response to DNA damage response. Selected CHEK2 germline mutations have been shown to confer an increased risk of breast cancer development. Multiple founder mutations in CHEK2 have been identified, and meta analyses have shown that CHEK2 truncating variants confer a higher breast cancer risk than missense variants. Here, we assessed the phenotype and repertoire of genetic alterations of breast cancers from 33 patients with CHEK2 pathogenic germline variants. Materials and methods: We performed targeted capture massively parallel sequencing (≥410 genes) of tumor and normal samples from 13 patients with CHEK2 pathogenic germline variants, and retrieved whole exome sequencing (WES) data (BAM files) of tumor and normal samples from 20 patients with CHEK2 germline pathogenic variants included in the TCGA breast cancer study. In addition, we retrieved WES data of BRCA1, BRCA2 and ATM associated breast cancers from TCGA and Weigelt et al. (JNCI 2018). Somatic mutations, copy number alterations, mutational signatures and large-scale transitions (LSTs) were defined using state-of-the-art bioinformatics algorithms. Results: Of the 33 CHEK2-associated breast cancers included in this study, 21 had missense and 12 had loss-of-function (LoF) germline mutations, and 81% were ER-positive and 12% HER2-positive. CHEK2-associated breast cancers statistically significantly less frequently displayed an ER-negative/HER2-negative phenotype (0%) than BRCA1- (80%) or BRCA2-associated (33%) breast cancers (BRCA1, p&amp;lt;0.0001 for both comparisons), but were similar to ATM-associated breast cancers. Biallelic inactivation of CHEK2 through loss of heterozygosity (LOH) of the wild-type allele was present in 17 of 33 samples (52%). LOH of the CHEK2 wild-type allele was significantly more frequent in tumors with LOF mutations than in those with missense mutations (78% vs 36%, respectively; p=0.0394). PIK3CA (36%) and GATA3 (33%) were the two most recurrently mutated genes in these samples. TP53 somatic mutations were detected in five cases, four of which harbored missense CHEK2 germline mutations. Unlike BRCA1- and BRCA2-associated breast cancers, but akin to ATM-associated breast cancers, CHEK2-associated breast cancers lacked the mutational signature associated with homologous recombination (HR) DNA repair defects (i.e. signature 3) and only five cases displayed high LST scores. Conclusion:CHEK2-associated breast cancers are phenotypically and genetically distinct from BRCA1- and BRCA2-associated breast cancers, but similar to ATM-associated breast cancers. Akin to ATM-associated breast cancers, CHEK2-associated breast cancers are preferentially ER-positive, lack genomics features consistent with defective HR, and have a repertoire of somatic genetic alterations similar to those of non-BRCA1/2 ER-positive breast cancers. Our results suggest that either CHEK2 germline mutations contribute to an increased risk of breast cancer independently of the HR DNA repair defects or that the mutational signatures caused by CHEK2 pathogenic germline mutations differ from those caused by pathogenic germline mutations affecting bona fide HR-related genes (e.g. BRCA1, BRCA2 and PALB2). Citation Format: Kumar R, Pei X, Selenica P, Wen HY, Powell S, Robson M, Riaz N, Reis-Filho JS, Weigelt B, Mandelker D. The landscape of somatic genetic alterations in breast cancers from CHEK2 germline mutation carriers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-04-01.