ATM Variants Research Articles

Comprehensive germline profiling of patients with breast cancer: initial experience from a Familial Cancer Clinic.

Breast cancer is the most common cancer among Indian females. There is limited data on germline profiling of breast cancer patients from India. The objective of the current study was to analyse the frequency and spectrum of germline variant profiles and clinicopathological characteristics of breast cancer patients referred to our Familial Cancer Clinic (FCC). It is a single-centre audit of patients with a confirmed diagnosis of breast carcinoma referred to our FCC from January 2017 to 2020. All patients underwent pretest counselling. Genetic testing was done by multigene panel testing by next-generation sequencing along with reflex multiplication ligation-dependent probe amplification for BRCA1 and 2. The variants were classified based on American College of Medical Genetics guidelines. Demographic and clinicopathological details were extracted from the case record files. One hundred and fifty-five patients were referred to the FCC and underwent pretest counselling. A total of 99 (63.9%) patients underwent genetic testing. Among them, 62 patients (62/99 = 62.6%) had a germline variant. A pathogenic/likely pathogenic (P/LP) germline variant was identified in 41 (41.4%) of the patients who underwent testing. Additional variants of unknown significance (VUS) were identified in seven patients who also carried a P/LP variant. VUS alone was detected in 21 patients (21/99 = 21.2%). Among the P/LP pathogenic variants (PV), BRCA 1 PV were seen in 27 patients (65.8%), BRCA 2 variants in 7 patients (17.1%), ATM variants in 3 patients (7.3%) and RAD51, TP53, CHEK2 and HMMR in 1 patient each. Variants were significantly more common in patients with a family history (FH) of malignancy than those without FH (58.5% versus 29.5%; p = 0.013). Age and triple-negative histology were not found to be significantly associated with the occurrence of P/LP PVs. We report a 41% P/LP variant rate in our selected cohort of breast cancer patients, with variants in BRCA constituting 83% and non-BRCA gene variants constituting 17%.

Abstract B001: Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer?

Abstract Breast cancer (BC) is the most common cancer and leading cause of cancer mortality in women. Average lifetime BC risk for a woman in the United States is 13%. However, pathogenic variants in BC susceptibility genes such as BRCA1/2, ATM, and RAD51C, or high polygenic risk scores substantially increase BC risk. Women with a family history of BC and/or ovarian cancer, BC < age 40, or BC and second primary tumor are considered high-risk with an increased likelihood of having a pathogenic variant in a BC susceptibility gene. Yet, more than 60% of women with high-risk BC test negative for coding variants. In these women, possible causal variation could be non-coding in deep intronic or regulatory regions, or cryptic structural variants (SV). To determine whether deep intronic splicing contributes to BC in high-risk women testing negative for pathogenic coding variants in BC genes, we analyzed the intronic sequences of 649 BRCA1/BRCA2 negative individuals with high-risk BC. The samples had undergone targeted capture sequencing of exons and introns of more than 100 target genes including ATM and RAD51C. From the 649 samples, we identified three intronic splicing variants in ATM and RAD51C in 10 samples (1.5%) that had SpliceAI delta scores >0.5 suggesting changes to splicing. One splicing donor gain variant, RAD51C c.145+246A>T, was found exclusively in women of self-reported African ancestry at a frequency of 0.047. In gnomAD, the variant was found exclusively in individuals of African ancestry at a frequency of 0.012. A 1.3-fold enrichment in variant frequency was observed in AFR women under 65 with breast cancer compared to age-matched cancer free controls in the All of Us dataset. Using lymphoblastoid-cell derived RNA from carriers of RAD51C c.145+246A>T, we performed RT-PCR and cDNA amplification targeting the exons surrounding the variant and observed a lengthened exon. Targeted amplicon sequencing also confirmed the presence of intronic sequence in the variant cDNA. The introduced intronic sequence contains a UGA stop codon 12 bp from the end of the exon which could led to loss of RAD51C function. We have created minigene vectors in the RHCglo background and are performing minigene splicing assays to validate the splicing changes in ATM due to intronic variants. Gene functional assays and association studies will be conducted to determine the functional effects and risk conferred by these variants. Understanding the level of contribution of deep intronic splicing in known BC susceptibility genes is critically important, as results impact sequencing offered to patients, and if positive, their medical management. Citation Format: Mwangala P. Akamandisa, Bradley Wubbenhorst, Kurt D'Andrea, Heather Symecko, Rhonda Gillette, Payal D. Shah, Angela R. Bradbury, Kara N. Maxwell, Susan M. Domchek, Katherine L. Nathanson. Does deep intronic splicing in breast cancer susceptibility genes contribute to breast cancer? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B001.

Uncovering the clinical relevance of unclassified variants in DNA repair genes: a focus on BRCA negative Tunisian cancer families.

Introduction: Recent advances in sequencing technologies have significantly increased our capability to acquire large amounts of genetic data. However, the clinical relevance of the generated data continues to be challenging particularly with the identification of Variants of Uncertain Significance (VUSs) whose pathogenicity remains unclear. In the current report, we aim to evaluate the clinical relevance and the pathogenicity of VUSs in DNA repair genes among Tunisian breast cancer families. Methods: A total of 67 unsolved breast cancer cases have been investigated. The pathogenicity of VUSs identified within 26 DNA repair genes was assessed using different in silico prediction tools including SIFT, PolyPhen2, Align-GVGD and VarSEAK. Effects on the 3D structure were evaluated using the stability predictor DynaMut and molecular dynamics simulation with NAMD. Family segregation analysis was also performed. Results: Among a total of 37 VUSs identified, 11 variants are likely deleterious affecting ATM, BLM, CHEK2, ERCC3, FANCC, FANCG, MSH2, PMS2 and RAD50 genes. The BLM variant, c.3254dupT, is novel and seems to be associated with increased risk of breast, endometrial and colon cancer. Moreover, c.6115G>A in ATM and c.592+3A>T in CHEK2 were of keen interest identified in families with multiple breast cancer cases and their familial cosegregation with disease has been also confirmed. In addition, functional in silico analyses revealed that the ATM variant may lead to protein immobilization and rigidification thus decreasing its activity. We have also shown that FANCC and FANCG variants may lead to protein destabilization and alteration of the structure compactness which may affect FANCC and FANCG protein activity. Conclusion: Our findings revealed that VUSs in DNA repair genes might be associated with increased cancer risk and highlight the need for variant reclassification for better disease management. This will help to improve the genetic diagnosis and therapeutic strategies of cancer patients not only in Tunisia but also in neighboring countries.

Abstract A002: Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy

Abstract The DNA damage response is crucial for the efficacy of radiotherapy (RT), prompting efforts to target this pathway with radiosensitizers to improve outcomes. Early clinical trials evaluating this approach, however, have failed to yield evidence of benefit in unselected populations. Here we demonstrate that a genotype-directed combination of DNA repair inhibition and RT can unleash the significant therapeutic benefit of this approach in pre-clinical models and present first experience from a Phase I/II trial (NCT05566574) testing this concept. Using CRISPR/Cas9, we created isogenic murine models (Atm-/- and WT) in 4T1 breast and CT26 colorectal cancer models, and then studied the synergy between camonsertib and RT across genotypes. In vitro, camonsertib radio-sensitized both Atm-/- and WT models, but was more potent and synergistic in Atm-/- models. In the orthotopically implanted 4T1 model, the combination of camonsertib (30 mg/kg, 3 days on; 4 days off) with RT (20Gy x 1) significantly delayed tumor growth in Atm-/- tumors compared to WT (p < 0.001; n=10 mice per group). Overall survival (OS) also improved in mice with Atm-/- tumors (20% alive at 150 days) vs. WT (0% alive; p < 0.001; log-rank test). Similar results were observed using other RT dose-fractionations (4Gy x 5 daily, 6Gy x 3, 8Gy x 2) with camonsertib. In the flank-implanted CT26 model, Atm-/- tumors had markedly improved response to combination therapy with 7/8 mice achieving a complete response (CR) compared to only 1/8 WT tumors (p<0.001). OS was also significantly improved with 87% of mice with CT26 Atm-/- tumors cured compared to 0% of CT26 WT tumors (p < 0.001). To evaluate the genotype-specificity for synergy from combination therapy, we performed similar experiments in a 4T1 Brca1-/- model, a key mediator of homologous recombination, and observed an absence of synergy, consistent with our hypothesis. Analysis of the microenvironment of 4T1 Atm-/- treated with combination therapy by flow cytometry did not reveal significant increases in CD4 or CD8 T-cells compared to vehicle nor increases in activated CD8 T-cells (Ki67+, PD1+, or GzmB+) suggesting the therapeutic benefit was primarily mediated by cell intrinsic effects. Similarly, re-challenge experiments in cured mice did not identify strong evidence of immunologic memory, again suggesting a modest immunologic role for efficacy of combination therapy in this setting. Based on these results, we initiated a Phase I/II study of camonsertib with RT (4Gy x 5) in patients with metastatic cancer that require palliative RT and have mutations in ATM (pathogenic or variant of unknown significance). The first two patients with pathogenic ATM mutant tumors enrolled in the Phase I portion of the study have demonstrated metabolic CR to combination therapy on PET scan. Overall, our findings suggest that genotype-directed concurrent radio-sensitization could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments. Further research to test this hypothesis is ongoing. Citation Format: Victor Ng, Sonali Sinha, Ardijana Novaj, Jennifer Ma, Niamh McDermott, Ana Longhini, Rui Gardner, Ezra Rosen, Simon Powell, Diana Mandelker, Atif Khan, Jeremy Setton, Anne Roulston, Stephen Morris, Maria Koehler, Nancy Lee, Jorge Reis-Filho, Nadeem Riaz. Genotype-directed synthetic cytotoxicity of ATR inhibition with radiotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A002.

Novel Pathogenic Variants in Hereditary Cancer Syndromes in a Highly Heterogeneous Cohort of Patients: Insights from Multigene Analysis.

Cancer is a major global public health challenge, affecting both quality of life and mortality. Recent advances in genetic research have uncovered hereditary cancer syndromes (HCS) that predispose individuals to malignant neoplasms. While traditional single-gene testing has focused on high-penetrance genes, the past decade has seen a shift toward multigene panels, which facilitate the analysis of multiple genes associated with specific HCS. This approach reveals variants in less-studied gene regions and improves our understanding of cancer predisposition. In a study composed of Russian patients with clinical signs of HCS, we used a multigene hereditary cancer panel and revealed 21.6% individuals with pathogenic or likely pathogenic genetic variants. BRCA1/BRCA2 mutations predominated, followed by the CHEK2 and ATM variants. Of note, 16 previously undescribed variants were identified in the MUTYH, GALNT12, MSH2, MLH1, MLH3, EPCAM, and POLE genes. The implications of the study extend to personalized cancer prevention and treatment strategies, especially in populations lacking extensive epidemiological data, such as Russia. Overall, our research provides valuable genetic insights that give the way for further investigation and advances in the understanding and management of hereditary cancer syndromes.

Cancer predisposition signaling in Beckwith-Wiedemann Syndrome drives Wilms tumor development.

Wilms tumor (WT) exhibits structural and epigenetic changes at chromosome 11p15, which also cause Beckwith-Wiedemann Syndrome (BWS). Children diagnosed with BWS have increased risk for WT. The aim of this study is to identify the molecular signaling signatures in BWS driving these tumors. We performed whole exome sequencing, methylation array analysis, and gene expression analysis on BWS-WT samples. Our data were compared to publicly available nonBWS data. We categorized WT from BWS and nonBWS patients by assessment of 11p15 methylation status and defined 5 groups- control kidney, BWS-nontumor kidney, BWS-WT, normal-11p15 nonBWS-WT, altered-11p15 nonBWS-WT. BWS-WT samples showed single nucleotide variants in BCORL1, ASXL1, ATM and AXL but absence of recurrent gene mutations associated with sporadic WT. We defined a narrow methylation range stratifying nonBWS-WT samples. BWS-WT and altered-11p15 nonBWS-WT showed enrichment of common and unique molecular signatures based on global differential methylation and gene expression analysis. CTNNB1 overexpression and broad range of interactions were seen in the BWS-WT interactome study. While WT predisposition in BWS is well-established, as are 11p15 alterations in nonBWS-WT, this study focused on stratifying tumor genomics by 11p15 status. Further investigation of our findings may identify novel therapeutic targets in WT oncogenesis.

Polygenic Risk Score Modifies Prostate Cancer Risk of Pathogenic Variants in Men of African Ancestry.

These findings highlight the importance of considering rare and common variants to comprehensively assess prostate cancer risk in men of African ancestry.

Clinicopathologic and genetic analysis of invasive breast carcinomas in women with germline CHEK2 variants

PurposeGermline pathogenic variants in checkpoint kinase 2 (CHEK2) are associated with a moderately increased risk of breast cancer (BC). The spectrum of clinicopathologic features and genetics of these tumors has not been fully established.MethodsWe characterized the histopathologic and clinicopathologic features of 44 CHEK2-associated BCs from 35 women, and assessed responses to neoadjuvant chemotherapy. A subset of cases (n = 23) was additionally analyzed using targeted next-generation DNA sequencing (NGS).ResultsMost (94%, 33/35) patients were heterozygous carriers for germline CHEK2 variants, and 40% had the c.1100delC allele. Two patients were homozygous, and five had additional germline pathogenic variants in ATM (2), PALB2 (1), RAD50 (1), or MUTYH (1). CHEK2-associated BCs occurred in younger women (median age 45 years, range 25–75) and were often multifocal (20%) or bilateral (11%). Most (86%, 38/44) were invasive ductal carcinomas of no special type (IDC-NST). Almost all (95%, 41/43) BCs were ER + (79% ER + HER2-, 16% ER + HER2 + , 5% ER-HER2 +), and most (69%) were luminal B. Nottingham grade, proliferation index, and results of multiparametric molecular testing were heterogeneous. Biallelic CHEK2 alteration with loss of heterozygosity was identified in most BCs (57%, 13/23) by NGS. Additional recurrent alterations included GATA3 (26%), PIK3CA (226%), CCND1 (22%), FGFR1 (22%), ERBB2 (17%), ZNF703 (17%), TP53 (9%), and PPM1D (9%), among others. Responses to neoadjuvant chemotherapy were variable, but few patients (21%, 3/14) achieved pathologic complete response. Most patients (85%) were without evidence of disease at time of study (n = 34). Five patients (15%) developed distant metastasis, and one (3%) died (mean follow-up 50 months).ConclusionAlmost all CHEK2-associated BCs were ER + IDC-NST, with most classified as luminal B with or without HER2 overexpression. NGS supported the luminal-like phenotype and confirmed CHEK2 as an oncogenic driver in the majority of cases. Responses to neoadjuvant chemotherapy were variable but mostly incomplete.

Abstract C038: BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia

Abstract One in 70 women will develop ovarian cancer over their lifetime in the US and epithelial ovarian cancer (EOC) remains the deadliest gynecological cancer. EOC is a heterogenous disease and high-grade serous carcinoma (HGSOC) is the most common histotype. It is genetically unstable due to pathogenic variants in BRCA1, BRCA2, PALB2, ATM, and RAD51 genes involved in DNA damage response and repair which are part of the hereditary breast and ovarian cancer syndrome (HBOC). In women with a BRCA1 or BRCA2 mutation undergoing risk-reducing surgery, 3-17% have pre-neoplastic and neoplastic lesions in their fallopian tubes. These lesions, known as serous tubal intra-epithelial carcinoma (STIC), display differential loss of heterozygosity of BRCA1/2. New insights into BRCA2 function could provide novel opportunities for preventative interventions in individuals with this mutation, and perhaps new treatment opportunities for HGSC associated with BRCA2 heterozygosity. We have identified distinct metabolic and mitochondrial deficits in BRCA2 mutation carriers compared to BRCA1 carriers and non-carriers. Fallopian tube epithelial cell lines derived from BRCA2 mutation carriers showed increased levels of mitochondrial respiration and activity followed by BRCA1 mutation carriers and non-carriers. Transmission electron microscopy of BRCA2 mutation carriers showed increased disrupted mitochondrial networks when compared to wild type, suggesting that mutation status may contribute to increased mitochondrial fission. These observations reveal an entirely new aspect of BRCA2 biology and further studies will disaggregate the effects of BRCA2 on mitochondrial function, versus DNA damage repair. Ultimately, this work will provide new insight into the adaptive changes within FT cells which promote neoplastic transformation and inform how high-grade serous carcinoma (HGSC) cells distinctively respond to microenvironmental stimuli. Citation Format: Jovanka Ravix, Iru Paudel, Ramlogan Sowamber, Gauthami Pulivendala, David Lombard, Sophia George. BRCA2 haploinsufficiency driven neoplastic events in the fallopian tube epithelia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C038.

Incidence and Prognosis of Primary Malignancies in Ataxia-Telangiectasia Patients: A Report from the Ataxia-Telangiectasia Clinical Center (ATCC)

Biallelic germline mutations in the ATM gene result in Ataxia-Telangiectasia (AT), a classic DNA repair disorder associated with a high incidence of primary malignancies. The lack of standardized cancer treatment approaches for patients with AT is believed to contribute to poor survival after diagnosis. Robust characterization of the incidence, outcomes, and ATM variant enrichment of large cohorts of individuals with AT who develop malignancies are needed to inform therapeutic decision-making. The AT Clinical Center (ATCC) at Johns Hopkins Hospital is a large, retrospective cohort that prospectively collects outcomes data on individuals diagnosed with AT in the US. Standardized incidence ratios (SIRs) were used to compare cancer incidence to that of age-, sex-, and year-matched US general population using the SEER database. Standardized mortality ratios (SMRs) and mortality rate ratios were used to estimate mortality risk compared to the general population. Among 508 total participants with AT, 84 developed cancers (16.5%): 39 non-Hodgkin lymphoma (NHL), 7 Hodgkin lymphoma, 16 leukemia (n=13 of T cell origin), and 22 carcinomas. SIRs were 355.9, 67.4, 45.6, and 18.0, respectively. Median (interquartile range) age at cancer diagnosis was 14.3 years (10.3-22.8), with NHL occurring in the youngest (12.4 years, IQR 7.3-16.7) and solid tumors in the oldest (26.3 years, IQR 18.7-29.0) individuals. The SMR was 24.6 (95% CI: 21.1-28.4) for the overall cohort of AT patients and 232.9 (95% CI: 178.1-299.2) among those with cancer. Median survival following a cancer diagnosis was 2.5 years (95% CI: 0.1-14.4). The post-cancer mortality rate ratio was 2.2 (95% CI: 1.1-4.3, p=0.025) comparing AT patients with cancer treated without dose reduction or for whom dosing was unknown (n=24) versus AT patients with cancer who received dose reduced chemotherapy (n= 38). Among those who received chemotherapy (n=62), 11/24 (45.8%, 95% CI: 25.6-67.2) treated with standard dosing and 24/38 (63%, 95% CI: 46.0-78.2) treated with reduced dosing experienced toxicity (p=0.20). ATM variant associations were detected by single variant analysis among 247 patients with AT who had confirmed biallelic ATM mutations via whole exome sequencing. Individuals with AT are at very high-risk of primary malignancies, of which NHL is most common. Prognosis is poor regardless of standard versus dose reduced chemotherapy regimens, which calls for novel therapies.

Characterizing ATM Aberrations in Chronic Lymphocytic Leukemia (CLL): Prognostic Implications and Sensitivity to PARP Inhibition

Introduction ATM mutations (muts) frequently occur in CLL, yet their influence on ATM protein function and DNA break repair is poorly elucidated. Our prior study demonstrated functional hypomorphism of the rare germline variant L2307F in ATM and a higher occurrence of 11q deletions (del(11q)) with ATM germline variants in CLL (Lampson et al, JCO 2023). However, the role of other ATM germline variants in association with del(11q) or somatic muts remains unknown. Here, we analyzed the characteristics of CLL patients (pts) with ATM aberrancies and evaluated ATM function in primary CLL cells. Methods We retrospectively analyzed 278 CLL pts with ATM aberrancy, evaluated by clinical targeted sequencing from 2014 to 2019, as per our recent study (Lampson et al, JCO 2023). ATM, KAP1, and H2AX phosphorylation levels were examined after etoposide or irradiation (IR) exposure, by western blot or flow cytometry, in primary CLL cells with or without ATM aberrancy. We assessed the inhibitory effect of talazoparib, a PARP inhibitor, on CLL proliferation over 14 days using flow cytometry and Cell Trace Violet dye. Results We identified 294 ATM variants, 146 unique (germline N=74, somatic N=72). In CLL pts, 58 had dual ATM allele loss (biallelic group), including del(11q) together with only germline ATM variant(s) (N=29), only somatic mut(s) (N=18), or both (N= 11). 168 pts had ATM muts or variants without del(11q), including only germline (N=137), somatic (N=19), or both (N=12) ( ATM group), and 52 pts had del(11q) alone (11q group). Complex karyotype increased with ATM allele deficiency, particularly with 5+ abnormalities (complex karyotype 5+, biallelic vs 11q vs ATM: 22.6% vs 10.6% vs 5.8%, P=0.002). Unmutated IGHV was significantly higher in the groups with del(11q) versus ATM alone (biallelic vs 11q vs ATM: 85.1% vs 87.8% vs 53.9%, p<0.001). Interestingly, when we compared IGHV status in the ATM alone group with or without somatic ATM muts, unmutated IGHV significantly increased in those with somatic muts ( ATM-somatic vs ATM-germline alone, 81.8% vs 43.6%, p<0.001), and karyotypic complexity tended to be higher in the somatic ATM mut group (P=0.077). This might suggest that pts with ATM somatic muts have clinical characteristics and pathogenicity more similar to del(11q) as compared to those with germline muts. SF3B1 muts were more common in those with del(11q) (biallelic vs 11q vs ATM: 20.7% vs 26.9% vs 12.5%, P=0.036). For functional evaluation, we compared the five groups of primary CLL samples: del(11q) with somatic ATM mut with/without germline (11q+ ATM-S), del(11q) alone with no ATM muts (11q), somatic ATM muts without del(11q) with/without germline ( ATM-S), germline ATM muts alone ( ATM-G), and no ATM aberrancies (wild type, WT). Western blot demonstrated significantly lower phosphorylated protein levels (pATM, pKAP1, γH2AX) in response to DNA damage with etoposide in ATM aberrancy groups compared to WT (WT vs 11q, vs ATM-S, vs 11q+ ATM-S, all P<0.001 except for WT vs 11q in γH2AX, P<0.05) (Figure 1A). Flow cytometry also showed significantly lower phosphorylation levels of ATM after IR in 11q, ATM-S and 11q+ ATM-S groups compared to WT, while ATM-G group didn't show significance. To assess ATM deficient cell sensitivity to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The 11q+ ATM-S group exhibited significantly greater sensitivity to talazoparib at lower dosage compared to the WT (talazoparib 0.1 μM, P<0.05; 0.5 μM, P=0.098) (Figure 1B). The monoallelic ATM deficient groups (del11q and ATM-G) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusions Pts with ATM somatic aberrancy including del11q had worse associated prognostic factors including unmutated IGHV and complex karyotype. Somatic events showed greater functional impact than some germline variants. CLL cells with ATM aberrancies had ATM pathway dysfunction, and those with biallelic ATM aberrancies were more sensitive to the PARP inhibitor, talazoparib, than WT. This work could potentially lead to additional avenues for treatment based on differential sensitivities of CLLs with ATM aberrancy. Further studies are needed to characterize specific ATM germline variants with or without somatic ATM events.

ATM Germline Pathogenic Variants Affect Treatment Outcomes in Children with Acute Lymphoblastic Leukemia/Lymphoma and Ataxia Telangiectasia

Introduction Ataxia telangiectasia (A-T) is a multisystem disorder caused by biallelic germline pathogenic variants (PV) in the ATM gene. An important feature of A-T is an increased predisposition to cancer with a reported incidence of 25%, primarily attributed to hematological malignancies. Patients with A-T and cancer are usually excluded from therapeutic clinical trials, limited information thus exists concerning their treatment outcomes and toxicity profiles and consequently, optimal management strategies are unclear and an unmet need. In this multinational study, we aimed to investigate the characteristics and outcomes of leukemia and lymphoma in a large cohort of children with A-T and to determine risk factors which impact treatment outcome in order to generate consensus and data-based prospective treatment recommendations. Methods This study of patients aged ≤25 years with A-T and hematological malignancies was conducted through the International BFM Study Group. Patient data were collected from medical records, including specific patient comorbidities. Each reported ATM PV identified in the cohort was classified as null (resulting in complete loss of ATM activity) or hypomorphic (allowing residual ATM activity) according to the expected functional activity of the ATM protein and published functional studies. Patients with reported ATM PV were then classified as Group A (two null PV) or Group B (at least one hypomorphic PV). Results We report 202 pediatric and adolescent/young adult patients with A-T and hematological malignancies from 25 countries. The cohort included 82 patients with ALL/LBL (41%), predominantly (85%) of T-cell lineage, 91 with mature B-cell lymphomas (45%), 21 with Hodgkin lymphoma (10%) and 8 with other hematological malignancies (4%) (Fig 1). Of 111 patients with classifiable germline ATM variants, 82 (74%) were classified as Group A and 29 as Group B (26%). The distribution of patients with Group A and Group B germline ATM PV differed considerably between tumor types with 44% of the patients with lymphoblastic leukemia/lymphoma classified as Group B vs. 5% of those with mature B cell lymphomas ( p<.001). In total, 185 patients (92%) treated with curative intent were included in the outcome analyses, 135 (73%) of whom were treated with attenuated therapy regimens. Four-year OS and EFS for the entire cohort were 50.8% (95% CI 43.6-59.1) and 47.9% (95% CI 40.8-56.2), respectively. Surprisingly, cure rates of patients with A-T and malignancy did not appear to improve significantly with therapy modernization over the last four decades with 4-year EFS rates of 41.6% (95% CI 26.6-65), 49.6% (95% CI 38.8-63.4) and 48.0% (95% CI 37.4-61.7) for those treated before 2000, between 2000-2009 and since 2010, respectively ( p=.54). The major cause of treatment failure for the entire cohort was treatment-related mortality (TRM) with a 4-year cumulative incidence of 32.8% (95% CI 19.5-32.4), followed by progressive cancer in 14.5% (95% CI 10-19.8) and second malignancy in 4.9% (95% CI 13.1-85.8) (Fig 2). We identified factors that were significantly associated with survival for this unique patient population. Older age had a significantly deleterious effect upon survival with 4-year EFS rates of 69.1% (95%CI 55.9-85.4), 45.3% (95% CI 34.5-59.4), 39.8% (95% CI 25-63.2), and 33.7% (95% CI 20.8-54.6) for children aged ≤5 years, 5-10 years, 10-15 years and ≥15 years, respectively ( p=.003). The type of germline ATM PV also had a significant impact: 4-year EFS for patients with Group B ATM PV was 78.7% (95% CI 63.7-97.2) vs. 39.4% (95% CI 29-53.3) for Group A ( p<.001). Group A PV were associated with an increased TRM (OR 9.3; 95% CI 1.6-180.1; p=.042) and decreased EFS (HR .371 95% CI 16.6-82.6; p=.009). Conclusions We demonstrate in this first comprehensive international study that leukemia and lymphoma in children with A-T are curable. While the standard treatment stratification system for patients with hematological malignancies without A-T focuses upon cancer relapse/progression as the main cause of treatment failure, TRM was the main cause of therapy failure in patients with A-T and was strongly associated with the underlying germline ATM variant type. This study further fulfills an unmet need for international collaboration and provides a platform for data-based guidelines for a novel risk stratification system and optimal therapy selection for this unique patient population.

Mosaic chromosomal alterations in blood across ancestries using whole-genome sequencing

Megabase-scale mosaic chromosomal alterations (mCAs) in blood are prognostic markers for a host of human diseases. Here, to gain a better understanding of mCA rates in genetically diverse populations, we analyzed whole-genome sequencing data from 67,390 individuals from the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine program. We observed higher sensitivity with whole-genome sequencing data, compared with array-based data, in uncovering mCAs at low mutant cell fractions and found that individuals of European ancestry have the highest rates of autosomal mCAs and the lowest rates of chromosome X mCAs, compared with individuals of African or Hispanic ancestry. Although further studies in diverse populations will be needed to replicate our findings, we report three loci associated with loss of chromosome X, associations between autosomal mCAs and rare variants in DCPS, ADM17, PPP1R16B and TET2 and ancestry-specific variants in ATM and MPL with mCAs in cis.

Corrigendum: A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: a case report

Corrigendum: A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: a case report

A New Set of in Silico Tools to Support the Interpretation of ATM Missense Variants Using Graphical Analysis

Establishing the pathogenic nature of variants in ATM, a gene associated with breast cancer and other hereditary cancers, is crucial for providing patients with adequate care. Unfortunately, achieving good variant classification is still difficult. To address this challenge, we extended the range of in silico tools with a series of graphical tools devised for the analysis of computational evidence by health care professionals. We propose a family of fast and easy-to-use graphical representations in which the impact of a variant is considered relative to other pathogenic and benign variants. To illustrate their value, the representations are applied to three problems in variant interpretation. The assessment of computational pathogenicity predictions showed that the graphics provide an intuitive view of prediction reliability, complementing and extending conventional numerical reliability indexes. When applied to variant of unknown significance populations, the representations shed light on the nature of these variants and can be used to prioritize variants of unknown significance for further studies. In a third application, the graphics were used to compare the two versions of the ATM-adapted American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines, obtaining valuable information on their relative virtues and weaknesses. Finally, a server [ATMision (ATM missense in silico interpretation online)] was generated for users to apply these representations in their variant interpretation problems, to check the ATM-adapted guidelines' criteria for computational evidence on their variant(s) and access different sources of information.

Analysis of Turkish Breast Cancer Patients With ATM-Heterozygous Germline Mutation According to Clinicopathological Features.

The ATM gene is one of the most common breast cancer (BC) susceptibility genes after BRCA1/2 and has been shown to be a moderate BC susceptibility gene. The association between ATM germline mutation and clinical features of BC is now unknown. In this article, clinicopathological features of BC patients with ATM germline heterozygous mutation were investigated. Patients admitted to the Medical Genetics departmentof a tertiary hospital between January 2020 and December 2022 were examined. Only invasive BC patients with pathogenic mutation, likely pathogenic mutation, or variants of uncertain significance (VUS) were included in the study. In all, 121 patients were included in the study. The median age at the first cancer diagnosis of the patients was 44 years. Of the total number of patients, 75.2% (91) had the histological subtype of infiltrating ductal carcinoma, and 43% (52) had Luminal B molecular subtype features. At a median follow-up of 16 months, 5.8% (7) of patients developed cancer in the contralateral breast. In addition, 7.4% (9) of the patients developed a second primary cancer during follow-up. When the patients were compared according to ATM variant classification, the localization, histologic types, and molecular subtypes of the BC were not different between all groups (respectively; p=0.68, p=0.65, p=0.32). To the best of our knowledge, this is the first publication that evaluates the clinical and pathological characteristics of BC patients with germline heterozygous ATM mutations in the Turkish population. When patients were compared according to variant classifications of ATM mutation, patients' histological and molecular subtypes were similar.

Breast Irradiation Is Well Tolerated in Carriers of a Pathogenic ATM Variant

There are mixed and limited data regarding radiation therapy (RT) tolerance in carriers of a germline pathogenic or likely pathogenic (P/LP)ATMvariant. We investigated RT-related toxic effects in carriers of an ATM variant who received treatment for breast cancer. We identified 71 patients treated with adjuvant RT for breast cancer who were carriers of a variant in ATM: 15 were classified as P/LP and 56 classified as variants of unknown significance (VUS). We additionally identified 205 consecutively treated patients during a similar timeframe who were either confirmed ATM wild type or had no prior genetic testing. RT plans were reviewed. Acute and chronic toxic effects were evaluated using Common Terminology Criteria for Adverse Events version 4.0 criteria. Fisher's exact tests for count data were performed to compare toxic effects between the cohorts (P/LP vs VUS vs control). Wilcoxon rank-sum testing was performed to assess for differences in patient characteristics. The median toxicity follow-up was 19.4 months; median follow-up for the subcohorts was 13.3 months (P/LP), 12.6 months (VUS), and 23.3 months (control). There were no significant differences in radiation plan heterogeneity, receipt of a boost, or size of breast/chest wall planning target volume. There was greater use of hypofractionated RT in the control cohort (P=.023). After accounting for patient- and treatment-related factors that may affect toxic effects, we found no significant differences with respect to acute dermatitis, hyperpigmentation, moist desquamation, breast/chest wall pain, or breast edema. Additionally, we found no significant differences with respect to chronic breast/chest wall pain, induration, telangiectasia, or cosmetic outcome. RT as part of the management of breast cancer was well tolerated in carriers of a P/LP ATM variant, with toxic effect profiles that were similar to those seen in patients without known ATM mutations. High rates of excellent or good cosmesis were observed in carriers of a P/LP ATM variant who underwent breast conservation.

Germline Sequencing Analysis to Inform Clinical Gene Panel Testing for Aggressive Prostate Cancer

Germline gene panel testing is recommended for men with advanced prostate cancer (PCa) or a family history of cancer. While evidence is limited for some genes currently included in panel testing, gene panels are also likely to be incomplete and missing genes that influence PCa risk and aggressive disease. To identify genes associated with aggressive PCa. A 2-stage exome sequencing case-only genetic association study was conducted including men of European ancestry from 18 international studies. Data analysis was performed from January 2021 to March 2023. Participants were 9185 men with aggressive PCa (including 6033 who died of PCa and 2397 with confirmed metastasis) and 8361 men with nonaggressive PCa. Sequencing data were evaluated exome-wide and in a focused investigation of 29 DNA repair pathway and cancer susceptibility genes, many of which are included on gene panels. The primary study outcomes were aggressive (category T4 or both T3 and Gleason score ≥8 tumors, metastatic PCa, or PCa death) vs nonaggressive PCa (category T1 or T2 and Gleason score ≤6 tumors without known recurrence), and metastatic vs nonaggressive PCa. A total of 17 546 men of European ancestry were included in the analyses; mean (SD) age at diagnosis was 65.1 (9.2) years in patients with aggressive PCa and 63.7 (8.0) years in those with nonaggressive disease. The strongest evidence of association with aggressive or metastatic PCa was noted for rare deleterious variants in known PCa risk genes BRCA2 and ATM (P ≤ 1.9 × 10-6), followed by NBN (P = 1.7 × 10-4). This study found nominal evidence (P < .05) of association with rare deleterious variants in MSH2, XRCC2, and MRE11A. Five other genes had evidence of greater risk (OR≥2) but carrier frequency differences between aggressive and nonaggressive PCa were not statistically significant: TP53, RAD51D, BARD1, GEN1, and SLX4. Deleterious variants in these 11 candidate genes were carried by 2.3% of patients with nonaggressive, 5.6% with aggressive, and 7.0% with metastatic PCa. The findings of this study provide further support for DNA repair and cancer susceptibility genes to better inform disease management in men with PCa and for extending testing to men with nonaggressive disease, as men carrying deleterious alleles in these genes are likely to develop more advanced disease.

Multi-gene panel testing and association analysis in Cypriot breast cancer cases and controls.

Introduction: It is estimated that around 5% of breast cancer cases carry pathogenic variants in established breast cancer susceptibility genes. However, the underlying prevalence and gene-specific population risk estimates in Cyprus are currently unknown. Methods: We performed sequencing on a population-based case-control study of 990 breast cancer cases and 1094 controls from Cyprus using the BRIDGES sequencing panel. Analyses were conducted separately for protein-truncating and rare missense variants. Results: Protein-truncating variants in established breast cancer susceptibility genes were detected in 3.54% of cases and 0.37% of controls. Protein-truncating variants in BRCA2 and ATM were associated with a high risk of breast cancer, whereas PTVs in BRCA1 and PALB2 were associated with a high risk of estrogen receptor (ER)-negative disease. Among participants with a family history of breast cancer, PTVs in ATM, BRCA2, BRCA1, PALB2 and RAD50 were associated with an increased risk of breast cancer. Furthermore, an additional 19.70% of cases and 17.18% of controls had at least one rare missense variant in established breast cancer susceptibility genes. For BRCA1 and PALB2, rare missense variants were associated with an increased risk of overall and triple-negative breast cancer, respectively. Rare missense variants in BRCA1, ATM, CHEK2 and PALB2 domains, were associated with increased risk of disease subtypes. Conclusion: This study provides population-based prevalence and gene-specific risk estimates for protein-truncating and rare missense variants. These results may have important clinical implications for women who undergo genetic testing and be pivotal for a substantial proportion of breast cancer patients in Cyprus.

Abstract A008: Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis

Abstract Wilms Tumor (WT), or nephroblastoma, is the most common pediatric renal cancer. The most prevalent epigenetic alterations in WT occur on chromosome 11p15, accounting for at least two-thirds of WT. These same changes on chromosome 11p15 also cause Beckwith-Wiedemann Syndrome (BWS). BWS is a pediatric overgrowth disorder affecting numerous tissues, including the kidney, and up to 28% of patients with BWS develop cancer. As patients with BWS have the same 11p15 epigenetic and structural changes that arise in some sporadic WT, BWS-WT provides a unique opportunity to investigate the specific molecular drivers by which 11p15 alterations induce oncogenesis. Using clinical data and patient samples from the International BWS Registry and Biorepository, we used whole exome sequencing, methylation array, and RNA-sequencing technologies to examine the genomic and epigenomic landscape of BWS-WT compared to control kidney samples. We found several large-scale recurrent copy number alterations (CNAs) in the BWS-WT cohort across cancer-associated chr1p36, WT-associated chr16q, and at a novel chr15q site. Two BWS-WT presented with an above average number of genomic alterations; one had mutations in telomere maintenance genes, and the other had mutations in mismatch repair genes. Recurrent mutations in genes associated with sporadic WT, such as TP53, CTNNB1, and WT1 were not present in BWS samples. We found other single nucleotide variants (SNVs) in BCORL1, ASXL1, ATM and AXL genes in the BWS cohort. We further compared the BWS data to publicly available non-syndromic/nonBWS data, stratified based on 11p15 status (normal or altered), and the molecular signaling differences were investigated. Differential methylation studies showed enrichment of biological processes related to the Wnt signaling, integrin signaling, insulin receptor signaling, and BMP signaling pathways in BWS-WT. Altered-11p15 nonBWS-WT showed enrichment of cell cycle and DNA damage checkpoint processes, as well as TORC1/TOR signaling and Wnt signaling pathways, as assayed by differential methylation. RNA sequencing data showed differential gene expression of Wnt signaling along with other distinct pathways including Notch, BMP, PPAR and, NIK/NF-kappaB signaling pathways in BWS-WT. Based on an interactome study, CTNNB1 exhibited the broadest range of interactions, and its overexpression was observed in BWS-WT, although not specifically mutated in the BWS samples. Weighted gene coexpression network analysis (WGCNA) showed that cell cycle-related processes and DNA repair mechanisms are shared features of WT with alterations in chromosome 11p15. While BWS-WT predisposition and 11p15 alterations in nonBWS-WT are well-established, this study is the first to focus on stratifying tumors by this characteristic. Data presented herein suggest a mechanism in which 11p15-altered genomes predispose cells to oncogenic transitions. Further investigation of our findings may serve to identify novel diagnostic or therapeutic targets in WT oncogenesis. Citation Format: Snehal Dinkar Nirgude, Natali Sobel Naveh, Sanam Kavai, Jennifer Kalish. Cancer predisposition signaling pathways drive Beckwith-Wiedemann syndrome Wilms tumor oncogenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A008.