ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers

CD137L dendritic cells induce potent response against cancer-associated viruses and polarize human CD8(+) T cells to Tc1 phenotype

10.1007/s00262-018-2144-xCANCER IMMUNOLOGY IMMUNOTHERAPY676893-90

COVID-19 vaccination uptake and safety profile among germline BRCA1 and BRCA2 pathogenic variant carriers in Singapore

Background: Although Singapore is one of the highest vaccinated countries in the world, vaccine hesitancy remains in a subpopulation, including individuals with cancer predisposition syndromes. At the Cancer Genetics Service National Cancer Centre Singapore, we see patients with germline genetic alterations, most being BRCA1/2 pathogenic/likely pathogenic variant (PV/LPV) carriers. While reported safe for cancer patients, there are limited studies addressing the safety profile and outcomes of COVID-19 vaccination among individuals with germline PV/LPV in cancer predisposition genes such as BRCA1/2. This study aims to evaluate the outcomes of COVID-19 vaccination among germline PV/LPV carriers in BRCA1/2. Methods: We conducted a phone call survey of COVID-19 vaccination uptake and toxicity in a prospective cohort of 189 participants with germline BRCA1/2 PV/LPV between 1st Sept 2021 and 30th Sept 2021. We collected demographics data including gender, race, age, history of cancer, types of cancer, and number of cancers. Statistical difference in baseline demographics between responders with history of cancer and those without were assessed using Chi-square, Fisher’s exact and independent t-test analysis. Logistic regression was used to evaluate effect of demographics on the occurrence of post-vaccination side effects. Results: Among 189 BRCA1/2 PV/LPV carriers responded, 97 carried PV/LPV in BRCA1 and 92 in BRCA2. Majority were vaccinated (89.5%) and had completed the two-dose vaccine schedule, with 7 (4.1%) received only one dose. The most common post-vaccination side effects was myalgia (56.5%) followed by fever (40.2%), headache (16.3%) and fatigue (11.2%). There were no major severe side events. Evaluation by logistic regression showed that the occurrence of side effects was not affected by PV/LPV gene (BRCA1 or BRCA2), gender, race, age or history of cancer. Conclusion: The post-vaccination side effects profile among individuals with germline PV/LPV in BRCA1/2 is consistent with the Singaporean general population, hence recommendations for COVID-19 vaccination for these individuals should not differ from non-carriers and should be encouraged by their healthcare providers.Published versio

Functional analysis of clinical BARD1 germline variants

Germline pathogenic variants in BRCA1/2 account for one-third of familial breast cancers. The majority of BRCA1 function requires heterodimerization with BARD1. In contrast to BRCA1, BARD1 is a low-penetrance gene with an unclear clinical relevance, partly because of limited functional evidence. Using patient-derived lymphoblastoid cells, we functionally characterized two pathogenic variants (c.1833dupT, c.2099delG) and three variants of uncertain significance (VUSs) (c.73G>C, c.1217G>A, c.1918C>A). Three of these patients had breast cancers, whereas the remaining had colorectal cancers (n = 3). Both patients with pathogenic variants (c.1833dupT, c.2099delG) developed breast cancers with aggressive disease phenotypes such as triple-negative breast cancer and high cancer grades. As BARD1 encompasses multiple functional domains, including those of apoptosis and homologous recombination repair, we hypothesized that the function being impaired would correspond with the domain where the variant was located. Variants c.1918C>A, c.1833dupT, c.1217G>A, and c.2099delG, located within and proximal to apoptotic domains of ankyrin and BRCT, were associated with impaired apoptosis. Conversely, apoptosis function was preserved in c.73G>C, which was distant from the ankyrin domain. All variants displayed normal BRCA1 heterodimerization and RAD51 colocalization, consistent with their location being distal to BRCA1-and RAD51-binding domains. In view of deficient apoptosis, VUSs (c.1217G>A and c.1918C>A) may be pathogenic or likely pathogenic variants. In summary, functional analysis of BARD1 VUSs requires a combination of assays and, more importantly, the use of appropriate functional assays with consideration to the variant's location.National Medical Research Council (NMRC)Published versionWe thank our sources of support: National Medical Research Council (CSA) (NMRC/CSAINV/0017/2017) and Singhealth Foundation Research Grant (SHF/PRISM002/2015) to J.N. and SingHealth (SMSTDA-Project FY2018) to M.R.T

Impact of variant reclassification in cancer predisposition genes on clinical care

PURPOSE: Genetic testing has clinical utility in the management of patients with hereditary cancer syndromes. However, the increased likelihood of encountering a variant of uncertain significance in individuals of non- European descent such as Asians may be challenging to both clinicians and patients. This study aims to evaluate the impact of variant reclassification in an Asian country with variants of uncertain significance reported in cancer predisposition genes. METHODS: A retrospective analysis of patients seen at the Cancer Genetics Service at the National Cancer Centre Singapore between February 2014 and March 2020 was conducted. The frequency, direction, and time to variant reclassification were evaluated by comparing the reclassified report against the original report. RESULTS: A total of 1,412 variants of uncertain significance were reported in 49.9% (845 of 1,695) of patients. Over 6 years, 6.7% (94 of 1,412) of variants were reclassified. Most variants of uncertain significance (94.1%, 80 of 85) were downgraded to benign or likely benign variant, with a smaller proportion of variants of uncertain significance (5.9%, 5 of 85) upgraded to pathogenic or likely pathogenic variant. Actionable variants of uncertain significance upgrades and pathogenic or likely pathogenic variant downgrades, which resulted in management changes, happened in 31.0% (39 of 126) of patients. The median and mean time taken for reclassification were 1 and 1.62 year(s), respectively. CONCLUSION: We propose a clinical guideline to standardize management of patients reported to have variants of uncertain significance. Management should be based on the patient's personal history, family history, and variant interpretation. For clinically relevant or suspicious variants of uncertain significance, follow-up is recommended every 2 years, as actionable reclassifications may happen during this period.Ministry of Health (MOH)National Medical Research Council (NMRC)National Research Foundation (NRF)Supported by the National Research Foundation Singapore under its Clinical Scientist Award (NMRC/CSA-INV/0017/2017) and administered by the Singapore Ministry of Health’s National Medical Research Council. This project was partially funded by NCC Research Fund, NCC Cancer Fund, and Terry Fox supporting funds. J.N. was funded by the National Medical Research Council Singapore and also received research funding from AstraZeneca

Using the actor-partner interdependence model to explore the psychological impact of COVID-19 on anxiety in dyads of patients with cancer and caregivers

Background: Delineating the compound psychological effect of the pandemic on cancer care, and the interdependency across cancer patient-caregiver dyads have yet to be explored. This study examines the levels of psychological impact of COVID-19 on patient-caregiver dyads anxiety, and the interdependent associations between their COVID-19 and cancer concerns, and risk perceptions. Method: There were 352 patients and caregivers (patient-caregiver dyads, N = 176) included in this study (43.2% spousal dyads). Generalized Anxiety Disorder-7 and questionnaires regarding risk perception, perceived confidence in healthcare system, COVID-19, and cancer-related concerns were administered. Actor-Partner Interdependence Model (APIM) analyses were used to determine the interdependent effects. Indirect effects were tested using mediation pathway analyses. Results: Patients reported significantly higher levels of risk perceptions and anxiety than their caregivers (p < 0.01). Anxiety rates (GAD-7 ≥ 10) were also significantly higher (26.7% vs 18.2%, p < 0.01). Dyads’ anxiety, “general COVID-19 concerns,” “cancer-related concerns,” and risk perceptions were correlated (ps < 0.01). APIM showed only actor effects of general COVID-19 concerns, cancer-related COVID-19 concerns, and risk perceptions on anxiety (βs = 0.19–0.53, ps < 0.01). No partner effects were observed. Similar results were found in the composite APIM. Indirect effects of the patient/caregiver’s variables on their partner’s anxiety were observed in the mediation analyses. Conclusion: Concerns about COVID-19 and cancer care could be indirectly associated in patient-caregiver dyads and need to be proactively addressed. As pandemic evolves into endemicity, engagement with patients and caregivers should strive to be sensitive to their differential needs and messages should be tailored to the informational needs of each.Ministry of Health (MOH)National Medical Research Council (NMRC)National Research Foundation (NRF)This study is supported by the National Research Foundation Singapore’s National Medical Research Council Clinician Scientist Award (NMRC/CSA-INV/0017/2017) and administered by the Singapore Ministry of Health’s National Medical Research Council. In addition, this research is supported by the National Cancer Centre Cancer Fund

Clinical relevance of screening checklists for detecting cancer predisposition syndromes in Asian childhood tumours

Assessment of cancer predisposition syndromes (CPS) in childhood tumours is challenging to paediatric oncologists due to inconsistent recognizable clinical phenotypes and family histories, especially in cohorts with unknown prevalence of germline mutations. Screening checklists were developed to facilitate CPS detection in paediatric patients; however, their clinical value have yet been validated. Our study aims to assess the utility of clinical screening checklists validated by genetic sequencing in an Asian cohort of childhood tumours. We evaluated 102 patients under age 18 years recruited over a period of 31 months. Patient records were reviewed against two published checklists and germline mutations in 100 cancer-associated genes were profiled through a combination of whole-exome sequencing and multiplex ligation-dependent probe amplification on blood-derived genomic DNA. Pathogenic germline mutations were identified in ten (10%) patients across six known cancer predisposition genes: TP53, DICER1, NF1, FH, SDHD and VHL. Fifty-four (53%) patients screened positive on both checklists, including all ten pathogenic germline carriers. TP53 was most frequently mutated, affecting five children with adrenocortical carcinoma, sarcomas and diffuse astrocytoma. Disparity in prevalence of germline mutations across tumour types suggested variable genetic susceptibility and implied potential contribution of novel susceptibility genes. Only five (50%) children with pathogenic germline mutations had a family history of cancer. We conclude that CPS screening checklists are adequately sensitive to detect at-risk children and are relevant for clinical application. In addition, our study showed that 10% of Asian paediatric solid tumours have a heritable component, consistent with other populations.NMRC (Natl Medical Research Council, S’pore)Published versio

Germline Mutations in Cancer Predisposition Genes are Frequent in Sporadic Sarcomas

Abstract Associations of sarcoma with inherited cancer syndromes implicate genetic predisposition in sarcoma development. However, due to the apparently sporadic nature of sarcomas, little attention has been paid to the role genetic susceptibility in sporadic sarcoma. To address this, we performed targeted-genomic sequencing to investigate the prevalence of germline mutations in known cancer-associated genes within an Asian cohort of sporadic sarcoma patients younger than 50 years old. We observed 13.6% (n = 9) amongst 66 patients harbour at least one predicted pathogenic germline mutation in 10 cancer-associated genes including ATM, BRCA2, ERCC4, FANCC, FANCE, FANCI, MSH6, POLE, SDHA and TP53. The most frequently affected genes are involved in the DNA damage repair pathway, with a germline mutation prevalence of 10.6%. Our findings suggests that genetic predisposition plays a larger role than expected in our Asian cohort of sporadic sarcoma, therefore clinicians should be aware of the possibility that young sarcoma patients may be carriers of inherited mutations in cancer genes and should be considered for genetic testing, regardless of family history. The prevalence of germline mutations in DNA damage repair genes imply that therapeutic strategies exploiting the vulnerabilities resulting from impaired DNA repair may be promising areas for translational research

ENIGMA CHEK2gether Project:A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ~ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers