High-resolution functional mapping of RAD51C by saturation genome editing.

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.

Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

Pathogenic Germline Variants in 10,389 Adult Cancers.

We conducted the largest investigation of predisposition variants in cancer to date, discovering 853 pathogenic or likely pathogenic variants in 8% of 10,389 cases from 33 cancer types. Twenty-one genes showed single or cross-cancer associations, including novel associations of SDHA in melanoma and PALB2 in stomach adenocarcinoma. The 659 predisposition variants and 18 additional large deletions in tumor suppressors, including ATM, BRCA1, and NF1, showed low gene expression and frequent (43%) loss of heterozygosity or biallelic two-hit events. We also discovered 33 such variants in oncogenes, including missenses in MET, RET, and PTPN11 associated with high gene expression. We nominated 47 additional predisposition variants from prioritized VUSs supported by multiple evidences involving case-control frequency, loss of heterozygosity, expression effect, and co-localization with mutations and modified residues. Our integrative approach links rare predisposition variants to functional consequences, informing future guidelines of variant classification and germline genetic testing in cancer.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

MHC-I Genotype Restricts the Oncogenic Mutational Landscape.

MHC-I molecules expose the intracellular protein content on the cell surface, allowing T cells to detect foreign or mutated peptides. The combination of six MHC-I alleles each individual carries defines the sub-peptidome that can be effectively presented. We applied this concept to human cancer, hypothesizing that oncogenic mutations could arise in gaps in personal MHC-I presentation. To validate this hypothesis, we developed and applied a residue-centric patient presentation score to 9,176 cancer patients across 1,018 recurrent oncogenic mutations. We found that patient MHC-I genotype-based scores could predict which mutations were more likely to emerge in their tumor. Accordingly, poor presentation of a mutation across patients was correlated with higher frequency among tumors. These results support that MHC-I genotype-restricted immunoediting during tumor formation shapes the landscape of oncogenic mutations observed in clinically diagnosed tumors and paves the way for predicting personal cancer susceptibilities from knowledge of MHC-I genotype.

miRNA biogenesis and inherited disorders: clinico-molecular insights.

MicroRNAs (miRNAs) play vital roles in the regulation of gene expression, a process known as miRNA-induced gene silencing. The human genome codes for many miRNAs, and their biogenesis relies on a handful of genes, including DROSHA, DGCR8, DICER1, and AGO1/2. Germline pathogenic variants (GPVs) in these genes cause at least three distinct genetic syndromes, with clinical manifestations that range from hyperplastic/neoplastic entities to neurodevelopmental disorders (NDDs). Over the past decade, DICER1 GPVs have been shown to lead to tumor predisposition. Moreover, recent findings have provided insight into the clinical consequences arising from GPVs in DGCR8, AGO1, and AGO2. Here we provide a timely update with respect to how GPVs in miRNA biogenesis genes alter miRNA biology and ultimately lead to their clinical manifestations.

Germline predisposition to pediatric Ewing sarcoma is characterized by inherited pathogenic variants in DNA damage repair genes.

More knowledge is needed regarding germline predisposition to Ewing sarcoma to inform biological investigation and clinical practice. Here, we evaluated the enrichment of pathogenic germline variants in Ewing sarcoma relative to other pediatric sarcoma subtypes, as well as patterns of inheritance of these variants. We carried out European-focused and pan-ancestry case-control analyses to screen for enrichment of pathogenic germline variants in 141 established cancer predisposition genes in 1,147 individuals with pediatric sarcoma diagnoses (226 Ewing sarcoma, 438 osteosarcoma, 180 rhabdomyosarcoma, and 303 other sarcoma) relative to identically processed cancer-free control individuals. Findings in Ewing sarcoma were validated with an additional cohort of 430 individuals, and a subset of 301 Ewing sarcoma parent-proband trios was analyzed for inheritance patterns of identified pathogenic variants. A distinct pattern of pathogenic germline variants was seen in Ewing sarcoma relative to other sarcoma subtypes. FANCC was the only gene with an enrichment signal for heterozygous pathogenic variants in the European Ewing sarcoma discovery cohort (three individuals, OR 12.6, 95% CI 3.0-43.2, p = 0.003, FDR = 0.40). This enrichment in FANCC heterozygous pathogenic variants was again observed in the European Ewing sarcoma validation cohort (three individuals, OR 7.0, 95% CI 1.7-23.6, p = 0.014), representing a broader importance of genes involved in DNA damage repair, which were also nominally enriched in individuals with Ewing sarcoma. Pathogenic variants in DNA damage repair genes were acquired through autosomal inheritance. Our study provides new insight into germline risk factors contributing to Ewing sarcoma pathogenesis.

Hereditary Colorectal Cancer and Polyposis Syndromes Caused by Variants in Uncommon Genes.

A substantial number of hereditary colorectal cancer (CRC) and colonic polyposis cannot be explained by alteration in confirmed predisposition genes, such as mismatch repair (MMR) genes, APC and MUTYH. Recently, a certain number of potential predisposition genes have been suggested, involving each a small number of cases reported so far. Here, we describe the detection of rare variants in the NTLH1, AXIN2, RNF43, BUB1, and TP53 genes in nine unrelated patients who were suspected for inherited CRC and/or colonic polyposis. Seven of them were classified as pathogenic or likely pathogenic variants (PV/LPV). Clinical manifestations of carriers were largely consistent with reported cases with, nevertheless, distinct characteristics. PV/LPV in these uncommon gene can be responsible for up to 2.7% of inherited CRC or colonic polyposis syndromes. Our findings provide supporting evidence for the role of these genes in cancer predisposition, and contribute to the determination of related cancer spectrum and cancer risk for carriers, allowing for the establishment of appropriate screening strategy and genetic counseling in affected families.

A high proportion of germline variants in pediatric chronic myeloid leukemia.

Chronic myeloid leukemia (CML) typically occurs in late adulthood. Pediatric CML is a rare form of leukemia. In all age groups, the characteristic genetic driver of the disease is the BCR::ABL1 fusion gene. However, additional genomic events contribute to leukemic transformation, which is not yet well-characterized in pediatric CML. We investigated the mutational landscape of pediatric CML to determine whether predisposing germline variants may play a role in early-age disease development. Whole exome sequencing and targeted sequencing were performed in pediatric and adult CML samples to identify age-related germline and somatic variants in addition to the BCR::ABL1 translocation. Germline variants were detected in about 60% of pediatric patients with CML, with predominantly hematopoietic genes affected, most frequently ASXL1, NOTCH1, KDM6B, and TET2. The number of germline variants was significantly lower in adult patients with CML. If only confirmed pathogenic variants were regarded as cancer-predisposing variants, the occurrence was ~ 10% of pediatric CML, which is comparable to other hematological malignancies and most childhood cancer entities in general. We hypothesize that the interaction with the strong oncogene BCR::ABL1 may also favor the development of leukemia by weaker variants in the same genes. In pediatric patients, the germline variants of genes associated with clonal hematopoiesis may increase the likelihood that an incidental BCR::ABL1 translocation triggers the early manifestation of CML.

Optical genome mapping identifies structural variants in potentially new cancer predisposition candidate genes in pediatric cancer patients.

Genetic predisposition is one of the major risk factors for pediatric cancer, with ~10% of children being carriers of a predisposing germline alteration. It is likely that this is the tip of the iceberg and many children are underdiagnosed, as most of the analysis focuses on single or short nucleotide variants, not considering the full spectrum of DNA alterations. Hence, we applied optical genome mapping (OGM) to our cohort of 34 pediatric cancer patients to perform an unbiased germline screening and analyze the frequency of structural variants (SVs) and their impact on cancer predisposition. All children were clinically highly suspicious for germline alterations (concomitant conditions or congenital anomalies, positive family cancer history, particular cancer type, synchronous or metachronous tumors), but whole exome sequencing (WES) had failed to detect pathogenic variants in cancer predisposing genes. OGM detected a median of 49 rare SVs (range 27-149) per patient. By analysis of 18 patient-parent trios, we identified three de novo SVs. Moreover, we discovered a likely pathogenic deletion of exon 3 in the known cancer predisposition gene BRCA2, and identified a duplication in RPA1, which might represent a new cancer predisposition gene. We conclude that optical genome mapping is a suitable tool for detecting potentially predisposing SVs in addition to WES in pediatric cancer patients.

Molecular analysis of cancer genomes in children with Lynch syndrome: Exploring causal associations.

Lynch syndrome (LS) predisposes to cancer in adulthood and is caused by heterozygous germline variants in a mismatch repair (MMR) gene. Recent studies show an increased prevalence of LS among children with cancer, suggesting a causal relationship. For LS-spectrum (LSS) cancers, including high-grade gliomas and colorectal cancer, causality has been supported by typical MMR-related tumor characteristics, but for non-LSS cancers, causality is unclear. We characterized 20 malignant tumors of 18 children with LS, including 16 non-LSS tumors. We investigated second hits, tumor mutational load, mutational signatures and MMR protein expression. In all LSS tumors and three non-LSS tumors, we detected MMR deficiency caused by second hit somatic alterations. Furthermore, these MMR-deficient tumors carried driver variants that likely originated as a consequence of MMR deficiency. However, in 13 non-LSS tumors (81%), a second hit and MMR deficiency were absent, thus a causal link between LS and cancer development in these children is lacking. These findings demonstrate that causality of LS in children with cancer, which can be determined by molecular tumor characterization, seems to be restricted to specific tumor types. Large molecular and epidemiological studies are needed to further refine the tumor spectrum in children with LS.

Identification of novel candidate predisposing genes in familial nonmedullary thyroid carcinoma implicating DNA damage repair pathways.

The genetic basis of nonsyndromic familial nonmedullary thyroid carcinoma (FNMTC) is still poorly understood, as the susceptibility genes identified so far only account for a small percentage of the genetic burden. Recently, germline mutations in DNA repair-related genes have been reported in cases with thyroid cancer. In order to clarify the genetic basis of FNMTC, 94 genes involved in hereditary cancer predisposition, including DNA repair genes, were analyzed in 48 probands from FNMTC families, through targeted next-generation sequencing (NGS). Genetic variants were selected upon bioinformatics analysis and in silico studies. Structural modeling and network analysis were also performed. In silico results of NGS data unveiled likely pathogenic germline variants in 15 families with FNMTC, in genes encoding proteins involved in DNA repair (ATM, CHEK2, ERCC2, BRCA2, ERCC4, FANCA, FANCD2, FANCF, and PALB2) and in the DICER1, FLCN, PTCH1, BUB1B, and RHBDF2 genes. Structural modeling predicted that most missense variants resulted in the disruption of networks of interactions between residues, with implications for local secondary and tertiary structure elements. Functional annotation and network analyses showed that the involved DNA repair proteins functionally interact with each other, within the same DNA repair pathway and across different pathways. MAPK activation was a common event in tumor progression. This study supports that rare germline variants in DNA repair genes may be accountable for FNMTC susceptibility, with potential future utility in patients' clinical management, and reinforces the relevance of DICER1 in disease etiology.

Hereditary Diffuse Gastric Cancer.

Hereditary diffuse gastric cancer (HDGC) is a dominantly inherited cancer syndrome characterized by a high incidence of diffuse gastric cancer (DGC) and lobular breast cancer (LBC). HDGC is caused by germline mutations in 2 genes involved in the epithelial adherens junction complex, CDH1 and CTNNA1. We discuss the genetics of HDGC and the variability of its clinical phenotype, in particular the variable penetrance of advanced DGC and LBC, both within and between families. We review the pathology of the disease, the mechanism of tumor initiation, and its natural history. Finally, we describe current best practice for the clinical management of HDGC, including emerging genetic testing criteria for the identification of new families, methods for endoscopic surveillance, the complications associated with prophylactic surgery, postoperative quality of life, and the emerging field of HDGC chemoprevention.

Parents' and patients' perspectives, experiences, and preferences for germline genetic or genomic testing of children with cancer: A systematic review.

PURPOSE: Germline testing in pediatric cancer presents opportunities and challenges. Understanding family perspectives, experiences, and preferences will optimize integration into routine care. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we searched 4 databases for studies exploring perspectives, experiences, and preferences of parents/caregivers and/or patients regarding germline testing of children with cancer. Qualitative and quantitative data were extracted, organized, and summarized by research question and themes. RESULTS: We identified 2286 unique articles, of which 24 were included. Interest in and uptake of testing was high. Families were motivated by altruism and a desire for inheritance/causation information. Testing barriers included psychological concerns, timing of the testing approach if offered at diagnosis or in a high-risk cancer setting and privacy/discrimination. Testing experiences highlighted challenges yet also positive impacts, with results providing psychological relief and informing proactive decision making. Timing preferences varied; however, allowing time to adjust to a new diagnosis was a common theme. Most wanted to receive as many germline sequencing-related results as possible. CONCLUSION: Findings underscore the importance of integrating germline analyses into pediatric cancer care with flexibility and support for families facing challenges. Where possible, consent should be provided at a time that suits each family's situation with access to information aligning with their needs and preferences. PROSPERO: CRD42023444890.

Parents' experiences with sequencing of all known pediatric cancer predisposition genes in children with cancer.

PURPOSE: Germline DNA sequencing is increasingly used within pediatric oncology, yet parental experiences remain underexplored. METHODS: Parents of children undergoing cancer predisposition gene panel sequencing (143 genes) were surveyed before and after disclosure of results. Questionnaires assessed knowledge, expectations, worries, satisfaction, and regret. Next to descriptives, linear mixed models and generalized mixed models were utilized to explore factors associated with knowledge and worries. RESULTS: Out of 325 eligible families, 310 parents (176 mothers and 128 fathers of 188 families) completed all after-consent questionnaires whereas 260 parents (150 mothers and 110 fathers of 181 families) completed all after disclosure questionnaires. Most parents hoped their participation would benefit others, although individual hopes were also common. Sequencing-related worries were common, particularly concerning whether their child would get cancer again, cancer risks for family members and psychosocial implications of testing. Parental satisfaction after disclosure was high and regret scores were low. Lower education was associated with lower knowledge levels, whereas foreign-born parents were at increased risk of experiencing worries. CONCLUSION: Germline sequencing of children with cancer is generally well received by their parents. However, careful genetic counseling is essential to ensure that parents are adequately informed and supported throughout the process.

Evaluation of Bayesian point-based system on the variant classification of hereditary cancer predisposition genes.

PURPOSE: To assess the differences in variant classifications using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology 2015 guidelines and the Bayesian point-based classification system (here referred to as the point system) in 115 hereditary cancer predisposition genes and explore variant sub-tiering by the point system. METHODS: Germline variant classifications for 721 pediatric patients from an in-house panel were retrospectively evaluated using the 2 scoring systems. RESULTS: A total of 2376 unique variants were identified, with ∼23.5% exhibiting discordant classifications. Unique variants classified by the point system demonstrated a lower rate of variants of uncertain significance (VUS; ∼15%) compared with American College of Medical Genetics and Genomics and the Association for Molecular Pathology 2015 guidelines (∼36%). This change is attributed to unique variants with 1 benign supporting evidence (∼12%) or 1 benign strong evidence (∼4%) being classified as likely benign by the point system. Additionally, variants with conflicting/modified evidence (∼5% of 2376) are also resolved by the point system. Sub-tiering unique variants classified by the point system as VUS (n = 354) indicates ∼77.4% were VUS-Low (0-1 points), whereas the remaining ∼22.6% were VUS-Mid (2-3 points) and VUS-High (4-5 points). CONCLUSION: The point system reduces the VUS rate and facilitates their sub-tiering. Future large-scale studies are warranted to explore the impact of the point system on improving VUS reporting and/or VUS clinical management.

Pediatric acute promyelocytic leukemia and Fanconi anemia: Case report and literature review.

Acute promyelocytic leukemia (APL) represents 5%-10% of childhood acute myeloid leukemia (AML) and is the most curable subtype of AML. Fanconi anemia (FA) is one of the most common inherited bone marrow failure syndromes caused by biallelic pathogenic variants (PV) in specific DNA-repair genes. Biallelic PVs in FANCD1/BRCA2 (FA-D1) account for 3% of FA and are associated with early-onset leukemia and a high risk of solid tumors. We report a 4 year-old boy from non-consanguineous parents diagnosed with standard risk APL. This child had café-au-lait spots and an extra thumb remnant. Genomic sequencing revealed two PV in FANCD1/BRCA2 confirming a diagnosis of FA-D1. Chromosomal breakage studies were compatible with FA. Each parent carried one variant and had no personal history of cancer. Morphological then molecular remissions were achieved with all-trans retinoic acid and Arsenic trioxide. This patient underwent haploidentical stem cell transplant. In addition to our patient, a literature search revealed four additional patients with APL/FA, with a total of three patients with FA-D1. This raises the possibility of an association between such rare disorders. Practical management of APL in the setting of FA-D1 is discussed with an overview of current evidence and knowledge gaps.

BUB1B monoallelic germline variants contribute to prostate cancer predisposition by triggering chromosomal instability.

BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.

Occurrence of cancer in Marfan syndrome: Report of two patients with neuroblastoma and review of the literature.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by pathogenic variants in FBN1, with a hitherto unknown association with cancer. Here, we present two females with MFS who developed pediatric neuroblastoma. Patient 1 presented with neonatal MFS and developed an adrenal neuroblastoma with unfavorable tumor genetics at 10 months of age. Whole genome sequencing revealed a germline de novo missense FBN1 variant (NP_000129.3:p.(Asp1322Asn)), resulting in intron 32 inclusion and exon 32 retention. Patient 2 was diagnosed with classic MFS, caused by a germline de novo frameshift variant in FBN1 (NP_000129.3:p.(Cys805Ter)). At 18 years, she developed high-risk neuroblastoma with a somatic ALK pathogenic variant (NP_004295.2:p.(Arg1275Gln)). We identified 32 reported cases of MFS with cancer in PubMed, yet none with neuroblastoma. Among patients, we observed an early cancer onset and high frequency of MFS complications. We also queried cancer databases for somatic FBN1 variants, finding 49 alterations reported in PeCan, and variants in 2% of patients in cBioPortal. In conclusion, we report the first two patients with MFS and neuroblastoma and highlight an early age at cancer diagnosis in reported patients with MFS. Further epidemiological and functional studies are needed to clarify the growing evidence linking MFS and cancer.

Osteopathia striata with cranial sclerosis as a cancer predisposition syndrome: The first report of neuroblastoma and review of all cancers in OSCS.

Osteopathia Striata with Cranial Sclerosis (OSCS) is a rare genetic condition primarily characterized by metaphyseal striations of long bones, bone sclerosis, macrocephaly, and other congenital anomalies. It is caused by pathogenic variants in AMER1, a tumor suppressor and a WNT signaling repressor gene with key roles in tissue regeneration, neurodevelopment, tumorigenesis, and other developmental processes. While somatic AMER1 pathogenic variants have frequently been identified in several tumor types (e.g., Wilms tumor and colorectal cancer), whether OSCS (i.e., with AMER1 germline variants) is a tumor predisposition syndrome is not clear, with only nine cases reported with tumors. We here report the first case of neuroblastoma diagnosed in a male child with OSCS, review all previously reported tumors diagnosed in individuals with OSCS, and discuss potential tumorigenic mechanisms of AMER1. Our report adds to the accumulating evidence suggesting OSCS is a tumor predisposition condition, highlighting the importance of maintaining a high index of suspicion for the associated tumors when evaluating patients with OSCS. Importantly, Wilms tumor stands out as the most commonly observed tumor in OSCS patients, underscoring the need for regular surveillance.