NBS1 Research Articles

Germline and somatic alterations in NBN and their putative impact on the pathogenesis of malignant neoplasms

Disruption of mechanisms that maintain genome stability is an essential factor of tumor progression. Accordingly, predisposition to the development of neoplasms is often associated with germline mutations in genes involved in DNA damage detection and repair. At the same time, impairment of DNA repair systems may be a predictor of antitumor treatment efficacy while overexpression of genes involved in DNA repair is a frequent event in various types of malignancies that can lead to development of tumor cells’ resistance to chemo- and radiotherapy. NBN (nibrin) gene encodes the subunit of the MRN complex which acts as a sensor of double-strand DNA breaks and participates in their repair by homologous recombination. Germline variants in NBN which are associated with increased risk of tumor development are generally represented by frameshift mutations that lead to the synthesis of truncated protein as well as by nonsense and some missense mutations which occur in functionally significant domains. These germline mutations result in partial loss of nibrin function and in increased frequency of spontaneous and induced chromosomal aberrations in the cells of the carriers. On the contrary, amplification of NBN locus is a predominant type of somatic mutations affecting this gene, which indicates a dual role of NBN protein in tumor progression. The results of several studies demonstrate the influence of NBN expression level and its mutational status on anti-tumor drug resistance in particular types of tumor cells and on the survival rate of patients. These data indicate that an in-depth study of different variants and their functional significance is necessary since NBN status may be essential for the choice of treatment tactics for some types of tumors.

6918 A Unique Case of Oncocytic Thyroid Cancer with an NBN Gene Pathogenic Variant

Abstract Disclosure: A. Grover: None. S. Jhawar: None. S. Shekhar: None. S.A. Avadhanula: None. G. Al-Naqeeb: None. G. Thota: None. P. Veeraraghavan: None. C. Cochran: None. M.Y. Kao-Hsieh: None. A. Gharib: None. F.E. Escorcia: None. J. Del Rivero: None. J. Klubo-Gwiezdzinska: None. S. Gubbi: None. Introduction: Oncocytic thyroid carcinoma (OTC), previously known as “Hürthle cell” carcinoma, makes up less than 5% of all thyroid cancers. OTCs are often aggressive with a high risk of metastasis, and a poorer prognosis compared to other forms of differentiated thyroid carcinoma (DTC). Molecular drivers of OTC include pathogenic variants (PVs) in NRAS, DAXX, CDKN1A, the mitochondrial genome and chromosomal alterations. PVs in the NBN gene in OTC have not been previously reported. Case: A 53-year-old male was noted to have a thyroid nodule on a routine physical examination. Patient denied any hyper/hypothyroid or compressive symptoms, and family history was unremarkable. Cytopathologic analysis of the fine needle aspiration biopsy of the nodule was consistent with a thyroid follicular neoplasm (Bethesda IV). A total thyroidectomy with central compartment neck dissection was performed. Histopathologic examination demonstrated a 7 cm x 4 cm x 4 cm OTC with capsular and lymphovascular invasion, but without extra-thyroidal extension or lymph node metastasis (pT3aN0Mx). Subsequently, adjuvant radioiodine (RAI) I131 therapy with 165 mCi was administered and post-therapy scan revealed neck uptake. One-year post-therapy RAI diagnostic scan was negative. Three years following the initial thyroid surgery, elevated serum thyroglobulin (Tg) levels were noted. A computed tomogram of the chest revealed multiple bilateral lung nodules. Further imaging ruled out involvement of other distant sites. A diagnostic lower lung wedge resection demonstrated metastatic OTC. The tumor stained positive for CK7, PAX8, TG, TTF-1 and negative for CK20 on immunohistochemistry. Next-generation sequencing of the tumor tissue (Oncomine Comprehensive Assay v3) revealed a PV in the NBN gene (c.2166_2167delGCinsAT; p.Trp722Ter). A year later, the serum Tg levels continued to rise, and the lung nodules had increased in size. Due to disease progression, lenvatinib was initiated. A [F18]-fluorodeoxyglucose positron emission tomography performed one year later demonstrated persistent pulmonary tumor burden with new osseous metastasis in the right scapula, left acetabulum, and the sternum. Zoledronic acid was initiated for the treatment of the skeletal metastases, and palliative external beam radiation (2000 cGy in 5 fractions) was administered to the sternal lesion to alleviate the bone pain. Conclusion: The NBN gene encodes for the protein nibrin, a member of the MRE11/RAD50 complex that is involved in DNA repair. PVs in NBN are commonly identified in non-thyroidal malignancies such as breast and lung carcinomas. While NBN PVs are rarely reported in DTCs, as per our knowledge, this is the first described case of OTC with a PV in NBN as the molecular driver, manifesting with an aggressive disease course. Identification of this novel PV therefore adds to the current knowledge of the molecular landscape of OTC. Presentation: 6/3/2024

Plasma Proteomics of Type 2 Diabetes, Hypertension, and Co-Existing Diabetes/Hypertension in Thai Adults.

The study explored proteomics to better understand the relationship between type 2 diabetes (T2DM) and hypertension (HT) in Thai adults, using shotgun proteomics and bioinformatics analysis. Plasma samples were taken from 61 subjects: 14 healthy subjects (mean age = 40.85 ± 7.12), 13 with T2DM (mean age = 57.38 ± 6.03), 16 with HT (mean age = 66.87 ± 10.09), and 18 with coexisting T2DM/HT (mean age = 58.22 ± 10.65). Proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein-protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 11.5. We identified six unique proteins in T2DM patients, including translationally controlled 1 (TPT1) and nibrin (NBN), which are associated with the DNA damage response. In HT patients, seven unique proteins were identified, among them long-chain fatty acid-CoA ligase (ASCL), which functions in the stimulation of triacylglycerol and cholesterol synthesis, and NADPH oxidase activator 1 (NOXA1), which is involved in high blood pressure via angiotensin II-induced reactive oxygen species (ROS)-generating systems. In coexisting T2DM/HT patients, six unique proteins were identified, of which two-microtubule-associated protein 1A (MAP1A)-might be involved in dementia via RhoB-p53 and diacylglycerol kinase beta (DGKB), associated with lipid metabolism. This study identified new candidate proteins that are possibly involved in the pathology of these diseases.

Genetic Characteristics of a Large Pediatric Cohort of Patients with Inborn Errors of Immunity: Single-Center Experience.

More than 450 genetic defects result in inborn errors of immunity (IEI). Their individual prevalence in specific cohorts is influenced by national characteristics and other factors. We present results of genetic testing conducted in 1809 Russian children with IEI. Genetic defects confirming IEI were found in 1112 out of 1809 (61.5%) probands. These defects included variants in 118 single genes (87.9% of patients) and aberrations in 6 chromosomes (11.8%). Notably, three patients harbored pathogenic variants in more than one IEI gene. Large deletions constituted 5% of all defects. Out of the 799 original variants, 350 (44%) have not been described previously. Rare genetic defects (10 or fewer patients per gene) were identified in 20% of the patients. Among 967 probands with germline variants, defects were inherited in an autosomal dominant manner in 29%, X-linked in 34%, and autosomal recessive in 37%. Four females with non-random X-inactivation exhibited symptoms of X-linked diseases (BTK, WAS, CYBB, IKBKG gene defects). Despite a relatively low rate of consanguinity in Russia, 47.9% of autosomal recessive gene defects were found in a homozygous state. Notably, 28% of these cases carried "Slavic" mutation of the NBN gene or known hot-spot mutations in other genes. The diversity of IEI genetic forms and the high frequency of newly described variants underscore the genetic heterogeneity within the Russian IEI group. The new variants identified in this extensive cohort will enrich genetic databases.

Germline pathogenic variants in the MRE11, RAD50, and NBN (MRN) genes in cancer predisposition: A systematic review and meta-analysis.

The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.

Rapid nanobody-based imaging of mesothelin expressing malignancies compatible with blocking therapeutic antibodies.

Mesothelin (MSLN) is overexpressed in a wide variety of cancers with few therapeutic options and has recently emerged as an attractive target for cancer therapy, with a large number of approaches currently under preclinical and clinical investigation. In this respect, developing mesothelin specific tracers as molecular companion tools for predicting patient eligibility, monitoring then response to mesothelin-targeting therapies, and tracking the evolution of the disease or for real-time visualisation of tumours during surgery is of growing importance. We generated by phage display a nanobody (Nb S1) and used enzymatic approaches were used to site-directed conjugate Nb S1 with either ATTO 647N fluorochrome or NODAGA chelator for fluorescence and positron emission tomography imaging (PET) respectively. We demonstrated that Nb S1 displays a high apparent affinity and specificity for human mesothelin and demonstrated that the binding, although located in the membrane distal domain of mesothelin, is not impeded by the presence of MUC16, the only known ligand of mesothelin, nor by the therapeutic antibody amatuximab. In vivo experiments showed that both ATTO 647N and [68Ga]Ga-NODAGA-S1 rapidly and specifically accumulated in mesothelin positive tumours compared to mesothelin negative tumours or irrelevant Nb with a high tumour/background ratio. The ex vivo biodistribution profile analysis also confirmed a significantly higher uptake of Nb S1 in MSLN-positive tumours than in MSLNlow tumours. We demonstrated for the first time the use of an anti-MSLN nanobody as PET radiotracer for same day imaging of MSLN+ tumours, targeting an epitope compatible with the monitoring of amatuximab-based therapies and current SS1-derived-drug conjugates.

Importance of Germline and Somatic Alterations in Human MRE11, RAD50, and NBN Genes Coding for MRN Complex

The MRE11, RAD50, and NBN genes encode for the nuclear MRN protein complex, which senses the DNA double strand breaks and initiates the DNA repair. The MRN complex also participates in the activation of ATM kinase, which coordinates DNA repair with the p53-dependent cell cycle checkpoint arrest. Carriers of homozygous germline pathogenic variants in the MRN complex genes or compound heterozygotes develop phenotypically distinct rare autosomal recessive syndromes characterized by chromosomal instability and neurological symptoms. Heterozygous germline alterations in the MRN complex genes have been associated with a poorly-specified predisposition to various cancer types. Somatic alterations in the MRN complex genes may represent valuable predictive and prognostic biomarkers in cancer patients. MRN complex genes have been targeted in several next-generation sequencing panels for cancer and neurological disorders, but interpretation of the identified alterations is challenging due to the complexity of MRN complex function in the DNA damage response. In this review, we outline the structural characteristics of the MRE11, RAD50 and NBN proteins, the assembly and functions of the MRN complex from the perspective of clinical interpretation of germline and somatic alterations in the MRE11, RAD50 and NBN genes.

Influence of dietary Bacillus coagulans and/or Bacillus licheniformis-based probiotics on performance, gut health, gene expression, and litter quality of broiler chickens

Probiotics are non-pathogenic microorganisms that are potentially important non-antibiotic alternatives. This study aimed to compare novel multi-strain and single-strain Bacillus probiotics and their respective influences on broiler chickens’ performance, gut health, litter quality, immune response, and NBN and TLR gene expression. A total of 1200 Arbor-Acres 1-day-old broiler chicks were randomly allocated into three treatments (T1 was a control, T2 was supplemented with a combined Bacillus coagulans (2 × 109 cfu/g) and Bacillus licheniformis (8 × 109 cfu/g) probiotic strains (0.2 kg/ton of feed), and T3 was supplemented with Bacillus licheniformis (3.2 × 109 cfu/g) probiotic (0.5 kg/ton of feed) with eight replicas of each. Supplementing the broiler diet with either the single-strain (T3) or the multi-strain (T2) Bacillus-based probiotic raised the overall birds’ body weight, body weight gain, feed conversion ratio, and European production efficiency factor compared to the control (T1), with a significant enhancement achieved by the multi-strain Bacillus product (P = 0.005). T2 and T3 exhibited significantly improved cholesterol, Alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase levels than the control (P ≤ 0.05). The transcript levels of both NBN and TLR genes were upregulated in the liver in the T2 and T3 groups. The T2 group experienced significant reductions in gut bacterial counts, especially for Clostridia, and recorded the lowest litter moisture and nitrogen. In conclusion, supplementing broiler diets with probiotics of multiple Bacillus strains increased production profitability by promoting bird growth, improving feed intake, enhancing gut mucosa and immune organs, and upregulating genes responsible for immunity. All these inhibit the overgrowth of enteric pathogens and sustain litter quality.

GENETIC PREDISPOSITION TO CANCER: REPORT OF THE FIRST CASE IN PERU OF THE 657DEL5 VARIANT IN THE NBN GENE

Background: Breast cancer has well known genetic mutations affecting the DNA-damage repair mechanism and predispose for the development of the disease. The heterozygous pathogenic variants found in the NBN gene has been linked to a higher likelihood of developing cancer; Methods: clinical description of 3 cases and review of the literature; Results: We describe a family cluster of 3 sisters affected by different cancer types. One of them had a breast cancer associated to the 657del5 pathogenic variant in the NBN gene and was handled with breast conserving therapy. Another had bilateral breast cancer and the third one had endometrial cancer; Conclusions: Breast cancer associated with the 657del5 pathogenic variant in the NBN gene has been described in various publications, mainly in populations other than Peru.

Abstract P056: Germline cancer susceptibility gene testing in unselected patients with soft tissue sarcoma: a multi-center prospective study

Abstract Sarcomas are rare heterogenous mesenchymal tumors with over 75 different subtypes, with varying biology and outcomes, with no clear inciting factor in the vast majority. The germline genetic susceptibility to sarcoma is not very well characterized. Identification of any underlying germline predisposition can have important implications for treatment of patients and screening recommendations for family members. To determine the prevalence of pathogenic germline variants (PGV) in patients with sarcomas, we undertook the Interrogating Cancer Etiology Using Proactive Genetic Testing (INTERCEPT) study, a prospective multi-site study of germline sequencing using a >80-gene next-generation sequencing platform. The study was done at the three Mayo Clinic Cancer Centers between April 1, 2018, and March 31, 2020. Patients were not selected based on stage, family cancer history, ethnicity, or age. Family cascade testing was offered at no charge. Results: 115 adult patients with soft tissue sarcoma were included in the study. The median age was 60 years, 58.5% were male, 81% were White. The anatomical location of the primary tumor included extremities (33.0%), retroperitoneum (18.2%), trunk (12.2%), head and neck (7.8%). Family history of cancer was present in 63% of the study population. Ten patients (8.7%) had a PGV. Thirty percent of the patients with PGV had stage IV sarcoma (3/10), and 70% (7/10) had earlier stage sarcoma (stages I-III). Among patients who were found to have a variant of uncertain significance (VUS), 40% (22/55) had stage IV sarcoma and 60% (33/55) had earlier stage disease. Of the 10 patients with PGV, high to medium penetrance gene abnormalities were identified in 8 patients (80%) involving TP53 (3), BRCA1 (1), SDHA (1), ATM (2) and heterozygous deletion of NBN (1) genes. Of these PGV, gene deletions were observed in 6/10 while missense variants comprised the remaining 4. The vast majority of the PGV (70%) would not have been identified using the current guidelines utilizing the 2018 or 2020 National Comprehensive Cancer Network (NCCN), National Society of Genetic Counselors (NSGC) or American College of Medical Genetics and Genomics. Conclusion: In this prospective, multi-site study comprising over 100 patients with diverse sarcomas, unselected germline testing identified a PGV in 1 in 11 patients (8.7%) with the majority of these not being detected using current standard of care guidelines for genetic testing. Because of the rarity of sarcomas, in addition to limited effective treatment options for advanced disease, large scale germline testing in sarcomas represents a potentially impactful strategy to assess therapeutic options and for assessment of familial risk. Citation Format: Isaak Ailts, Michael A. Golafshar, Katie L. Kunze, Margaret Klint, Kathleen Barrus, Samuel Nussbaum, Edward Esplin, Brandie Leach, Sarah Young, Niloy Jewel Samadder, Mahesh Seetharam. Germline cancer susceptibility gene testing in unselected patients with soft tissue sarcoma: a multi-center prospective study. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P056.

Associations of genes of DNA repair systems with Parkinson’s disease

Background. Approximately 5–10 % of cases of Parkinson’s disease (PD) are monogenic, in other cases the pathology has a multifactorial etiology. One of recognized pathogenetic pathways of PD is mitochondrial dysfunction, in particular the accumulation of damage in mitochondrial DNA. Hence, the genes of DNA repair proteins are promising candidate genes for multifactorial forms of PD.The aim. To study the involvement of genes of DNA repair proteins in the development of Parkinson’s disease.Materials and methods. The associative analysis was carried out while comparing a group of patients with PD (n = 133) with a Tomsk population sample (n = 344). SNaPshot analysis was used to study 8 SNPs in genes of DNA repair proteins (rs560191 (TP53BP1); rs1805800 and rs709816 (NBN); rs473297 (MRE11A); rs1189037 and rs1801516 (ATM); rs1799977 (MLH1); rs1805321 (PMS2)).Results. Common alleles and homozygous rs1801516 genotypes in the ATM gene predispose the development of PD (odds ratio (OR) – 3.27 (p = 0.000004) and OR = 3.46 (p = 0.00008) for risk alleles and genotype respectively) and rs1799977 in the MLH1 gene (OR = 1.88 (p = 0.0004) and OR = 2.42 (p = 0.00007) respectively); heterozygotes have a protective effect (OR = 0.33 (p = 0.0007) and OR = 0.46 (p = 0.0007) for ATM and MLH1, respectively). The rare rs1805800 allele in the NBN gene (OR = 1.62 (p = 0.019)) and a homozygous genotype for it (OR = 2.28 (p = 0.016)) also predispose to PD. Associations with PD of the ATM, MLH1, NBN genes were revealed for the first time.Conclusion. Mitochondrial dysfunction is one of the key factors in the pathogenesis of PD, while at least two of the three protein products of associated genes are involved in the development of mitochondrial dysfunction. Accordingly, it can be assumed that associated genes are involved in the pathogenesis of PD precisely through mitochondrial dysfunction.

Cell-Free DNA Sequencing Reveals Gene Variants in DNA Damage Repair Genes Associated with Prognosis of Prostate Cancer Patients.

In the present study, we further analyzed the data obtained in our previous study, where we investigated the cell-free DNA (cfDNA) of 34 progressive prostate cancer patients via targeted sequencing. Here, we studied the occurrence and prognostic impact of sequence variants according to their clinical pathological significance (CPS) or their functional impact (FI) in 23 DNA damage repair (DDR) genes with a focus on the ATM serine/threonine kinase gene (ATM). All patients had at least one DDR gene with a CPS or FI variant. Kaplan-Meier analysis indicated that the group with a higher number of CPS variants in DDR genes had a shorter time to treatment change (TTC) compared to the group with a lower number of CPS variants (p = 0.038). Analysis of each DDR gene revealed that CPS variants in the ATM gene and FI variants in the nibrin (NBN) gene showed a shorter TTC (p = 0.034 and p = 0.042). In addition, patients with CPS variants in the ATM gene had shorter overall survival (OS; p = 0.022) and disease-specific survival (DSS; p = 0.010) than patients without these variants. Interestingly, patients with CPS variants in seven DDR genes possessed a better OS (p = 0.008) and DSS (p = 0.009), and patients with FI variants in four DDR genes showed a better OS (p = 0.007) and DSS (p = 0.008). Together, these findings demonstrated that the analysis of cfDNA for gene variants in DDR genes provides prognostic information that may be helpful for future temporal and targeted treatment decisions for advanced PCa patients.

Ex vivo γH2AX assay for tumor radiosensitivity in primary prostate cancer patients and correlation with clinical parameters

BackroundAccurate surrogate parameters for radio resistance are warranted for individualized radiotherapy (RT) concepts in prostate cancer (PCa). The purpose of this study was to assess intertumoral heterogeneity in terms of radio resistance using an ex-vivo γH2AX assay after irradiation of prostate biopsy cores and to investigate its correlation with clinical features of respective patients as well as imaging and genomic features of tumor areas.MethodsTwenty one patients with histologically-proven PCa and pre-therapeutic multiparametric resonance imaging and prostate-specific membrane antigen positron emission tomography were included in the study. Biopsy cores were collected from 26 PCa foci. Residual γH2AX foci were counted 24 h after ex-vivo irradiation (with 0 and 4 Gy) of biopsy specimen and served as a surrogate for radio resistance. Clinical, genomic (next generation sequencing) and imaging features were collected and their association with the radio resistance was studied.ResultsIn total 18 PCa lesions from 16 patients were included in the final analysis. The median γH2AX foci value per PCa lesion was 3.12. According to this, the patients were divided into two groups (radio sensitive vs. radio resistant) with significant differences in foci number (p < 0.0001). The patients in the radio sensitive group had significantly higher prostate specific antigen serum concentration (p = 0.015), tumor areas in the radio sensitive group had higher SUV (standardized uptake values in PSMA PET)-max and -mean values (p = 0.0037, p = 0.028) and lower ADC (apparent diffusion coefficient-mean values, p = 0.049). All later parameters had significant (p < 0.05) correlations in Pearson’s test. One patient in the radio sensitive group displayed a previously not reported loss of function frameshift mutation in the NBN gene (c.654_658delAAAAC) that introduces a premature termination codon and results in a truncated protein.ConclusionIn this pilot study, significant differences in intertumoral radio resistance were observed and clinical as well as imaging parameters may be applied for their prediction. After further prospective validation in larger patient cohorts these finding may lead to individual RT dose prescription for PCa patients in the future.

A gene dosage-dependent effect unveils NBS1 as both a haploinsufficient tumour suppressor and an essential gene for SHH-medulloblastoma.

AimsInherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB‐prone animal model.Materials and methodsWe generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)‐dependent MB‐prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1‐depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA‐Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs.ResultsWe found that monoallelic Nbn deletion increases SmoA1‐dependent MB incidence. In addition to a defective DDR, Nbn +/− GCPs show increased clonogenicity compared to Nbn +/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full Nbn KO impairs MB development both in SmoA1 mice and in an SHH‐driven tumour allograft.ConclusionsOur study indicates that Nbn is haploinsufficient for SHH‐MB development whereas full Nbn KO is epistatic on SHH‐driven MB development, thus revealing a gene dosage‐dependent effect of Nbn inactivation on SHH‐MB development.

The NBN founder mutation-Evidence for a country specific difference in age at cancer manifestation.

Nijmegen breakage syndrome (NBS) is an autosomal-recessive chromosome instability disorder characterized by, among others, hypersensitivity to X-irradiation and an exceptionally high risk for lymphoid malignancy. The vast majority of NBS patients is homozygous for a common Slavic founder mutation, c.657del5, of the NBN gene, which is involved in the repair of DNA double-strand breaks (DSBs). The founder mutation also predisposes heterozygous carriers to cancer, apparently however, with a higher risk in the Czech Republic/Slovakia (CS) than in Poland. To examine whether the age of cancer manifestation and cancer death of NBN homozygotes is different between probands from CS and Poland. The study is restricted to probands born until 1989, before replacement of the communist regime by a democratic system in CS and Poland, and a substantial transition of the health care systems. Moreover, all patients were recruited without knowledge of their genetic status since the NBN gene was not identified until 1998. Here, we show that cancer manifestation of NBN homozygotes is at a significantly earlier age in probands from CS than from Poland. This is explained by the difference in natural and medical radiation exposure, though within the permissible dosage. It is reasonable to assume that this finding also sheds light on the higher cancer risk of NBN heterozygotes in CS than in Poland. This has implications for genetic counseling and individualized medicine also of probands with other DNA repair defects.

Abstract 5757: Germline and somatic mutational landscape of Chinese early-onset colorectal cancers

Abstract Background: The incidence of colorectal cancer (CRC) in young people, described as early-onset colorectal cancer (EOCRC) has been rising up in decades. Apart from definite familial genetic disease, a considerable proportion of EOCRC remained unknown of the mechanism. Characterizing the difference in genomic features of EOCRC and late-onset CRC (LOCRC) would help us to understand it. Methods: A total of 1777 Chinese CRC patients diagnosed in 2019-2020 were included. 489 patients under 50 years were defined as EOCRC. A 733-gene panel next-generation sequencing (NGS) testing and measurement of tumor mutational burden (TMB) levels based on the panel were conducted in all patients’ tumor specimen. Lynch syndrome (LS) was defined as patients harboring at least one non-benign germline mutation in four LS MMR genes (MLH1, MSH2, MSH6, PMS2). Results: 43 of 489(8.8%) cases and 54 of 1288 (4.2%) cases were MSI-H in EOCRC and LOCRC, separately. Excluded 97 LS patients (84.3% in MSI-H EOCRC and 72.0% in MSI-H LOCRC), the proportion of non-LS MSI-H in EOCRC and in LOCRC were 1.8% and 1.7%, separately. Compared to non-LS MSI-H LOCRC, EOCRC has higher germline mutational frequencies in APC, CD74, ATM, BCL2L11, CHEK2, and RECQL4 genes and somatic mutational frequencies in APC, ERCC4, CHD2, ARHGAP5, CASP8, and ATM genes. In non-LS MSS patients, germline mutations in CHEK2, NBN and POLE genes, somatic SNV/indels in RNF43, ATR, AFF3, JAK2, POLG, CHD2 genes and somatic copy number gain in genes on chromosome 8 (ZNF703, FGFR1, MYC, PTK2) occurred more frequently in EOCRC. KEGG enrichment analysis in somatic mutations indicated significant overexpressed MAPK signaling pathway. The TMB level was significantly lower in non-LS MSS EOCRC than in LOCRC. Conclusions: The proportion of non-LS MSI-H in Chinese EOCRC and LOCRC were approximate. Non-LS MSS EOCRC had marked chromosome 8 somatic copy number gain and MAPK signaling pathway, indicating the potential mechanism. The comparative lower TMB level in EOCRC was concordance with previous studies and the correlations between low TMB level and high CNV burden also corroborate the etiopathogenesis. Citation Format: Xiancheng Zeng, Junhao Liu, Yurong Luo, Hailiang Li, Xinyi Liu, Mengli Huang. Germline and somatic mutational landscape of Chinese early-onset colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5757.

15 years of experience of an oncogenetics service in the northeast of Brazil.

e22522 Background: The Oncogenetics service at the Federal University of Bahia was implemented in 2007 and since then has been offering pre- and post-test genetic counseling for cancer susceptibility genes, mainly tumors associated with King's syndrome in the population of the state of Bahia. This is a mixed population of about 80% of African ancestry. Methods: Between the years 2007 to 2021, 462 probands and their families have already been evaluated by NGS analysis, Sanger sequencing. Large rearrangements were detected by multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 and BRCA2 genes. Results: The mean age at diagnosis was 41 years. Pathogenic/probably pathogenic mutations were founded in 118 patients: 67 had breast cancer, 09 with ovarian cancer, 26 with more than one tumor, 15 without cancer, and 01 with another tumor type. Most of the pathogenic variants were located in the BRCA1 gene (27 variants), the two most frequent variants being: c.211G &gt; A (13 probands) and c.3331_3334delCAAG (18 probands), both of Iberian origin. Of patients with mutation, 9.8% had large rearrangements in BRCA1 and 11.5% in BRCA2. Bilateral breast cancer appears predominantly in patients with BRCA1 (4) and BRCA2 (1) variants. In this study, only two mutations in BRCA described as of African origin were found in two patients. Other pathogenic mutations appear in ATM, BARD1, BRIP1, CDH1, CHEK2, FAM175A, FANCM, MSH6, NBN, NF2, PALB2, RAD51, SDHB, SLX4, and TP53 genes. Variants of uncertain significance were identified in 71 patients. Conclusions: The population of the state of Bahia has a peculiar mutational profile, containing mutations that are not very frequent in other regions of the country. Despite being a population with high African ancestry, most mutations are of European origin.

Mutational spectrum of breast cancer susceptibility genes among women ascertained in a cancer risk clinic in Northeast Brazil

PurposeThere is a paucity of data on the spectrum and prevalence of pathogenic variants among women of African ancestry in the Northeast region of Brazil.MethodsWe performed BROCA panel sequencing to identify inherited loss-of-function variants in breast cancer susceptibility genes among 292 Brazilian women referred to a single institution cancer risk assessment program.ResultsThe study included a convenient cohort of 173 women with invasive breast cancer (cases) and 119 women who were cancer-free at the time of ascertainment. The majority of the women self-reported as African-descended (67% for cases and 90.8% for unaffected volunteers). Thirty-seven pathogenic variants were found in 36 (20.8%) patients. While the spectrum of pathogenic variants was heterogeneous, the majority (70.3%) of the pathogenic variants were detected in high-risk genes BRCA1, BRCA2, PALB2, and TP53. Pathogenic variants were also found in the ATM, BARD1, BRIP1, FAM175A, FANCM, NBN, and SLX4 genes in 6.4% of the affected women. Four recurrent pathogenic variants were detected in 11 patients of African ancestry. Only one unaffected woman had a pathogenic variant in the RAD51C gene. Different risk assessment models examined performed well in predicting risk of carrying germline loss-of-function variants in BRCA1 and/or BRCA2 in breast cancer cases.ConclusionThe high prevalence and heterogenous spectrum of pathogenic variants identified among self-reported African descendants in Northeast Brazil is consistent with studies in other African ancestry populations with a high burden of aggressive young onset breast cancer. It underscores the need to integrate comprehensive cancer risk assessment and genomic testing in the management of newly diagnosed Black women with breast cancer across the African Diaspora, enabling improved cancer control in admixed underserved and understudied populations.

Impaired p53-Mediated DNA Damage Response Contributes to Microcephaly in Nijmegen Breakage Syndrome Patient-Derived Cerebral Organoids.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive genetic disorder caused by mutations within nibrin (NBN), a DNA damage repair protein. Hallmarks of NBS include chromosomal instability and clinical manifestations such as growth retardation, immunodeficiency, and progressive microcephaly. We employed induced pluripotent stem cell-derived cerebral organoids from two NBS patients to study the etiology of microcephaly. We show that NBS organoids carrying the homozygous 657del5 NBN mutation are significantly smaller with disrupted cyto-architecture. The organoids exhibit premature differentiation, and Neuronatin (NNAT) over-expression. Furthermore, pathways related to DNA damage response and cell cycle are differentially regulated compared to controls. After exposure to bleomycin, NBS organoids undergo delayed p53-mediated DNA damage response and aberrant trans-synaptic signaling, which ultimately leads to neuronal apoptosis. Our data provide insights into how mutations within NBN alters neurogenesis in NBS patients, thus providing a proof of concept that cerebral organoids are a valuable tool for studying DNA damage-related disorders.

Abstract P2-09-14: Characteristics of TNBC patients carrying other than BRCA gene mutations

Abstract Triple-negative breast cancer (TNBC) represents approximately 10-20% of Breast Cancer (BC) diagnosed worldwide and in accordance with the current guideline it is defined by the absence of expression of the of expression of the estrogen receptor (ER&amp;lt;1%), progesterone receptor (PR&amp;lt;1%) and human epidermal growth factor 2-receptor (HER-2) amplification or overexpression. Up to 10-20% of TNBC patients (pts) are found to carry deleterious germline BRCA1/2 mutations and germline genetic testing is common practice in Modena Family Cancer Clinic (MFCC) since 1998. Many of the other genes involved in HR repair are now recognized to contribute to hereditary BC and/or ovarian cancer (OC) risk. Nowadays, the Italian Association of Medical Oncology (AIOM) guidelines recommend BRCA1/2 testing for patients meeting hereditary BC and/or OC or personal history of TNBC diagnosed before 60y while recommendations for testing of other genes are not fully established by National Guidelines. The aim of our study was to explore the high and moderate penetrance BC genes mutation rate in TNBC identified in the MFCC. Between 1998 and 2017, the MFCC offered BRCA1/2 genetic testing, while on January 2018, the Clinical Genomics Laboratory of the MFCC started to provide a Next Generation Sequencing (NGS) multigene panel (MGP) testing for 22 actionable genes (APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53).For the purpose of our study, we retrospectively evaluated 274 TNBC patients underwent MGP testing in the last 42 months. In our study, 35 non-BRCA pathogenic or likely-pathogenic variant (PVs) were identified (12.8%). In details, we observed 16 PALB2 mutations (5.8%), 5 RAD51C/D mutations (1.8%), 3 ATM and MUTYH mutations (1.1%), 2 BRIP mutations (0.7%) and one mutation of PSM2, BARD1, CHECK2, TP53, NBN and PTEN genes (0.4%). Two pts carried 2 PVs together: BRCA and MUTYH, BRIP1 and TP53, respectively. Finally, 7 out of 34 pts (20.5%) were diagnosed at age &amp;gt;=60yThe majority of BC was diagnosed at an early stage. All TNBCs present ductal histotype, mostly with high proliferation rate and grading 3. Characteristics of tumors are shown in Table 1. In our cohort, PALB2 mutations were the most frequent PVs in non-BRCA genes, in line with several previous studies. Interesting, we identified RAD51C/D mutations as second PVs non-BRCA genes, in support of certain studies that suggest an association with an increased risk for TNBC and OC. In conclusion, our results support the MGP using, opening the question about the 60-year cut off for genetic testing in TNBC, regardless of family history. Table 1.Characteristics of TNBC tumors in patients carrying other than BRCA gene mutationsPALB2RAD51C/DATMMUTYHBRIPPMS2PTENNBNBARD1CHECK2TP53Number of patients165332111111Mean age at diagnosis (y)61.4 (44-94)48 (33-62)52.3 (49-54)44.3(40-50)42.5 (36-49)394958485036Ki 67 (%)&amp;lt;405(45.5%)01(50%)1 (50%)0011000&amp;gt;=406(54.5%)4(100%)1(50%)1 (50%)1(100%)100100unknown51111000011GradingG1-23 (25%)0000001000G39 (75%)4 (100%)3 (100%)3(100%)2(100%)110101Unknown41000000010Histotypeductal14 (100%)4(100%)3(100%)3(100%)2(100%)110111others00000001000unknown21000000000Stage at diagnosisI7 (63.6%)3 (60%)01 (100%)1 (100%)010000II4 (36.4%)1 (20%)2 (100%)00001000III01 (20%)000300000unknown50121000111 Citation Format: Laura Cortesi, Marta Venturelli, Angela Toss, Claudia Piombino, Elena Barbieri, Elisabetta Razzaboni, Luca Moscetti, Federico Piacentini, Claudia Omarini, Federica Domati, Massimo Dominici. Characteristics of TNBC patients carrying other than BRCA gene mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-09-14.