Prevalence and clinical implications of germline pathogenic variants in cancer predisposing genes in young patients across sarcoma subtypes.

BACKGROUND: Sarcomas are a rare and diverse group of cancers occurring mainly in young individuals for which an underlying germline genetic cause remains unclear in most cases. METHODS: Germline DNA from 177 children, adolescents and young adults with soft tissue or bone sarcomas was tested using multigene panels with 113 or 126 cancer predisposing genes (CPGs) to describe the prevalence of germline pathogenic/likely pathogenic variants (GPVs). Subsequent testing of a subset of tumours for loss of heterozygosity (LOH) evaluation was performed to investigate the clinical and molecular significance of these variants. RESULTS: GPVs were detected in 21.5% (38/177) of the patients (15.8% in children and 21.6% in adolescents and young adults), with dominant CPGs being altered in 15.2% overall. These variants were found in genes previously associated with the risk of developing sarcomas (TP53, RB1, NF1, EXT1/2) but also in genes where that risk is still emerging/limited (ERCC2, TSC2 and BRCA2) or unknown (PALB2, RAD50, FANCM and others). The detection rates of GPVs varied from 0% to 33% across sarcoma subtypes and GPV carriers were more likely to present more than one primary tumour than non-carriers (21.1%×6.5%; p=0.012). Loss of the wild-type allele was detected in 48% of tumours from GPV carriers, mostly in genes definitively associated with sarcoma risk. CONCLUSION: Our findings reveal that a high proportion of young patients with sarcomas presented a GPV in a CPG, underscoring the urgency of establishing appropriate genetic screening strategies for these individuals and their families.

Xeroderma pigmentosum: low prevalence of germline XPA mutations in a Brazilian XP population.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by DNA repair defects that cause photophobia, sunlight-induced cancers, and neurodegeneration. Prevalence of germline mutations in the nucleotide excision repair gene XPA vary significantly in different populations. No Brazilian patients have been reported to carry a germline mutation in this gene. In this study, the germline mutational status of XPA was determined in Brazilian patients exhibiting major clinical features of XP syndrome. The study was conducted on 27 unrelated patients from select Brazilian families. A biallelic inactivating transition mutation c.619C>T (p.Arg207Ter) was identified in only one patient with a history of neurological impairment and mild skin abnormalities. These findings suggest that XP syndrome is rarely associated with inherited disease-causing XPA mutations in the Brazilian population. Additionally, this report demonstrates the effectiveness of genotype-phenotype correlation as a valuable tool to guide direct genetic screening.

Misclassification of a frequent variant from PMS2CL pseudogene as a PMS2 loss of function variant in Brazilian patients.

PMS2, a Lynch Syndrome gene, presents challenges in genetic testing due to the existence of multiple pseudogenes. This study aims to describe a series of cases harboring a variant in the PMS2CL pseudogene that has been incorrectly assigned to PMS2 with different nomenclatures. We reviewed data from 647 Brazilian patients who underwent multigene genetic testing at a single center to identify those harboring the PMS2 V1:c.2186_2187delTC or V2:c.2182_2184delACTinsG variants, allegedly located at PMS2 exon 13. Gene-specific PCR and transcript sequencing was performed. Among the 647 individuals, 1.8% (12) carried the investigated variants, with variant allele frequencies ranging from 15 to 34%. By visually inspecting the alignments, we confirmed that both V1 and V2 represented the same variant and through gene-specific PCR and PMS2 transcript analysis, we demonstrated that V1/V2 is actually located in the PMS2CL pseudogene. Genomic databases (ExAC and gnomAD) report an incidence of 2.5 - 5.3% of this variant in the African population. Currently, V1 is classified as "uncertain significance" and V2 as "conflicting" in ClinVar, with several laboratories classifying them as "pathogenic". We identified a frequent African PMS2CL variant in the Brazilian population that is misclassified as a PMS2 variant. It is likely that V1/V2 have been erroneously assigned to PMS2 in several manuscripts and by clinical laboratories, underscoring a disparity-induced matter. Considering the limitations of short-read NGS differentiating between certain regions of PMS2 and PMS2CL, using complementary methodologies is imperative to provide an accurate diagnosis.

Germline pathogenic variants in patients with early-onset neuroendocrine neoplasms.

Neuroendocrine neoplasms (NENs) are a rare group of cancers with heterogeneous behaviour and mostly of unknown aetiology. Excluding some infrequent hereditary cancer syndromes, the extent and clinical significance of mutations in other cancer predisposing genes (CPGs) are not known. We aimed to investigate the frequency of pathogenic and likely germline pathogenic variants (GPVs) in known CPGs in young adults with NEN and the clinical and molecular characteristics of these patients. We recruited 108 patients with lung or digestive NEN diagnosed between 18 and 50 years and performed targeted sequencing of 113 CPGs on germline DNA. For some patients, tumour features such as loss of heterozygosity (LOH), tumour mutation burden and microsatellite instability were evaluated. GPVs were detected in 17 patients (15.7%). Median age, sex, stage at diagnosis, family history of NENs or any personal history of neoplasm were similar between patients with or without GPVs. GPV carriers had more gastric (P = 0.084), functioning NEN (P = 0.041), positive family history of cancer (P = 0.015) and exclusively well-differentiated histology. Genes affected were mostly involved in DNA repair (CHEK2, ERCC2, ERCC3, XPC, MSH6, POLE and SLX4), with most GPVs found in MUTYH (four cases). LOH was performed in eight tumours and detected only in an SLX4-positive case. Overall, our findings indicate a role of inherited genetic alterations, particularly in DNA repair genes, in NEN carcinogenesis in young adults. These patients more often had a family history of cancer and functioning NENs.

Risk of metastasis in BRCA2 carriers diagnosed with triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is the neoplasia most associated with BRCA1 germline pathogenic variants (PV) and is more likely to develop metastases than the other breast cancer (BC) subtypes, mainly in the lungs and the central nervous system (CNS). Recently, BRCA2 carriers were shown to have a higher risk for developing CNS metastases. However, the patterns of recurrence and metastases of BRCA2 carriers with TNBC are unknown. METHODS: TNBC patient data attending the A.C. Camargo Cancer Center, from 1998 through 2020, were verified either by medical records or by BRCA1/2 genetic testing carried out. Multivariable logistic regression models were fit to the data to assess the independent factors for bone and CNS metastases. Adjustment was done using all independent variables with p < 0.2 in the univariable Cox model to describe the relationship between the independent variables until time of death. RESULTS: A total of 388 TNBC patients were evaluated. We identified PV in BRCA1/2 genes in 21% (82/388), being 17.7% (69/388) in BRCA1 and only 3.3% (13/388) in BRCA2. A total of 120 patients (31%) developed distant metastases. Bone or CNS metastases were observed in 40% and 60% of BRCA2 PV carriers (p = 0.155), respectively. The BRCA2 carriers tended to have a higher likelihood of developing bone metastases (OR, 4.06; 95% CI, 0.82-20.01; p = 0.085), when compared to BRCA1 carriers (OR, 0.6; 95% CI, 0.12-2.87; p = 0.528). BRCA2 carriers had an OR of 1.75 (95% CI, 0.33-9.14; p = 0.503) for CNS metastasis development, while BRCA1 carriers had an OR of 0.72 (95% CI, 0.23-2.23; p = 0.574). CONCLUSIONS: Patients with TNBC and PV in the BRCA2 gene had higher frequencies of secondary bone involvement and CNS in the course of the disease. However, the BRCA2 PV did not represent an independent outcome predictor of metastases and overall survival. Efforts to increase the number of BRCA2 carriers among TNBC patients are crucial for determining their risk of developing bone and CNS metastases compared to BRCA2 noncarriers.

Characterization of genetic predisposition to molecular subtypes of breast cancer in Brazilian patients.

Introduction: BRCA1 and BRCA2 germline pathogenic variants (GPVs) account for most of the 5-10% of breast cancer (BC) that is attributable to inherited genetic variants. BRCA1 GPVs are associated with the triple negative subtype, whereas BRCA2 GPVs are likely to result in higher grade, estrogen-receptor positive BCs. The contribution of other genes of high and moderate risk for BC has not been well defined and risk estimates to specific BC subtypes is lacking, especially for an admixed population like Brazilian. Objective: The aim of this study is to evaluate the value of a multigene panel in detecting germline mutations in cancer-predisposing genes for Brazilian BC patients and its relation with molecular subtypes and the predominant molecular ancestry. Patients and methods: A total of 321 unrelated BC patients who fulfilled NCCN criteria for BRCA1/2 testing between 2016-2018 were investigated with a 94-genes panel. Molecular subtypes were retrieved from medical records and ancestry-specific variants were obtained from off-target reads obtained from the sequencing data. Results: We detected 83 GPVs in 81 patients (positivity rate of 25.2%). Among GPVs, 47% (39/83) were identified in high-risk BC genes (BRCA1/2, PALB2 and TP53) and 18% (15/83) in moderate-penetrance genes (ATM, CHEK2 and RAD51C). The remainder of the GPVs (35% - 29/83), were identified in lower-risk genes. As for the molecular subtypes, triple negative BC had a mutation frequency of 31.6% (25/79), with predominance in BRCA1 (12.6%; 10/79). Among the luminal subtypes, except Luminal B HER2-positive, 18.7% (29/155) had GPV with BRCA1/2 genes contributing 7.1% (11/155) and non-BRCA1/2 genes, 12.9% (20/155). For Luminal B HER2-positive subtype, 40% (16/40) had GPVs, with a predominance of ATM gene (15% - 6/40) and BRCA2 with only 2.5% (1/40). Finally, HER2-enriched subtype presented a mutation rate of 30.8% (4/13) with contribution of BRCA2 of 7.5% (1/13) and non-BRCA1/2 of 23% (3/13). Variants of uncertain significance (VUS) were identified in 77.6% (249/321) of the patients and the number of VUS was increased in patients with Asian and Native American ancestry. Conclusion: The multigene panel contributed to identify GPVs in genes other than BRCA1/2, increasing the positivity of the genetic test from 9.6% (BRCA1/2) to 25.2% and, considering only the most clinically relevant BC predisposing genes, to 16.2%. These results indicate that women with clinical criteria for hereditary BC may benefit from a multigene panel testing, as it allows identifying GPVs in genes that directly impact the clinical management of these patients and family members.

MIR605 rs2043556 is associated with the occurrence of multiple primary tumors in TP53 p.(Arg337His) mutation carriers.

Li-Fraumeni and Li-Fraumeni-like (LFS/LFL) Syndrome are cancer predisposition syndromes caused by germline pathogenic variants in TP53 and are associated with an increased risk of multiple early-onset cancers. In Southern and Southeastern Brazil, a germline founder variant with partial penetrance located in the oligomerization domain of TP53, c.1010G>A p.(Arg337His, commonly known as R337H), has been detected in 0.3% of the general population. Recently, the functional MIR605 variant rs2043556 (A>G) has been identified as a novel LFS phenotype modifier in families with germline TP53 DNA binding variants. In this study, our goal was to verify MIR605 rs2043556 allele frequencies and further explore its possible effects on the phenotype of 238 Brazilian individuals carrying TP53 p.(Arg337His). The MIR605 rs2043556 G allele was detected in 136 (57.1%) individuals, including 25 homozygotes (10.5%), and although it had been previously associated with an earlier mean age of tumor onset, this effect was not observed in this study (p = 0.8). However, in p.(Arg337His) mutation carriers, the GG genotype was significantly associated with the occurrence of multiple primary tumors (p = 0.005). We provide further evidence of MIR605 rs2043556 G allele's effect as a phenotype modulator in carriers of germline TP53 pathogenic variants.

Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer.

Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.

Baseline Surveillance in Li-Fraumeni Syndrome Using Whole-Body Magnetic Resonance Imaging: A Meta-analysis.

Importance: Guidelines for clinical management in Li-Fraumeni syndrome, a multiple-organ cancer predisposition condition, are limited. Whole-body magnetic resonance imaging (WBMRI) may play a role in surveillance of this high-risk population. Objective: To assess the clinical utility of WBMRI in germline TP53 mutation carriers at baseline. Data Sources: Clinical and research surveillance cohorts were identified through the Li-Fraumeni Exploration Research Consortium. Study Selection: Cohorts that incorporated WBMRI for individuals with germline TP53 mutations from January 1, 2004, through October 1, 2016, were included. Data Extraction and Synthesis: Data were extracted by investigators from each cohort independently and synthesized by 2 investigators. Random-effects meta-analysis methods were used to estimate proportions. Main Outcomes and Measures: The proportions of participants at baseline in whom a lesion was detected that required follow-up and in whom a new primary malignant neoplasm was detected. Results: A total of 578 participants (376 female [65.1%] and 202 male [34.9%]; mean [SD] age, 33.2 [17.1] years) from 13 cohorts in 6 countries were included in the analysis. Two hundred twenty-five lesions requiring clinical follow-up were detected by WBMRI in 173 participants. Sixty-one lesions were diagnosed in 54 individuals as benign or malignant neoplasms. Overall, 42 cancers were identified in 39 individuals, with 35 new localized cancers treated with curative intent. The overall estimated detection rate for new, localized primary cancers was 7% (95% CI, 5%-9%). Conclusions and Relevance: These data suggest clinical utility of baseline WBMRI in TP53 germline mutation carriers and may form an integral part of baseline clinical risk management in this high-risk population.

Perfil genômico da síndrome do xeroderma pigmentoso no Brasil: avaliação de alterações germinativas e somáticas / Genomic profile of xeroderma pigmentosum syndrome in Brazil: assessment of germline and somatic alterations

A síndrome do Xeroderma Pigmentoso (XP) ocorre frente à herança monogênica e bialélica de variantes germinativas patogênicas de perda ou redução de função em genes das vias de reparo por excisão de nucleotídeos ou síntese translesão. Consequentemente, é estabelecida deficiência na correção de lesões induzidas, principalmente por radiação ultravioleta, favorecendo alta sensibilidade à radiação solar e risco aumentado para o desenvolvimento de múltiplas lesões cutâneas pré-malignas e malignas. Visto que a heterogeneidade na manifestação clínica da síndrome é uma questão em discussão na literatura, para investigar este aspecto propusemos avaliar o perfil de variantes germinativas e variantes somáticas de tumores cutâneos e não cutâneos de indivíduos portadores de XP. Foi realizado o sequenciamento de alto desempenho utilizando a plataforma NextSeq (Illumina) para avaliar as regiões codificantes de 114 genes selecionados pela sua relevância em desordens dermatológicas, tumorigênese e fisiologia cutânea e resposta de dano ao DNA. Seis pacientes com fenótipo clínico da síndrome do XP e portadores de variantes germinativas clinicamente relevantes nos genes XPC ou POLH/XPV foram avaliados no estudo. Variantes germinativas de significado incerto foram identificadas, em heterozigose, no DNA de leucócito de cinco dos seis pacientes avaliados ocorrendo nos genes DNAH11, PCDHB3, RGS22, SLC27A5, TTN e UGT2B10 e nenhuma das variantes identificadas apresentou perda de heterozigose do alelo selvagem nos tecidos tumorais. O polimorfismo de risco para carcinoma basocelular de pele (CBC) rs3769823[A] no gene CASP8 não foi identificado em apenas um caso do estudo, o qual desenvolveu o menor número de tumores. O polimorfismo de risco rs1126809[A] no gene TYR foi detectado apenas no caso que apresentou o maior número de CBC. Amostras de DNA de nove CBCs de tecido armazenado em parafina e duas amostras de tumor gástrico de uma mesma peça cirúrgica, de tecido armazenado em parafina e congelado a fresco, foram avaliadas de forma pareada com o DNA de leucócito correspondente, para pesquisa de variantes somáticas. Variantes somáticas não foram identificadas na amostra de CBC da paciente XP-C com fenótipo menos agressivo da síndrome. O total de 235 variantes missense e 29 variantes de perda de função foram identificadas em 71 genes para sete amostras de CBC, mínimo de 11 e máximo de 127 variantes por amostra, com 85,2% destas apresentando frequência alélica ≥20%. Com exceção de um CBC, mais de 95% das variantes somáticas identificadas representam alterações tipicamente fotoinduzidas (C:G>T:A e G:C>T:A). Embora pacientes XP acumulem maior número de mutações devido deficiência no mecanismo de reparo, não observamos carga mutacional diferente do observado em CBCs esporádicos. Vinte e sete genes apresentaram variantes somáticas em mais de uma amostra de CBC. Nenhum gene foi compartilhado entre as sete amostras de CBC. Entre os genes alterados em maior número de tumores estão incluídos genes drivers de CBC (LATS1, NOTCH2, PTCH1, PTPN14 e TP53), bem como genes não clássicos na carcinogênese do CBC (APC, FLG e TTN). Uma variante driver em SMO foi recorrente em três CBCs de um mesmo paciente. Duas variantes somáticas foram identificadas no tumor gástrico de tecido congelado a fresco ocorrendo nos genes GLI3 e RB1, não sendo as mesmas detectadas no tecido armazenado em parafina. Nesse trabalho, ressalta-se a heterogeneidade na manifestação clínica da síndrome do XP e a identificação de dois polimorfismos de risco, bem como destaca-se o papel central das vias Sonic Hedgehog e Hippo na carcinogênese do CBC de pacientes XP (AU)

The Xeroderma Pigmentosum (XP) syndrome occurs on base of biallelic inheritance of pathogenic germline variants of loss of function or function reduction in genes that plays role in nucleotide excision repair and translesion synthesis. Consequently, patients are deficient in correct DNA lesions mainly induced by ultraviolet radiation, present high sensitivity to solar radiation and increased risk for the development of multiple premalignant and malignant skin lesions. Since the heterogeneity in the clinical manifestation is under constantly discussion in the literature, to investigate it we proposed to explore the profile of germline variants and somatic variants in skin and non-skin tumors from XP patients. High-performance sequencing using the NextSeq (Illumina) platform was performed to assess the coding regions of 114 genes selected for their relevance in dermatological disorders, skin carcinogenesis, cutaneous physiology and DNA damage response. Six patients with clinical phenotype of XP syndrome and carriers of clinically relevant germline variants in the XPC or POLH/XPV genes were evaluated in the study. Heterozygous germline variants of uncertain significance were identified in the leukocyte DNA from five of the six patients occurring in DNAH11, PCDHB3, RGS22, SLC27A5, TTN and UGT2B10 genes. None of the identified variants showed loss of heterozygosity of the wild allele in tumor tissues. The CASP8 risk polymorphism for basal cell carcinoma of the skin (BCC) rs3769823[A] was not identified in only one case of the study which developed the minor number of tumors. The TYR risk polymorphism rs1126809[A] was detected only in the case with the highest number of BCC. Somatic variants were investigated in DNA from nine samples of BCCs (tissue stored in paraffin) and two samples of gastric tumor from the same surgical (tissue stored in paraffin and fresh frozen), all paired with the corresponding leukocyte DNA. Somatic variants were not identified in the BCC sample of XP-C patient with a less aggressive syndrome phenotype. A total of 235 missense variants and 29 loss of function variants were identified in 71 genes for seven BCC samples. A minimum of 11 and a maximum of 127 variants per sample were detected, with 85.2% showing an allelic frequency ≥20%. Except for one BCC, more than 95% of the identified somatic variants represented typically photoinduced mutations (C:G>T:A and G:C>T:A). Although XP patients accumulate a greater number of mutations due to deficiency in the repair mechanism, we did not observe different mutational load compared with sporadic BCCs. Twenty-seven genes showed somatic variants in more than one BCC sample. Genes shared between the seven BCC samples were not found. Among the altered genes in a greater number of tumors, it was identified BCC driver genes (LATS1, NOTCH2, PTCH1, PTPN14 and TP53), as well as genes non-classical for BCC carcinogenesis (APC, FLG and TTN). A driver variant in SMO was recurrent in three BCCs from the same patient. Two somatic variants in GLI3 and RB1 genes were identified occurring only in the fresh frozen tissue of gastric tumor, not in the tissue stored in paraffin. In this work, the heterogeneous clinical manifestation of XP syndrome is highlighted, as well as the identification of two risk polymorphisms. In addition, this work emphasizes the central role of the Sonic Hedgehog and Hippo pathways in BCC carcinogenesis of XP patients.