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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.

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Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

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Hepatoblastoma stands as the most prevalent liver cancer in the pediatric population. Characterized by a low mutational burden, chromosomal and epigenetic alterations are key drivers of its tumorigenesis. Transcriptome analysis is a powerful tool for unraveling the molecular intricacies of hepatoblastoma, shedding light on the effects of genetic and epigenetic changes on gene expression. In this study conducted in Brazilian patients, an in-depth whole transcriptome analysis was performed on 14 primary hepatoblastomas, compared to control liver tissues. The analysis unveiled 1,492 differentially expressed genes (1,031 upregulated and 461 downregulated), including 920 protein-coding genes (62%). Upregulated biological processes were linked to cell differentiation, signaling, morphogenesis, and development, involving known hepatoblastoma-associated genes (DLK1, MEG3, HDAC2, TET1, HMGA2, DKK1, DKK4), alongside with novel findings (GYNG4, CDH3, and TNFRSF19). Downregulated processes predominantly centered around oxidation and metabolism, affecting amines, nicotinamides, and lipids, featuring novel discoveries like the repression of SYT7, TTC36, THRSP, CCND1, GCK and CAMK2B. Two genes, which displayed a concordant pattern of DNA methylation alteration in their promoter regions and dysregulation in the transcriptome, were further validated by RT-qPCR: the upregulated TNFRSF19, a key gene in the embryonic development, and the repressed THRSP, connected to lipid metabolism. Furthermore, based on protein-protein interaction analysis, we identified genes holding central positions in the network, such as HDAC2, CCND1, GCK, and CAMK2B, among others, that emerged as prime candidates warranting functional validation in future studies. Notably, a significant dysregulation of non-coding RNAs (ncRNAs), predominantly upregulated transcripts, was observed, with 42% of the top 50 highly expressed genes being ncRNAs. An integrative miRNA-mRNA analysis revealed crucial biological processes associated with metabolism, oxidation reactions of lipids and carbohydrates, and methylation-dependent chromatin silencing. In particular, four upregulated miRNAs (miR-186, miR-214, miR-377, and miR-494) played a pivotal role in the network, potentially targeting multiple protein-coding transcripts, including CCND1 and CAMK2B. In summary, our transcriptome analysis highlighted disrupted embryonic development as well as metabolic pathways, particularly those involving lipids, emphasizing the emerging role of ncRNAs as epigenetic regulators in hepatoblastomas. These findings provide insights into the complexity of the hepatoblastoma transcriptome and identify potential targets for future therapeutic interventions.

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Ependymomas (EPN) are central nervous system neoplasms that exhibit an ependymal phenotype. In particular, supratentorial EPN (ST-EPN) must be differentiated from more aggressive entities such as glioblastoma, IDH-wildtype. This task is frequently addressed with the use of immunohistochemistry coupled with clinical presentation and morphological features. Here we describe the case of a young adult presenting with migraine-like symptoms and a temporoinsular-based expansile mass that was first diagnosed as a GBM, mostly based on strong and diffuse oligodendrocyte transcription factor 2 (OLIG2) expression. Molecular characterization revealed a ZFTA::RELA fusion, supporting the diagnosis of ST-EPN, ZFTA fusion-positive. OLIG2 expression is rarely reported in tumors other than GBM and oligodendrocyte-lineage committed neoplasms. The patient was treated with radiotherapy and temozolomide after surgery and was alive and well at follow-up. This report illustrates the need to assess immunostains within a broader clinical, morphological and molecular context to avoid premature exclusion of important differential diagnoses.

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PURPOSE: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. METHODS: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. RESULTS: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. CONCLUSIONS: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

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Increased genetic risk for melanoma can occur in the context of germline pathogenic variants in high-penetrance genes, such as CDKN2A and CDK4, risk variants in low- to moderate-penetrance genes (MC1R and MITF), and possibly due to variants in emerging genes, such as ACD, TERF2IP, and TERT. We aimed to identify germline variants in high- and low- to moderate-penetrance melanoma risk genes in Brazilian patients with clinical criteria for familial melanoma syndrome. We selected patients with three or more melanomas or melanoma patients from families with three tumors (melanoma and pancreatic cancer) in first- or second-degree relatives. Genetic testing was performed with a nine-gene panel (ACD, BAP1, CDK4, CDKN2A, POT1, TERT, TERF2IP, MC1R, and MITF). In 36 patients, we identified 2 (5.6%) with germline pathogenic variants in CDKN2A and BAP1 and 4 (11.1%) with variants of uncertain significance in the high-penetrance genes. MC1R variants were found in 86.5%, and both red hair color variants and unknown risk variants were enriched in patients compared to a control group. The low frequency of germline pathogenic variants in the high-penetrance genes and the high prevalence of MC1R variants found in our cohort show the importance of the MC1R genotype in determining the risk of melanoma in the Brazilian melanoma-prone families.

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BACKGROUND: Multiple primary melanoma (MPM) is a diagnostic challenge even with ancillary imaging technologies available to dermatologists. In selected patients' phenotypes, the use of imaging approaches can help better understand lesion characteristics, and aid in early diagnosis and management. METHODS: Under a 5-year prospective single-center follow-up, 58 s primary melanomas (SPMs) were diagnosed in two first-degree relatives, with fair skin color, red hair, green eyes, and personal history of one previous melanoma each. Patients' behavior and descriptive demographic data were collected from medical records. The information on the first two primary melanomas (PMs) were retrieved from pathology reports. The characteristics of 60 melanomas were collected from medical records, video dermoscopy software, and pathology reports. Reflectance confocal microscopy (RCM) was performed prior to excision of 22 randomly selected melanomas. RESULTS: From February 2018 to May 2023, two patients underwent a pooled total of 214 excisional biopsies of suspect lesions, resulting in a combined benign versus malignant treatment ratio (NNT) of 2.0:1.0. The number of moles excised for each melanoma diagnosed (NNE) was 1.7:1.0 and 6.9:1.0 for the female and male patient respectively. The in-situ melanoma/invasive melanoma ratio (IIR) demonstrated a higher proportion of in-situ melanomas for both patients. From June 2018 to May 2023, a total of 58 SPMs were detected by the combination of total body skin exam (TBSE), total body skin photography (TBSP), digital dermoscopy (DD), and sequential digital dermoscopy imaging (SDDI) via comparative approach. The younger patient had her PM one month prior to the second and third cutaneous melanomas (CMs), characterizing a case of synchronous primary CM. The male older relative had a total of 7 nonsynchronous melanomas. CONCLUSIONS: This CM cohort is composed of 83.3% in-situ melanoma and 16.7% invasive melanoma. Both patients had a higher percentage of SPM with clinical nevus-like morphology (84.5%), global dermoscopic pattern of asymmetric multiple component (60.3%) and located on the lower limbs (46.6%). When RCM was performed prior to excision, 81% of SPM had features suggestive of malignancy. As well, invasive melanomas were more frequent in the lower limbs (40%). In the multivariate model, for the two high-risk patients studied, the chance of a not associated with nevus ("de novo") invasive SPM diagnosis is 25 times greater than the chance of a diagnosis of a nevus-associated invasive SPM.

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Circulating tumor cells (CTCs) and/or circulating tumor microemboli (CTM) from non-small cell lung cancer (NSCLC) patients may be a non-invasive tool for prognosis, acting as liquid biopsy. CTCs interact with platelets through the transforming growth factor-ß/transforming growth factor-ß receptor type 1 (TGF-ß/TGFßRI) forming clusters. CTCs also may express the Cluster of Differentiation 47 (CD47) protein, responsible for the inhibition of phagocytosis, the "don't eat me" signal to macrophages. OBJECTIVES: To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFßRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). METHODS: Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET®. Immunocytochemistry was conducted to evaluate TGFßRI/CD47 expression. RESULTS: 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33-11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33-9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFßRI expression in CTCs/CTMs was not associated with PFS. CONCLUSION: In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFßRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS.

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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.

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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.

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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.

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Detecting MLH1 promoter methylation is highly relevant to differentiate between possible Lynch syndrome patients or patients with sporadic causes of MLH1/PMS2 deficiency in colorectal (CRC) and endometrial cancers. Here, we aimed to develop a test for assessing MLH1 promoter methylation based in next generation sequencing (NGS), and to evaluate the concordance of MLH1 methylation and BRAF-V600 mutation status in CRC. For that, we performed a series of experiments with DNA from tumor, saliva and commercial control samples and our in house developed amplicon-based NGS test. In patients' samples, MLH1 methylation above 10% was only observed in tumors with MLH1/PMS2 loss. We confirmed the reproducibility and accuracy of MLH1 promoter analysis performing a serial dilution experiment with completely methylated and unmethylated control DNAs and a comparison between two NGS platforms (Ion Proton and Illumina). In MLH1/PMS2 deficient tumors, the MLH1 methylation status was concordant with the BRAF mutation status in 90% (18/20) of the cases. Our amplicon-based NGS test showed a great sensitivity and specificity for detecting MLH1 methylation in CRC samples, with a high agreement with the evaluation of BRAF mutation. This simple and affordable test could be used as a reflex test to identify patients with sporadic causes of MLH1/PMS2 deficiency in CRC, aiding to genetic test referral and identification of Lynch syndrome patients.

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Purpose: To determine molecular events involved in the tumorigenesis of phyllodes tumors (PT) and the role of each stromal (SC) and epithelial (EC) cell. Methods: Frozen breast samples enriched with epithelial and stromal cells from three fibroadenomas and 14 PT were retrieved and laser microdissected. Sanger and polymerase chain reaction-based sequencing of exon 2 MED12 and TERT promoter hotspot mutations were performed; 44K microarray platform was used to analyze gene expression. Results: All three fibroadenomas (FAs) presented mutations in MED12, but not in TERT, whose mutation was observed in five of the 14 PTs. EC and SC of each affected tumor displayed identical alterations. Of the total differentially expressed genes (DEG) (EC = 1,543 and SC = 850), 984 were EC-eDEGs and 291 were SC-eDEGs. We found a high similarity of diseases and functions enriched by both cell types, but dissimilarity in the number of enriched canonical pathways. Three signaling canonical pathways overlapping with EC and SC were predicted to be activated in one cell type and inactivated in the other, while no overlap in eDEGs was assigned to them. We also identified 13 EC-eDEGs and five SC-eDEGs enriched networks, in which the SC-eDEGs were able to segregate FA from PT samples. Conclusions: Identical TERT mutations from both SC and ES origins might affect the PTs tumorigenesis. Gene expression differences suggest coordinated molecular processes between these components with determinant differences acquired by SC, able to fully distinguish PTs from FAs lesions.

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Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues.

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Scalp melanoma (SM) has a worse prognosis than melanoma in other locations likely because of late diagnosis due to hair coverage, difficulties in interpreting dermoscopy findings, and its unique molecular profile. We aimed to describe the clinical, histopathological, molecular, and dermoscopic patterns of SM and its relation to androgenetic alopecia/elastosis at the tumor site. Through a retrospective cross-sectional study, we identified all SM diagnosed at the A.C.Camargo Cancer Center between 2008 and 2018. In all, 48 SM were analyzed: 45.8% of which exhibited moderate/severe androgenetic alopecia and 54.1% exhibited elastosis. Androgenetic alopecia/elastosis at the site of the SM was associated with older age (p < 0.001), chronic sun damage (p < 0.001), lentigo maligna subtype (p = 0.029), and photodamaged dermoscopic pattern (p < 0.001). Additionally, 41 cases were evaluated with a 14-gene panel: 53.7% displayed mutations and 46.3% were wild-type. BRAF mutations were most common (77%), with BRAF V600K being more frequent (50%) than BRAF V600E (31.2%). The NF1 gene was evaluated in 40 samples, of which 20% exhibited mutations. SM presents differently in areas covered by hair compared to in areas with androgenetic alopecia. Patients without alopecia may have higher Breslow thickness due to late diagnosis because of hair concealment. The high frequency of detrimental mutations can also explain the poor prognosis of SM.

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Salivary gland neoplasms comprise a heterogeneous group of lesions with multiple histological subtypes, each with distinct growth patterns, resulting in a spectrum of tumor-specific prognoses; pleomorphic adenoma (PA) and mucoepidermoid carcinoma (MEC) are the most common representatives of these neoplasms. Many studies have associated specific profiles of membrane and adhesion molecules in salivary gland tissues; these profiles appear to be relevant in tumor biology as well as be interpreted as fingerprints for tumor classification, diagnostic prognostic and therapeutic targets. One of these membrane molecule complexes are the tight junctions, composed by various proteins, in which claudins are protagonists. The aim of this study was to evaluate the expressions of genes that encode tight junction proteins (CLDN-1, -3, -4, -5, -7, and -11, occludin [OCLN], zonula occludens [TJP1, TJP2, and TJP3] and junctional adhesion molecule A [F11R]) in MEC and PA using real time RT-PCR. We observed high expression of CLDN-1 and -7 and low expression of CLDN-3, -11 and TJP2 in MEC compared to PA. PA samples demonstrated high OCLN expression when compared to MEC. CRTC1::MAML2 fusion was detected in 12 of 20 (60.0%) MEC samples and was associated with CLDN7 expression, while the absence of fusion was associated with high histological grade. Increased CLDN5 expression was associated with submandibular gland tumors. This study demonstrated differential expressions of genes encoding tight junction constituent proteins and their associations with tumor characteristics, suggesting their potential future role as diagnostic and prognostic markers.

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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.

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Circular RNAs (circRNAs) are a class of long non-coding RNAs that have the ability to sponge RNA-Binding Proteins (RBPs). Triple-negative breast cancer (TNBC) has very aggressive behavior and poor prognosis for the patient. Here, we aimed to characterize the global expression profile of circRNAs in TNBC, in order to identify potential risk biomarkers. For that, we obtained RNA-Seq data from TNBC and control samples and performed validation experiments using FFPE and frozen tissues of TNBC patients and controls, followed by in silico analyses to explore circRNA-RBP interactions. We found 16 differentially expressed circRNAs between TNBC patients and controls. Next, we mapped the RBPs that interact with the top five downregulated circRNAs (hsa_circ_0072309, circ_0004365, circ_0006677, circ_0008599, and circ_0009043) and hsa_circ_0000479, resulting in a total of 16 RBPs, most of them being enriched to pathways related to cancer and gene regulation (e.g., AGO1/2, EIF4A3, ELAVL1, and PTBP1). Among the six circRNAs, hsa_circ_0072309 was the one that presented the most confidence results, being able to distinguish TNBC patients from controls with an AUC of 0.78 and 0.81, respectively. This circRNA may be interacting with some RBPs involved in important cancer-related pathways and is a novel potential risk biomarker of TNBC.