Diffuse hemispheric glioma with H3 p.K28M (K27M) mutation: Unusual non-midline presentation of diffuse midline glioma, H3 K27M-altered?

Diffuse midline glioma, H3 K27-altered (DMG-H3 K27) is an aggressive group of diffuse gliomas that predominantly occurs in pediatric patients, involves midline structures, and displays loss of H3 p.K28me3 (K27me3) expression by immunohistochemistry and characteristic genetic/epigenetic profile. Rare examples of a diffuse glioma with an H3 p.K28M (K27M) mutation and without involvement of the midline structures, so-called "diffuse hemispheric glioma with H3 p.K28M (K27M) mutation" (DHG-H3 K27), have been reported. Herein, we describe 2 additional cases of radiologically confirmed DHG-H3 K27 and summarize previously reported cases. We performed histological, immunohistochemical, molecular, and DNA methylation analysis and provided clinical follow-up in both cases. Overall, DHG-H3 K27 is an unusual group of diffuse gliomas that shows similar clinical, histopathological, genomic, and epigenetic features to DMG-H3 K27 as well as enrichment for activating alterations in MAPK pathway genes. These findings suggest that DHG-H3 K27 is closely related to DMG-H3 K27 and may represent an unusual presentation of DMG-H3 K27 without apparent midline involvement and with frequent MAPK pathway activation. Detailed reports of additional cases with clinical follow-up will be important to expand our understanding of this unusual group of diffuse gliomas and to better define the clinical outcome and how to classify DHG-H3 K27.

Characterization of Potential Melanoma Predisposition Genes in High-Risk Brazilian Patients.

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.

Strong OLIG2 expression in supratentorial ependymoma, ZFTA fusion-positive: A potential diagnostic pitfall.

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.

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.

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.

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.

Amplicon-based NGS test for assessing MLH1 promoter methylation and its correlation with BRAF mutation in colorectal cancer patients.

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.

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.

Tight junction gene expression in salivary gland tumors.

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.

Dihydropyrimidine dehydrogenase (DPD) polymorphisms knocking on the door.

Identifying polymorphisms in the dihydropyrimidine dehydrogenase (DPYD) genes is gaining importance as predictors of fluoropyrimidine-associated toxicity. The recommendation of dose adjustment for chemotherapy guided by the presence of polymorphisms of the DPYD gene can potentially improve treatment safety for a large number of patients, saving lives, avoiding complications and reducing health care costs. This article discusses how personalisation of fluoropyrimidine treatment based on the identification of DPYD variants can mitigate toxicities and be cost effective.

Case Report of Small Cell Carcinoma of the Ovary, Hypercalcemic Type (Ovarian Rhabdoid Tumor) with SMARCB1 Mutation: A Literature Review of a Rare and Aggressive Condition.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive condition that is associated with the SMARCA4 mutation and has a dismal prognosis. It is generally diagnosed in young women. Here, we report a case of a young woman with SCCOHT harboring a rare molecular finding with a highly aggressive biological behavior. The patient had a somatic SMARCB1 mutation instead of an expected SMARCA4 alteration. Even though the patient was treated with high-dose chemotherapy followed by stem cell transplantation, she evolved with disease progression and died 11 months after her first symptoms appeared. We present a literature review of this rare disease and discuss the findings in the present patient in comparison to expected molecular alterations and options for SCCOHT treatment.

Family-based whole-exome sequencing identifies rare variants potentially related to cutaneous melanoma predisposition in Brazilian melanoma-prone families.

Genetic predisposition accounts for nearly 10% of all melanoma cases and has been associated with a dozen moderate- to high-penetrance genes, including CDKN2A, CDK4, POT1 and BAP1. However, in most melanoma-prone families, the genetic etiology of cancer predisposition remains undetermined. The goal of this study was to identify rare genomic variants associated with cutaneous melanoma susceptibility in melanoma-prone families. Whole-exome sequencing was performed in 2 affected individuals of 5 melanoma-prone families negative for mutations in CDKN2A and CDK4, the major cutaneous melanoma risk genes. A total of 288 rare coding variants shared by the affected relatives of each family were identified, including 7 loss-of-function variants. By performing in silico analyses of gene function, biological pathways, and variant pathogenicity prediction, we underscored the putative role of several genes for melanoma risk, including previously described genes such as MYO7A and WRN, as well as new putative candidates, such as SERPINB4, HRNR, and NOP10. In conclusion, our data revealed rare germline variants in melanoma-prone families contributing with a novel set of potential candidate genes to be further investigated in future studies.

Genetic Landscape of Male Breast Cancer.

Male breast cancer (MBC) is now considered molecularly different from female breast cancer (FBC). Evidence from studies indicates that common genetic and epigenetic features of FBC are not shared with those diagnosed in men. Genetic predisposition is likely to play a significant role in the tumorigenesis of this rare disease. Inherited germline variants in BRCA1 and BRCA2 account for around 2% and 10% of MBC cases, respectively, and the lifetime risk of breast cancer for men harboring BRCA1 and BRCA2 mutations is 1.2% and 6.8%. As for FBC, pathogenic mutations in other breast cancer genes have also been recently associated with an increased risk of MBC, such as PALB2 and CHEK2 mutations. However, while multigene germline panels have been extensively performed for BC female patients, the rarity of MBC has resulted in limited data to allow the understanding of the magnitude of risk and the contribution of recently identified moderate penetrance genes of FBC for MBC predisposition. This review gathers available data about the germline genetic landscape of men affected by breast cancer, estimated risk associated with these genetic variants, and current guidelines for clinical management.

Desmoplastic Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging Therapy.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from the serosal surface of the abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1-WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within three years. The dismal survival makes DSRCT an orphan disease with an urgent need for new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors was evaluated by small trials. Recent studies using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.

The mutational repertoire of uterine sarcomas and carcinosarcomas in a Brazilian cohort: A preliminary study.

OBJECTIVES: The present study aimed to contribute to the catalog of genetic mutations involved in the carcinogenic processes of uterine sarcomas (USs) and carcinosarcomas (UCSs), which may assist in the accurate diagnosis of, and selection of treatment regimens for, these conditions. METHODS: We performed gene-targeted next-generation sequencing (NGS) of 409 cancer-related genes in 15 US (7 uterine leiomyosarcoma [ULMS], 7 endometrial stromal sarcoma [ESS], 1 adenosarcoma [ADS]), 5 UCS, and 3 uterine leiomyoma (ULM) samples. Quality, frequency, and functional filters were applied to select putative somatic variants. RESULTS: Among the 23 samples evaluated in this study, 42 loss-of-function (LOF) mutations and 111 missense mutations were detected, with a total of 153 mutations. Among them, 66 mutations were observed in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. TP53 (48%), ATM (22%), and PIK3CA (17%) were the most frequently mutated genes. With respect to specific tumor subtypes, ESS showed mutations in the PDE4DIP, IGTA10, and DST genes, UCS exhibited mutations in ERBB4, and ULMS showed exclusive alterations in NOTCH2 and HER2. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS. In silico pathway analyses demonstrated that many genes mutated in ULMS and ESS have functions associated with the cellular response to hypoxia and cellular response to peptide hormone stimulus. In UCS and ADS, genes with most alterations have functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process. CONCLUSION: This preliminary study observed pathogenic mutations in US and UCS samples. Further studies with a larger cohort and functional analyses will foster the development of a precision medicine-based approach for the treatment of US and UCS.

Impact of BRCA1/2 Mutations on the Efficacy of Secondary Cytoreductive Surgery.

BACKGROUND: Phase III trials evaluating the role of secondary cytoreductive surgery (SCS) in recurrent ovarian cancer have pointed to the importance of patient selection. Two studies showed conflicting results regarding the benefit of SCS in BRCA1/2 mutation carriers. Our aim was to evaluate the impact of SCS on recurrent ovarian cancer according to BRCA1/2 status. METHODS: All patients with ovarian carcinoma with platinum-sensitive recurrent disease and tested for BRCA1/2 germline mutations were included. Cox regression and log rank test were used to evaluate the impact of SCS on progression-free survival (PFS) and the influence of BRCA1/2 mutations on the effect of SCS. RESULTS: 127 patients were included, 45.6% were treated with SCS and chemotherapy and 54.3% treated with chemotherapy only. Patients treated with SCS were younger, presented better performance status, had lower CA125, and had a longer platinum-free interval. In multivariate analysis SCS was associated with longer PFS (HR 0.42, 95% CI 0.25-0.72, p = 0.002). BRCA1/2 mutations were found in 35 patients (27.5%), and 11.8% of patients were treated with PARP inhibitors. Although not statistically significant, both BRCA1/2 wild type patients (PFS: 21.6 vs 18.4 months; p = 0.114) and BRCA1/2 mutation carriers (PFS: 23.1 vs 18.2 months, p = 0.193) appeared to derive benefit from SCS. DISCUSSION: The present study suggests a benefit of SCS irrespective of BRCA1/2 status among patients mostly not treated with PARP inhibitor. Further data on post hoc analysis from the phase III trials are warranted to confirm whether BRCA1/2 mutated patients should be selected for SCS.

The mutational repertoire of uterine sarcomas and carcinosarcomas in a Brazilian cohort: A preliminary study

OBJECTIVES: The present study aimed to contribute to the catalog of genetic mutations involved in the carcinogenic processes of uterine sarcomas (USs) and carcinosarcomas (UCSs), which may assist in the accurate diagnosis of, and selection of treatment regimens for, these conditions. METHODS: We performed gene-targeted next-generation sequencing (NGS) of 409 cancer-related genes in 15 US (7 uterine leiomyosarcoma [ULMS], 7 endometrial stromal sarcoma [ESS], 1 adenosarcoma [ADS]), 5 UCS, and 3 uterine leiomyoma (ULM) samples. Quality, frequency, and functional filters were applied to select putative somatic variants. RESULTS: Among the 23 samples evaluated in this study, 42 loss-of-function (LOF) mutations and 111 missense mutations were detected, with a total of 153 mutations. Among them, 66 mutations were observed in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. TP53 (48%), ATM (22%), and PIK3CA (17%) were the most frequently mutated genes. With respect to specific tumor subtypes, ESS showed mutations in the PDE4DIP, IGTA10, and DST genes, UCS exhibited mutations in ERBB4, and ULMS showed exclusive alterations in NOTCH2 and HER2. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS. In silico pathway analyses demonstrated that many genes mutated in ULMS and ESS have functions associated with the cellular response to hypoxia and cellular response to peptide hormone stimulus. In UCS and ADS, genes with most alterations have functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process. CONCLUSION: This preliminary study observed pathogenic mutations in US and UCS samples. Further studies with a larger cohort and functional analyses will foster the development of a precision medicine-based approach for the treatment of US and UCS.