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Background: The Prospective Lynch Syndrome Database (PLSD) collates information on carriers of pathogenic or likely pathogenic MMR variants (path_MMR) who are receiving medical follow-up, including colonoscopy surveillance, which aims to the achieve early diagnosis and treatment of cancers. Here we use the most recent PLSD cohort that is larger and has wider geographical representation than previous versions, allowing us to present mortality as an outcome, and median ages at cancer diagnoses for the first time.Methods: The PLSD is a prospective observational study without a control group that was designed in 2012 and updated up to October 2022. Data for 8500 carriers of path_MMR variants from 25 countries were included, providing 71,713 years of follow up. Cumulative cancer incidences at 65 years of age were combined with 10-year crude survival following cancer, to derive estimates of mortality up to 75 years of age by organ, gene, and gender.Findings: Gynaecological cancers were more frequent than colorectal cancers in path_MSH2, path_MSH6 and path_PMS2 carriers [cumulative incidence: 53.3%, 49.6% and 23.3% at 75 years, respectively]. Endometrial, colon and ovarian cancer had low mortality [8%, 13% and 15%, respectively] and prostate cancers were frequent in male path_MSH2 carriers [cumulative incidence: 39.7% at 75 years]. Pancreatic, brain, biliary tract and ureter and kidney and urinary bladder cancers were associated with high mortality [83%, 66%, 58%, 27%, and 29%, respectively]. Among path_MMR carriers undergoing colonoscopy surveillance, particularly path_MSH2 carriers, more deaths followed non-colorectal Lynch syndrome cancers than colorectal cancers.Interpretation: In path_MMR carriers undergoing colonoscopy surveillance, non-colorectal Lynch syndrome cancers were associated with more deaths than were colorectal cancers. Reducing deaths from non-colorectal cancers presents a key challenge in contemporary medical care in Lynch syndrome. Background: The Prospective Lynch Syndrome Database (PLSD) collates information on carriers of pathogenic or likely pathogenic MMR variants (path_MMR) who are receiving medical follow-up, including colonoscopy surveillance, which aims to the achieve early diagnosis and treatment of cancers. Here we use the most recent PLSD cohort that is larger and has wider geographical representation than previous versions, allowing us to present mortality as an outcome, and median ages at cancer diagnoses for the first time. Methods: The PLSD is a prospective observational study without a control group that was designed in 2012 and updated up to October 2022. Data for 8500 carriers of path_MMR variants from 25 countries were included, providing 71,713 years of follow up. Cumulative cancer incidences at 65 years of age were combined with 10-year crude survival following cancer, to derive estimates of mortality up to 75 years of age by organ, gene, and gender. Findings: Gynaecological cancers were more frequent than colorectal cancers in path_MSH2, path_MSH6 and path_PMS2 carriers [cumulative incidence: 53.3%, 49.6% and 23.3% at 75 years, respectively]. Endometrial, colon and ovarian cancer had low mortality [8%, 13% and 15%, respectively] and prostate cancers were frequent in male path_MSH2 carriers [cumulative incidence: 39.7% at 75 years]. Pancreatic, brain, biliary tract and ureter and kidney and urinary bladder cancers were associated with high mortality [83%, 66%, 58%, 27%, and 29%, respectively]. Among path_MMR carriers undergoing colonoscopy surveillance, particularly path_MSH2 carriers, more deaths followed non-colorectal Lynch syndrome cancers than colorectal cancers. Interpretation: In path_MMR carriers undergoing colonoscopy surveillance, non-colorectal Lynch syndrome cancers were associated with more deaths than were colorectal cancers. Reducing deaths from non-colorectal cancers presents a key challenge in contemporary medical care in Lynch syndrome. Funding: We acknowledge funding from the Norwegian Cancer Society, contract 194751-2017.

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.

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.

The current study aimed to identify new breast and/or ovarian cancer predisposition genes. For that, whole‐exome sequencing (WES) was performed in the germline DNA of 52 non‐BRCA1/BRCA2/TP53 mutation carrier women at high‐risk for hereditary breast and ovarian cancer (HBOC). All variants were classified using information from population and disease specific databases, in silico prediction tools and the American College of Medical Genetics and Genomics (ACMG) criteria. Loss of heterozygosity (LOH) of tumor samples and segregation analyses were performed whenever possible. The variants identified were investigated in a second, independent cohort of 17 BC cases. Pathogenic/Likely Pathogenic variants were identified in known cancer genes such as CHEK2, MUTYH, PMS2, and RAD51C. Rare and potentially pathogenic variants were identified in DNA repair genes (FAN1, POLQ, and RAD54L) and other cancer‐related genes such as DROSHA and SLC34A2. Interestingly, the variant c.149T>G in the FAN1 gene was identified in two unrelated families, and exhibited LOH in the tumor tissue of one of them. In conclusion, this is the largest Brazilian WES study involving families at high‐risk for HBOC which has brought novel insights into the role of potentially new genetic risk factors for hereditary breast and ovarian cancer., The current study aimed to identify new breast and/or ovarian cancer predisposition genes. Rare and potentially pathogenic variants were identified in DNA repair genes (FAN1, POLQ, and RAD54L) and other cancer‐related genes such as DROSHA and SLC34A2. This is the largest Brazilian WES study involving families at high‐risk for hereditary breast and ovarian cancer which has brought novel insights into the role of potentially new genetic risk factors for hereditary breast and ovarian cancer.

Approximately 15% of colorectal cancers (CRCs) are deficient in DNA mismatch repair proteins (dMMR), a characteristic that can occur in both sporadic and hereditary CRC. Due to sparse studies on dMMR CRC in the Brazilian population, we conducted a retrospective analysis of referral rates for Genetic Cancer Risk Assessment of this population and also describing clinical and molecular characterization of these tumors. A retrospective, longitudinal, and unicenter study that included patients with dMMR CRC detected by IHC analysis from Pathology Database of our institution, from January 2015 to July 2017. MMR IHC testing was performed in 998 CRC tumors, and 78 tumors (7.8%) had dMMR. The mean age at diagnosis was 56.8 years (17–90), and most patients were female (41 out of 78, 52.6%). Of the 52 patients with right-sided CRC, 40 tumors (77%) had loss of the MLH1 and/or PMS2 expression, and 12 tumors (23%) had loss of MSH2 and/or MSH6 expression (p = 0.005). From 78 patients with dMMR CRC, only 43 patients (55.1%) were referred for genetic counseling (GC), and of them, only 33 patients (76.7%) really went to GC consultation. A total of 21 patients with dMMR CRC performed genetic testing. Overall, genetic referral was less than expected in our population. Most of dMMR CRC patients did not receive GC, even in a cancer center, either due to the absence of referral or personal decision and few patients who pursued genetic counseling performed genetic testing.

Germline pathogenic variants in the DNA mismatch repair genes (MMR): MLH1, MSH2, MSH6, and PMS2, are causative of Lynch syndrome (LS). However, many of the variants mapping outside the invariant splice site positions (IVS ± 1, IVS ± 2) are classified as variants of unknown significance (VUS). Three such variants (MLH1 c.588+5G>C, c.588+5G>T and c.677+5G>A) were identified in 8 unrelated LS families from Argentina, Brazil and Chile. Herein, we collected clinical information on these families and performed segregation analysis and RNA splicing studies to assess the implication of these VUS in LS etiology. Pedigrees showed a clear pattern of variant co-segregation with colorectal cancer and/or other LS-associated malignancies. Tumors presented deficient expression of MLH1-PMS2 proteins in 7/7 of the LS families, and MSI-high status in 3/3 cases. Moreover, RNA analyses revealed that c.588+5G>C and c.588+5G>T induce skipping of exon 7 whereas c.677+5G>A causes skipping of exon 8. In sum, we report that the combined clinical findings in the families and the molecular studies provided the evidences needed to demonstrate that the three MLH1 variants are causative of LS and to classify c.588+5G>C and c.677+5G>A as class 5 (pathogenic), and c.588+5G>T as class 4 (likely-pathogenic). Our findings underline the importance of performing clinical and family analyses, as well as RNA splicing assays in order to determine the clinical significance of intronic variants, and contribute to the genetic counseling and clinical management of patients and their relatives.

Background Lung cancer represents around 3.6% of all Li-Fraumeni Syndrome (LFS)-associated tumors. A high prevalence of p.R337H germline mutation in the TP53 gene has been observed in Brazil and family histories of cancer associated with this variant range from isolated cases to families fulfilling Li-Fraumeni-like or Li-Fraumeni criteria. We aimed to evaluate the frequency of EGFR mutations in lung adenocarcinoma (LA) diagnosed in Brazilian patients carrying the TP53 founder mutation p.R337H, as well as the frequency of TP53 p.R337H mutation among LA cases with EGFR mutation. Methods Patients diagnosed with LA were selected from a total of 164 TP53 p.R337H mutation carriers followed at A.C. Camargo Cancer Center. Tumor samples were submitted to EGFR hotspot mutations sequencing. TP53 p.R337H was genotyped in a cohort of 257 LA tumor samples, 114 with and 143 without EGFR activating mutations. Sequencing was performed using Ion Torrent. Results The frequency of LA among TP53 p.R337H carriers was 5.4% (9/164), and EGFR mutations were detected in 89% (8/9) of these patients. The prevalence of TP53 variants at codon 337 in patients with LA harboring EGFR activating mutations was 5.3% (6/114 – 5 p.R337H and 1 p.R337C) and 12.5% (4/32) in LA tumors diagnosed at any age and before the age of 50 years, respectively. This prevalence was significantly higher than that in the cohort of LA tumors with no EGFR activating mutation (1/143 in LA diagnosed at any age, 0/34 in LA diagnosed before the age of 50 years). Conclusions There is a higher than expected frequency of EGFR activating mutations in LA Brazilian patients with TP53 p.R337H mutation. The high frequency of p.R337H/C carriers among patients with EGFR-mutated LA indicates that TP53 genetic testing should be recommended to these patients, regardless of whether they fulfill LFS/LFL criteria or no defined cancer risk criteria.

Variants of unknown significance (VUS) occur in approximately 10% of BRCA1 and BRCA2 genetic testing. Definition of VUS as pathogenic or of benign potential is a crucial step in the reporting and counseling process. Thus limiting the clinical utility of a genetic test and impairs the correct patient management. BRCA1 and 2 are tumor suppressor genes and loss of heterozygosity (LoH) as a second hit for inactivating wildtype allele is a relatively common event in tumor. Previously, in a multi-institutional effort, AC Camargo Cancer Center (Brazil), Barretos Cancer Hospital (Brazil) and UTMD Anderson Cancer Center (USA) compiled a total of 332 BRCA1/2 VUS supposedly from unrelated carriers (46 of BRCA1 and 286 of BRCA2). From them, 256 were distinct VUS (37 in BRCA1 and 219 in BRCA2). Breast cancer sub-classification were collected from medical records and in silico analyzes were performed using four software (SIFT, Polyphen2, CADD and REVEL). Based on this analysis, VUS were categorized into five classes: very high, high, medium, low and very low risk of pathogenicity, according to agreement level among the four in silico software. However, all these analysis are not sufficient for supporting the reclassification of VUS. Therefore we proposed to analyze LoH in matched tumor tissue to assess the pathogenic potential of BRCA1 and 2 VUS. Normal (leucocytes or normal breast tumor tissue) samples, when available, were used for correcting PCR amplification bias between the two alleles. DNA was extracted and subjected to PCR reaction. Amplicons libraries were performed according to manufacturing protocol (Ion Plus Fragment Library Kit) and sequenced on the Ion Proton™ System (ThermoFisher Scientific). Specific variants were called in the TVC program (ThermoFisher Scientific) with standard criteria. A total of 12 VUS were investigated (5 in BRCA1 and 7 in BRCA2). LoH was detected in 7 (60%) tumor samples (3 in BRCA1 and 4 in BRCA2). Of these, 4 VUS were classified as very high or high risk of pathogenicity [2 in BRCA1 (BR1p.Y1703C; BR1p.S1655P) and 2 in BRCA2 (BR2p.S2670L; BR2p.E3002K)]. Variant of allele frequency (VAF) of 94, 65, 70 and 75%, respectively, were detected in tumor tissues. Additionally, recent functional assays described these 4 VUS as non-functional or deleterious variants showing complete agreement with the current LoH results. According to ACMG guidelines considering all available data, LoH, deleterious function and concordant pathogenicity probability. These 4 VUS might be reclassified as pathogenic variants. On the other hand, the same combined analysis did not allow reclassification of 3 other VUS. Although VAF were 74, 73 and 70% respectively, they were previously classified as very or low risk of pathogenicity and there are no functional assays describing them until now. Our results showed that LoH analyses can substantially contribute to VUS reclassification, improving the management and counseling of BRCA1/2 VUS-carriers. Citation Format: Dirce M Carraro, Edenir I Palmero, Carolina M Berra, Giovana T Torrezan, Rafael C Brianese, Gabriela C Fernandes, Angelica M Gutierrez Barrera, Henrique C Galvo, Maria NC Formiga, Banu K Arun. Reclassification of variant of unknown significance in BRCA1 and BRCA2 genes based on loss of heterozigosity assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-09-04.

1 Background: Adrenocortical carcinoma (ACC) is a rare (1,02 per million population) disease with unfavorable prognosis. In Brazil, the incidence is about 15 times higher than in the rest of the world, mainly due to significant prevalence of the TP53-R337H germline mutation in this population. Methods: We performed a retrospective analysis of 66 patients diagnosed with ACC at AC Camargo Cancer Center. Clinical, pathological, epidemiological, treatment and survival data were collected from the patient’s chart and described. Results: Between 2004 and 2021, 66 patients were diagnosed with ACC. The median age was 45 years and 56% were female. 23 (35%) patients had at least one first-degree relative with a history of cancer and 8 (12%) had a second primary tumor. Ten patients were tested for Li-Fraumeni Syndrome and 30% went positive (TP53- R337H germline mutation). Two of them had first-degree relative with cancer. In our cohort, 22 (33%) patients were stage IV, 14 (21%) stage III, 19 (29%) stage II, 6 (9%) stage I and 5 (7%) unknown stage. 36% of tumors were considered functional (cortisol and/or androgens). At 50 months the estimated OS was 25,9% in the metastatic group and 83% in the localized group (p10%) group versus 75% in the low ki67 index (≤10%) group (p = 0.47). In metastatic scenario 31 (47%) underwent systemic treatment and the most common (71%) first line chemotherapy was EDP (etoposide-doxorubicin-cisplatin) plus mitotane with an objective response rate of 27% and 56% of them had progression of disease as the best response to treatment. 14 patients received second line and 7 received third line treatment. Gemcitabine-capecitabine,cisplatin-based and Pembrolizumab were the most common regimens used in advanced lines. Conclusions: ACC has a poor prognosis and a significant prevalence of personal and family history of cancer. EDP-mitotane was the preferred regimen used as the first line treatment. As a rare disease, worldwide data can add evidence for future improvements in adrenal cancer.

704 Background: Treatment of advanced clear cell renal carcinoma (ccRCC) improved dramatically in the last 20 years, but biomarkers development lagged behind. Circulating tumor cell (CTC) is used as a prognostic and predictive tool in many solid tumors but is poorly studied in ccRCC. Objective: Our aim was to evaluate CTC counts in serial blood samples from patients with advanced ccRCC that started first-line treatment and analyze the protein expression of PBRM1, BAP1, PD-L1 and CD133 in these cells. Methods: Blood samples (10mL) were collected in EDTA tubes at three different timepoints, 30 days apart, after treatment start. We used a filtration technique (ISET system, Rarecells/France) to isolate and collect CTCs. Protein expression was evaluated by immunocytochemistry. Results: Twelve patients were included. All had detectable CTCs at baseline (1st blood draw), with a median of 1.5 CTCs/mL. Patients with CTCs above the median had ≥2 metastatic sites (p=0.015) and worse progression free survival (PFS) (19.7 vs 31.1 months, p=0.35), although the difference was not statistically different. Favorable CTCs kinetics at the time of first follow-up (2nd blood draw) indicated better PFS (24.76 months) versus unfavorable (6.65 months; p=0.014). PBRM-1, BAP-1 and PD-L1 expression in CTCs was associated with better overall survival (OS), although without statistical significance. CD133 expression in CTCs at baseline was associated with worse OS (p=0.08). Conclusions: CTCs isolation was feasible in advanced ccRCC patients starting first-line treatment and were frequently detected by ISET method. CTC counts at baseline and at 30 days after treatment initiation had prognostic implication, as well as its dynamic evaluation after 30 days of treatment with a favorable kinetics associated with better outcome.

Objective: Only 5–10% of breast cancer (BC) is related to inherited genetic variants, and BRCA1 and BRCA2 mutations are responsible for the majority of cases. BRCA1 is more associated with triple-negative and BRCA2 to the luminal subtype. The contribution of other genes of high and moderate risk for BC, such as TP53, STK11, CDH1, PTEN, ATM, CHEK2, and PALB2, are not well defined, and risk estimates to specific BC subtype are lacking, especially for an admixed population like Brazilian. The aim of this study was to evaluate the contribution of the multigene panel in detecting germline mutations in Brazilian BC patients and their relationship with molecular subtypes and predominant ancestry. Methods: A 94-gene panel was performed on 321 patients with BC fulfilling NCCN criteria who were referred for BRCA1/2 testing between August 2016 and May 2018. Molecular subtypes were retrieved from medical records, and ancestry-specific variants were obtained from the sequencing data. Results: A panel analysis of 321 patients resulted in a total of 83 pathogenic/likely pathogenic (P/LP) variants identified in 81 patients, leading to a positivity rate of 25%. Of the total P/LP variants, 47% were identified in high-risk BC genes (BRCA1/2, PALB2, and TP53) and 17% in moderate-penetrance genes (ATM and CHEK2). The remainders of the variants were identified in low-risk genes and were considered unexpected findings. Variants of uncertain significance were identified in 77.6% of the patients. Regarding the molecular subtype, triple-negative BC had a mutation frequency of 32% (25/79), with predominance in BRCA1 (40%). Among the luminal subtype, 19% (29/155) had P/LP variants, with BRCA1/2 genes contributing to 38% of mutated cases. For the Luminal B HER2-positive subtype, 40% (16/40) had P/LP variants, with a predominance of the ATM gene (37%). Finally, the HER2-enriched subtype presented a mutation rate of 31% (4/13; 1 BRCA2 and 3 non-BRCA1/2). We did not detect any association of ancestry with P/LP variants or molecular subtypes. Conclusion: The multigene panel contributed to identify P/LP variants in other actionable genes besides BRCA1/2, increasing 7.2% of the positivity of the genetic test. Additionally, our results highlight the distinct contributions of BC genes in each molecular subtype. These results indicate that women with clinical criteria for hereditary BC may benefit from multigene panel testing as it allows them to identify P/LP variants in other BC susceptibility genes, including actionable genes, which directly impact the clinical management of these patients and family members.

Objective: Loss-of-function germline mutation in BRCA1 increases breast cancer risk, especially in the triple-negative breast cancer (TNBC) subtype. BRCA1 impairment may confer benefit from the treatment with DNA damage-inducing drugs and PARP1 inhibitors. Patients who respond to neoadjuvant chemotherapy tend to have good outcomes. The aim of this study was to characterize the resistance to chemotherapy in patients with germline-characterized TNBC by investigating somatic mutations in ctDNA. Methods: Germline genetic testing was done using cancer-predisposing gene panels (26–126 genes) to classify TNBC as hereditary or sporadic. Somatic mutation identification in tumor (409 cancer-related gene panel) and screening of ctDNA in plasma samples during treatment were performed. Results: We enrolled 96 TNBC patients of which 88 were tested for germline variants: 23% (20/88) of cases were hereditary – BRCA1 (16%), BRCA2 (4%), PALB2 (1%), RAD51D (1%), and TP53 (1%). Tumor mutation burden (TMB) analysis (43 cases) showed that 11.6% had high and 89.4% had low TMB, not associated with hereditary status. We found, on average, 3 somatic variants per tumor (range 1–7) and used them as tumor marks for screening ctDNA in plasma. Somatic mutations in TP53 were identified in most tumors (71%). In ctDNA before treatment, detection of at least one tumor mutation was observed in 24 out of 30 patients (80%), and no association was observed between hereditary status and TMB score. Although ctDNA was not associated with the residual cancer burden score, ctDNA-positive patients were associated with clinical progression, either at baseline or during monitoring (post-neoadjuvant chemo), and ctDNA identification anticipated progression detected by imaging. Conclusion: Hereditary tumors, markedly due to germline variants in BRCA1, are frequent in TNBC. Tumor-mark identification using gene panels and ctDNA screening in plasma samples provide valuable information regarding the clinical progression of patients treated with preoperative chemotherapy.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive condition that is associated with the

Neurotrophic tropomyosin receptor kinase (NTRK) fusion positive neoplasms were first identified in colorectal carcinoma almost 40 years ago1 . The availability of tyrosine kinase inhibitors specifically targeting receptors encoded by NTRK genes (Trk receptors), together with a considerable clinical response and favourable safety profile has sparked interest in improving the detection of such fusions in the clinical setting, including in sarcomas with unresectable/metastatic disease2 . In this context, the identification of morphological patterns that should lead to prompt assessment of NTRK rearrangements may aid the pathologist to improve detection of patients who might benefit from targeted therapy.

Introduction: In Brazil, genetic testing for hereditary cancer predisposition syndromes, including Hereditary Breast Cancer (HBC), is not covered by the public health system (SUS) and was only incorporated in most private health insurance (PHI) plans after 2013. Nonetheless, considering that only 22% of the Brazilian population has access to private health system and the high cost of genetic testing in private laboratories, the majority of Brazilian women at risk for HBC do not have access to a genetic diagnosis. In this context, we have implemented at A.C.Camargo Cancer Center (ACCCC – a reference Cancer Center in Brazil which serves both the private and public health systems) a pilot program that offers genetic testing for SUS patients at risk for HBC. The goal of this study is to present the adopted clinical criteria and the corresponding detection rate of pathogenic variants in HBC genes observed in our cohort in comparison to detection rates observed in private health scenario. Methods: Since 2018, 34 cases of breast cancer patients from SUS under medical follow-up at ACCCC and at risk for HBC were discussed in multidisciplinary Tumor Board meetings. The Tumor Board team consists of health care professionals from different specialties, including medical oncologists, surgeons, pathologists, molecular biologists, and clinical geneticists. The clinical criteria defined for referring patients to genetic testing were: (1) personal history of triple negative breast cancer (TNBC) in any age; (2) personal history of non-TNBC developed before 30 years old, positive family history of cancer, and a PENN II model prediction of >15% risk in carry a BRCA1/2 mutation; and (3) personal history of BC in any age and a PENN II model prediction of >30% risk in carry a BRCA1/2 mutation. Detection rates of SUS patients were compared to BRCA1/2 results obtained during the same period from the ACCCC Laboratory of Genomics Diagnostics that serves manly PHI and private patients. Genetic tests were performed using different gene panels containing 26 or 94 genes. Results: Pathogenic (P) and likely pathogenic (LP) variants were detected in 38% of SUS patients (13/34), with a higher proportion of BRCA1 carriers (69% - 9 patients) than of BRCA2 (1 patient). Aside BRCA1/2 mutations that were detected in 10 patients (29%), P/LP variants were also identified in BRIP1, CHEK2, and PALB2 (1 patient each). The rate for BRCA1/2 P/LP variants in PHI cohort was 9.8% (23/234 patients), with a lower proportion of BRCA1 carriers (22%) than of BRCA2 (40%). It was identified a high rate of P/LP variants in TNBC from SUS cohort (62% - 10/16 cases) and a marked association of BRCA1 and TNBC, with 80% (8/10) of hereditary TNBC having BRCA1 mutations. The mean turnaround time for genetic test results was 4 months, varying from 2 to 8 months. From 11 patients with P/LP variants that already received the genetic test result, 5 underwent prophylactic contralateral mastectomy and/or bilateral salpingo-oophorectomy. Conclusions: The introduction of genetic testing for HBC made possible to tailor preventative measures for women at high risk, including prophylactic surgery and screening protocols. However, in many developing countries, the high cost of genetic tests and the lack of national policies prevent its application in clinical practice for the majority of the population. By defining more stringent clinical criteria for HBC genetic test, we detected a mutation rate three times as high as that detected in the private health scenario. With the continuity our study, we expect to contribute to the development of national standards for HBC screening and to encourage the implementation of consistent and cost-effective genetic testing under the real-life conditions of our public health system, maximizing the benefit for the population with the available resources. Citation Format: Dirce M Carraro, Giovana T Torrezan, Karina M Santiago, Maria\ Nirvana da Cruz Formiga, Solange M Sanches, Fabiana BA Makdissi, Breast Cancer Tumor Board Team. Genetic testing for hereditary breast cancer in Brazilian public health system: The experience of tumor board reference in a cancer center [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-34.

Background Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF

Recently, two studies demonstrated that a relevant percentage of primary antiphospholipid syndrome (PAPS) patients had an upregulation of interferon (IFN) genes. However, 20%-28% of these patients had anti-dsDNA, a highly specific systemic lupus erythematosus (SLE) autoantibody. This study aimed to determine the prevalence of the type I IFN signature in the peripheral blood mononuclear cells of PAPS patients without specific SLE autoantibodies and search for its clinical associations.Fifty-three PAPS patients (Sydney's criteria) were consecutively selected and age-matched with 50 healthy controls. A third group of nonimmune-mediated thrombophilia patients was also included. The expression of 41 IFN-induced genes was analyzed using real time quantitative PCR. A principal component analysis determined which genes composed the IFN signature, and the z-score was calculated. An ROC curve defined the signature cut-off.Six genes remained in the IFN signature DNAJA1, IFIT5, IFI27, MX1, IFI6, and TYK2. The ROC cutoff was 3.9-fold (AUC = 0.706, S = 0.49, E = 0.86, PPV = 0.79, NPV = 0.61). The type I IFN signature was present in 49% of the patients with PAPS compared with 14.0% of the healthy controls and 17% of the nonimmune-mediated thrombophilia patients (p .0001). The IFN signature was associated with a younger age at the first antiphospholipid syndrome event (p = .023) and with preeclampsia (p = .032).Our results indicate that PAPS patients without lupus-specific antibodies have an enhanced type I IFN gene signature that is not observed in nonimmune-mediated thrombophilia. Also, this overexpression of type I IFN-regulated genes associated with an earlier onset of antiphospholipid syndrome event and preeclampsia.

Simple Summary Male breast cancer is a rare disease, representing around 0.5% of the malignances in men. Although they receive the same treatment as women with breast cancer, there is increasing knowledge showing that both have a distinct genetic background. Pathogenic variants in cancer predisposing genes are a likely etiology for male breast cancer in 4% to 40% of the cases, and it is currently recommended that all men diagnosed with breast cancer be offered genetic counseling followed by genetic testing. Even though, men are still less likely to undergo the test than women for many reasons, which include an unfamiliarity with the issue by health professionals. The purpose of this article is to review the current knowledge of the germline genetic background of male breast cancer and its impact in the management of the patients and their families. Abstract 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.

Introdução: Uma pequena parcela dos pacientes portadores de síndromes de predisposição hereditária ao câncer (SPHC) apresenta variantes patogênicas em mais de um gene de predisposição ao câncer (GPC), condição recentemente descrita como síndrome de neoplasia hereditária multilocus (MINAS) e com implicações clínicas ainda desconhecidas. Objetivo: O presente estudo tem como objetivo descrever as características clínicas de pacientes/famílias com MINAS e avaliar características moleculares dos tumores desses indivíduos, buscando definir relações de etiologia entre variantes e tumores, efeitos sinérgicos e riscos modificados nesses indivíduos. Metodologia: Foi realizada coleta de dados clínicos, histopatológicos e de histórico familiar de 27 pacientes portadores de MINAS identificados na casuística do A.C.Camargo. Estão sendo realizadas análises de co-segregação em familiares com câncer e, em material tumoral, análises de perda de heterozigose (LoH) através de NGS de amplicon. Resultados: Observa-se um predomínio de pacientes mulheres (22/27) e com histórico de câncer de mama (14/27) e prevalência de síndrome de câncer de mama e ovário hereditários (HBOC). Os genes com variantes patogênicas/provavelmente patogênicas identificados em um maior número de pacientes foram: BRCA2 (13), MUTYH (6), BRCA1 (5), CHEK2 (5), TP53 (3). Sessenta e quatro por cento dos pacientes apresentam variantes em dois genes de herança autossômica dominante, 30% apresentaram 1 gene de herança autossômica dominante e 1 de autossômica recessiva (monoalélico) e 7% apresentaram variantes em dois genes de herança autossômica recessiva (monoalélico). Os resultados preliminares de perda de heterozigose demonstram que houve perda somática do alelo selvagem por deleção em 3 dos 5 tumores avaliados, nos genes BRCA1, BRCA2, ATM. Conclusão: Esperamos descrever as consequências fenotípicas clínicas e moleculares de um grupo raro de pacientes portadores de MINAS, colaborando para o conhecimento científico da área e contribuindo com o banco de dados de MINAS.

Introdução: A Polipose Associada ao gene MUTYH (MAP) é uma síndrome recessiva causada por mutações bialélicas em MUTYH, as quais inativam o gene e levam a uma alta ocorrência de transversões G:C à T:A, gerando a mutação KRAS c.34G>T; G12C. A mutação ocorre em menos de 5% dos casos de câncer colorretal (CCR) esporádicos, mas é altamente frequente em CCR de pacientes MAP. Objetivo: O objetivo do estudo é avaliar a taxa de detecção de variantes germinativas patogênicas/provavelmente patogênicas em MUTYH em pacientes com CCR e mutação somática KRAS G12C. Metodologia: Avaliaremos 3 variantes patogênicas de MUTYH (p.Tyr179Cys, p.Gly396Asp e deleção dos éxons 4 a 16) por PCR multiplex, eletroforese e sequenciamento de nova geração (NGS). Pacientes com 1 variante em heterozigose serão submetidos ao sequenciamento completo do MUTYH. As características clínicas e anatomopatológicas dos pacientes serão correlacionadas com resultados que serão descritos estatisticamente. Resultados preliminares: Até o momento foram identificados 121 pacientes com CCR mutação KRAS c.34G>T; G12C com média de idade de 59 anos, dos quais 51 apresentavam história familiar positiva (42,14%) e 46 apresentavam ao menos 1 pólipo (38,01%). Para 39 pacientes identificamos disponibilidade de material no Biobanco da instituição. Em análises prévias, avaliamos 9 pacientes com CCR antes dos 50 anos e identificamos 3 (33%) com alteração em MUTYH, sendo 2 com variantes patogênicas em ambos os alelos e 1 com uma variante patogênica e uma de significado incerto (VUS). Conclusão: Esperamos verificar se pacientes com CCR mutação somática KRAS G12C apresentam maior risco de detecção de variantes germinativas patogênicas no gene MUTYH, podendo-se utilizar dos resultados da detecção da mutação em KRAS como um teste pré-triagem para diagnóstico da MAP.

ImportanceSARS-CoV-2 genomic variants impacts the overall sensitivity of COVID-19 diagnosis, leading to false-negative diagnosis and the continued spread of the virus.ObjectiveTo evaluate how nucleotide variability in target primer binding sites of the SARS-CoV-2 genomes may impact diagnosis using different recommended primer/probe sets, as well as to suggest the best primer/probes for diagnosis.DesignWe downloaded 105,118 public SARS-CoV-2 genomes from GISAID (Sept, 25th, 2020), removed genomes of apparent worst quality (genome length 5% ambiguous bases) and missing metadata, and performed an analysis of complementarity for the 13 most used diagnostic primers/probe sets for RT-PCR detection. We calculated the N rate and % of genome recovery, with all primer/probe-sets considering viral origin and clade. Results: Our findings indicate that currently, the Paris_nCoV-IP2, -IP4 and WHO|E_Sarbeco primer/probe sets for COVID-19, to perform the best diagnostically worldwide, recovering >99.5% of the good quality SARS-CoV-2 genomes from GISAID, with no mismatches. The Chinese_CDC|2019-nCoV-NP primer/probe set, among the first to be designed during the pandemic, was the most susceptible to currently most abundant SARS-CoV-2 variants. Mismatches encompassing the binding sites for this set are more frequent in Clade-GR and are highly prevalent in over 30 countries globally, including Brazil and India, two of the hardest hit countries. Conclusions: Detection of SARS-CoV-2 in patients may be hampered by significant variability in parts of the viral genome that are targeted by some widely used primer sets. The geographic distribution of different viral clades indicates that continuous assessment of primer sets via sequencing-based surveillance and viral evolutionary analysis is critical to accurate diagnostics. This study highlights sequence variance in target regions that may reduce the efficiency of primer:target hybridization that in turn may lead to the undetected spread of the virus. As such, due to this variance, the Chinese_CDC|2019-nCoV-NP-set should be used with caution, or avoided, especially in countries with high prevalence of the GR clade.Key PointsQuestionHow variable are the binding-sites of primers/probes used for COVID-19 diagnosis?FindingsWe investigated nucleotide variations in primer-binding sites used for COVID-19 diagnosis, in 93,143 SARS-CoV-2 genomes, and found primer sets targeting regions of increasingly nucleotide variance over time, such as the Chinese_CDC|2019-nCoV-NP. The frequency of these variations is higher in Clade-GR whose frequency is increasing worldwide. Paris_nCoV-IP2, IP4 and WHO|E_Sarbeco performed best.MeaningWe suggest the use of some sets to be halted and reinforce the importance of a continuous surveillance of SARS-CoV-2 variations to prompt the use of the best primers.

Lynch syndrome (LS) is a hereditary cancer-predisposing syndrome associated most frequently with epithelial tumors, particularly colorectal (CRC) and endometrial carcinomas (EC). The aim of this study was to investigate the relationship between sarcomas and LS by performing clinical and molecular characterization of patients presenting co-occurrence of sarcomas and tumors from the LS spectrum. We identified 27 patients diagnosed with CRC, EC, and other LS-associated tumors who had sarcomas in the same individuals or families. Germline genetic testing, mismatch repair (MMR) protein immunohistochemistry, microsatellite instability (MSI), and other molecular analyses were performed. Five LS patients presenting personal or family history of sarcomas were identified (3 MSH2 carriers and 2 MLH1), with 2 having Muir&ndash, Torre phenotypes. For two MSH2 carriers we confirmed the etiology of the sarcomas (one liposarcoma and two osteosarcomas) as LS-related, since the tumors were MSH2/MSH6-deficient, MSI-high, or presented a truncated MSH2 transcript. Additionally, we reviewed 43 previous reports of sarcomas in patients with LS, which revealed a high frequency (58%) of MSH2 alterations. In summary, sarcomas represent a rare clinical manifestation in patients with LS, especially in MSH2 carriers, and the analysis of tumor biological characteristics can be useful for definition of tumor etiology and novel therapeutic options.

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. 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. 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. 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.

Lynch syndrome (LS) is a hereditary cancer-predisposing syndrome associated most frequently with epithelial tumors, particularly colorectal (CRC) and endometrial carcinomas (EC). The aim of this study was to investigate the relationship between sarcomas and LS by performing clinical and molecular characterization of patients presenting co-occurrence of sarcomas and tumors from the LS spectrum. We identified 27 patients diagnosed with CRC, EC, and other LS-associated tumors who had sarcomas in the same individuals or families. Germline genetic testing, mismatch repair (MMR) protein immunohistochemistry, microsatellite instability (MSI), and other molecular analyses were performed. Five LS patients presenting personal or family history of sarcomas were identified (3 MSH2 carriers and 2 MLH1), with 2 having Muir–Torre phenotypes. For two MSH2 carriers we confirmed the etiology of the sarcomas (one liposarcoma and two osteosarcomas) as LS-related, since the tumors were MSH2/MSH6-deficient, MSI-high, or presented a truncated MSH2 transcript. Additionally, we reviewed 43 previous reports of sarcomas in patients with LS, which revealed a high frequency (58%) of MSH2 alterations. In summary, sarcomas represent a rare clinical manifestation in patients with LS, especially in MSH2 carriers, and the analysis of tumor biological characteristics can be useful for definition of tumor etiology and novel therapeutic options.

Colorectal cancer (CRC) is one of the most common cancers in Latin America and the Caribbean, with the highest rates reported for Uruguay, Brazil and Argentina. We provide a global snapshot of the CRC patterns, how screening is performed, and compared/contrasted to the genetic profile of Lynch syndrome (LS) in the region. From the literature, we find that only nine (20%) of the Latin America and the Caribbean countries have developed guidelines for early detection of CRC, and also with a low adherence. We describe a genetic profile of LS, including a total of 2,685 suspected families, where confirmed LS ranged from 8% in Uruguay and Argentina to 60% in Peru. Among confirmed LS, path_MLH1 variants were most commonly identified in Peru (82%), Mexico (80%), Chile (60%), and path_MSH2/EPCAM variants were most frequently identified in Colombia (80%) and Argentina (47%). Path_MSH6 and path_PMS2 variants were less common, but they showed important presence in Brazil (15%) and Chile (10%), respectively. Important differences exist at identifying LS families in Latin American countries, where the spectrum of path_MLH1 and path_MSH2 variants are those most frequently identified. Our findings have an impact on the evaluation of the patients and their relatives at risk for LS, derived from the gene affected. Although the awareness of hereditary cancer and genetic testing has improved in the last decade, it is remains deficient, with 39%–80% of the families not being identified for LS among those who actually met both the clinical criteria for LS and showed MMR deficiency. Radium Hospital Foundation (Oslo, Norway)

Background Hereditary breast and ovarian cancer is characterized by mutations in BRCA1 or BRCA2 genes and PCR-based screening techniques, such as capillary sequencing and next-generation sequencing (NGS), are considered gold standard methods for detection of pathogenic mutations in these genes. Single-nucleotide polymorphisms (SNPs) constitute a vast source of variation in the human genome and represent a risk for misdiagnosis in genetic testing, since the presence of a SNP in primer-annealing sites may cause false negative results due to allele dropout. However, few reports are available and the frequency of this phenomenon in diagnostic assays remains unknown. Methods and Results In this article, we investigated the causes of a false negative capillary sequencing result in BRCA1 involving a mother-daughter dyad. Using several molecular strategies, including different DNA polymerases, primer redesign, allele-specific PCR and NGS, we established that the initial misdiagnosis was caused by a SNP located in the primer-annealing region, leading to allele dropout of the mutated allele. Conclusion Assuming that this problem can also occur in any PCR-based method that are widely used in diagnostic settings, the clinical report presented here draws attention for one of the limitations of genetic testing in general, for which medical and laboratory communities need to be aware.

Background BRCA1 associated-protein 1 (BAP1) tumor predisposition syndrome is associated with an increased risk for malignant mesotheliomas, uveal and cutaneous melanomas, renal cell carcinomas, and singular cutaneous lesions. The latter are referred to as BAP1-inactivated melanocytic tumors (BIMTs). When multiple BIMTs manifest, they are considered potential markers of germline BAP1 mutations. Case presentation Here, we report a novel pathogenic BAP1 germline variant in a family with a history of BIMTs, cutaneous melanomas, and mesotheliomas. We also describe singular pathological aspects of the patient’s BIMT lesions and their correlation with dermoscopic and reflectance confocal microscopy findings. Conclusions This knowledge is crucial for the recognition of BIMTs by dermatologists and pathologists, allowing the determination of appropriate management for high-risk patients, such as genetic investigations and screening for potentially aggressive tumors.

Objectives: Approximately 60% of lung adenocarcinomas (LAs) carry mutations that can guide treatment with tyrosine-kinase inhibitors (TKI) and other targeted therapies. Data on activating mutations in EGFR and other tyrosine-kinase receptor (TKR) genes in highly admixed populations, such as that of Brazil, are scarce. In this study, we comprehensively analyzed the actionable alteration profile of LA in Brazilian patients. Materials and Methods: EGFR driver mutation data were collected from a large Brazilian LA cohort covering an 8-year period of molecular testing in a single institution. Tests were performed using three distinct methods, and demographic and histopathological data were analyzed. For a subset of patients, driver mutations in KRAS, NRAS, and BRAF and gene fusions involving TKR genes (before TKI treatment) and EGFR T790M (after TKI treatment) were assessed. Results: EGFR mutations were detected in 25% of 1,316 LAs evaluated, with exon 19 deletions and exon 21 L858R TKI sensitizing mutations representing 72.5% of all mutations. Mutation rates were higher in women and non-smokers (p < 0.001). Next-generation sequencing was very sensitive, with a lower rate of inconclusive results compared with Sanger sequencing and pyrosequencing. EGFR/RAS/BRAF hotspot gene panels were applied in 495 LA cases and detected oncogenic mutations in 51.3% of samples, most frequently in EGFR (22.4%) and KRAS (26.9%). In subgroups of 36 and 35 patients, gene fusions were detected in 11.1% of tumors and EGFR T790M resistance mutations were detected in 59% of plasma samples from patients previously treated with TKI, respectively. Conclusion: This report provides the first comprehensive actionable alteration portrait of LA in Brazil. The high rate of actionable alterations in EGFR and other driver genes in LA reinforces the need to incorporate TKI guided by molecular diagnostics into clinical routines for patients in both public and private healthcare systems.

Gastric cancer (GC) is the fifth most common type of cancer worldwide with high incidences in Asia, Central, and South American countries. This patchy distribution means that GC studies are neglected by large research centers from developed countries. The need for further understanding of this complex disease, including the local importance of epidemiological factors and the rich ancestral admixture found in Brazil, stimulated the implementation of the GE4GAC project. GE4GAC aims to embrace epidemiological, clinical, molecular and microbiological data from Brazilian controls and patients with malignant and pre-malignant gastric disease. In this letter, we summarize the main goals of the project, including subject and sample accrual and current findings.

Folate, vitamin B2, vitamin B6, vitamin B12, choline, and betaine are nutrients involved in the 1-carbon cycle that can alter the levels of DNA methylation and influence genesis and/or tumor progression. Thus, the objective of this study was to evaluate the association of folate and vitamins involved in the 1-carbon cycle and MTHFR polymorphisms in global DNA methylation in patients with colorectal cancer gene. The study included 189 patients with colorectal adenocarcinoma answering a clinical evaluation questionnaire and the Food Frequency Questionnaire (FFQ) validated for patients with colon and rectal cancer. Blood samples were collected for evaluation of MTHFR gene polymorphisms in global DNA methylation in blood and in tumor. The values for serum folate were positively correlated with the equivalent total dietary folate (total DFE) (rho = 0.51, p = 0.03) and global DNA methylation (rho = 0.20, p = 0.03). Individuals aged over 61 years (p = 0.01) in clinicopathological staging III and IV (p = 0.01) and with + heterozygous mutated homozygous genotypes for the MTHFR A1298C gene had higher levels of global DNA methylation (p = 0.04). The association between dietary intake of folate, serum folate, and tumor stage were predictive of global DNA methylation in patients&rsquo, blood. The levels of serum folate, the dietary folate and the status of DNA methylation can influence clinicopathological staging.

6076 Background: Germline BRCA pathogenic variants are present in 15% to 25% of ovarian carcinoma patients. These tumors are more sensitive to platinum and PARP inhibitor therapy and have a better prognosis. Two retrospective studies with limited number of patients have shown conflicting results regarding the benefit of secondary cytoreductive surgery (SCS) in patients with BRCA mutations. Our aim was to evaluate the impact of SCS in recurrent ovarian cancer according to BRCA status. Methods: All patients with ovarian carcinoma with recurrent disease and who were tested for BRCA pathogenic variants treated at a tertiary Cancer Center in Brazil were included. Patients characteristics were compared between patients treated with SCS and not treated with SCS. Cox regression analysis was used to evaluate the impact of SCS on progression free survival (PFS) and the influence of BRCA pathogenic variants on the effect of SCS. Results: One hundred and forty patients were included, 49.6% were treated with SCS and chemotherapy and 50.4% treated with chemotherapy only. Patients treated with SCS were younger, presented better performance status, lower CA 125 and longer platinum free interval. After adjusting for relevant covariables SCS was associated with longer PFS (HR 0.53, 95%CI 0.29-0.97, p = 0.039). Germline BRCA pathogenic variants were found in 37 patients (26.4%). No patient was treated with PARP inhibitors. Among non-carriers of pathogenic variants in BRCA, SCS lead to a longer PFS (HR 0.48, 95%CI 0.28-0.81, p = 0.006) but among carriers there was no benefit of SCS (HR 0.84, 95%CI 0.30-2.34, p = 0.735). Test for interaction was not statistically significant (p = 0.359). Conclusions: Our study is the second to demonstrate no benefit of SCS among patients with BRCA pathogenic variants and not treated with PARP inhibitor. The only other study to show a benefit of SCS in this group of patients included a limited number of patients and all of them were treated with PARP inhibitors. BRCA germline status might influence the efficacy of SCS, and should be evaluated as a potential biomarker to be assessed together with clinical factors to better select patients for SCS.

Background BRCA1/2 pathogenic (P) and likely pathogenic (LP) germline variants are frequent among patients with ovarian carcinoma. However, these variants have not been extensively characterized in patients with ovarian cancer in Brazil. Methods In this retrospective study we evaluated clinical characteristics and BRCA1/2 genetic test results from patients with ovarian carcinoma who underwent genetic counseling at A.C.Camargo Cancer Center (Brazil) between 2015 and 2017 and had performed germline genetic testing of BRCA1/2 genes. Results Among 158 patients, 33 P and LP variants and were found (20.8%), 27 in BRCA1 and six in BRCA2, and six variants of unknown clinical significance (VUS). Thirteen percent of the patients did not have Multiplex Ligation-dependent Probe Amplification (MLPA) results. Three P variants in BRCA1 were found in more than one patient: c.5266dupC (p.Gln1756Profs*74), c.3331_3334delCAAG (p.Gln1111Asnfs5*), and c.211A > G (p.Arg71Gly). One LP variant in BRCA1 had not been previously described, c.4153_4154delCT (p.Leu1385Ilefs*5). Patients with previous diagnosis of breast cancer were carriers of P or LP variant in 8 of 12 cases (66.7%), and patients with a family history of ovarian or breast cancer in first- or second-degree relatives were carriers of P or LP variant in 26.7% of cases compared to 16.9% for patients without family history (p = 0.166). Conclusion Prevalence of BRCA1/2 germline P and LP variants is slightly higher than previously described by the largest occidental studies, with a high prevalence of variant c.5266dupC (p.Gln1756Profs*74) in BRCA1 observed. Moreover, we identified a new LP variant.

Background: Triple-negative breast cancer (TNBC), a very aggressive breast cancer subtype, exhibits high variability at both molecular and clinical aspects. Loss of function (LOF) germline mutation in BRCA1 has a very close association with this subtype of breast cancer. BRCA1 impairment is associated with homologous recombination deficiency (HRD) and tumor with HRD may benefit from treatment with drugs that induces DNA damage and also with PARP1 inhibitors. We previously detected that a significant fraction of TNBC diagnosed in young Brazilian women exhibits BRCA1 impairment by mechanisms including germline pathogenic mutation and promoter hypermethylation and that this group of tumors presented better overall survival. In the current study our aim is to comprehensively characterize the resistance to chemotherapy (DNA-damage agents) in patients with Hereditary or Sporadic TNBC by investigating somatic mutations in circulating tumor DNA (ctDNA). Methods: A series of 34 TNBC patients were subjected to germline genetic testing using a 26- or 96-gene panel, including homologous recombination-related genes, for classifying the TNBC as Hereditary or Sporadic. Tumor mutation burden (TMB) analysis was assessed in 20 cases using a panel containing 409 cancer-associated genes. Tumor somatic mutations were detected by evaluating DNA from tumor biopsies (Formalin-fixed, paraffin-embedded tissue) and matched leucocyte and were screened in cell-free DNA from 6 serial plasma samples during neoadjuvant treatment and for 6 months after surgery. Results: Pathogenic germline mutations were identified in BRCA1 (17.6% - 6/34), BRCA2 (2.9% -1/34) and TP53 (2.9% - 1/34) genes. Additionally, variants of uncertain significance (VUS) were identified in 21 patients (61.7% - 21/34), being ATM, BUB1B and PMS2 the most affected genes by VUS. Based on the 34 patients that underwent pre-operative chemotherapy and surgery, 47% exhibited pathological complete response (pCR), 50% in hereditary and 48% in sporadic. In terms of somatic variants, tumor mutation burden (TMB) analysis showed that 20% had high and 80% low TMB, with no association with hereditary or sporadic status. Also, we found, on average, three somatic variants per tumor (range 0-7) and used them as tumor marks in the screening of ctDNA in plasma. Somatic mutations in TP53 were identified in 80% (16 of 21) tumor biopsy samples investigated. In DNA from plasma before treatment, confident detection of at least one tumor mutation (ctDNA) was observed in 60% of patients (10 of 16). Persistent ctDNA detection in plasma during treatment was observed in 6 out 10 (60%) patients with residual disease after surgery and only in 1 out of 6 (16%) in patients with pCR. For 3 patients persistent ctDNA was detected after surgery at increasing levels showing clear anticipation of disease progression or metastasis. Serial plasma ctDNA samples showed great association with the clinical response data suggesting that the chemotherapy-resistance mechanisms can be investigated by ctDNA in TNBC. Conclusions: Germline and somatic evaluation of TNBC can provide valuable information for improving the clinical management of patients. For somatic evaluation, development of gene panels optimized for FFPE samples and low DNA input is enabling a comprehensive detection of potentially clinically-actionable somatic variants with better success rate in cancer treatment. Although still challenging, investigation of somatic variants in ctDNA from plasma samples is feasible and presents huge potential in monitoring patients during chemotherapy, especially regarding both the presence of residual disease and monitoring of disease progression. Citation Format: Dirce M Carraro, Rafael C Brianese, Giovana T Torrezan, Marina de Brot, Claudia AA de Paula, Maria NC Formiga, Solange M Sanches, Vladmir CC de Lima, Fabiana BA Makdissi. Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-19.