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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.

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.

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 p p = 0.029), and photodamaged dermoscopic pattern (p 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.

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.

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.

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.

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.

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.

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.

Renal cell carcinoma (RCC) accounts for 2–3% of all malignant disease in adults. Hereditary RCC represents 5 to 8% of kidney tumors. Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) represents an autosomal dominant syndrome that results from a germline mutation in fumarate hydratase gene (FH). HLRCC patients typically present with skin or uterine leiomyomas and renal neoplasms. HLRCC was recently recognized as a distinct renal tumor subtype by the WHO 2016 classification. Many morphological patterns such as papillary, solid, tubular, and cystic had been described as part of morphological aspects of HLRCC. In this study, we describe a case of a patient that had a history of persistence of ductus arteriosus (PDA) and cryptorchidism. In addition, the renal tumor showed a very unusual hystiocytoid morphological aspect. We confirmed the presence of a FH germline mutation both in the patient and his mother.

This abstract was not presented at the conference. Citation Format: Carraro DM, Palmero EI, Galvao HC, Berra CM, Brianese RC, Torrezan GT, da Cruz Formiga MN, de Lima FT, Fernandes GC, de Paula AE, Michelli RD, Gutierrez Barrera AM, Arun BK. Not presented [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-03-05.

Background: Uterine sarcomas are the most common gynecological sarcomas, comprising around 90% of all of the cases. These tumors can arise either from the endometrial stroma or from the muscle itself. They are genetically heterogeneous tumors and have no specific molecular and radiological diagnosis, or treatment. Histologically, uterine sarcomas are classified according to their cell of origin and are divided into: carcinosarcomas (CCS), leiomyosarcomas (LMS), endometrial stromal sarcoma (ESS) and adenosarcomas (ADS). There are many factors that can be attributed to uterine sarcomas etiopathogeny such as ethnic, hormonal and hereditary factors. However, few studies were performed searching for specific molecular signature in these tumors, especially concerning a singular population. Objective: To assess the oncogenic profile of Brazilian patients with different histological types of uterine sarcomas. Methods: Until now, we performed the Next Generation Sequencing for 13 samples of uterine sarcomas (comprising: 5 CCS, 3 LMS, 4 SEE and 1 ADS) using the Ion Proton System (ThermoFisher) with the Comprehensive Cancer Panel (containing 409 genes related to carcinogenesis). Results: The genetic sequencing profile analysis showed 140 specific mutations in our samples. Among them, the most common were TP53 (58%), JAK3 (33%), NF1 (33%), KMT2D (33%), MTOR (25%) and PIK3CA (25%). Some findings corroborate the literature on uterine sarcomas, with an exception of the KMT2D gene, that despite of being known and described in other tumors, including the ones of the genital tract, such as vulva, ovary and endometrium, has never been described in uterine sarcomas Conclusion: We found the most frequent mutations in 6 genes in our samples. Only one mutation had not been described before for uterine sarcomas (KMT2D). These are preliminary results, and further analyzes are ongoing.Key words: Uterine Sarcomas, Carcinosarcomas, Next Generation Sequencing Citation Format: Leonardo T. Costa, Giovana T. Torrezan, Claudia A. Paula, Glauco B. Neto, Edmund C. Baracat, Dirce M. Carraro, Kátia C. Carvalho. Uterine sarcomas: A preliminary study for characterization of a specific molecular signature for Brazilian patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5374.

Background: Melanoma is a highly aggressive skin cancer; approximately 10% of melanomas are caused by germline mutations, mainly affecting the p16 isoform of the CDKN2A gene (responsible for up to 40% of the familial melanoma). Other genes exhibiting moderate- to high-penetrant mutations have been recently associated with melanoma susceptibility but in low frequency. Therefore, the majority of the melanoma-prone patients remain without the identification of genetic etiology. Objective: In order to find new genes potentially related to melanoma predisposition, we performed whole-exome sequencing of 10 affected individuals from 5 unrelated families. All patients were negative for CDKN2A/CDK4/TERT promoter/MITF (E318K) mutations. Materials and Methods: Melanoma-prone patients were ascertained based on either the classical criteria for familial melanoma syndrome or based on evidence of a strong genetic predisposition (occurrence of ≥2 primary melanomas). Whole-exome sequencing was performed using the Ion Proton Sequencer (Life Technologies). Our experimental design was performed using at least 20x each base pair sequencing as confidence pattern and the most interesting variants were prioritized, when identified on the two probands from the same family, using the VarSeq software 1.4.2. Once we obtained the most interesting germline rare variants we designed a panel for the validation of these variants through the target sequencing (Ion Proton Thermo Fisher Scientific Inc). Results: We identified 214 rare nonsynonymous variants. We selected a set with 63 rare germline variants potentially damaging the protein function and associated to important cellular pathways to perform validation (4 LoF variants classified as frameshift). All variants were validated by resequencing using probes specifically designed for the rare selected variants; some of them already associated to melanoma, skin pigmentation, melanoma, and/or cancer susceptibility. Conclusion: This study provides new knowledge about melanoma predisposition in the Brazilian population, in which the risk is smaller to develop this disease than in other populations, and also highlighted possible new risk factor or predisposition genes for hereditary melanoma syndrome. Citation Format: Felipe Fidalgo, Luciana Facure, Bruna Barros, Renan Valieris, Giovana Torrezan, Bianca de Sá, Amanda Nóbrega, Maria Isabel Achatz, João Duprat, Carla Rosenberg, Sandro Souza, Ana Krepischi, Dirce Maria Carraro. Identification of new promising germline variants in melanoma-prone patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A20.

When considering family history, hereditary breast cancer (HBC) syndromes correspond to nearly 5-10% of all breast cancer (BC) cases. However, pathogenic variants in known moderate- and high-risk BC genes explain only ~30% of familial BC cases. Recent advances in sequencing technology resulted in an increasing number of BC genes being revealed in previously negative families by using extended gene panels and whole-exome sequencing (WES). WES offers the opportunity to concomitantly investigate several rare risk genes as well as to identify new BC-predisposing genes. Thus, in this study we used WES to disclose variants contributing to BC increased risk in patients that were negative for mutations in three major BC genes (BRCA1/2 and TP53) and met stringent clinical criteria indicating a genetic predisposition to BC. We selected 17 women (two of them sisters) who fulfilled one or more of the following criteria: early onset BC (2, FS 0.01 or present in five BRCA1 mutated patients sequenced in the same platforms. Next, using a functional-based variant prioritization, we selected candidate variants according to the predicted impact in the protein function, the affected gene, and segregation with the phenotype (in the family-based study of the two sisters). Candidate variants included all loss-of-function variants as well as missense and in-frame indels occurring in a specific list of 651 genes of interest (DNA repair and cancer-related genes) and if predicted to be damaging by at least 3/6 prediction software. The resulting 244 selected variants were submitted for technical validation by targeted NGS and, of these, 220 were validated (89%). Using the same custom panel, we evaluated 25 control samples of healthy Brazilian women for filtering common polymorphisms in our population, resulting in a final 139 variants in 129 genes (89 LOF and 50 missense variants). For two families---one with WES of two affected sisters and target NGS of one affected aunt; one with WES of the affected daughter and target NGS of the affected mother---we were able to perform cosegregation analysis in other family members and 11 out of 23 variants were found to be segregating with the disease. These 11 distinct genes as well as 82 genes harboring LOF variants were evaluated in an independent cohort of 34 WES of high-risk BC patients. Several rare, possibly damaging variants were identified in this cohort, providing additional evidence of the potential role in BC predisposition of some new genes. Of those, we highlight ATRX, FANCC, TET2, ERCC1, PTPRD, and ROS1 genes, which are involved in DNA repair and other tumorigenic pathways. Overall, our results provide a set of putative BC-predisposing genes and underpin WES as a useful tool for assessing the complex landscape of HBC predisposition. The discovery and validation of new HBC genes in different populations will continue to provide insights into disease mechanisms, eventually leading to the development of more effective therapies and improved management of affected families. Citation Format: Giovana T. Torrezan, Fernanda GSR Almeida, Marcia CP Figueiredo, Bruna DF Barros, Claudia A. Paula, Renan Valieris, Jorge ES Souza, Elisa N. Ferreira, Felipe CC Silva, Amanda F. Nobrega, Maria Isabel Achatz, Edenir I. Palmero, Rodrigo F. Ramalho, Sandro J. Souza, Dirce M. Carraro. Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A37.

Germline mutations in known colorectal cancer (CRC) predisposing genes accounts for less than half of the genetic cause of hereditary CRC cases. The absence of mutations in the remaining patients suggests that other yet unknown CRC predisposing genes might exist. However, recently studies with large cohorts probably discounts the existence of major unknown genes and it is thought that rare or even family private variants and genes may contribute to familial CRC. Thus, the aim of our study was to identify novel CRC susceptibility genes by whole exome sequencing (WES) of a family presenting hereditary CRC. The studied family fulfilled Amsterdam I criteria and appears to segregate a dominant cancer syndrome with a high risk of colon and rectal cancer, with four affected members in three generations. The index patient’s tumor was microsatellite stable and positive for the four mismatch repair proteins at immunohistochemistry. Genetic testing of the proband was negative for the following genes: MLH1, MSH2, MSH6, PMS2, PMS1, TP53, CHEK2. We performed WES in three family members: two with rectal cancer (onset 26 and 56 years) and one with colon cancer (onset 63 years). Using a functional-based variant prioritization, we identified six novel or very rare (MAFA) is located 8 base pairs from the 3’ end of exon 13, and is predicted it to disrupt an exonic splicing enhancer by in silico tools (ESR finder and Human Splice Finder), possibly affecting the splice of this gene. Pyrosequencing of the KIT variant in tumor cDNA revealed an allelic imbalance between mutated and wild type alleles, with the mutated allele being three times more expressed. Transcript analysis using a customized heteroduplex enrichment-based method identified a novel KIT transcript in the index patient’s tumor, presenting an exon skipping event of exon 12. The resulting transcript (c.1775_1879del) is predicted to produce an in-frame deletion of 35 amino acids (p.Gly592_Pro627delinsAla) at the beginning of the protein kinase domain. The functional impact of this transcript and its relevance for CRC predisposition will be further evaluated. Moreover, experiments evaluating allelic imbalance, loss of heterozygosity and alternative spliced transcripts for the other identified variants are ongoing. Our results provide a set of putative CRC predisposing genes and underpin that WES of highly informative CRC families, ascertained on the basis of highly selected phenotypes and followed by functional validation, are likely to continue to prove an effective strategy for gene discovery. Citation Format: Giovana T. Torrezan, Anna Smirnova, Felipe C. Silva, Erika M. Santos, Renan Valieris, Jorge E. de Souza, Samuel Aguiar, Benedito M. Rossi, Sandro J. de Souza, Dirce M. Carraro. Identification of putative cancer susceptibility genes through whole exome sequencing of a family presenting hereditary colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2017-4281

Background: Germ line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1). Methods: Capillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes. Results: The positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively. Conclusions: In summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria for HBOC and negative for BRCA1/2 genes should be tested for the TP53 R337H variant. Furthermore, the presence of genomic structural rearrangement resulting in CNVs in other genes that predispose breast cancer in conjunction with BRCA2 point mutations demonstrated a highly complex genetic etiology in Brazilian breast cancer families.

Wilms tumour (WT) is an embryonal kidney neoplasia for which very few driver genes have been identified. Here we identify DROSHA mutations in 12% of WT samples (26/222) using whole-exome sequencing and targeted sequencing of 10 microRNA (miRNA)-processing genes. A recurrent mutation (E1147K) affecting a metal-binding residue of the RNase IIIb domain is detected in 81% of the DROSHA-mutated tumours. In addition, we identify non-recurrent mutations in other genes of this pathway (DGCR8, DICER1, XPO5 and TARBP2). By assessing the miRNA expression pattern of the DROSHA-E1147K-mutated tumours and cell lines expressing this mutation, we determine that this variant leads to a predominant downregulation of a subset of miRNAs. We confirm that the downregulation occurs exclusively in mature miRNAs and not in primary miRNA transcripts, suggesting that the DROSHA E1147K mutation affects processing of primary miRNAs. Our data underscore the pivotal role of the miRNA biogenesis pathway in WT tumorigenesis, particularly the major miRNA-processing gene DROSHA., Wilms tumour (WT) is the most common paediatric kidney cancer and few driver genes related to its development have been identified. Here, the authors identify DROSHA mutations that may contribute to WT tumorigenesis through their effect on primary microRNA processing.

Background Patients with multiple colorectal adenomas are currently screened for germline mutations in two genes, APC and MUTYH. APC-mutated patients present classic or attenuated familial adenomatous polyposis (FAP/AFAP), while patients carrying biallelic MUTYH mutations exhibit MUTYH-associated polyposis (MAP). The spectrum of mutations as well as the genotype-phenotype correlations in polyposis syndromes present clinical impact and can be population specific, making important to obtain genetic and clinical data from different populations. Methods DNA sequencing of the complete coding region of the APC and MUTYH genes was performed in 23 unrelated Brazilian polyposis patients. In addition, mutation-negative patients were screened for large genomic rearrangements by multiplex ligation-dependent probe amplification, array-comparative genomic hybridization, and duplex quantitative PCR. Biallelic MUTYH mutations were confirmed by allele-specific PCR. Clinical data of the index cases and their affected relatives were used to assess genotype–phenotype correlations. Results Pathogenic mutations were identified in 20 of the 23 probands (87%): 14 in the APC gene and six in the MUTYH gene; six of them (30%) were described for the first time in this series. Genotype-phenotype correlations revealed divergent results compared with those described in other studies, particularly regarding the extent of polyposis and the occurrence of desmoid tumors in families with mutations before codon 1444 (6/8 families with desmoid). Conclusions This first comprehensive investigation of the APC and MUTYH mutation spectrum in Brazilian polyposis patients showed a high detection rate and identified novel pathogenic mutations. Notably, a significant number of APC-positive families were not consistent with the predicted genotype-phenotype correlations from other populations.

We wish to draw the attention of cancer geneticists to a particular genetic variant of the Prolactin-Induced Protein (PIP) gene that may be an important predisposing factor to cancer because of its high frequency and significant association with cancer, as determined in this study. In an initial copy number variation (CNV) screen for germ-line deletions in 123 Brazilian cancer patients selected as high risk either because of early age of onset (pediatric cancer) or a positive family history (TP53-negative Li–Fraumeni and APC/MUTYH-negative Familial Adenomatous Polyposis patients), a previously undescribed microdeletion was discovered in four patients. The deletion carried in these patients was apparently identical (Figure 1), with a size of 69 kb and similar base-pair position in chromosome region 7q34. All four deletions were validated by qPCR and found to harbor only the PIP and TAS2R39 (taste receptor type 2 member 39) genes. TAS2R39 is a member of ∼30 TAS2R bitter taste receptors,1 several of which are known to exhibit variation in copy number. It is unlikely that TAS2R39 has a role in tumorigenesis, and will not be considered further in this submission. We here refer to the deletion encompassing the PIP gene as PIP-Sao Paulo, following the convention of adding the place of discovery to the gene name. This deletion was not detected in a control group of 260 non-related individuals from the Sao Paulo urban area that had attended our genetic service for reasons other than cancer (normal relatives of patients with intellectual disabilities). The difference in deletion frequency between patients and controls was significant at the 0.01 level (Fisher's exact test). Another example of the PIP-Sao Paulo deletion was serendipitously detected in a TP53-mutated patient who was not part of these cohorts, but had been investigated by aCGH for presenting with an atypically severe course of cancer that commenced at 4 years of age with the diagnosis of a rhabdomyosarcoma, followed by choroid plexus tumor (7 years of age), liposarcoma and osteochondroma (10 years of age), and finally passed away at 11 years. This patient was not included in the statistics of the patient–control comparison; however, a possible interaction between the TP53 mutation and PIP-Sao Paulo deletion may have contributed to the severity of the cancer progression. Figure 1 PIP-Sao Paulo microdeletion at 7q34, detected by array-CGH in cancer patients. Array-CGH profile of a chromosome region at 7q34 (microarray platform Agilent 180K—Agilent Technologies, Santa Clara, CA, USA), showing heterozygous losses ... Following our initial observations, we interrogated the 1000 Genomes Project database and found a comparable deletion to be present in 13 out of 1097 individuals: 9 of European and 4 of Latin-American origin (see Supplementary Table 1). Manual review of the sequence data in this region indicated that 7 out of the 13 individuals had sufficient sequence depth to determine that they shared identical breakpoints. From this information, we designed PCR primers and were able to amplify across the breakpoints in all the five Brazilian deletion cases. Subsequent sequencing of the PCR fragments (see Supplementary Figure 1) demonstrated that all identified PIP-Sao Paulo deletions were identical to the deletions present in the 1000 Genomes Project data set. In a replicate study, we determined by breakpoint PCR the frequency of PIP deletions in an independent Brazilian cancer group (219 individuals) that had either presented with more than one primary cancer prior to 60 years of age (166 individuals) or were probands of hereditary melanoma families (53 individuals with no CDKN2A or CDK4 mutations). This identified a further 6 individuals, who had had cancer and carried the PIP deletion (2.6%), as opposed to 10 out of 847 individuals (1.2%) from a second control group. As in the initial study, the deletion frequency in the replicate study in cancer patients was higher than that in controls, although this was not significant at the 5% level (P=0.11). However, when cancer and control samples from both studies were pooled, statistical significance was attained (P=0.04, Fisher's exact test). Importantly, heterogeneity tests demonstrated that no significant differences existed at the 0.05 level between the two control and two patient samples, respectively. We conclude that the relatively frequent germ-line deletion of the PIP gene has more than a two-fold increase in frequency in the Brazilian cancer patients compared with the Brazilian controls. The clinical phenotype, mutation status, type of tumor and age of diagnosis of all patients carrying the PIP-Sao Paulo deletion are summarized in Table 1, demonstrating that several types of cancer are involved in this study and that no single type or group of cancers predominates. Presence of the PIP-Sao Paulo deletion was investigated in paraffin blocks from several different primary tumors taken from four of the deleted patients. In all samples, except for one from patient 7, the PIP-Sao Paulo deletion was detected (Supplementary Figure 1); additionally, at least one exon of the PIP gene was amplified from each of the tested samples, indicating that one allele was retained, similar to blood. Unfortunately, DNA quality did not allow sequence screening for point mutations or other deletions that may have occurred in the remaining allele. We conclude that if the PIP-Sao Paulo deletion is directly responsible for tumor induction, this has not occurred by induction of homozygosity of the deletion itself in the cases studied. Table 1 Characteristics of the PIP-Sao Paulo deletion patients: mutation status, clinical phenotype, type of tumor and age at diagnosis To date, several publications have reported association between PIP expression and tumor progression, particularly for prostate and breast cancers.2, 3 The gene has been implicated in multiple functions, including apoptosis, cell proliferation and migration,4 but mutations have only been studied in tumor cell lines and never investigated as germ-line mutations. According to the TCGA database (http://cancergenome.nih.gov/), PIP gains in tumors are more frequent than deletions; however, data in different type of tumors are very heterogeneous and difficult to interpret. We have no specific insights into the mechanisms through which the PIP-Sao Paulo deletion could be oncogenic. It is evident from the foregoing that the PIP-Sao Paulo deletion is not specific to Brazil. Inspection of the HapMap3 database shows that the haplotype adjacent to the deletion (Table 2) is either absent or extremely rare in African populations. Interestingly, although about ∼25% of the 1094 individuals sequenced in data set 1 of the 1000 Genome Project were of African origin, none of the 13 individuals carrying the deletion were African and, except for one Colombian and two Mexican individuals from Los Angeles, CA, USA, all others were of European origin. Ethnic certainty of Brazilian cancer patients and controls is obscured by miscegenation and ethnic diversity; however, they are most likely to be mainly Caucasians, given the relatively high Caucasian composition of Sao Paulo, and through hospital attendance preferences and other socioeconomic differences. It is probable that PIP-Sao Paulo arose in an European population as a founder mutation that was subsequently exported to the New World. Table 2 SNPs, genomic positions (Hg19) and the common haplotype deduced from the proximal region of the patients with 7q34 deletion The crucial question of whether the PIP-Sao Paulo deletion constitutes a widespread cancer risk can only be answered by similar investigations in other countries, which we hope will be stimulated by this report. However, maintenance of a deletion variant associated with cancer at such high frequency is surprising and probably implies that, if the cancer association is confirmed by independent studies, PIP-Sao Paulo appears to have a relatively low cancer penetrance rate so that many carriers either do not manifest cancer or do so at later age, following transmission of the deletion to their offspring, similar to the well-documented Brazilian TP53 variant R337H4. Whereas R337H has an estimated population frequency of ∼0.3% in Southern Brazil and exhibits an ∼8% penetrance rate in families with adrenocortical cancer,5 PIP-Sao Paulo is practically an order of magnitude more frequent (estimated at ∼2% in European populations from the 1000 Genome Project data set) and appears as a very frequent factor associated with increased cancer risk. Further, if the PIP-Sao Paulo deletion frequency is at least two-fold higher in European cancer patients than in controls, as in the Sao Paulo study, then even allowing for a low penetrance rate, there would be an increased cancer risk for Europeans. In the case of PIP-Sao Paulo, it is the high frequency and not the penetrance that constitutes the main risk parameter. This completely unanticipated aspect deserves a rapid, coordinated response to confirm whether indeed such an increased risk exists, and if so, to establish its magnitude and specificity. Rapid and cost-effective detection of the deletion can be performed using either PCR across the breakpoints, or copy number estimates by RT-PCR, MLPA and so on. The exact genome location of PIP-Sao Paulo is chr7: 142824847-142893913 (Genome build GRCh37/hg19).

e20611Background: Lung adenocarcinoma in never-smokers (LANS) is a disease largely defined by targetable oncogenic mutations. High frequency of actionable genomic alterations has been reported in c...

The cytochrome P450 family (CYPs) and the glutathione S-transferase (GSTs) enzymes play an important role in the metabolism of environmental carcinogens and of oestrogen and can affect breast cancer risk. In this study we examine the role of the genes CYP1A1, CYP17, CYP2D6, GSTM1, GSTP1 and GSTT1 in breast cancer risk in Brazilian women. The study population consisted of 102 incident breast cancer cases and 102 healthy controls. Genotyping analyses were performed by PCR-based methods. A significant finding was observed between GSTP1 Ile-Val polymorphism and breast cancer risk (OR = 1.81; CI 95% = 1.04-3.16). A significant association was observed between women with 0-2 risk genotypes and those with 4 or more risk genotypes (OR = 2.42; CI 95% = 1.13-5.18) when the potential combined effects of the risk genotypes were examined. No significant differences between cases and controls were found correlating the genotypes and the clinical-histopathological parameters. In conclusion, in our population only GSTP1 was associated with breast cancer risk. However, when the genes were tested in combination, a significant association in the breast cancer risk was observed.

Lynch syndrome (LS), former known as Hereditary Non Polyposis Colorectal cancer (HNPCC), accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autossomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous and metachronous tumors, and accelerated transition adenoma-carcinoma (2-3 years). Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five Mismatch repair genes, such as MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), post-meiotic segregation increased 1 (PMS1) or post-meiotic segregation increased 2 (PMS2). In order to characterize Brazilian patients suspect for LS, we assessed the frequency of germline point mutations in MSH6, PMS1 and PMS2 in patients negative for point mutations in MLH1 and MSH2 by capillary sequencing. We also assessed chromosomal deletions in MLH1 and MSH2 and MSH6 generating a complete characterization of MMR genes. The analysis of the five MMR genes revealed 46 carriers of pathogenic mutations, including 25 in MSH2 (54%), 16 in MLH1 (35%), 4 in MSH6 (9%) and one in PMS2 (2%) gene In addition, from the 70 negative patients, we selected one mutation negative family that met the stringent Amsterdam criteria (AC) for whole exome sequencing using the SOLID platform. For this family, we selected 5 close blood relatives, including 3 affected and 2 unaffected individuals in order to facilitate the identification of new susceptibility genes with autosomal dominant pattern. The results revealed two candidate genes, c-KIT and WDR27 that segregated with the disease. Additional analysis are under evaluation. Citation Format: Felipe C. Silva, Giovana T. Torrezan, Márcia CP Figueiredo, Jose Roberto O. Ferreira, Érika M. Santos, Wilson T. Nakagawa, Samuel Aguiar-Junior, Benedito M. Rossi, Fabio O. Ferreira, Dirce M. Carraro. Molecular characterization of Brazilian patients suspected for Lynch syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 18. doi:10.1158/1538-7445.CANSUSC14-18