Publications by authors named "Giovana Tardin Torrezan"

19 Publications

  • Page 1 of 1

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

Cancers (Basel) 2021 Jan 28;13(3). Epub 2021 Jan 28.

Department of Surgery, A.C.Camargo Cancer Center, Sao Paulo 01509-010, Brazil.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare, aggressive sarcoma affecting adolescents and young adults with male predominance. Generally, it originates from the serosal surface of the abdominal cavity. The hallmark characteristic of DSRCT is the EWSR1-WT1 gene fusion. This translocation up-regulates the expression of PDGFRα, VEGF and other proteins related to tumor and vascular cell proliferation. Current management of DSRCT includes a combination of chemotherapy, radiation and aggressive cytoreductive surgery plus intra-peritoneal hyperthermic chemotherapy (HIPEC). Despite advances in multimodal therapy, outcomes remain poor since the majority of patients present disease recurrence and die within three years. The dismal survival makes DSRCT an orphan disease with an urgent need for new drugs. The treatment of advanced and recurrent disease with tyrosine kinase inhibitors, such as pazopanib, sunitinib, and mTOR inhibitors was evaluated by small trials. Recent studies using comprehensive molecular profiling of DSRCT identified potential therapeutic targets. In this review, we aim to describe the current studies conducted to better understand DSRCT biology and to explore the new therapeutic strategies under investigation in preclinical models and in early phase clinical trials.
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http://dx.doi.org/10.3390/cancers13030498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865637PMC
January 2021

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

Clinics (Sao Paulo) 2021 20;76:e2324. Epub 2021 Jan 20.

Laboratorio de Ginecologia Estrutural e Molecular, Disciplina de Ginecologia, Hospital das Clinicas (HCFMUSP), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

Objectives: The present study aimed to contribute to the catalog of genetic mutations involved in the carcinogenic processes of uterine sarcomas (USs) and carcinosarcomas (UCSs), which may assist in the accurate diagnosis of, and selection of treatment regimens for, these conditions.

Methods: We performed gene-targeted next-generation sequencing (NGS) of 409 cancer-related genes in 15 US (7 uterine leiomyosarcoma [ULMS], 7 endometrial stromal sarcoma [ESS], 1 adenosarcoma [ADS]), 5 UCS, and 3 uterine leiomyoma (ULM) samples. Quality, frequency, and functional filters were applied to select putative somatic variants.

Results: Among the 23 samples evaluated in this study, 42 loss-of-function (LOF) mutations and 111 missense mutations were detected, with a total of 153 mutations. Among them, 66 mutations were observed in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. TP53 (48%), ATM (22%), and PIK3CA (17%) were the most frequently mutated genes. With respect to specific tumor subtypes, ESS showed mutations in the PDE4DIP, IGTA10, and DST genes, UCS exhibited mutations in ERBB4, and ULMS showed exclusive alterations in NOTCH2 and HER2. Mutations in the KMT2A gene were observed exclusively in ULM and ULMS. In silico pathway analyses demonstrated that many genes mutated in ULMS and ESS have functions associated with the cellular response to hypoxia and cellular response to peptide hormone stimulus. In UCS and ADS, genes with most alterations have functions associated with phosphatidylinositol kinase activity and glycerophospholipid metabolic process.

Conclusion: This preliminary study observed pathogenic mutations in US and UCS samples. Further studies with a larger cohort and functional analyses will foster the development of a precision medicine-based approach for the treatment of US and UCS.
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http://dx.doi.org/10.6061/clinics/2021/e2324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798418PMC
April 2021

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

Ann Surg Oncol 2021 Jul 21;28(7):3637-3645. Epub 2020 Nov 21.

Department of Medical Oncology, A.C. Camargo Cancer Center, São Paulo, SP, Brazil.

Background: Phase III trials evaluating the role of secondary cytoreductive surgery (SCS) in recurrent ovarian cancer have pointed to the importance of patient selection. Two studies showed conflicting results regarding the benefit of SCS in BRCA1/2 mutation carriers. Our aim was to evaluate the impact of SCS on recurrent ovarian cancer according to BRCA1/2 status.

Methods: All patients with ovarian carcinoma with platinum-sensitive recurrent disease and tested for BRCA1/2 germline mutations were included. Cox regression and log rank test were used to evaluate the impact of SCS on progression-free survival (PFS) and the influence of BRCA1/2 mutations on the effect of SCS.

Results: 127 patients were included, 45.6% were treated with SCS and chemotherapy and 54.3% treated with chemotherapy only. Patients treated with SCS were younger, presented better performance status, had lower CA125, and had a longer platinum-free interval. In multivariate analysis SCS was associated with longer PFS (HR 0.42, 95% CI 0.25-0.72, p = 0.002). BRCA1/2 mutations were found in 35 patients (27.5%), and 11.8% of patients were treated with PARP inhibitors. Although not statistically significant, both BRCA1/2 wild type patients (PFS: 21.6 vs 18.4 months; p = 0.114) and BRCA1/2 mutation carriers (PFS: 23.1 vs 18.2 months, p = 0.193) appeared to derive benefit from SCS.

Discussion: The present study suggests a benefit of SCS irrespective of BRCA1/2 status among patients mostly not treated with PARP inhibitor. Further data on post hoc analysis from the phase III trials are warranted to confirm whether BRCA1/2 mutated patients should be selected for SCS.
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http://dx.doi.org/10.1245/s10434-020-09366-wDOI Listing
July 2021

Using Co-segregation and Loss of Heterozygosity Analysis to Define the Pathogenicity of Unclassified Variants in Hereditary Breast Cancer Patients.

Front Oncol 2020 2;10:571330. Epub 2020 Oct 2.

Molecular Oncology Research Center, Barretos Cancer Hospital, São Paulo, Brazil.

The use of gene panels introduces a new dilemma in the genetics field due to the high frequency of variants of uncertain significance (VUS). The objective of this study was to provide evidence that may help in the classification of these germline variants in terms of their clinical impact and association with the disease in question. A total of 52 unrelated women at-risk for HBOC and negative for pathogenic variants were evaluated through a gene panel comprising 14 breast and/or ovarian cancer susceptibility genes. Of the 453 germline variants identified, 15 variants (classes 3, 4, and 5) in the , and genes were evaluated via databases, co-segregation studies and loss of heterozygosity in the tumor. The co-segregation analysis allowed the establishment of an association with the presence of variants and the risk of cancer for variant c.316C>T in the gene. Four variants of uncertain significance showed loss of heterozygosity in the tumor ( c.4709T>C; c.1036C>T; c.1001A>G, and c.281T>C), which is an indication of pathogenicity. Thus, the present study provides novel evidence that favors the association of variants in moderate-risk genes with the development of hereditary breast cancer.
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http://dx.doi.org/10.3389/fonc.2020.571330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566163PMC
October 2020

Mutational Portrait of Lung Adenocarcinoma in Brazilian Patients: Past, Present, and Future of Molecular Profiling in the Clinic.

Front Oncol 2020 2;10:1068. Epub 2020 Jul 2.

Genomics and Molecular Biology Group, International Research Center, A.C. Camargo Cancer Center, São Paulo, Brazil.

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 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. 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 , and and gene fusions involving TKR genes (before TKI treatment) and T790M (after TKI treatment) were assessed. 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 ( < 0.001). Next-generation sequencing was very sensitive, with a lower rate of inconclusive results compared with Sanger sequencing and pyrosequencing. hotspot gene panels were applied in 495 LA cases and detected oncogenic mutations in 51.3% of samples, most frequently in (22.4%) and (26.9%). In subgroups of 36 and 35 patients, gene fusions were detected in 11.1% of tumors and T790M resistance mutations were detected in 59% of plasma samples from patients previously treated with TKI, respectively. This report provides the first comprehensive actionable alteration portrait of LA in Brazil. The high rate of actionable alterations in 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.
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http://dx.doi.org/10.3389/fonc.2020.01068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343968PMC
July 2020

Clinical and Molecular Assessment of Patients with Lynch Syndrome and Sarcomas Underpinning the Association with Germline Pathogenic Variants.

Cancers (Basel) 2020 Jul 9;12(7). Epub 2020 Jul 9.

Genomics and Molecular Biology Group, International Research Center/CIPE, A.C.Camargo Cancer Center, São Paulo 01508-010, Brazil.

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 carriers and 2 ), with 2 having Muir-Torre phenotypes. For two 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 alterations. In summary, sarcomas represent a rare clinical manifestation in patients with LS, especially in carriers, and the analysis of tumor biological characteristics can be useful for definition of tumor etiology and novel therapeutic options.
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http://dx.doi.org/10.3390/cancers12071848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408879PMC
July 2020

MLH1 intronic variants mapping to + 5 position of splice donor sites lead to deleterious effects on RNA splicing.

Fam Cancer 2020 10;19(4):323-336

Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB) [HIBA-IUHI-CONICET], C1199ABH, Buenos Aires, Argentina.

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.
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http://dx.doi.org/10.1007/s10689-020-00182-5DOI Listing
October 2020

BAP1 tumor predisposition syndrome case report: pathological and clinical aspects of BAP1-inactivated melanocytic tumors (BIMTs), including dermoscopy and confocal microscopy.

BMC Cancer 2019 Nov 9;19(1):1077. Epub 2019 Nov 9.

Laboratory of Genomics and Molecular Biology, A.C. Camargo Cancer Center, Rua Taguá, 440, São Paulo, SP, CEP: 0508-010, Brazil.

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.
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http://dx.doi.org/10.1186/s12885-019-6226-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842488PMC
November 2019

Association of Folate and Vitamins Involved in the 1-Carbon Cycle with Polymorphisms in the Methylenetetrahydrofolate Reductase Gene (MTHFR) and Global DNA Methylation in Patients with Colorectal Cancer.

Nutrients 2019 Jun 18;11(6). Epub 2019 Jun 18.

Department of Pelvic Surgery, A.C. Camargo Cancer Center, Rua Professor Antônio Prudente, 211, Liberdade, São Paulo, São Paulo CEP 01509-010, Brazil.

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, = 0.03) and global DNA methylation (rho = 0.20, = 0.03). Individuals aged over 61 years ( = 0.01) in clinicopathological staging III and IV ( = 0.01) and with + heterozygous mutated homozygous genotypes for the MTHFR A1298C gene had higher levels of global DNA methylation ( = 0.04). The association between dietary intake of folate, serum folate, and tumor stage were predictive of global DNA methylation in patients' blood. The levels of serum folate, the dietary folate and the status of DNA methylation can influence clinicopathological staging.
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http://dx.doi.org/10.3390/nu11061368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627304PMC
June 2019

Enhanced type I interferon gene signature in primary antiphospholipid syndrome: Association with earlier disease onset and preeclampsia.

Autoimmun Rev 2019 Apr 14;18(4):393-398. Epub 2019 Feb 14.

Rheumatology Division, Faculdade de Medicina da Universidade de São Paulo (USP), Brazil.

Objective: 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.

Methods: 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.

Results: 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).

Conclusion: 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.
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http://dx.doi.org/10.1016/j.autrev.2018.11.004DOI Listing
April 2019

Prevalence of BRCA1 and BRCA2 pathogenic and likely pathogenic variants in non-selected ovarian carcinoma patients in Brazil.

BMC Cancer 2019 Jan 3;19(1). Epub 2019 Jan 3.

Department of Medical Oncology, AC Camargo Cancer Center, Rua Professor Antonio Prudente 211, São Paulo, CEP: 01509-900, Brazil.

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.
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http://dx.doi.org/10.1186/s12885-018-5235-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319008PMC
January 2019

From colorectal cancer pattern to the characterization of individuals at risk: Picture for genetic research in Latin America.

Int J Cancer 2019 07 5;145(2):318-326. Epub 2018 Dec 5.

AC Camargo Cancer Center, Sao Paulo, Brazil.

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.
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http://dx.doi.org/10.1002/ijc.31920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587543PMC
July 2019

Expanding morphological and clinical aspects of hereditary leiomyomatosis and renal cell carcinoma (HLRCC): a case report in a patient with unusual morphology and clinical presentation.

Virchows Arch 2018 Dec 31;473(6):775-779. Epub 2018 Aug 31.

Department of Pathology, AC Camargo Cancer Center, Rua Professor Antônio Prudente 211, São Paulo, 01509-900, Brazil.

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.
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http://dx.doi.org/10.1007/s00428-018-2420-3DOI Listing
December 2018

Pitfalls in genetic testing: a case of a SNP in primer-annealing region leading to allele dropout in .

Mol Genet Genomic Med 2017 Jul 11;5(4):443-447. Epub 2017 May 11.

Laboratory of Genomics and Molecular BiologyInternational Research CenterAC Camargo Cancer CenterSão PauloBrazil.

Background: Hereditary breast and ovarian cancer is characterized by mutations in or 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 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.
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http://dx.doi.org/10.1002/mgg3.295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511807PMC
July 2017

Germline Mutations in MLH1 Leading to Isolated Loss of PMS2 Expression in Lynch Syndrome: Implications for Diagnostics in the Clinic.

Am J Surg Pathol 2017 06;41(6):861-864

*Laboratory of Genomics and Molecular Biology, International Research Center/CIPE †Anatomic Pathology Department ‡Colorectal Tumors Department, AC Camargo Cancer Center §National Institute of Science and Technology in Oncogenomics (INCITO) São Paulo, Brazil.

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http://dx.doi.org/10.1097/PAS.0000000000000827DOI Listing
June 2017

A genomic case study of desmoplastic small round cell tumor: comprehensive analysis reveals insights into potential therapeutic targets and development of a monitoring tool for a rare and aggressive disease.

Hum Genomics 2016 11 18;10(1):36. Epub 2016 Nov 18.

International Research Center/CIPE, A.C. Camargo Cancer Center, São Paulo, SP, Brazil.

Background: Genome-wide profiling of rare tumors is crucial for improvement of diagnosis, treatment, and, consequently, achieving better outcomes. Desmoplastic small round cell tumor (DSRCT) is a rare type of sarcoma arising from mesenchymal cells of abdominal peritoneum that usually develops in male adolescents and young adults. A specific translocation, t(11;22)(p13;q12), resulting in EWS and WT1 gene fusion is the only recurrent molecular hallmark and no other genetic factor has been associated to this aggressive tumor. Here, we present a comprehensive genomic profiling of one DSRCT affecting a 26-year-old male, who achieved an excellent outcome.

Methods: We investigated somatic and germline variants through whole-exome sequencing using a family based approach and, by array CGH, we explored the occurrence of genomic imbalances. Additionally, we performed mate-paired whole-genome sequencing for defining the specific breakpoint of the EWS-WT1 translocation, allowing us to develop a personalized tumor marker for monitoring the patient by liquid biopsy.

Results: We identified genetic variants leading to protein alterations including 12 somatic and 14 germline events (11 germline compound heterozygous mutations and 3 rare homozygous polymorphisms) affecting genes predominantly involved in mesenchymal cell differentiation pathways. Regarding copy number alterations (CNA) few events were detected, mainly restricted to gains in chromosomes 5 and 18 and losses at 11p, 13q, and 22q. The deletions at 11p and 22q indicated the presence of the classic translocation, t(11;22)(p13;q12). In addition, the mapping of the specific genomic breakpoint of the EWS-WT1 gene fusion allowed the design of a personalized biomarker for assessing circulating tumor DNA (ctDNA) in plasma during patient follow-up. This biomarker has been used in four post-treatment blood samples, 3 years after surgery, and no trace of EWS-WT1 gene fusion was detected, in accordance with imaging tests showing no evidence of disease and with the good general health status of the patient.

Conclusions: Overall, our findings revealed genes with potential to be associated with risk assessment and tumorigenesis of this rare type of sarcoma. Additionally, we established a liquid biopsy approach for monitoring patient follow-up based on genomic information that can be similarly adopted for patients diagnosed with a rare tumor.
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http://dx.doi.org/10.1186/s40246-016-0092-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116179PMC
November 2016

Clinical and Molecular Characterization of Brazilian Patients Suspected to Have Lynch Syndrome.

PLoS One 2015 5;10(10):e0139753. Epub 2015 Oct 5.

Laboratory of Genomics and Molecular Biology, International Research Center/CIPE-A. C. Camargo Cancer Center, São Paulo, Brazil; National Institute of Science and Technology in Oncogenomics (INCITO), Brazil.

Lynch syndrome (LS) accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autosomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous/metachronous tumors and extra-colonic tumors. Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five mismatch repair genes (MMR), mostly in MLH1 and MSH2, but also in MSH6, PMS1 and PMS2. To comprehensively characterize 116 Brazilian patients suspected for LS, we assessed the frequency of germline mutations in the three minor genes MSH6, PMS1 and PMS2 in 82 patients negative for point mutations in MLH1 and MSH2. We also assessed large genomic rearrangements by MLPA for detecting copy number variations (CNVs) in MLH1, MSH2 and MSH6 generating a broad characterization of MMR genes. The complete analysis of the five MMR genes revealed 45 carriers of pathogenic mutations, including 25 in MSH2, 15 in MLH1, four in MSH6 and one in PMS2. Eleven novel pathogenic mutations (6 in MSH2, 4 in MSH6 and one in PMS2), and 11 variants of unknown significance (VUS) were found. Mutations in the MLH1 and MSH2 genes represented 89% of all mutations (40/45), whereas the three MMR genes (MSH6, PMS1 and PMS2) accounted for 11% (5/45). We also investigated the MLH1 p.Leu676Pro VUS located in the PMS2 interaction domain and our results revealed that this variant displayed no defective function in terms of cellular location and heterodimer interaction. Additionally, we assessed the tumor phenotype of a subset of patients and also the frequency of CRC and extra-colonic tumors in 2,365 individuals of the 116 families, generating the first comprehensive portrait of the genetic and clinical aspects of patients suspected of LS in a Brazilian cohort.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139753PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593564PMC
June 2016

Mutational spectrum of the APC and MUTYH genes and genotype-phenotype correlations in Brazilian FAP, AFAP, and MAP patients.

Orphanet J Rare Dis 2013 Apr 5;8:54. Epub 2013 Apr 5.

Laboratory of Genomics and Molecular Biology, A.C. Camargo Hospital, São Paulo, SP, Brazil.

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.
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http://dx.doi.org/10.1186/1750-1172-8-54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623842PMC
April 2013

A novel SYBR-based duplex qPCR for the detection of gene dosage: detection of an APC large deletion in a familial adenomatous polyposis patient with an unusual phenotype.

BMC Med Genet 2012 Jul 16;13:55. Epub 2012 Jul 16.

CIPE - International Center of Research and Training - A, C, Camargo Hospital, Rua Taguá, São Paulo, Brazil.

Background: Familial adenomatous polyposis (FAP) is a hereditary colorectal cancer syndrome caused by a loss of function of the APC gene. Large deletions in APC are a common cause of FAP; despite the existence of a variety of gene dosage detection methodologies, most are labor intensive and time and resource consuming.

Methods: We describe a new duplex qPCR method for gene dosage analysis based on the coamplification of a target and a reference gene in a SYBR Green reaction, followed by a comparison of the ratio between the target and the reference peaks of the melting curve for the test (patient) and control samples. The reliability of the described duplex qPCR was validated for several genes (APC, HPRT1, ATM, PTEN and BRCA1).

Results: Using this novel gene dosage method, we have identified an APC gene deletion in a FAP patient undergoing genetic testing. Comparative genomic hybridization based on microarrays (aCGH) was used to confirm and map the extent of the deletion, revealing a 5.2 MB rearrangement (5q21.3-q22.3) encompassing the entire APC and 19 additional genes.

Conclusion: The novel assay accurately detected losses and gains of one copy of the target sequences, representing a reliable and flexible alternative to other gene dosage techniques. In addition, we described a FAP patient harboring a gross deletion at 5q21.3-q22.3 with an unusual phenotype of the absence of mental impairment and dysmorphic features.
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http://dx.doi.org/10.1186/1471-2350-13-55DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458979PMC
July 2012