Elisa Napolitano Ferreira

Recurrent somatic mutation in DROSHA induces microRNA profile changes in Wilms tumour

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.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts

The importance of epithelial‐stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA‐MB231) basal cell lines, with fibroblasts isolated from breast benign‐disease adjacent tissues (NAF) or with carcinoma‐associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA‐MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA‐MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA‐MB231 by a decrease in the expression of genes induced by TGFβ1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling.

TGIF1 splicing variant 8 is overexpressed in oral squamous cell carcinoma and is related to pathologic and clinical behavior.

OBJECTIVE Possible differences in splicing variants of TGIF1 in oral squamous cell carcinoma (OSCC) have not yet been reported. This study analyzed the expression levels of different splicing variants of the TGIF1 gene in OSCC compared with nontumoral epithelium (NT) and the relationship with clinical-pathologic features of tumors. STUDY DESIGN Forty-eight frozen samples of OSCC and 17 of NT were analyzed using quantitative reverse transcription polymerase chain reaction. RESULTS TGIF1v2 and v8 are overexpressed in OSCC, whereas TGIF1v5 is underexpressed when compared with NT. Low TGIF1v8 expression was correlated with lower cellular differentiation, positive blood vascular invasion, advanced pathologic stage, and positive vascular lymphatic invasion of OSCC. TGIF1v8 is also related to overall survival over time, with lower values associated with an increased risk of cancer-related death. CONCLUSIONS These data suggest that alternative splicing of TGIF1 is deregulated in OSCC, with overexpression of some splicing variants, especially TGIF1v8, which is associated with advanced stages of OSCC.

Upregulated genes at 2q24 gains as candidate oncogenes in hepatoblastomas

AIM Cytogenetic data of hepatoblastomas, a rare embryonal tumor of the liver, mostly consist of descriptions of whole-chromosome aneuploidies and large chromosome alterations. High-resolution cytogenetics may provide clues to hepatoblastoma tumorigenesis and indicate markers with clinical significance. PATIENTS & METHODS We used array-CGH (180K) to screen for genomic imbalances in nine hepatoblastomas. Additionally, we investigated the expression pattern of selected genes exhibiting copy number changes. RESULTS Analysis showed mainly whole-chromosome or chromosome-arm aneuploidies, but some focal aberrations were also mapped. Expression analysis of 48 genes mapped at one 10 Mb amplification at 2q24 revealed upregulation of DAPL1, ERMN, GALNT5, SCN1A and SCN3A in the set of tumors compared with differentiated livers. CONCLUSION These genes appear as candidates for hepatoblastoma tumorigenesis.

Mutational spectrum of WTX, WT1, CTNNB1, APC and PLCG2 genes in Wilms tumor defined by massive parallel resequencing

Background The identification of molecular alterations that trigger Wilms tumor (WT) development is crucial to understanding the tumorigenesis of this malignancy. Currently, it is estimated that WTX and WT1 genomic losses together with CTNNB1 point mutations occur in about 30% of WTs. However, the majority of cases remain without any identified driver mutation. Results from a previous study by our group pointed to APC and PLCG2 as candidate genes altered in WT [1]. Given the advent of modern DNA sequencing technologies, it is now feasible to evaluate large genomic regions spanning complete genes (exons and introns), allowing the description of the mutation patterns occurring in tumor cells. Thus, the aim of this study was to identify point mutations and indels in the complete sequence of APC, CTNNB1, WT1, WTX and PLCG2 genes in order to characterize both the exonic mutational spectrum and the intronic nucleotide substitution pattern. Material and methods The complete genomic regions of the selected genes, spanning a total of 430 kb, were amplified by long-range PCR in 15 WTs and 3 non-neoplasic control samples, giving a total of 60 amplicons per sample (10 kb on average). The resulting amplicons were mixed at equimolar concentrations and, for each sample, the Ion PGM library preparation protocol was performed. The libraries of the 18 barcoded samples were combined in four sequencing pools that were individually submitted to an Ion PGM™ Sequencer run on an Ion 316™ Chip. Point mutation and indels not present in the non-neoplasic controls were selected for capillary sequencing validation. The validated

Complex landscape of germline variants in Brazilian patients with hereditary and early onset breast cancer

Pathogenic variants in known breast cancer (BC) predisposing genes explain only about 30% of Hereditary Breast Cancer (HBC) cases, whereas the underlying genetic factors for most families remain unknown. Here, we used whole-exome sequencing (WES) to identify genetic variants associated to HBC in 17 patients of Brazil with familial BC and negative for causal variants in major BC risk genes (BRCA1/2, TP53, and CHEK2 c.1100delC). First, we searched for rare variants in 27 known HBC genes and identified two patients harboring truncating pathogenic variants in ATM and BARD1. For the remaining 15 negative patients, we found a substantial vast number of rare genetic variants. Thus, for selecting the most promising variants we used functional-based variant prioritization, followed by NGS validation, analysis in a control group, cosegregation analysis in one family and comparison with previous WES studies, shrinking our list to 23 novel BC candidate genes, which were evaluated in an independent cohort of 42 high-risk BC patients. Rare and possibly damaging variants were identified in 12 candidate genes in this cohort, including variants in DNA repair genes (ERCC1 and SXL4) and other cancer-related genes (NOTCH2, ERBB2, MST1R, and RAF1). Overall, this is the first WES study applied for identifying novel genes associated to HBC in Brazilian patients, in which we provide a set of putative BC predisposing genes. We also underpin the value of using WES for assessing the complex landscape of HBC susceptibility, especially in less characterized populations.

Abstract A49: BRCA1 deficiency is a recurrent event in early onset triple-negative breast cancer (TNBC): A comprehensive analysis of germline mutations and somatic promoter methylation

Background: Triple-negative breast cancer (TNBC) is a breast cancer subtype that accounts for 15-20% of all breast cancer cases. TNBC treatment is challenging because it does not respond to conventional hormonal and available target therapies, which are effective in other subtypes, making systemic chemotherapy the mainstay treatment. Moreover, TNBC displays higher aggressiveness and distinct metastatic pattern compared to other breast tumors, resulting in worse prognosis and survival. We and others have identified high prevalence of BRCA1 germline mutation in TNBC. However, BRCA1 deficiency may also be caused by somatic gene promoter methylation among other mechanisms. BRCA1/2 deficiency may lead to impairment of DNA repair and tumor development. Hence, understanding the mechanisms of BRCA deficiency in driving this tumor subtype, in both hereditary and sporadic scenery, is of great clinical and biologic interest. Methods: We included in this study TNBC samples unselected for age or family history and attending A. C. Camargo Cancer Center. Tumor tissues were screened for point mutation in BRCA1/2 using next-generation sequencing (NGS). Sanger sequencing was performed in both tumor and normal tissue/leucocyte for categorizing the pathogenic mutations in germline or somatic. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to investigate BRCA1 copy number alterations (CNA) resulting from large rearrangements in tumor and leucocyte. Additionally, for BRCA1 gene silencing investigation a customized bisulfite NGS approach was performed to assess the BRCA1 promoter methylation in tumor tissue. Results: Point mutations were screened in 131 TNBC tumor samples, detecting a total of 18 pathogenic mutations (13.7%)---16 (88.8%) in BRCA1 and 2 (11.2%) in BRCA2. No large rearrangement was detected by MLPA in tumor tissue or leucocyte. Mutations classified as germline accounted for the majority of the pathogenic mutations detected in tumor tissue (93.75% - 15/16)---81.25% (13/16) in BRCA1 and 12.5% (2/16) in BRCA2. Only one somatic mutation was detected (6.25% - 1/16) in BRCA1 gene. Considering early onset TNBC (≤40 year of age) the germline mutation rate increased to 25% (8/32), mainly in BRCA1 gene (22% - 7/32). BRCA1 promoter hypermethylation in tumor was detected in 20.6%, all in sporadic TNBC, with a slight increase to 28% in early onset cases. Ultimately, BRCA impairment by either constitutive or somatic events was identified in 34% (45/131) of the cases and was significantly more frequent in young women (56% in ≤40; 33% in 41-50; 23% in >50 year of age; p=0.007) and associated with better overall and disease-free survival rates in this group of patients. Conclusions: We observed that a high number of the TNBC cases are hereditary and BRCA1-related, especially in young women. For the sporadic cases, BRCA1 gene silencing was also a recurrent event. Taken together, our data showed that BRCA1 impairment (either by germline mutation or somatic promoter hypermethylation) was present at high frequency in the early-onset cases and was associated with better survival for these patients. With the new treatment modalities including Poly (ADP-ribose) polymerase (PARP) inhibitors being investigated, our results shed light that a significant proportion of young women with TNBC might benefit from PARP-inhibitor and future investigation in this subject is warranted. Citation Format: Rafael Canfield Brianese, Kivvi D. M. Nakamura, Fernanda G. S. R. Almeida, Rodrigo F. Ramalho, Elisa N. Ferreira, Bruna D. F. Barros, Maria N. C. Formiga, Victor P. Andrade, Vladmir C. C. Lima, Dirce M. Carraro. BRCA1 deficiency is a recurrent event in early onset triple-negative breast cancer (TNBC): A comprehensive analysis of germline mutations and somatic promoter methylation [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; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A49.

Epithelial cells captured from ductal carcinoma in situ reveal a gene expression signature associated with progression to invasive breast cancer

Breast cancer biomarkers that can precisely predict the risk of progression of non-invasive ductal carcinoma in situ (DCIS) lesions to invasive disease are lacking. The identification of molecular alterations that occur during the invasion process is crucial for the discovery of drivers of transition to invasive disease and, consequently, biomarkers with clinical utility. In this study, we explored differences in gene expression in mammary epithelial cells before and after the morphological manifestation of invasion, i.e., early and late stages, respectively. In the early stage, epithelial cells were captured from both pre-invasive lesions with distinct malignant potential [pure DCIS as well as the in situ component that co-exists with invasive breast carcinoma lesions (DCIS-IBC)]; in the late stage, epithelial cells were captured from the two distinct morphological components of the same sample (in situ and invasive components). Candidate genes were identified using cDNA microarray and rapid subtractive hybridization (RaSH) cDNA libraries and validated by RT-qPCR assay using new samples from each group. These analyses revealed 26 genes, including 20 from the early and 6 from the late stage. The expression profile based on the 20 genes, marked by a preferential decrease in expression level towards invasive phenotype, discriminated the majority of DCIS samples. Thus, this study revealed a gene expression signature with the potential to predict DCIS progression and, consequently, provides opportunities to tailor treatments for DCIS patients.

Abstract A32: Urine as a potential liquid biopsy for detecting tumor DNA in Wilms tumor patient: Detection of somatic mutations in urine opens perspectives of monitoring chemotherapy response in WT patients

Detection of tumor DNA in urine has been shown as viable method of diagnosis not only for urinary tract (e.g., kidney and bladder) cancers but also for other types, e.g. prostate. Applications of such method, resemble those which use plasma DNA and include cancer detection, monitoring of tumor growth or recurrence and response to chemotherapy or radiation therapy. As far as we know, there is a lack of studies applying urine DNA detection in Wilms tumor (WT), an embryonary kidney cancer type. Here we show evidences of detection for two somatic variants in the urine DNA of one WT patient. By using NGS exome sequencing of tumor tissue and leukocytes samples of the same patient we were able to find new somatic variants: a frame-shift indel in TNRC18 and a misssense SNV in INTS1 gene. Target sequencing applied to the DNA from the patient9s urine revealed the presence of these two somatic variants. More interestingly, both variants could not be found in the urine DNA of this patient after treatment (chemotherapy and nephrectomy). Additionally, these genes are poorly characterized in WT contributing for the comprehension of the cellular processes that are operating in tumorigenesis of WT. Altogether, our findings contribute with the mutational repertoire of WT and reveals the potential of using urine DNA sequencing as a noninvasive cancer screening approach. Citation Format: Ana C. K. Miguez, Rodrigo F. Ramalho, Elisa N. Ferreira, Bruna D. F. Barros, Claudia A. A. de Paula, Renan Valieris, Louise D. C. Mota, Jorge E. Souza, Isabela W. Cunha, Cecilia L. Costa, Sandro J. de Souza, Dirce M. Carraro. Urine as a potential liquid biopsy for detecting tumor DNA in Wilms tumor patient: Detection of somatic mutations in urine opens perspectives of monitoring chemotherapy response in WT patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A32.

Identification of Biomarkers and Expression Signatures

Recently, molecular biology has been substantially improved by the development of new technologies that allow the assessment of the genome, transcriptome and proteome on a high-throughput scale and at reasonable costs. The translation of all the information generated by these technologies into new biomarkers is an enormous challenge for the biomedical community, and vast efforts have been made in this arena. The practice of personalized medicine based on DNA/RNA information used for clinical decision-making has led to considerable advances in different areas of medicine and is now a reality at several medical centers worldwide. The aspiration is that in the near future, the medical community will have more and more available biomarkers to properly classify patients and to allow them to offer efficient and tailored treatment for a broader range of diseases, resulting in a high cure rate and minimal side effects. In this chapter, we discuss the identification of biomarkers by primarily examining gene expression. Two of the most important approaches, microarrays and RNA sequencing (RNA-Seq), and strategies for defining gene expression signatures are addressed. We also present important aspects involved in the validation of gene expression signatures as biomarkers, the bottlenecks and difficulties for their broader use in clinical practice and some good examples of signatures representing aspects of human diseases.