Tumor heterogeneity in Esophageal Adenocarcinoma: a genomic approach

INTRODUCTION: Esophageal adenocarcinoma (EAC) is a severe malignancy in terms of prognosis and mortality rate. Because its great genetic heterogeneity, disputes regarding classification, prevention and treatments are still unsolved. AIM: We investigated intra- and inter-EAC heterogeneity by defining EAC’s somatic mutational profile and the role of candidate microRNAs, to correlate the molecular profile of tumors to clinical outcomes and to identify biomarkers for classification. METHODS: 38 EAC cases were analyzed via high-throughput cell sorting technology combined with targeted sequencing and whole genome low-pass sequencing. Targeted sequencing of further 169 cases was performed to widen the study. miR221 and miR483-3p expression was profiled via qPCR in 112 EACs and correlation with clinical outcomes was investigated. RESULTS: 35/38 EACs carried at least one somatic mutation absent in stromal cells. TP53 was found mutated in 73.7% of cases. Selective sorting revealed tumor subclones with different mutational loads and copy number alterations, confirming the high intra-tumor heterogeneity of EAC. Mutations were in most cases at homozygous state, and we identified alterations that were missed with the whole-tumor analysis. Mutations in HNF1A gene, not previously associated with EAC, were identified in both cohorts. Higher expression of miR483-3p and miR221 was associated with poorer cancer specific survival (P=0.0293 and P=0.0059), and recurrence in the Lauren intestinal subtype (P=0.0459 and P=0.0002). Median expression levels of miRNAs were higher in patients with advanced tumor stages. The loss of SMAD4 immunoreactivity was significantly associated with poorer cancer specific survival and recurrence (P=0.0452; P=0.022 respectively). CONCLUSION: Combining selective sorting technology and next generation sequencing allowed to better define EAC inter- and intra-tumor heterogeneity. We identified HNF1A as a new mutated gene associated to EAC that could be involved in tumor progression and promising biomarkers such as SMAD4, miR221 and miR483-3p to identify patients at higher risk for more aggressive tumors.

Identification of molecular biomarkers in esophageal adenocarcinoma

Esophageal adenocarcinoma (EAC) is a severe cancer that has been on the rise in Western nations over the past few decades. It has a high mortality rate and the 5-year survival rate is only 35%–45%. EAC has been included in a group of tumors with one of the highest rates of copy number alterations (CNAs), somatic structural rearrangements, high mutation frequency, with different mutational signatures, and with epigenetic mechanisms. The vast heterogeneity of EAC mutations makes it challenging to comprehend the biology that underlies tumor onset and development, identify prognostic biomarkers, and define a molecular classification to stratify patients. The only way to resolve the current disagreements is through an exhaustive molecular analysis of EAC. We examined the genetic profile of 164 patients' esophageal adenocarcinoma samples (without chemo-radiotherapy). The included patients did not receive neoadjuvant therapies, which can change the genetic and molecular composition of the tumor. Using next-generation sequencing technologies (NGS) at high coverage, we examined a custom panel of 26 cancer-related genes. Over the entire cohort, 337 variants were found, with the TP53 gene showing the most frequent alteration (67.27%). Poorer cancer-specific survival was associated with missense mutations in the TP53 gene (Log Rank P=0.0197). We discovered HNF1alpha gene disruptive mutations in 7 cases that were also affected by other gene changes. We started to investigate its role in EAC cell lines by silencing HNF1alpha to mimic our EAC cohort and we use Seahorse technique to analyze its role in the metabolism in esophageal cell. No significant changes were found in transfected cell lines. We conclude by finding that a particular class of TP53 mutations (missense changes) adversely impacted cancer-specific survival in EAC. HNF1alpha, a new EAC-mutated gene, was found, but more research is required to fully understand its function as a tumor suppressor gene.

A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.

arrayMap 2014: an updated cancer genome resource.

Somatic copy number aberrations (CNA) represent a mutation type encountered in the majority of cancer genomes. Here, we present the 2014 edition of arrayMap (http://www.arraymap.org), a publicly accessible collection of pre-processed oncogenomic array data sets and CNA profiles, representing a vast range of human malignancies. Since the initial release, we have enhanced this resource both in content and especially with regard to data mining support. The 2014 release of arrayMap contains more than 64,000 genomic array data sets, representing about 250 tumor diagnoses. Data sets included in arrayMap have been assembled from public repositories as well as additional resources, and integrated by applying custom processing pipelines. Online tools have been upgraded for a more flexible array data visualization, including options for processing user provided, non-public data sets. Data integration has been improved by mapping to multiple editions of the human reference genome, with the majority of the data now being available for the UCSC hg18 as well as GRCh37 versions. The large amount of tumor CNA data in arrayMap can be freely downloaded by users to promote data mining projects, and to explore special events such as chromothripsis-like genome patterns.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situhybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.

Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. © 2013 Spandidos Publications Ltd. All rights reserved.

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma

Objective: To understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients. Methods: Tumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses-all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region. Results and Conclusions: HR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods free-of-neoplasia. Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis. © 2013 Elsevier Inc.

Identificação de marcadores moleculares de resposta à quimioterapia neoadjuvante e associação de radioterapia e quimioterapia em pacientes com carcinoma epidermóide de orofaringe

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações genômicas e perfil de expressão gênica global em leiomiomas uterinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genomic screening of testicular germ cell tumors from monozygotic twins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações epigenéticas e do número de cópias do gene SFN em carcinomas mamários

Pós-graduação em Ciências Biológicas (Genética) - IBB