Increased Levels of Genomic Instability and Mutations in Homologous Recombination Genes in Locally Advanced Rectal Carcinomas

Pre-operative 5-fluoracil-based chemoradiotherapy (nCRT) is the standard treatment for patients with locally advanced rectal cancer (LARC). Patients with pathological complete response (pCR–0% of tumor cells in the surgical specimen after nCRT) have better overall survival and lower risk of recurrence in comparison with incomplete responders (pIR). Predictive biomarkers to be used for new therapeutic strategies and capable of stratifying patients to avoid overtreatment are needed. We evaluated the genomic profiles of 33 pre-treatment LARC biopsies using SNP array and targeted-next generation sequencing (tNGS). Based on the large number of identified genomic alterations, we calculated the genomic instability index (GII) and three homologous recombination deficiency (HRD) scores, which have been reported as impaired DNA repair markers. We observed high GII in our LARC cases, which was confirmed in 165 rectal cancer cases from TCGA. Patients with pCR presented higher GII compared with pIR. Moreover, a negative correlation between GII and the fraction of tumor cells remaining after surgery was observed (ρ = –0.382, P = 0.02). High HRD scores were detected in 61% of LARC, of which 70% were incomplete responders. Using tNGS (105 cancer-related genes, 13 involved in HR and 5 in mismatch repair pathways), we identified 23% of cases with mutations in HR genes, mostly in pIR cases (86% of mutated cases). In agreement, the analysis of the TCGA dataset (N = 145) revealed 21% of tumors with mutations in HR genes. The HRD scores were shown to be predictive of better response to PARP-inhibitors and platinum-based chemotherapy in breast and ovarian cancer. Our results suggest that the same strategy could be applied in a set of LARC patients with HRD. In conclusion, we identified high genomic instability in LARC, which was related to alterations in the HR pathway, especially in pIR. These findings suggest that patients with impaired HRD would clinically benefit from PARP-inhibitors and platinum-based therapy

Transcription profiling in papillary thyroid carcinoma reveals potential diagnostic markers and drug targets

Financial support FAPESP and CAPES

Integration of miRNA and mRNA expression profles reveals microRNA-regulated networks during muscle wasting in cardiac cachexia

Cardiac cachexia (CC) is a common complication of heart failure (HF) associated with muscle wasting and poor patient prognosis. Although different mechanisms have been proposed to explain muscle wasting during CC, its pathogenesis is still not understood. Here, we described an integrative analysis between miRNA and mRNA expression profiles of muscle wasting during CC. Global gene expression profiling identified 1,281 genes and 19 miRNAs differentially expressed in muscle wasting during CC. Several of these deregulated genes are known or putative targets of the altered miRNAs, including miR-29a-3p, miR-29b-3p, miR-210-5p, miR-214, and miR-489. Gene ontology analysis on integrative mRNA/miRNA expression profiling data revealed miRNA interactions affecting genes that regulate extra-cellular matrix (ECM) organization, proteasome protein degradation, citric acid cycle and respiratory electron transport. We further identified 11 miRNAs, including miR-29a-3p and miR-29b-3p, which target 21 transcripts encoding the collagen proteins related to ECM organization. Integrative miRNA and mRNA global expression data allowed us to identify miRNA target genes involved in skeletal muscle wasting in CC. Our functional experiments in C2C12 cells confirmed that miR-29b down-regulates collagen genes and contributes to muscle cell atrophy. Collectively, our results suggest that key ECM-associated miRNAs and their target genes may contribute to CC in HF

CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

BACKGROUND: The E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression. METHODS: The aim of our study was to assess, by Methylation-Specific Polymerase Chain Reaction (MSP), the methylation pattern of the CDH1 gene and its possible correlation with the expression of E-cadherin and other standard immunohistochemical parameters (Her-2, ER, PgR, p53, and K-67) in a series of 79 primary breast cancers (71 infiltrating ductal, 5 infiltrating lobular, 1 metaplastic, 1 apocrine, and 1 papillary carcinoma). RESULTS: CDH1 hypermethylation was observed in 72% of the cases including 52/71 ductal, 4/5 lobular carcinomas and 1 apocrine carcinoma. Reduced levels of E-cadherin protein were observed in 85% of our samples. Although not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association was observed between CDH1 methylation and ER expression (p = 0.0301, Fisher's exact test). However, this finding was not considered significant after Bonferroni correction of p-value. CONCLUSION: Our preliminary findings suggested that abnormal CDH1 methylation occurs in high frequencies in infiltrating breast cancers associated with a decrease in E-cadherin expression in a subgroup of cases characterized by loss of expression of other important genes to the mammary carcinogenesis process, probably due to the disruption of the mechanism of maintenance of DNA methylation in tumoral cells

Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

<p>Abstract</p> <p>Background</p> <p><it>HER-2 </it>gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH). These procedures permit correlation between <it>HER-2 </it>expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic <it>in situ </it>hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.</p> <p>Methods</p> <p>To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.</p> <p>Results</p> <p>The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of <it>HER-2 </it>status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and <it>HER-2 </it>transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and <it>HER-2 </it>downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between <it>HER-2 </it>downexpression and the involvement of less than four lymph nodes (<it>P </it>= 0.0350).</p> <p>Conclusion</p> <p>Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the <it>HER-2 </it>gene.</p

Comparative genomic hybridization analysis of benign and invasive male breast neoplasms

Comparative genomic hybridization (CGH) analysis was performed for the identification of chromosomal imbalances in two benign gynecomastias and one malignant breast carcinoma derived from patients with male breast disease and compared with cytogenetic analysis in two of the three cases. CGH analysis demonstrated overrepresentation of 8q in all three cases. One case of gynecomastia presented gain of 1p34.3similar topter. 11p14similar toq12. and 17p11.2similar toqter, and loss of 1q41similar toqter and 4q33similar toqter. The other gynecomastia presented del(1)(q41) as detected by both cytogenetic and CGH analysis. CGH analysis of the invasive ductal carcinoma confirmed a gain of 17p11.2similar toqter previously detected by cytogenetic analysis. These regions showed some similarity in their pattern of imbalance to the chromosomal alterations described in female and male breast cancer. (C) 2002 Elsevier B.V. All rights reserved

Genotyping of AR and PSA polymorphisms in a patient with Klinefelter syndrome, non-Hodgkin lymphoma, and adenocarcinoma of the prostate

It has been hypothesized that the AR (androgen receptor) gene binds the two PSA (prostate-specific antigen) alleles with differing affinities and may differentially influence prostate cancer risk. In this article, we report a case of adenocarcinoma of the prostate in a 56-year-old man with Klinefelter syndrome (47,XXY) and non-Hodgkin lymphoma, as well as the AR and PSA genotype. AR and PSA gene polymorphisms were analyzed by polymerase chain reaction-based methods using DNA from peripheral white blood cells and the prostate cancer. We determined the methylation status of the AR gene on the X chromosome. The patient presents with the AG genotype for the ARE-I (androgen response element) region of the PSA gene. We detect the presence of two short AR alleles with 19 and 11CAG repeats each. Unmethylated alleles were demonstrated for both. The shorter allele was inactive in more than 60% of total DNA in both control blood and prostate cancer cells. The presence of short AR alleles and the G allele of the PSA gene may contribute to the development of prostate cancer in a 47,XXY patient. (C) 2004 Elsevier B.V. All rights reserved

Independent clonal origin of multiple uterine leiomyomas that was determined by X chromosome inactivation and microsatellite analysis

Objective: In an attempt to clarify the clonality and genetic relationships that are involved in the tumorigenesis of uterine leiomyomas, we used a total of 43 multiple leiomyomas from 14 patients and analyzed the allelic status with 15 microsatellite markers and X chromosome inactivation analysis.Study design: We have used a set of 15 microsatellite polymorphism markers mapped on 3q, 7p, 11, and 15q by automated analysis. The X chromosome inactivation was evaluated by the methylation status of the X-linked androgen receptor gene.Results: Loss of heterozygosity analysis showed a different pattern in 7 of the 8 cases with allelic loss for at least 1 of 15 microsatellite markers that were analyzed. A similar loss of heterozygosity findings at 7p22-15 was detected in 3 samples from the same patient. X chromosome inactivation analysis demonstrated the same inactivated allele in all tumors of the 9 of 12 informative patients;. different inactivation patterns were observed in 3 cases.Conclusion: Our data support the concept that uterine leiomyomas are derived from a single cell but are generated independently in the uterus. Loss of heterozygosity findings at 7p22-15 are consistent with previous data that suggested the relevance of chromosomal aberrations at 7p that were involved in individual uterine leiomyomas. (C) 2005 Mosby, Inc. All rights reserved