BackgroundHER-2 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 in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.MethodsTo 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.ResultsThe concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 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 HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 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 HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350).ConclusionBased 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 HER-2 gene.
BackgroundUndifferentiated Pleomorphic Sarcoma (UPS) and high-grade Leiomyosarcoma (LMS) are soft tissue tumors with an aggressive clinical behavior, frequently developing local recurrence and distant metastases. Despite several gene expression studies involving soft tissue sarcomas, the potential to identify molecular markers has been limited, mostly due to small sample size, in-group heterogeneity and absence of detailed clinical data.Materials and MethodsGene expression profiling was performed for 22 LMS and 22 UPS obtained from untreated patients. To assess the relevance of the gene signature, a meta-analysis was performed using five published studies. Four genes (BAD, MYOCD, SRF and SRC) selected from the gene signature, meta-analysis and functional in silico analysis were further validated by quantitative PCR. In addition, protein-protein interaction analysis was applied to validate the data. SRC protein immunolabeling was assessed in 38 UPS and 52 LMS.ResultsWe identified 587 differentially expressed genes between LMS and UPS, of which 193 corroborated with other studies. Cluster analysis of the data failed to discriminate LMS from UPS, although it did reveal a distinct molecular profile for retroperitoneal LMS, which was characterized by the over-expression of smooth muscle-specific genes. Significantly higher levels of expression for BAD, SRC, SRF, and MYOCD were confirmed in LMS when compared with UPS. SRC was the most value discriminator to distinguish both sarcomas and presented the highest number of interaction in the in silico protein-protein analysis. SRC protein labeling showed high specificity and a positive predictive value therefore making it a candidate for use as a diagnostic marker in LMS.ConclusionsRetroperitoneal LMS presented a unique gene signature. SRC is a putative diagnostic marker to differentiate LMS from UPS.
Undifferentiated high-grade pleomorphic sarcomas (UPSs) display aggressive clinical behavior and frequently develop local recurrence and distant metastasis. Because these sarcomas often share similar morphological patterns with other tumors, particularly leiomyosarcomas (LMSs), classification by exclusion is frequently used. In this study, array-based comparative genomic hybridization (array CGH) was used to analyze 20 UPS and 17 LMS samples from untreated patients. The LMS samples presented a lower frequency of genomic alterations compared with the UPS samples. The most frequently altered UPS regions involved gains at 20q13.33 and 7q22.1 and losses at 3p26.3. Gains at 8q24.3 and 19q13.12 and losses at 9p21.3 were frequently detected in the LMS samples. Of these regions, gains at 1q21.3, 11q12.2-q12.3, 16p11.2, and 19q13.12 were significantly associated with reduced overall survival times in LMS patients. A multivariate analysis revealed that gains at 1q21.3 were an independent prognostic marker of shorter survival times in LMS patients (HR = 13.76; P = 0.019). Although the copy number profiles of the UPS and LMS samples could not be distinguished using unsupervised hierarchical clustering analysis, one of the three clusters presented cases associated with poor prognostic outcome (P = 0.022). A relative copy number analysis for the ARNT, SLC27A3, and PBXIP1 genes was performed using quantitative real-time PCR in 11 LMS and 16 UPS samples. Gains at 1q21-q22 were observed in both tumor types, particularly in the UPS samples. These findings provide strong evidence for the existence of a genomic signature to predict poor outcome in a subset of UPS and LMS patients.
A similar pattern of gene expression and chromosomal imbalances in both exponents would suggest a common mechanism of functional regulation. AURKB, FGF18, and SUPT16H were identified as potential molecular markers in JNA.
Cytogenetic analyses were accomplished in two populations of Astyanax altiparanae Garutti & Britzki, 2000 and one population of Hyphessobrycon eques Steindachner, 1882, considered incertae sedis in Characidae family. Two populations of Astyanax altiparanae (Mogi-Guaçu and Tietê rivers) presented 2n=50, with the same karyotype formula: 6M+12SM+20ST+12A (FN=88). Hyphessobrycon eques from Capivara river presented 2n=52 and karyotype formula 14M+16SM+4ST+18A (FN=86). In each karyotype, the nucleolus organizer regions were detected at the end of the short arm of a single medium-sized subtelocentric chromosome. The Chromomycin A3 (CMA3) marking is coincident for the NORs in chromosomes of the two species and present additionally in two different chromosomes of Astyanax altiparanae thus showinginterpopulation differences in this species. In Hyphessobrycon eques, weak heterochromatic blocks in the position of centromeres and telomeres of most chromosomes and negative C-banding for the NOR bearing chromosome were visualized. The obtained results contribute both to the understanding of karyotype evolution of these species and to the clarifying their phylogenetic relationships.
BackgroundTesticular germ cell tumors (TGCTs) account for 1-2% of all tumors in young and middle aged men. A 75-fold increase in TCGT development has been reported for monozygotic (MZ) twins. Therefore, the occurrence of simultaneous tumors in MZ twins emphasizes the importance of genetic factors that influence the risk of developing these tumors. Genomic screening was performed for one family containing MZ twins with testicular germ cell tumors, in order to define alterations associated with risk of tumor development.MethodsCopy number alterations were evaluated using array-CGH (4x44K, Agilent Technologies) in one seminoma and one embryonal carcinoma (EC) from MZ twins. In addition, genomic alterations from the tumors and peripheral blood cells of the twins were compared to the parental genomes via their peripheral blood cells.ResultsEmbryonal carcinoma (Twin-1 t) presented a lower frequency of genomic alterations compared to the seminoma (Twin-2 t). One minimal common region of loss was observed in 9p13.1-p12 in the comparison between DNA from blood samples for Twin-1 and Twin-2. In this region is mapped the CNTNAP3 gene which was confirmed as involved in losses by qPCR. Comparative analysis of novel CNVs between the Twin-1 t and Twin-2 t showed five minimal common regions involving gain at chromosomes 12 (12p12.3-p11.1 and 12p13.33-p12.3), while losses were observed at 10p15.3-p15.2, 13q21.1-q21.2 and 15q11.1-q11.2. In addition, one exclusive rare copy number alteration was detected in Twin-1 t and Twin-2 t, and 19 novel alterations were identified in the Twin-2 t.ConclusionDistinct genomic profiles for MZ twins with phenotypically different TGCT were described. Of particular interest, 12p gains were detected exclusively in tumor samples. In peripheral blood samples, loss of 9p13.1-p12 was the unique novel CNV shared by the twins, confirming the involvement of CNTNAP3 gene in TGCTs development. Although similar CNV profiles were shared by both the peripheral blood and tumor samples of the twins, tumor-specific CNV loci were identified for seminoma and non-seminomatous tumors. These findings suggest the presence of de novo germline structural alterations and TGCT predisposition.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0181-x) contains supplementary material, which is available to authorized users.
The dog can spontaneously develop prostate cancer and consequently can be used as an experimental model for prostatic diseases associated with aging, including benign prostate hyperplasia (BPH) and prostate carcinoma (PCa). DNA copy number variations (CNVs) have been used to identify genes associated with cancer development and progression. DNA microarray based comparative genomic hybridization (aCGH) is a technique that allows to identify copy number of thousands of genes throughout the genome. aCGH was used to identify genomic regions with significantly different DNA copy number in three benign prostatic hyperplasia (BPH), four proliferative inflammatory atrophy (PIA), and 14 canine prostate carcinoma (PCa). Five histologically normal prostate tissue were used as reference. Genomic DNA was extracted from formalin fixed and paraffin embedded samples and CNVs data was evaluated in Canine Genome CGH Microarray 4x44K (G2519F, Design ID021193, Agilent). Data analysis was performed using Genomic Workbench Standard Edition 5.0.14 (Agilent). PCa showed higher number of altered genes related to canonical diseases process, cellular functions and molecular pathways as well as greater inter-relationship between genes, compared with PIA and BPH. In conclusion, PCa showed a more complex genotype, being losses the most frequent genomic changes. Some discrepancies between genomic alterations in human and canine carcinomas may indicate the different clinical behavior of these tumors in these two species. In addition, it was observed was an ascending pattern of genomic complexity in BPH, PIA and CA consistent with a model of multistep tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5260. doi:1538-7445.AM2012-5260
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