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

BACKGROUND: HER-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. METHODS: 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. RESULTS: The 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). CONCLUSION: 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 HER-2 gene.

Gene expression profiling in leiomyosarcomas and undifferentiated pleomorphic sarcomas: SRC as a new diagnostic marker.

BACKGROUND: Undifferentiated 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 METHODS: Gene 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. RESULTS: We 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. CONCLUSIONS: Retroperitoneal LMS presented a unique gene signature. SRC is a putative diagnostic marker to differentiate LMS from UPS.

Cytogenetic analysis in the incertae sedis species Astyanax altiparanae Garutti and Britzki, 2000 and Hyphessobrycon eques Steindachner, 1882 (Characiformes, Characidae) from the upper Paraná river basin.

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.