An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer.

Nowadays, HER2 testing in breast cancer represents a necessity for both prognostic and therapy. Despite widespread use of immunohistochemistry (IHC) for assessing HER2 status, there are some limitations to identify truly negative or positive HER2 cases. Fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) could solve the equivocal HER2 IHC cases but there is no consensus on which is the best method. Consequently, finding a sensitive method for HER2 testing is critical for the management of the disease. In addition, tumor heterogeneity is an important factor which could affect accuracy of molecular diagnostics. Laser capture micro-dissection (LCM) is used to isolate pure cell populations from heterogeneous tumor tissue. The combination between LCM and quantitative polymerase chain reaction (Q-PCR), the gold standard in molecular biology for quantifying gene amplification levels, could define an important tool to improve the molecular diagnostics of HER2 status.In our pilot study we used LCM and Q-PCR to evaluate HER2 gene amplification for invasive breast carcinoma samples. The samples were selected based on HER2 status assessed by IHC and CISH. Our results demonstrated high sensitivity of Q-PCR for assessing HER2 DNA amplification as well as a good concordance between Q-PCR and IHC/ CISH assay.

Gene expression profiling reveals activation of the FA/BRCA pathway in advanced squamous cervical cancer with intrinsic resistance and therapy failure.

BACKGROUND: Advanced squamous cervical cancer, one of the most commonly diagnosed cancers in women, still remains a major problem in oncology due to treatment failure and distant metastasis. Antitumor therapy failure is due to both intrinsic and acquired resistance; intrinsic resistance is often decisive for treatment response. In this study, we investigated the specific pathways and molecules responsible for baseline therapy failure in locally advanced squamous cervical cancer. METHODS: Twenty-one patients with locally advanced squamous cell carcinoma were enrolled in this study. Primary biopsies harvested prior to therapy were analyzed for whole human gene expression (Agilent) based on the patient's 6 months clinical response. Ingenuity Pathway Analysis was used to investigate the altered molecular function and canonical pathways between the responding and non-responding patients. The microarray results were validated by qRT-PCR and immunohistochemistry. An additional set of 24 formalin-fixed paraffin-embedded cervical cancer samples was used for independent validation of the proteins of interest. RESULTS: A 2859-gene signature was identified to distinguish between responder and non-responder patients. 'DNA Replication, Recombination and Repair' represented one of the most important mechanisms activated in non-responsive cervical tumors, and the 'Role of BRCA1 in DNA Damage Response' was predicted to be the most significantly altered canonical pathway involved in intrinsic resistance (p = 1.86E-04, ratio = 0.262). Immunohistological staining confirmed increased expression of BRCA1, BRIP1, FANCD2 and RAD51 in non-responsive compared with responsive advanced squamous cervical cancer, both in the initial set of 21 cervical cancer samples and the second set of 24 samples. CONCLUSIONS: Our findings suggest that FA/BRCA pathway plays an important role in treatment failure in advanced cervical cancer. The assessment of FANCD2, RAD51, BRCA1 and BRIP1 nuclear proteins could provide important information about the patients at risk for treatment failure.

Early transcriptional pattern of angiogenesis induced by EGCG treatment in cervical tumour cells.

The major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been shown to exhibit antitumour activities in several tumour models. One of the possible mechanisms by which EGCG can inhibit cancer progression is through the modulation of angiogenesis signalling cascade. The tumour cells' ability to tightly adhere to endothelium is a very important process in the metastatic process, because once disseminated into the bloodstream the tumour cells must re-establish adhesive connections to endothelium in order to extravasate into the target tissues. In this study, we investigated the anti-angiogenic effects of EGCG treatment (10 µM) on human cervical tumour cells (HeLa) by evaluating the changes in the expression pattern of 84 genes known to be involved in the angiogenesis process. Transcriptional analysis revealed 11 genes to be differentially expressed and was further validated by measuring the induced biological effects. Our results show that EGCG treatment not only leads to the down-regulation of genes involved in the stimulation of proliferation, adhesion and motility as well as invasion processes, but also to the up-regulation of several genes known to have antagonist effects. We observed reduced proliferation rates, adhesion and spreading ability as well as invasiveness of HeLa tumour cells upon treatment, which suggest that EGCG might be an important anti-angiogenic therapeutic approach in cervical cancers.

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Expression of VEGF, VEGFR, EGFR, COX-2 and MVD in cervical carcinoma, in relation with the response to radio-chemotherapy.

INTRODUCTION: Despite the improvement in the treatment results due to modern irradiation techniques and to the association of chemo-radiotherapy, cervical cancer remains an unsolved problem of oncology both due to the increased rate of local failures and of the distant metastasis. Efforts to implement new therapeutic strategies in order to obtain better results in patients with cervical cancer appear justified. Neovascularization is an important step in the tumor progression and the therapeutic targeting of the tumor blood vessels appears to be a good strategy to follow in the anti-cancer treatment. Thus, even in an incipient phase of the clinical research process, the combination between the anti-angiogenic aimed therapies and the current radio-chemotherapy seems to represent a new, feasible and promising approach. The aim of the present study was to determine the prognostic and/or predictive value of some biological markers of tumor angiogenesis and of their implication in increasing the efficacy of current treatments for this cancer. MATERIALS AND METHODS: So far, 54 women were included in a prospective trial: 44 having an advanced cervical carcinoma and 10 healthy women, as controls. A tumor biopsy and a blood sample were obtained from each patient before the start of therapy. The density of microvascularization was assessed using CD34 monoclonal antibody (hot spot technique), the expression of angiogenic factors VEGFR, EGFR and COX-2 were determined in tumor biopsies by specific immunohistochemistry techniques, using primary antibodies anti-EGFR, anti-VEGF and anti-COX-2 respectively. The quantitative polymerase chain reaction (Real Time PCR) was employed for assessing the expression level of the genes involved. Serum VEGF was determined by quantitative ELISA technique. RESULTS: Among the studied clinical and molecular factors, we found to be predictive for the type of response the following factors: tumor size at diagnosis (p=0.01), VEGFR2 expression (p=0.02) and a tendency to significance for patients' age (p=0.06). From the large panel of studied markers it was observed correlation between MVD expression with stromal COX-2 (p=0.01) and a tendency with epithelial COX-2 (p=0.06). Stromal COX-2 has higher correlation with VEGFR2 (p=0.01) and MVD (p=0.01) and also has a lower correlation with tumor size (p=0.08). CONCLUSIONS: Univariate analysis demonstrates that the response to radio-chemotherapy in cervical cancer is related to a set of clinical and molecular factors as: the tumor size, the expression of VEGFR2 as mRNA level and the patients' age. Unfortunately, the multivariate analysis by logistic model selects only VEGFR2 expression for prediction of tumor response. The interrelations between the different biomarkers demonstrate the complexity of the tumor progression process and the necessity of further studies to identify new therapeutic targets.