Transcript expression and genetic variability analysis of caspases in breast carcinomas suggests CASP9 as the most interesting target.

BACKGROUND: Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. METHODS: Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. RESULTS: CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). CONCLUSIONS: Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

Genetic and functional analyses do not explain the association of high PRC1 expression with poor survival of breast carcinoma patients.

Microtubules are vitally important for eukaryotic cell division. Therefore, we evaluated the relevance of mitotic kinesin KIF14, protein-regulating cytokinesis 1 (PRC1), and citron kinase (CIT) for the prognosis of breast carcinoma patients. Transcript levels were assessed by quantitative real-time PCR in tissues from two independent groups of breast carcinoma patients and compared with clinical data. Tissue PRC1 protein levels were estimated using immunoblotting, and the PRC1 tagged haplotype was analyzed in genomic DNA. A functional study was performed in MDA-MB-231 cells in vitro. KIF14, PRC1, and CIT transcripts were overexpressed in tumors compared with control tissues. Tumors without expression of hormonal receptors or high-grade tumors expressed significantly higher KIF14 and PRC1 levels than hormonally-positive or low-grade tumors. Patients with high intra-tumoral PRC1 levels had significantly worse disease-free survival than patients with low levels. PRC1 rs10520699 and rs11852999 polymorphisms were associated with PRC1 transcript levels, but not with patients survival. Paclitaxel-induced PRC1 expression, but PRC1 knockdown did not modify the paclitaxel cytotoxicity in vitro. PRC1 overexpression predicts poor disease-free survival of patients with breast carcinomas. Genetic variability of PRC1 and the protein interaction with paclitaxel cytotoxicity do not explain this association.

The role of cytochromes p450 and aldo-keto reductases in prognosis of breast carcinoma patients.

Metabolism of anticancer drugs affects their antitumor effects. This study has investigated the associations of gene expression of enzymes metabolizing anticancer drugs with therapy response and survival of breast carcinoma patients. Gene expression of 13 aldo-keto reductases (AKRs), carbonyl reductase 1, and 10 cytochromes P450 (CYPs) was assessed using quantitative real-time polymerase chain reaction in tumors and paired adjacent nonneoplastic tissues from 68 posttreatment breast carcinoma patients. Eleven candidate genes were then evaluated in an independent series of 50 pretreatment patients. Protein expression of the most significant genes was confirmed by immunoblotting. AKR1A1 was significantly overexpressed and AKR1C1-4, KCNAB1, CYP2C19, CYP3A4, and CYP3A5 downregulated in tumors compared with control nonneoplastic tissues after correction for multiple testing. Significant association of CYP2B6 transcript levels in tumors with expression of hormonal receptors was found in the posttreatment set and replicated in the pretreatment set of patients. Significantly higher intratumoral levels of AKR1C1, AKR1C2, or CYP2W1 were found in responders to neoadjuvant chemotherapy compared with nonresponders. Patients with high AKR7A3 or CYP2B6 levels in the pretreatment set had significantly longer disease-free survival than patients with low levels. Protein products of AKR1C1, AKR1C2, AKR7A3, CYP3A4, and carbonyl reductase (CBR1) were found in tumors and those of AKR1C1, AKR7A3, and CBR1 correlated with their transcript levels. Small interfering RNA-directed knockdown of AKR1C2 or vector-mediated upregulation of CYP3A4 in MDA-MB-231 model cell line had no effect on cell proliferation after paclitaxel treatment in vitro. Prognostic and predictive roles of drug-metabolizing enzymes strikingly differ between posttreatment and pretreatment breast carcinoma patients. Mechanisms of action of AKR1C2, AKR7A3, CYP2B6, CYP3A4, and CBR1 should continue to be further followed in breast carcinoma patients and models.

Non-coding polymorphisms in nucleotide binding domain 1 in ABCC1 gene associate with transcript level and survival of patients with breast cancer.

OBJECTIVES: ATP-Binding Cassette (ABC) transporters may cause treatment failure by transporting of anticancer drugs outside of the tumor cells. Multidrug resistance-associated protein 1 coded by the ABCC1 gene has recently been suggested as a potential prognostic marker in breast cancer patients. This study aimed to explore tagged haplotype covering nucleotide binding domain 1 of ABCC1 in relation with corresponding transcript levels in tissues and clinical phenotype of breast cancer patients. METHODS: The distribution of twelve ABCC1 polymorphisms was assessed by direct sequencing in peripheral blood DNA (n = 540). RESULTS: Tumors from carriers of the wild type genotype in rs35623 or rs35628 exhibited significantly lower levels of ABCC1 transcript than those from carriers of the minor allele (p = 0.003 and p = 0.004, respectively). The ABCC1 transcript levels significantly increased in the order CT-GT>CC-GT>CC-GG for the predicted rs35626-rs4148351 diplotype. Chemotherapy-treated patients carrying the T allele in rs4148353 had longer disease-free survival than those with the GG genotype (p = 0.043). On the other hand, hormonal therapy-treated patients with the AA genotype in rs35628 had significantly longer disease-free survival than carriers of the G allele (p = 0.012). CONCLUSIONS: Taken together, our study shows that genetic variability in the nucleotide binding domain 1 has a significant impact on the ABCC1 transcript level in the target tissue and may modify survival of breast cancer patients.

Breast cancer characteristics-comparison of preoperative and postoperative values.

Breast cancer characteristics obtained at the time of diagnosis are important for setting the basic strategy of the treatment. Reliability of preoperative investigation differs for various features of the disease. The aim of this study was to ascertain the agreements and differences between preoperative and postoperative values. This retrospective study analyzed the results of 617 women with primary surgery of the breast and axilla. Cohen's kappa coefficient has been employed to measure the degree of agreement between preoperative and postoperative values. Substantial or "almost perfect" agreement has been documented for the histological type of the tumors, their histopathological grade, proliferation index Ki67, as well as for estrogen, progesterone, and HER-2/neu receptors. Substantial differences exist between preoperative and postoperative diagnoses of invasiveness of the tumor, determination of the size of the tumors, and the number of tumor foci. Preoperative imaging and clinical examination of lymph nodes exhibited unacceptably high false negative rates. Heterogeneity of breast cancer cell population, methodology of histology examinations, and insufficient imaging of lymph nodes are the major limitations precluding satisfactory accuracy of preoperative diagnosis. Preoperatively diagnosed in situ carcinomas, as well as multifocal lesions, were the most often sources of diagnostic failures.

The expression profile of ATP-binding cassette transporter genes in breast carcinoma.

AIM: ATP-binding cassette (ABC) transporters contribute to development of resistance to anticancer drugs via ATP-dependent drug efflux. A major goal of our study was to investigate associations between the expression of ABC transporters and outcome of breast carcinoma patients. PATIENTS & METHODS: Transcript levels of all 49 human ABC transporters were determined in post-treatment tumor and non-neoplastic tissue samples from 68 breast carcinoma patients treated by neoadjuvant chemotherapy. Six ABC transporters were then evaluated in independent series of 100 pretreatment patients. RESULTS: ABCA5/6/8/9/10, ABCB1/5/11, ABCC6/9, ABCD2/4, ABCG5 and ABCG8 were significantly downregulated and ABCA2/3/7/12, ABCB2/3/8/9/10, ABCC1/4/5/10/11/12, ABCD1/3, ABCE1, ABCF1/2/3 and ABCG1 were upregulated in post-treatment tumors compared with non-neoplastic tissues. Significant associations of intratumoral levels of ABCC1 and ABCC8 with grade and expression of hormonal receptors were found in both sets of patients. ABCA12, ABCA13 and ABCD2 levels were significantly associated with the response to neoadjuvant chemotherapy in post-treatment patients. Protein expression of ABCA12, ABCC8 and ABCD2 in tumor tissues of patients with breast carcinoma was observed by immunoblotting for the first time. CONCLUSION: ABCA12, ABCA13, ABCC1, ABCC8 and ABCD2 present potential modifiers of progression and response to the chemotherapy of breast carcinoma.

Importance of transcript levels of caspase-2 isoforms S and L for breast carcinoma progression.

AIM: A role of caspase-2 in chemotherapy-induced apoptosis has been suggested. Our study aimed to evaluate the prognostic and predictive importance of caspase-2 isoforms in breast cancer patients. MATERIALS & METHODS: Caspase-2L and -2S transcript levels were determined in paired tumor and non-malignant control tissues from 64 patients after neoadjuvant chemotherapy and 100 pretreatment patients (general set) by real-time PCR with absolute quantification. RESULTS: Low but statistically significant upregulation of caspase-2L in tumor versus control tissues was observed in both sets. Significant associations of the levels of caspase-2L, -2S or S/L ratio with clinical prognostic factors were observed. However, none of these associations were confirmed in both sets. Levels of caspase-2 isoforms or the S/L ratio did not significantly associate with progression-free survival in the general set or with chemotherapy response in the neoadjuvant set. CONCLUSION: Our results suggest that the role of caspase-2 isoforms in the progression of breast cancer may considerably differ between pre- and post-chemotherapy patients.

Detection of frequent ABCB1 polymorphisms by high-resolution melting curve analysis and their effect on breast carcinoma prognosis.

BACKGROUND: The ABCB1 gene encodes P-glycoprotein implicated in the development of cellular drug resistance. The aim of this study was to develop high-resolution melting (HRM) analysis for determination of ABCB1 polymorphisms and evaluate their associations with clinical data of breast carcinoma patients. METHODS: HRM analysis was designed to assess five single nucleotide polymorphisms (SNPs) in ABCB1 (rs2214102, rs1128503, rs2032582, rs2032583 and rs1045642) in genomic DNA from 103 breast carcinoma patients. Results were confirmed by direct DNA sequencing. RESULTS: HRM analysis revealed distinct patterns of melting curves for the respective genotypes of all followed SNPs. Sensitivity of HRM analysis compared with direct DNA sequencing was superior (97.1% vs. 93.9%). The overall accuracy of HRM was 97.6%. The coefficients of variation in replicate experiments encompassed the range 0.002%-0.038%. On the basis of the examined SNPs, one strong haplotype block containing rs2032582 and rs1128503 SNPs was identified. Significant associations of rs2032582 SNP with tumor size, negative HER-2/neu status, and family history of breast carcinoma were found. Patients carrying the ancestral homozygous genotype (GG) in rs2214102 had significantly worse progression-free survival in comparison with carriers of the non-ancestral allele (A) in the adjuvant set (p=0.005). CONCLUSIONS: A rapid, accurate, low-cost and time-effective method for screening ABCB1 SNPs was developed. Significant associations of ABCB1 rs2032582 and rs2214102 SNPs with prognostic factors and survival of patients were found.

Sentinel lymph node biopsy in breast cancer patients treated with neoadjuvant chemotherapy.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is a widely used staging method for patients with early breast cancer. Neoadjuvant chemotherapy modifies the anatomical conditions in the breast and axilla, and thus SLNB remains controversial in patients treated preoperatively. The aim of this study was to demonstrate the reliability and accuracy of this procedure in this particular group of patients. METHODS: The retrospective study analyzed medical records of patients diagnosed with primary breast cancer between the years 2005 and 2009. Of the patients treated by neoadjuvant therapy, 343 underwent lymphatic mapping to identify sentinel lymph nodes, and these were included in the analysis. RESULTS: The overall detection rate of sentinel lymph nodes was 80.8%. It was strongly influenced by clinical lymph node status (significantly higher success rate in lymph node-negative patients); higher detection rates were also associated with age <50 years, estrogen receptor positivity, lower proliferation index, and absent lymphovascular space invasion. The false-negative rate was 19.5% and was only marginally significantly dependent on lymphovascular space invasion. The overall accuracy of the method was 91.5%. CONCLUSIONS: By using the present technique, sentinel lymph node biopsy cannot be recommended as a reliable predictor of axillary lymph node status when performed at the authors' institution after neoadjuvant chemotherapy. Infrequent use of blue dye for lymphatic mapping, low number of resected sentinel lymph nodes, and absence of any selection among patients included in the study could be the main factors responsible for the low detection rate and high false-negative rate.