Gene expression differences in primary colorectal tumors and matched liver metastases: chemotherapy related or tumoral heterogeneity?

Background: Treatment of metastatic colorectal cancer (mCRC) is generally based on genetic testing performed in primary tumor biopsies, but whether the genomic status of primary tumors is identical to that of metastases is not well known. We compared the gene expression profiles of formalin-fixed paraffin-embedded (FFPE) biopsies of colorectal primary tumors and matched liver metastases. Patients and methods: We compared the expression of 18 genes in FFPE CRC tumors and their matched liver metastases from 32 patients. The expression of each gene in CRC primary tumors and their matched liver metastases was tested using Student’s t test for paired samples. Pairwise correlations of each gene in the primary tumors and matched liver metastases were evaluated by Pearson’s correlation coefficient. Results: The expression of six genes was significantly different in primary tumors compared with their matched liver metastases [CXCR4 (p < 0.001), THBS1 (p = 0.007), MMP 9 (p = 0.048), GST Pi (p = 0.050), TYMP(p = 0.042) and DPYD (p < 0.001)]. For the remaining genes, where no significant differences were observed, only SMAD4 (r s = 0.447, p = 0.010), ERCC1 (r s = 0.423, p = 0.016) and VEGF A (r s = 0.453, p = 0.009) showed significant correlation in expression between the two tissues. Therefore, we only detected similar gene expression levels between the tumor and the metastases in these three markers. Conclusions: We only found similar gene expression levels between the tumor and the metastases in three genes (SMAD4, ERCC1, and VEGF A). However, our study could not assess whether the differences in gene expression were secondary to tumoral heterogeneity or to molecular changes induced by previous chemotherapy (AU)

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SMAD4 and TS expression might predict the risk of recurrence after resection of colorectal liver metastases

Purpose. Colorectal liver metastases (CLM) have significant molecular heterogeneity, which contributes to the risk of recurrence following surgery. Most of the traditional scores intended to predict recurrence is based on clinicopathological variables and it is unclear whether incorporating molecular biomarkers might improve our assessment of the risk of recurrence. Our aim was to determine if molecular biomarkers might be associated with the risk of recurrence after surgery of CLM. Patients and methods. A total of 121 patients diagnosed with colorectal cancer (CRC) with resected liver metastases were included. The role of several clinicopathological variables to predict patient’s outcome after resection of liver metastases was analyzed. Eighteen genes related to CRC pathogenesis were also included in the analyses. Univariate and multivariate stepwise Cox regression analyses were performed to identify factors associated with recurrence and the risk of death. Results. Eight prognostic factors for progression-free survival and nine factors for overall survival were identified in the univariate analyses. After adjusting for other risk factors, only the expression of two molecular factors was associated with the risk of recurrence: TS (HR 0.631, 95 % CI 0.422–0.944) and SMAD4 (HR 1.680, 95 % CI 1.047–2.695). None of the variables was significantly associated with the risk of death in the multivariate analyses. Conclusions. The prognostic significance of most traditional clinicopathological variables might be insufficient to define patients at risk for recurrence after liver metastases resection. Molecular biomarkers might improve the identification of patients with higher risk of recurrence (AU)

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SMAD4 and TS expression might predict the risk of recurrence after resection of colorectal liver metastases.

PURPOSE: Colorectal liver metastases (CLM) have significant molecular heterogeneity, which contributes to the risk of recurrence following surgery. Most of the traditional scores intended to predict recurrence is based on clinicopathological variables and it is unclear whether incorporating molecular biomarkers might improve our assessment of the risk of recurrence. Our aim was to determine if molecular biomarkers might be associated with the risk of recurrence after surgery of CLM. PATIENTS AND METHODS: A total of 121 patients diagnosed with colorectal cancer (CRC) with resected liver metastases were included. The role of several clinicopathological variables to predict patient's outcome after resection of liver metastases was analyzed. Eighteen genes related to CRC pathogenesis were also included in the analyses. Univariate and multivariate stepwise Cox regression analyses were performed to identify factors associated with recurrence and the risk of death. RESULTS: Eight prognostic factors for progression-free survival and nine factors for overall survival were identified in the univariate analyses. After adjusting for other risk factors, only the expression of two molecular factors was associated with the risk of recurrence: TS (HR 0.631, 95 % CI 0.422-0.944) and SMAD4 (HR 1.680, 95 % CI 1.047-2.695). None of the variables was significantly associated with the risk of death in the multivariate analyses. CONCLUSIONS: The prognostic significance of most traditional clinicopathological variables might be insufficient to define patients at risk for recurrence after liver metastases resection. Molecular biomarkers might improve the identification of patients with higher risk of recurrence.

Gene expression differences in primary colorectal tumors and matched liver metastases: chemotherapy related or tumoral heterogeneity?

BACKGROUND: Treatment of metastatic colorectal cancer (mCRC) is generally based on genetic testing performed in primary tumor biopsies, but whether the genomic status of primary tumors is identical to that of metastases is not well known. We compared the gene expression profiles of formalin-fixed paraffin-embedded (FFPE) biopsies of colorectal primary tumors and matched liver metastases. PATIENTS AND METHODS: We compared the expression of 18 genes in FFPE CRC tumors and their matched liver metastases from 32 patients. The expression of each gene in CRC primary tumors and their matched liver metastases was tested using Student's t test for paired samples. Pairwise correlations of each gene in the primary tumors and matched liver metastases were evaluated by Pearson's correlation coefficient. RESULTS: The expression of six genes was significantly different in primary tumors compared with their matched liver metastases [CXCR4 (p < 0.001), THBS1 (p = 0.007), MMP 9 (p = 0.048), GST Pi (p = 0.050), TYMP (p = 0.042) and DPYD (p < 0.001)]. For the remaining genes, where no significant differences were observed, only SMAD4 (r s = 0.447, p = 0.010), ERCC1 (r s = 0.423, p = 0.016) and VEGF A (r s = 0.453, p = 0.009) showed significant correlation in expression between the two tissues. Therefore, we only detected similar gene expression levels between the tumor and the metastases in these three markers. CONCLUSIONS: We only found similar gene expression levels between the tumor and the metastases in three genes (SMAD4, ERCC1, and VEGF A). However, our study could not assess whether the differences in gene expression were secondary to tumoral heterogeneity or to molecular changes induced by previous chemotherapy.

Pharmacogenetic predictors of severe peripheral neuropathy in colon cancer patients treated with oxaliplatin-based adjuvant chemotherapy: a GEMCAD group study.

BACKGROUND: Oxaliplatin-based chemotherapy (CT), widely used as adjuvant therapy for stage III and selected high-risk stage II colon cancer (CC) patients, is often associated with cumulative peripheral neuropathy. Our aim is to identify single-nucleotide polymorphisms (SNPs) in genes involved in oxaliplatin metabolism, DNA repair mechanisms, cell cycle control, detoxification or excretion pathways to predict severe (grade 2-3) oxaliplatin-induced peripheral neuropathy (OXPN) among CC patients treated with oxaliplatin and fluoropyrimidine-based adjuvant CT. PATIENTS AND METHODS: Genomic DNA was extracted from formalin-fixed-paraffin-embedded peritumoral samples from 206 high-risk stage II and stage III CC patients receiving oxaliplatin-based adjuvant CT from January 2004 to December 2009. Genotyping was carried out for 34 SNPs in 15 genes using MassARRAY (SEQUENOM) technology. A total of 181 stage II-III CC patients treated with the same CT regimens were enrolled as a validation set. RESULTS: The rs2230641 cyclin H (CCNH) rs2230641 C/C [odd ratio (OR)=5.03, 95% confidence interval (CI) 1.061-2.41, P=0.042] and the ATP-binding cassette subfamily G, member 2 (ABCG2) rs3114018 A/A genotypes (OR=2.67; 95% CI 0.95-4.41; P=0.059) were associated with a higher risk of severe OXPN. In addition, patients harboring the combination of CCNH C/C and/or the ABCG2 rs3114018 A/A genotypes had a higher risk of grade 2-3 OXPN than those with the CCNH any T and ABCG2 any C genotypes (37.73% versus 19.42%; OR=2.46; 95% CI 1.19-5.07; P=0.014) in the logistic regression analysis using age, gender, adjuvant CT regimen and cumulative dose of oxaliplatin as covariates. The ability to predict severe OXPN of this combined analysis was independently validated in the second cohort (58% versus 33.33%; OR=2.99; 95% CI 1.45-6.13; P=0.002). CONCLUSIONS: Our results suggest that SNPs in CCNH and ABCG2 can modulate the development of severe OXPN among stage II-III CC patients who received oxaliplatin-based CT, thus enabling the individualization of adjuvant treatment.

Management of colorectal cancer patients after resection of liver metastases: can we offer a tailored treatment?

Surgical resection remains the only option of cure for patients with colorectal liver metastases, and no patient should be precluded from surgery. There is much controversy not only regarding the most appropriate therapeutic approach in the neoadjuvant setting but also after surgery is performed. Many patients will experience early relapses but others will be long survivors. We need to establish reliable prognostic and predictive factors to offer a tailored treatment. Several prognostic factors after metastasectomy have been identified: high C-reactive protein levels, a high neutrophil-lymphocyte ratio, elevated neutrophil count and low serum albumin are related to a worst outcome. Elevated CEA and Ki 67 levels, intrahepatic and perihepatic lymph node invasion are also some of the markers related to a worst outcome. In contrast, the administration of preoperative chemotherapy has been associated with a better prognosis after hepatectomy. The administration of adjuvant chemotherapy should be done taking in consideration these factors. Regarding predictive factors, determination of ERCC1, TS, TP and DPD and UGT1 polymorphisms assessment could be considered prior to chemotherapy administration. This would avoid treatment related toxicities and increase this population quality of life (AU)

Analysis of the concordance in the EGFR pathway status between primary tumors and related metastases of colorectal cancer patients:implications for cancer therapy.

Patients with metastatic Colorectal Cancer (mCRC), in which primary tumors are KRAS mutated, have no response to anti-EGFR therapy. However, less than half of mCRC patients with KRAS wild-type primary tumors respond to anti-EGFR therapy. Other downstream effectors of the EGFR pathway are being analyzed to fine-tune KRAS predictive value. However, as the primary tumor is the tissue of analysis that determines the use of anti-EGFR therapy in advanced disease, a high concordance in the status of these effectors between primary tumors and related metastases is required. We analyzed the concordances of downstream EGFR effectors in tumoral pairs of primaries and related metastases in a series of KRAS wild-type patients. One hundred seventeen tumoral pairs from patients with CRC were tested for KRAS mutational status. The level of concordance in the presence of KRAS mutations was 91% between the primary tumor and related metastases. The 70 pairs with KRAS wild-type primary tumors were further analyzed for BRAF and PIK3CA mutational status and for EGFR, PTEN and pAKT expression, and the number of concordant pairs was 70 (100%), 66 (94%), 43 (61%), 46 (66%) and 36 (54%), respectively. Our findings suggest that the mutational status of KRAS, BRAF and PIK3CA in the primary tumor is an adequate surrogate marker of the status in the metastatic disease. On the other hand, the immunohistochemical analysis of EGFR, PTEN and pAKT showed a much higher degree of discordance between primaries and related metastases.

Immunohistochemical analysis of tumour regression grade for rectal cancer after neoadjuvant chemoradiotherapy.

AIM: Tumour regression grade (TRG) as defined by Rödel et al. has been used as an independent prognostic factor for rectal carcinoma after preoperative treatment by chemoradiotherapy (CRT). Determination of TRG 2 and 3, semiquantitatively defined as more or less than 50% tumour regression, respectively, does not appear to correlate with prognosis. The purpose of this study was to find an immunohistochemical pattern to permit improved stratification of intermediate responders defined by disease free (DFS) and overall survival (OS). METHOD: Immunohistochemistry of EGFR (epidermal growth factor receptor), VEGF (vascular endothelial growth factor), CD133 antibody, p53 antibody and Ki67 antibody was evaluated using tissue microarrays (TMA) on post-treatment surgical specimens from 88 patients. CD133 expression was confirmed in the whole section when available. RESULTS: At a median follow-up of 40 months, TRG was found to be an independent predictor of DFS (P = 0.05) and OS (P = 0.001) but no differences were found between TRG 2 and 3 in terms of DFS (P = 0.74) or OS (P = 0.41). The results of TMA showed an immunohistochemically poor prognostic profile for intermediate responders configured by negativity of CD133 expression. However, when examining CD133 expression in the whole section, there was an intermediate correlation with TMA and the prognostic significance was lost. CONCLUSION: The results did not confirm the value of immunohistochemistry in predicting the prognosis of patients with rectal cancer following neoadjuvant chemoradiotherapy. This questions the accuracy of TMA in detecting CD133 expression in this setting.

IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer.

Cisplatin-based chemotherapy is the paradigm of non-small-cell lung cancer (NSCLC) treatment; however, it also induces de novo DNA-hypermethylation, a process that may be involved in the development of drug-resistant phenotypes by inactivating genes required for drug-cytotoxicity. By using an expression microarray analysis, we aimed to identify those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Results revealed specific silencing by promoter hypermethylation of IGFBP-3 in CDDP resistant cells, whereas IGFBP-3 siRNA interference, induced resistance to CDDP in sensitive cells (P<0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (P<0.001). Thus, stage I patients, whose tumors harbor an unmethylated promoter, had a trend towards increased disease-free survival (DFS). We report that a loss of IGFBP-3 expression, mediated by promoter-hypermethylation, results in a reduction of tumor cell sensitivity to cisplatin in NSCLC. Basal methylation status of IGFBP-3 before treatment may be a clinical biomarker and a predictor of the chemotherapy outcome, helping to identify patients who are most likely to benefit from CDDP therapy alone or in combination with epigenetic treatment.

A critical role for choline kinase-alpha in the aggressiveness of bladder carcinomas.

Bladder cancer is one of the most common causes of death in industrialized countries. New tumor markers and therapeutic approaches are still needed to improve the management of bladder cancer patients. Choline kinase-alpha (ChoKalpha) is a metabolic enzyme that has a role in cell proliferation and transformation. Inhibitors of ChoKalpha show antitumoral activity and are expected to be introduced soon in clinical trials. This study aims to assess whether ChoKalpha plays a role in the aggressiveness of bladder tumors and constitutes a new approach for bladder cancer treatment. We show here that ChoKalpha is constitutively altered in human bladder tumor cells. Furthermore, in vivo murine models, including an orthotopic model to mimic as much as possible the physiological conditions, revealed that increased levels of ChoKalpha potentiate both tumor formation (P< or =0.0001) and aggressiveness of the disease on different end points (P=0.011). Accordingly, increased levels of ChoKalpha significantly reduce survival of mice with bladder cancer (P=0.05). Finally, treatment with a ChoKalpha-specific inhibitor resulted in a significant inhibition of tumor growth (P=0.02) and in a relevant increase in survival (P=0.03).

Prognostic value of carcinoembryonic antigen level in rectal cancer treated with neoadjuvant chemoradiotherapy.

BACKGROUND: The purpose of this study was to identify clinical and pathological parameters to improve prediction of disease-free survival (DFS) and overall survival (OS) in patients treated with neoadjuvant chemoradiotherapy for rectal cancer. METHODS: Between July 1995 and May 2007, 148 patients with primary rectal adenocarcinoma received neoadjuvant chemoradiotherapy followed by mesorectal excision. Preoperative treatment included various protocols, UFT and leucovorin (28%) and oxaliplatin-based chemotherapy (72%). Clinical and pathological variables were evaluated in relation to patient outcomes. RESULTS: Thirteen percent of patients achieved a complete pathologic response. No response or minimal response as defined by Dworak (Tumor Regression Grade 0/1) was observed in 30 patients (20%). At a median follow-up of 37 months, the 3-year DFS and OS were 64% and 83%, respectively. Pre-treatment serum carcinoembryonic antigen (CEA) level

Molecular markers in colorectal cancer: genetic bases for a customised treatment.

Colorectal cancer (CRC) is the second leading cause of cancer death in Western countries. CRC treatment is based on the employment of three chemotherapeutic drugs, including 5-fluorouracil, oxaliplatin and irinotecan, and the use of recently incorporated targeted agents directed to vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). The approval of these biologicals and of others to come holds great promise for the improvement of patient outcome. The molecular bases for this lethal disease have been extensively investigated, laying the foundations for a rational and customised treatment approach, expanding the therapeutic index of current drugs and easing the incorporation of new molecules. Individual markers have been mainly investigated based on drug targets and metabolism. Also, the increasing availability of highthroughput technologies has prompted the opportunity for blind studies capable of screening new markers and of identifying the specific oncogenic pathways responsible for drug resistance in a given patient. An updated review of the field is presented in this article.

Molecular markers in colorectal cancer: genetic bases for a customised treatment

Colorectal cancer (CRC) is the second leading cause of cancer death in Western countries. CRC treatment is based on the employment of three chemotherapeutic drugs, including 5-fluorouracil, oxaliplatin and irinotecan, and the use of recently incorporated targeted agents directed to vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). The approval of these biologicals and of others to come holds great promise for the improvement of patient outcome. The molecular bases for this lethal disease have been extensively investigated, laying the foundations for a rational and customised treatment approach, expanding the therapeutic index of current drugs and easing the incorporation of new molecules. Individual markers have been mainly investigated based on drug targets and metabolism. Also, the increasing availability of highthroughput technologies has prompted the opportunity for blind studies capable of screening new markers and of identifying the specific oncogenic pathways responsible for drug resistance in a given patient. An updated review of the field is presented in this article (AU)

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) expression is downregulated in poorly differentiated breast invasive ductal carcinoma.

Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx) is the only known enzyme able to reduce lipid peroxides bound to cell membranes. Moreover it has been involved in apoptosis and can influence intracellular signaling. To investigate the possible relationship between PHGPx and human cancer we have quantified PHGPx expression levels by real-time quantitative PCR and immunohistochemistry in tissue samples of human breast invasive ductal carcinoma from 34 patients compared with their own controls of benign breast tissue. PHGPx expression levels were compared with the clinical and pathological data of these patients. The results showed that PHGPx expression levels are downregulated in poorly differentiated (grade 3) breast invasive ductal carcinoma (P = 0.0043). PHGPx expression levels decreased gradually with tumor grade from grade 1 to grade 3. We also found a downregulation of PHGPx in cases that showed p53 accumulation compared with cases without p53 immunostaining (P = 0.0011). PHGPx was also downregulated in cases without progesterone receptors (PR) immunostaining compared with cases with PR immunostaining (P = 0.0165). Grade 3, p53 immunostaining and absence of PR immunostaining are poor prognostic factors. These results suggest that PHGPx downregulation could be related with a poorer prognosis in breast invasive ductal carcinoma.

MKP1/CL100 controls tumor growth and sensitivity to cisplatin in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) represents the most frequent and therapy-refractive sub-class of lung cancer. Improving apoptosis induction in NSCLC represents a logical way forward in treating this tumor. Cisplatin, a commonly used therapeutic agent in NSCLC, induces activation of N-terminal-c-Jun kinase (JNK) that, in turn, mediates induction of apoptosis. In analysing surgical tissue samples of NSCLC, we found that expression of MKP1/CL100, a negative regulator of JNK, showed a strong nuclear staining for tumor cells, whereas, in normal bronchial epithelia, MKP1 was localized in the cytoplasm as well as in nuclei. In the NSCLC-derived cell lines H-460 and H-23, we found that MKP1 was constitutively expressed. Expressing a small-interfering RNA (siRNA) vector for MKP1 in H-460 cells resulted in a more efficient activation by cisplatin of JNK and p38 than in the parental cells, and this correlated with a 10-fold increase in sensitivity to cisplatin. A similar response was also observed in H-460 and H-23 cells when treated with the MKP1 expression inhibitor RO-31-8220. Moreover, expression of a siRNA-MKP2, an MKP1-related phosphatase, had no effect on H-460 cell viability response to cisplatin. Tumors induced by H-460 cells expressing MKP1 siRNA grew slower in nu(-)/nu(-) mice and showed more susceptibility to cisplatin than parental cells, and resulted in an impaired growth of the tumor in mice. On the other hand, overexpression of MKP1 in the H-1299 NSCLC-derived cell line resulted in further resistance to cisplatin. Overall, the results showed that inhibition of MKP1 expression contributes to a slow down in cell growth in mice and an increase of cisplatin-induced cell death in NSCLC. As such, MKP1 can be an attractive target in sensitizing cells to cisplatin to increase the effectiveness of the drug in treating NSCLC.

Immunohistochemical expression of p53, Bcl-2, COX-2, C-erb-B2, EPO-R, beta-catenin, and E-cadherin in non tumoral gastric mucous membrane.

Different authors have investigated the immunohistochemical expression of some proteins in the adenocarcinoma of the stomach, including cell cycle regulators proteins like p53 and Bcl-2; growth factors (c-erb-B2 and EPO-R); angiogenesis-related markers such as COX-2 and cellular adhesion molecules (beta-catenin and E-cadherin). While these proteins have been studied in gastric adenocarcinoma, their immunophenotyping in non tumoral gastric mucous membrane remains unexplored. In the present study, we investigated the expression, function and behavior of these proteins in normal gastric mucous membrane to contribute to gain further knowledge on the significance of their loss or overexpression in malignant gastric tumors.

Breast cancer prognosis determined by gene expression profiling: a quantitative reverse transcriptase polymerase chain reaction study.

PURPOSE: We sought to reproduce with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) the results obtained with a 70-gene expression profile that has been described previously in breast cancer. PATIENTS AND METHODS: Frozen breast cancer samples from patients who were operated on were used to isolate tumor RNA. Ninety-six patients with stage I to II disease were included. Median age was 57 years (range, 27 to 80 years). Forty-eight patients had lymph node-negative and 48 lymph node-positive disease. qRT-PCR amplifications were performed and the results were correlated with clinical data. RESULTS: After a minimum follow-up of 5 years, 25 patients had a relapse. The gene profile divided patients into two groups with poor and good prognosis. Significant differences with regard to grade of differentiation, size and hormone receptors were seen between the two groups. The gene profile was significantly associated with relapse-free survival and overall survival in the whole group of 96 patients. Multivariate analysis showed that only lymph node status and gene profile were significantly correlated to overall survival. CONCLUSION: qRT-PCR reproduced the results obtained with microarrays for a prognostic gene profile in women with early-stage breast cancer.

Localisation of COX-2 protein is different in breast ductal carcinoma and adjacent non-tumour ductal epithelium.

INTRODUCTION: A number of findings suggest that cyclooxygenase-2 (COX-2) is overexpressed in breast tumours. However, there is a lack of consensus in the literature regarding the pattern of expression of this protein in invasive breast ductal carcinoma and in the adjacent non-tumour ductal epithelium. This study compares the expression of COX-2 mRNA and protein in breast ductal carcinoma relative to non-tumour breast tissue. MATERIAL AND METHODS: We analysed the expression of COX-2 mRNA by quantitative PCR, and COX-2 protein by immunohistochemistry in invasive ductal carcinoma as well as in non-tumour adjacent ductal epithelium from 34 breast biopsies diagnosed as being invasive ductal carcinoma. As control, we analysed expression of COX-2 protein by immunohistochemistry in surgically-resected benign breast lesions. RESULTS: Our results show that COX-2 mRNA and protein are overexpressed in non-tumour ductal epithelium compared with invasive ductal carcinoma. However, the pattern of the protein expression is different in tumour and non-tumour tissue: COX-2 protein is expressed predominantly in the membrane of the non-tumour ductal epithelium (including in benign breast lesions) while, in invasive ductal carcinoma cells, it is localised in the cytoplasm. CONCLUSIONS: The non-tumour ductal epithelium adjacent to invasive ductal carcinoma shows a higher COX-2 expression than does the invasive ductal carcinoma. However, the different localisation of the immunohistochemically-detected protein suggests a possible post-translational regulation of the protein.

Localisation of COX-2 protein is different in breast ductal carcinoma and adjacent non-tumour ductal epithelium

Introduction. A number of findings suggest that cyclooxygenase-2 (COX-2) is overexpressed in breast tumours. However, there is a lack of consensus in the literature regarding the pattern of expression of this protein in invasive breast ductal carcinoma and in the adjacent non-tumour ductal epithelium. This study compares the expression of COX-2 mRNA and protein in breast ductal carcinoma relative to non-tumour breast tissue. Material and methods. We analysed the expression of COX-2 mRNA by quantitative PCR, and COX-2 protein by immunohistochemistry in invasive ductal carcinoma as well as in non-tumour adjacent ductal epithelium from 34 breast biopsies diagnosed as being invasive ductal carcinoma. As control, we analysed expression of COX-2 protein by immunohistochemistry in surgically-resected benign breast lesions. Results. Our results show that COX-2 mRNA and protein are overexpressed in non-tumour ductal epithelium compared with invasive ductal carcinoma. However, the pattern of the protein expression is different in tumour and non-tumour tissue: COX-2 protein is expressed predominantly in the membrane of the non-tumour ductal epithelium (including in benign breast lesions) while, in invasive ductal carcinoma cells, it is localised in the cytoplasm. Conclusions. The non-tumour ductal epithelium adjacent to invasive ductal carcinoma shows a higher COX-2 expression than does the invasive ductal carcinoma. However, the different localisation of the immunohistochemically-detected protein suggests a possible post-translational regulation of the protein

New insights in beta-tubulin sequence analysis in non-small cell lung cancer.

Scarce data are available regarding the molecular mechanisms implicated in paclitaxel resistance. There is controversial data about beta-tubulin mutations role in paclitaxel resistance. We have conducted this trial to address the influence of beta-tubulin mutations in paclitaxel resistance in advanced non-small cell lung cancer (NSCLC). A group of 15 patients were biopsied and diagnosed of stages IIIB and IV NSCLC. Tumor specimens were used for DNA isolation and exon 4 of HM40 beta-tubulin isotype was amplified and automatically sequenced, using both intronic and exonic primers. Next, the chemotherapy schedule consisted of weekly paclitaxel (100 or 150 mg/m(2) x 6) followed 2 weeks later by cisplatin 100 mg/m(2) on day 1, gemcitabine 1000 mg/m(2) on days 1 and 14, and vinorelbine 25 mg/m(2) on days 1 and 14, every 28 days. Using exonic primers, gene sequence alterations were found in 13/15 (87%) patients, including transitions (codons 180 and 182) and one silent transversion (codon 195). Also, three transversions (codons 231, 234, and 235) were found in all patients and controls. All alterations disappeared when sequenced with intronic primers. Our results suggest that point mutations demonstrated with exonic primers but not with intronic ones are probably due to beta-tubulin pseudogenes present in advanced NSCLC specimens. Even so, when these beta-tubulin pseudogenes are found there is a clear relation with clinical response. Although these changes could be relevant in paclitaxel resistance, this observation must be proven in future clinical trials to resolve "the tubulin dilemma".