Javier de Castro

Implications of oxidative stress and cell membrane lipid peroxidation in human cancer (Spain)

Reactive Oxygen Species (ROS) result from cell metabolism as well as from extracellular processes. ROS exert some functions necessary for cell homeostasis maintenance. When produced in excess they play a role in the causation of cancer. ROS mediated lipid peroxides are of critical importance because they participate in chain reactions that amplify damage to biomolecules including DNA. DNA attack gives rise to mutations that may involve tumor suppressor genes or oncogenes, and this is an oncogenic mechanism. On the other hand, ROS production is a mechanism shared by many chemotherapeutic drugs due to their implication in apoptosis control. The ROS mediated cell responses depend on the duration and intensity of the cells exposing to the increased ROS environment. Thus the statusredox is of great importance for oncogenetic process activation and it is also implicated in tumor susceptibility to specific chemotherapeutic drugs. Phospholipid Hydroperoxide Glutathione Peroxidase (PH-GPx) is an antioxidant enzyme that is able to directly reduce lipid peroxides even when they are bound to cellular membranes. This article will review the relevance of oxidative stress, particularly of lipid peroxidation, in cell response with special focus in carcinogenesis and cancer therapy that suggests PH-GPx as a potentially important enzyme involved in the control of this processes.

β-Catenin expression pattern in primary oesophageal squamous cell carcinoma. Relationship with clinicopathologic features and clinical outcome

Abstract. β-Catenin has an essential role in intercellular adhesion and signal transduction. β-catenin functions as a transcriptional activator downstream in the Wnt signalling pathway. Cytoplasmic stabilisation of β-catenin, mainly due to inactivating mutations of the adenomatous polyposis coli (APC) tumour suppressor gene or activating mutations in exon 3 of the β-catenin gene, can activate this important pathway in the development of several carcinomas. To determine whether this pathway for malignant transformation is important in oesophageal cancer, we analysed 39 primary oesophageal squamous cell carcinomas (OSCC). Immunohistochemical expression of β-catenin was studied in formalin-fixed, paraffin-embedded tissue samples. Results were correlated with clinicopathological parameters and immunohistochemical expression of the proteins p53, E-cadherin, bcl-2 and Ki-67. All examined OSCC had β-catenin expression localised in the cellular membrane, frequently with a heterogeneous pattern. Seven (18%) cases also showed immunoexpression in the cytoplasm and nuclei of the tumour cells. These seven tumours were localised in the upper (three) or in the middle third (four) of the oesophagus. Only one patient had p53 expression and all had bcl-2 expression. The consensus sequence for glycogen synthase kinase (GSK) 3β phosphorylation in exon 3 of the β-catenin gene was studied using polymerase chain reaction and direct sequencing in the seven cases with nuclear β-catenin expression. No genetic alteration was found. These results suggest that β-catenin expression may characterise a subset of OSCC.

Randomized Phase IIIb Trial Evaluating the Continuation of Bevacizumab Beyond Disease Progression in Patients with Advanced Non-Squamous Non–Small-Cell Lung Cancer after First-Line Treatment with Bevacizumab Plus Platinum-Based Chemotherapy: Treatment Rationale and Protocol Dynamics of the AvaALL (MO22097) Trial

We present the treatment rationale and study design of the AvaALL (MO22097; ClinicalTrials: NCT01351415) trial, a multicenter, open-label, randomized, two-arm, phase IIIb study. Patients with advanced non-squamous non-small-cell lung cancer (NSCLC) whose disease has progressed after four to six cycles of first-line treatment with bevacizumab plus a platinum-based doublet and a minimum of two cycles of bevacizumab (monotherapy) maintenance treatment will be randomized in a 1:1 ratio to one of two study arms. Patients treated on arm A will receive bevacizumab 7.5 or 15 mg/kg intravenously (I.V.) on day 1, every 21 days plus, investigators choice of agents indicated for use in second-line (limited to pemetrexed, docetaxel, or erlotinib) and subsequent lines of treatment. Patients treated on arm B, will receive investigators choice of agents alone indicated for use in second-line and subsequent lines of treatment, but no further bevacizumab treatment. The primary endpoint of this study is overall survival (OS). Secondary endpoints include the 6-month, 12-month, and 18-month OS rates, progression-free survival, and time to progression at second and third progressive disease (PD), response rate, disease control rates, and duration of response at second and third PD. Additionally, efficacy in the subgroup of patients with adenocarcinoma, and the safety of bevacizumab treatment across multiple lines of treatment will be assessed. Exploratory objectives include assessment of the quality of life through multiple lines of treatment, comparison of the efficacy between Asian and non-Asian patients, and correlation of biomarkers with efficacy outcomes, disease response, and adverse events.

Accurate identification of ALK positive lung carcinoma patients: Novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry

Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioviews automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

DICER1, DROSHA and miRNAs in patients with non-small cell lung cancer: implications for outcomes and histologic classification.

The clinical and functional significance of RNA-interference machinery in lung cancer is poorly understood. Besides, microRNAs (miRNA) have the potential to serve both as biomarkers and therapeutic agents, by personalizing diagnosis and therapy. In this study, we investigated whether the expression levels of DICER1 and DROSHA, components of the RNA-interference machinery, can predict survival, and whether the miRNA expression profiles can differentiate histologic subtypes in non-small cell lung cancer (NSCLC). Levels of DICER1, DROSHA and five different miRNAs were measured in NSCLC specimens (N = 115) by qRT-PCR assay and correlated with clinical outcomes. Low expression of DROSHA was associated with an increased median survival (154.2 versus 39.8 months, P = 0.016). Also, high DROSHA expression was associated with decreased median survival in the following subgroups: adenocarcinoma (P = 0.011), grade III tumors (P = 0.038) and low-stage patients (P = 0.014). In multivariate analyses, we found two independent predictors of reduced disease-specific survival: high DROSHA expression [hazards ratio = 2.24; P = 0.04] and advanced tumor stage (hazards ratio = 1.29, P = 0.02). In general, the overall tumor miRNA expression was downregulated in our cohort compared with normal tissues. Expression levels of hsa-let-7a (P = 0.005) and miR-16 (P = 0.003) miRNA were significantly higher in squamous cell carcinoma than in adenocarcinoma samples. This study supports the value of the expression profiling of the components of the miRNA-processing machinery in the prognosis of NSCLC patients, especially DROSHA expression levels. In addition, differential expression of miRNAs, such as hsa-let-7a and miR-16 may be helpful tools in the histologic subclassification of NSCLC.

MALDI profiling of human lung cancer subtypes.

Background Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time. Methodology/Principal Findings In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree–based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non–small cell lung cancer histological subtypes. Conclusions/Significance A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer.

Relevance of insulin-like growth factor 1 receptor gene expression as a prognostic factor in non-small-cell lung cancer

AbstractPurpose Signalling through the insulin-like growth factor 1 receptor (IGF-1R) is implicated in carcinogenesis, metastasis, and resistance to cytotoxic cancer therapies. The purpose of this study was to investigate the prognostic role of IGF-1R expression in surgically resected non-small-cell lung cancer (NSCLC), and responses to IGF-1R tyrosine kinase inhibitor NVP-ADW742 in a panel of lung cancer cell lines. MethodsInsulin-like growth factor 1 receptor (IGF-1R) expression was evaluated by quantitative RT-PCR in 115 NSCLC samples and in a panel of 6 NSCLC cell lines. Cytotoxicity experiments with IGF-1R inhibitor and conventional systemic drugs such as paclitaxel in cell lines were realised.ResultsInsulin-like growth factor 1 receptor (IGF-1R) was differentially expressed across histologic subtypes, with the lowest levels observed in squamous cell tumours. Median survival was longer in patients with squamous tumour histology expressing low IGF-1R levels. In multivariable analysis, ageing and high tumour stage were significant predictors of worse overall survival. The hazard of death was lower in patients with squamous histology and low IGF-1R gene expression. There was no correlation between IGF-1R expression and response to tyrosine kinase inhibitor in cell lines tested. However, combination drug treatment resulted in synergistically enhanced antiproliferative effects on several cell lines.ConclusionsThese findings suggest that IGF-1R is a potential target for therapy in NSCLC patients. Combination therapies will have an important role in treatment.

Strategies for maintenance therapy in advanced non-small cell lung cancer: current status, unanswered questions and future directions.

Systemic chemotherapy (CT) with platinum-based doublets result in modest improvements in both overall survival (OS) and quality of life in good performance status patients with advanced non-small cell lung cancer (NSCLC). However, although substantial progress has been made in the therapeutic options currently available for these patients, the overall outcome remains poor. Maintenance therapy for patients who achieved at least stable disease after first-line treatment has been an area of intense investigation in recent years as a way of improving outcomes in metastatic NSCLC. Several alternative strategies for prolongation of initial treatment have been evaluated. These include the prolongation of the initial combination CT regimen until disease progression, unacceptable toxicity or a predefined greater number of cycles, continuation with a lower intensity version of the first-line CT regimen or administration of a new active agent immediately after completion of the first-line therapy (switch-maintenance or early second-line therapy). Treatments that have been studied in randomized trials to date include CT, molecularly targeted agents, and immunotherapy approaches. Phase III trials have not revealed a survival benefit for extended first-line CT with combination regimens for more than 4-6 cycles. Nevertheless, early second-line therapy with pemetrexed in nonsquamous tumours and erlotinib have demonstrated to improve OS results, especially in select patient groups characterized by histology and/or molecular profile. This article reviews recent data with maintenance therapy in advanced NSCLC and discusses the implications for routine patient care and future drug development.

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

IntroductionA 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 methodsWe 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 54 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.ResultsOur 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.ConclusionsThe 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.

Chemotherapy-associated anemia in patients with lung cancer: an epidemiological, retrospective and multicenter study.

AIM Providing epidemiological data and treatment of anemia in lung cancer patients undergoing first-line chemotherapy. METHODS Epidemiological, observational, retrospective and multicenter study carried out at 30 sites throughout Spain. RESULTS The prevalence of anemia (hemoglobin [Hb] level <12 g/dl) was 18.3% and the incidence 80.7%. Mean Hb levels were 13.4 g/dl (95% Cl: 13.2-13.6) and 11.5 g/dl (95% Cl: 11.3-11.7) at starting and at the end of chemotherapy, respectively. Of the 294 patients with anemia, 174 (59.2%) were treated. Erythropoiesis-stimulating agents were given to 90.2% patients, alone in 31.6% and combined iron in 39.7%, transfusion in 9.2% and iron and transfusion in 9.8%. CONCLUSION These results suggest an appropriate and rational use of erythropoiesis-stimulating agents in the treatment of chemotherapy-associated anemia in lung cancer patients. [corrected].