Identification of a new HLA-C allele, HLA-C*08:76 in a Caucasian individual.

HLA-C*08:76 differs from HLA-C*08:02:01 by one nonsynonymous nucleotide change at the codon 144 (CAG to AAG) in exon 3.

Identification of predictive circulating biomarkers of bevacizumab-containing regimen efficacy in pre-treated metastatic colorectal cancer patients.

BACKGROUND: To identify whether circulating levels of angiogenesis-related factors may be predictive of bevacizumab efficacy in pre-treated metastatic colorectal cancer (mCRC) patients. METHODS: Pre-treatment serum levels of 24 cytokines were measured using a multiplex bead assay (MBA) in 32 pre-treated mCRC patients treated with irinotecan plus bevacizumab-based salvage therapy. Macrophage-derived chemokine (MDC), interleukins (ILs) 8 and 6 levels were also validated by enzyme-linked immunosorbent assay (ELISA) at different time points during therapy. RESULTS: Higher epidermal growth factor (EGF) and MDC baseline levels (2.2- and 1.4-fold, respectively) and lower IL-10, IL-6 and IL-8 levels (0.2-, 0.6-, and 0.6-fold, respectively, P<0.05) were observed in patients responding to therapy. Baseline levels of these five serum factors compose a risk signature that may define the subset of patients most likely to benefit from bevacizumab-based therapy in terms of response rate and survival times. A positive correlation was found between MBA and ELISA results (P<0.01). Treatment exposure increased MDC and had opposite effects on IL-8 levels, which were decreased (P<0.05). CONCLUSION: This study suggests that a set of inflammatory and angiogenesis-related serum markers may be associated with the efficacy of bevacizumab-containing regimen.

Association of RRM1 -37A>C polymorphism with clinical outcome in colorectal cancer patients treated with gemcitabine-based chemotherapy.

BACKGROUND: To investigate whether single nucleotide polymorphisms (SNPs) in gemcitabine (GMB) metabolism genes were associated with clinical outcome in pre-treated metastatic colorectal cancer (mCRC) patients. PATIENTS AND METHODS: SNPs of hCNT1, hENT1, CDA, dCTD and RRM1 genes were evaluated in 95 mCRC patients and detected using TaqMan genotyping assays. Association of genotypes with overall response rate (ORR), time to progression (TTP) and overall survival (OS) was tested by univariate and multivariate analysis. RRM1 -37A>C polymorphism was correlated with GMB IC50 value and with the RRM1 gene expression level in CRC cell lines. RESULTS: The ORR was 38.9%. The median TTP and OS were 4 and 14.3 months, respectively. By multivariate analysis, patients carrying the RRM1 -37CC genotype or the CDA A-76 C-containing allele had a significantly higher likelihood of achieving a tumour response. RRM1 -37A>C polymorphism remained associated with clinical efficacy (TTP). In vitro experiments, in CRC cell lines, showed that the RRM1 A-37C genotype was associated with the levels of RRM1 expression and with GMB IC50 values. Finally, the down-regulation of RRM1 with a specific siRNA strongly influenced GMB sensitivity. CONCLUSION: RRM1 -37A>C polymorphism may represent a useful biomarker to select mCRC patients most likely to benefit from GMB-based salvage therapy.

Role of primary miRNA polymorphic variants in metastatic colon cancer patients treated with 5-fluorouracil and irinotecan.

MicroRNAs are non-coding RNAs that can block mRNA translation and influence mRNA stability. Recent evidence indicates that miRNA variations can affect drug resistance, efficacy, and metabolism, opening new avenues of pharmacogenomics research. We investigated associations between polymorphisms in both miRNA-containing genomic regions (primary and precursor miRNA) and in genes related to miRNA biogenesis with clinical outcome in metastatic colorectal cancer (mCRC) patients treated with 5-fluorouracil and irinotecan (CPT-11). Eighteen single-nucleotide polymorphisms (SNPs) were analyzed in 61 patients. A significant association with tumor response and time to progression (TTP) was found for SNP rs7372209 in pri-miR26a-1 (P=0.041 and P=0.017, respectively). The genotypes CC and CT were favorable when compared with the TT variant genotype. In addition, SNP rs1834306, located in the pri-miR-100 gene, significantly correlated with a longer TTP (P=0.04). In the miRNA-biogenesis pathway, a trend was identified between SNP rs11077 in the exportin-5 gene and disease control rate (P=0.076). This study is the first to suggest a relationship between treatment outcome and SNPs in the miRNA-biogenesis machinery, in both primary and precursor miRNAs. Our results suggest that miRNA polymorphic variants might be useful predictors of clinical outcome in mCRC patients treated with 5-fluorouracil and CPT-11 combination.

DUSP1/MKP1 promotes angiogenesis, invasion and metastasis in non-small-cell lung cancer.

DUSP1/MKP1 is a dual-specific phosphatase that regulates MAPKs activity, with an increasingly recognized role in tumor biology. To understand more about the involvement of DUSP1 in lung cancer, we performed gene expression analyses of parental and DUSP1-interfered H460 non-small-cell lung cancer (NSCLC) cells. Downregulation of DUSP1 induced changes in the expression levels of genes involved in specific biological pathways, including angiogenesis, MAP kinase phosphatase activity, cell-cell signaling, growth factor and tyrosine-kinase receptor activity. Changes in the expression of some of these genes were due to modulation of c-Jun-N-terminal kinase and/or p38 activity by DUSP1. Complementary functional assays were performed to focus on the implication of DUSP1 in angiogenesis and metastasis. In H460 cells, interference of DUSP1 resulted in a diminished capacity to invade through Matrigel, to grow tumors in nude mice and also to induce metastasis through tail-vein injection. Furthermore, the angiogenic potential of H460 cells was also impaired, correlating with a decrease in VEGFC production and indicating that DUSP1 could be required to induce angiogenesis. Finally, we studied whether a similar relationship occurred in patients. In human NSCLC specimens, DUSP1 was mainly expressed in those tumor cells close to CD31 vascular structures and a statistically significant correlation was found between VEGFC and DUSP1 expression. Overall, these results provide evidence for a role of DUSP1 in angiogenesis, invasion and metastasis in NSCLC.

Dose-finding study and pharmacogenomic analysis of fixed-rate infusion of gemcitabine, irinotecan and bevacizumab in pretreated metastatic colorectal cancer patients.

BACKGROUND: To determine the dose-limiting toxicity (DLT), maximum tolerated dose, recommended dose (RD) and preliminary evidence of activity of escalating doses of irinotecan (CPT-11) fixed-dose-rate infusional gemcitabine (FDR-GMB) and bevacizumab in pretreated metastatic colorectal cancer (mCRC) patients. Pharmacogenomic analysis was performed to investigate the association between VEGF single-nucleotide polymorphisms and clinical outcome. PATIENTS AND METHODS: A total of 89 mCRC patients were recruited in a two-step study design; 28 were included in the dose-finding study and 59 in the pharmacogenomic analysis. The FDR-GMB of 1000 mg m⁻², bevacizumab 5 mg kg⁻¹ and CPT-11 doses ranging from 100 to 160 mg m⁻² were explored. The VEGF protein serum levels were quantified by EIA. Allelic discrimination was performed to genotype polymorphisms in the VEGF gene. RESULTS: CPT-11 RD was 150 mg m⁻². Diarrhoea and neutropenia were the DLT. After a median follow-up of 42 months, the median time to progression (TTP) and overall survival were 5.2 and 19.9 months, respectively. VEGF levels were significantly correlated with VEGF-2578AA and VEGF-460CC genotypes, and a trend was observed with VEGF+405GG genotype. The presence of any of these genotypes correlated with a longer median TTP (8.8 vs 4.5 months, P=0.04). CONCLUSION: The triplet combination tested in this study is effective and well tolerated. A possible predictive role for VEGF gene polymorphisms and baseline VEGF circulating levels is suggested.

EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2.

BACKGROUND: The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans. METHODS: We performed QT-RT-PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT. RESULTS: Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines. CONCLUSIONS: Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

Oxaliplatin, irinotecan and capecitabine as first-line therapy in metastatic colorectal cancer (mCRC): a dose-finding study and pharmacogenomic analysis.

BACKGROUND: A dose-finding study was performed to evaluate the dose-limiting toxicity (DLT), maximum-tolerated dose (MTD) and the recommended dose (RD) of escalating the doses of capecitabine and fixed doses of irinotecan and oxaliplatin on a biweekly schedule for metastatic colorectal cancer patients (mCRC). A pharmacogenomic analysis was performed to investigate the association between SNPs and treatment outcome. METHODS: Eighty-seven chemotherapy-naïve mCRC patients were recruited through a two-step study design; 27 were included in the dose-finding study and 60 in the pharmacogenomic analysis. Oxaliplatin (85 mg m(-2)) and CPT-11 (150 mg m(-2)), both on day 1, and capecitabine doses ranging from 850 to 1500 mg m(-2) bid on days 1-7 were explored. Peripheral blood samples were used to genotype 13 SNPs in 10 genes related to drug metabolism or efficacy. Univariate and multivariate Cox analysis was performed to examine associations between SNPs, ORR and PFS. RESULTS: The capecitabine RD was 1000 mg m(-2) bid. Diarrhoea and neutropenia were the DLTs. After a median follow-up of 52.5 months, the median PFS and OS were 12 (95% CI; 10.6-13.4) and 27 months (95% CI; 17.2-36.8), respectively.The GSTP1-G genotype, the Köhne low-risk category and use of a consolidation approach strongly correlated with decreased risk of progression. Patients with all favourable variables showed a median PFS of 42 months vs 3.4 months in the group with all adverse factors. A superior clinical response was obtained in patients with one GSTP1-G allele as compared with GSTP1-AA carriers (P=0.004). CONCLUSION: First-line therapy with oxaliplatin, irinotecan and capecitabine is efficient and well-tolerated. The GSTP1 polymorphism A>G status was significantly associated with ORR and PFS in mCRC treated with this triplet therapy.

MicroRNAs as a new potential therapeutic opportunity in gastrointestinal cancer.

MicroRNAs (miRNAs) comprise an abundant class of small non-coding RNAs that act as important post-transcriptional regulators of gene expression. Accumulating evidence shows that aberrantly expressed miRNAs play important roles in human cancers and underscores them as potential targets for therapeutic intervention. Basic research has provided strong evidence about the role of miRNA as oncogenes and as tumor suppressor genes in cancer. In accord with this finding, miRNA-based cancer therapy is a very interesting field of investigation that offers the appeal of targeting multiple gene networks controlled by a single, aberrantly expressed miRNA. A new door in cancer research is opening and it may lead to the modulation of miRNAs.

Epigenetic events in normal colonic mucosa surrounding colorectal cancer lesions.

Gene inactivation by promoter hypermethylation has been demonstrated in the colonic mucosa of colorectal cancer (CRC) patients. However, current data do not prove direct involvement of this epigenetic modification in the early stages of CRC. Promoter methylation profiles of E-cadherin, hMLH1, MGMT, p16(INK4a), p15(INK4b) and p14(ARF); mutations of K-ras, B-raf and TP53 and microsatellite instability (MSI) were examined in normal and cancerous colonic mucosal tissue in 82 CRC patients using methylation-specific PCR assays. Methylation of hMLH1 and MGMT in normal mucosa correlated significantly with MSI and K-ras activation in neighbouring cancerous mucosal tissues. Similarly, poorly differentiated tumours were associated with methylated p16(INK4a) and E-cadherin in neighbouring normal colonic tissues (NCTs). Our results indicate that epigenetic changes in mucosa surrounding colorectal neoplastic lesions may describe a 'field cancerisation' phenomenon that may occur previous to genetic alterations in early stages of carcinogenesis.

Combining chemotherapy and targeted therapies in metastatic colorectal cancer.

Colorectal cancer remains one of the major causes of cancer death worldwide. During the past years, the development of new effective treatment options has led to a considerable improvement in the outcome of this disease. The advent of agents such as capecitabine, irinotecan, oxaliplatin, erbitux and bevacizumab has translated into median survival times in the range of 2 years. Intense efforts have focused on identifying novel agents targeting specific growth factor receptors, critical signal transduction pathways or mediators of angiogenesis. In addition, several clinical trials have suggested that some of these molecularly targeted drugs can be safely and effectively used in combination with conventional chemotherapy. In this article we review various treatment options combining cytotoxic and targeted therapies currently available for patients with metastatic colorectal cancer.

GSTP1 and MTHFR polymorphisms are related with toxicity in breast cancer adjuvant anthracycline-based treatment.

We have analyzed several members of drug-metabolizing enzymes (DMEs) and other polymorphisms in genes implicated in tumor aggressivity regarding possible links between specific genetic variability in systemic drug bioavailability and toxicity in breast cancer patients treated with adjuvant anthracycline-based treatment. PCR-RFLP and sequencing analyses technique were used for evaluating fourteen previously identified polymorphisms in 94 patients. GSTP1A>G and MTHFR 1298A>C genotypes remained as significant predictors in a multivariate logistic regression analysis. GSTP1 polymorphism was linked to haematological GIII-IV toxicity (P = 0.044, HR= 6.4, 95% CI = 1.05 to 39. Increased and significant HR was obtained for MTHFR-1298 AC+CC group when non-haematological toxicities GIII-IV toxicities were evaluated (HR = 24; 95% CI = 2.3 to 254), P = 0.008. Our results suggest that GSTP1 and MTHFR genotypes may be consider relevant and independent factors of toxicity in adjuvant anthracycline-based treatment of breast cancer.

Xeroderma pigmentosum group D 751 polymorphism as a predictive factor in resected gastric cancer treated with chemo-radiotherapy.

AIM: To evaluate the potential association of xeroderma pigmentosum group D (XPD) codon 751 variant with outcome after chemo-radiotherapy in patients with resected gastric cancer. METHODS: We used PCR-RFLP to evaluate the genetic XPD Lys751Gln polymorphisms in 44 patients with stage III (48%) and IV (20%) gastric cancer treated with surgery following radiation therapy plus 5-fluorouracil/leucovorin based chemotherapy. RESULTS: Statistical analysis showed that 75% (12 of 16) of relapse patients showed Lys/Lys genotype more frequently (P = 0.042). The Lys polymorphism was an independent predictor of high-risk relapse-free survival from Cox analysis (HR: 3.07, 95% CI: 1.07-8.78, P = 0.036) and Kaplan-Meir test (P = 0.027, log-rank test). CONCLUSION: XPD Lys751Gln polymorphism may be an important marker in the prediction of clinical outcome to chemo-radiotherapy in resected gastric cancer patients.

Potential application of GSTT1-null genotype in predicting toxicity associated to 5-fluouracil irinotecan and leucovorin regimen in advanced stage colorectal cancer patients.

Our aim was to evaluate the role of C-69T in GSTA1, Ile105Val in GSTP1, null allele in GSTT1 and GSTM1 in the prediction of toxicity in patients treated with 5-Fu/CPT-11/Lv regimens in metastatic CRC patients. Fifty-one patients with CRC metastatic disease were analysed. All patients had bidimensionally measurable disease according to WHO criteria. The gender distribution was 37 (74%) males and 13 (26%) females; age ranged from 41 to 71 years; performance status was in all patients > or = 80 (Karnofsky index). The analysis of gene polymorphism was performed in lymphocytes by using PCR-RFLP (GSTA1, GSTP1), PCR (GSTT1, GSTM1) and sequencing analysis (UGT1A1 *28). An appreciable significant association was observed between the GSTT1-null and toxicity: 57% developed gastrointestinal toxicity grade III versus 23% of patients with GSTT1-present genotype (p = 0.053). The other polymorphisms analysed did not show any significant relation with toxicity. Our data suggest that GSTT1-null is associated with a greater probability of developing toxicity to 5-Fu/CPT-11/Lv treatments, indicating a potential application of this genetic analysis in predicting adverse effects of this regimen.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Association between bone mineral density and polymorphisms of the VDR, ERalpha, COL1A1 and CTR genes in Spanish postmenopausal women.

Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a strong genetic component, and several candidate gene polymorphisms have been implicated in the regulation of this process. In view of the reported associations between the BMD and polymorphisms in the collagen type I alpha 1 gene (COL1A1), vitamin D receptor (VDR), estrogen receptor (ER)alpha and calcitonin receptor (CTR) genes, an association study was performed between VDR, COL1A1, CTR and ER genotypes and lumbar spine, femoral neck and Ward's triangle BMD in postmenopausal Spanish women. We statistically controlled for many confounding factors, such as height, weight, age, years since menopause, use of hormone replacement therapy (HRT), tobacco consumption, use of oral contraceptives, calcium dietary intake or exercise practice. No association between COL1A1 or ER genotypes and BMD was detected. However, we described a statistically significant association between a personal history of fractures and COL1A1 genotype. The ss genotype was found to be over-represented between those women who had a personal history of fractures. The analyses of the VDR polymorphisms showed that FF subjects reached the highest values of BMD at the three measured sites, whereas Ff individuals had an intermediate BMD and ff women had the lowest values. However, the VDR-BsmI gene polymorphism was not found to be associated with adjusted BMD. For the CTR polymorphisms, our study showed that women with the aa genotype had a lower adjusted BMD at the femoral neck. In conclusion, in our postmenopausal Spanish women cohort we found a statistically significant association beteween the VDR and CTR gene polymorphisms and the BMD. However, we did not find any association between the ER and COL1A1 gene and the BMD. The COL1A1 gene was found to be associated with the prevalence of osteoporotic fractures. Of all the studied gene polymorphisms, the FokI VDR gene polymorphism seems to be the strongest BMD genetic determinant of postmenopausal Spanish women.

Factores moleculares predictivos de respuesta y toxicidad para seleccionar el tratamiento adyuvante / No disponible

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Conceptos básicos en biología molecular del cáncer. Susceptibilidad genética / Basic concepts in the molecular biology of cancer. Genetic susceptibility

En los últimos años se han producido cambios en la biología molecular que nos permiten optimizar el tratamiento de cáncer, minimizando los efectos secundarios, y actuar a nivel de la prevención a partir del estudio de los procesos biológicos que subyacen en la enfermedad. Uno de los aspectos más destacados se dirige a la capacidad con la que contamos actualmente de poder analizar el ADN y/o ARN fruto de los descubrimientos y progresos en la segunda mitad del siglo XX. Todas estas innovaciones tecnológicas y en el campo de la genética molecular preparan el camino para la comprensión y el análisis funcional que se deriva de la información contenida en el genoma humano. Asimismo el estudio de algunos de los múltiples eventos moleculares que acontecen en el proceso multifactorial del cáncer, permiten ofrecer una nueva visión de los factores pronósticos al tiempo que identifican individuos con una gran susceptibilidad de desarrollar determinados tipos de cáncer. Algunos de estos genes asociados a cáncer pueden alterarse como consecuencia de mutágenos endógenos, mutaciones a nivel de línea germinal, que ocurren durante la replicación celular y que pueden incrementar la inestabilidad genómica en células precancerosas. En la misma línea, los avances de la biotecnología ofrecen la posibilidad de contar con una visión amplia de un amplio espectro de eventos moleculares involucrados en el proceso neoplásico. (AU)

The increase of IFN-gamma production through aging correlates with the expanded CD8(+high)CD28(-)CD57(+) subpopulation.

The use of flow cytometry to detect intracellular cytokines at the single cell level has the potential to quantify cytokine production together with the possibility of phenotypic identification of the cell population concerned. The unbalanced presence of intracellular cytokines produced by T cells has been recognized in some pathological conditions. To better address this issue, we studied the production of IFN-gamma and IL-4 in CD4(+) and CD8(+high) T cells in healthy donors of a broad range of age (17-62 years). Given that an increase of IFN-gamma and IL-4 with aging had been reported by some authors in healthy controls, we have performed a multivariate analysis to assess the intrinsic role of aging or of other external factors, such as chronic antigenic exposures (i.e., viruses), over the cytokine production of phenotypically characterized T cells. In this respect we show that, mainly in CD8(+high) T cells, the production of IFN-gamma is directly correlated with age. Besides, the cytokine production correlates with the CD8(+high)CD28(-)CD57(+) T-cell population, which we have recently reported elevated in aged individuals. Perhaps this T-cell subpopulation plays a regulatory role as a Tc1 response in aging individuals.