Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Prognostic role of genetic biomarkers in clinical progression of prostate cancer.

The aim of this study was to analyze the use of 12 single-nucleotide polymorphisms in genes ELAC2, RNASEL and MSR1 as biomarkers for prostate cancer (PCa) detection and progression, as well as perform a genetic classification of high-risk patients. A cohort of 451 men (235 patients and 216 controls) was studied. We calculated means of regression analysis using clinical values (stage, prostate-specific antigen, Gleason score and progression) in patients and controls at the basal stage and after a follow-up of 72 months. Significantly different allele frequencies between patients and controls were observed for rs1904577 and rs918 (MSR1 gene) and for rs17552022 and rs5030739 (ELAC2). We found evidence of increased risk for PCa in rs486907 and rs2127565 in variants AA and CC, respectively. In addition, rs627928 (TT-GT), rs486907 (AG) and rs3747531 (CG-CC) were associated with low tumor aggressiveness. Some had a weak linkage, such as rs1904577 and rs2127565, rs4792311 and rs17552022, and rs1904577 and rs918. Our study provides the proof-of-principle that some of the genetic variants (such as rs486907, rs627928 and rs2127565) in genes RNASEL, MSR1 and ELAC2 can be used as predictors of aggressiveness and progression of PCa. In the future, clinical use of these biomarkers, in combination with current ones, could potentially reduce the rate of unnecessary biopsies and specific treatments.

Clinical intervals and diagnostic characteristics in a cohort of prostate cancer patients in Spain: a multicentre observational study.

BACKGROUND Little is known about the healthcare process for patients with prostate cancer, mainly because hospital-based data are not routinely published. The main objective of this study was to determine the clinical characteristics of prostate cancer patients, the, diagnostic process and the factors that might influence intervals from consultation to diagnosis and from diagnosis to treatment. METHODS We conducted a multicentre, cohort study in seven hospitals in Spain. Patients' characteristics and diagnostic and therapeutic variables were obtained from hospital records and patients' structured interviews from October 2010 to September 2011. We used a multilevel logistic regression model to examine the association between patient care intervals and various variables influencing these intervals (age, BMI, educational level, ECOG, first specialist consultation, tumour stage, PSA, Gleason score, and presence of symptoms) and calculated the odds ratio (OR) and the interquartile range (IQR). To estimate the random inter-hospital variability, we used the median odds ratio (MOR). RESULTS 470 patients with prostate cancer were included. Mean age was 67.8 (SD: 7.6) years and 75.4 % were physically active. Tumour size was classified as T1 in 41.0 % and as T2 in 40 % of patients, their median Gleason score was 6.0 (IQR:1.0), and 36.1 % had low risk cancer according to the D'Amico classification. The median interval between first consultation and diagnosis was 89 days (IQR:123.5) with no statistically significant variability between centres. Presence of symptoms was associated with a significantly longer interval between first consultation and diagnosis than no symptoms (OR:1.93, 95%CI 1.29-2.89). The median time between diagnosis and first treatment (therapeutic interval) was 75.0 days (IQR:78.0) and significant variability between centres was found (MOR:2.16, 95%CI 1.45-4.87). This interval was shorter in patients with a high PSA value (p = 0.012) and a high Gleason score (p = 0.026). CONCLUSIONS Most incident prostate cancer patients in Spain are diagnosed at an early stage of an adenocarcinoma. The period to complete the diagnostic process is approximately three months whereas the therapeutic intervals vary among centres and are shorter for patients with a worse prognosis. The presence of prostatic symptoms, PSA level, and Gleason score influence all the clinical intervals differently.

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer

BACKGROUND Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk. METHODS A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area. RESULTS Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09-2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09-2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms. CONCLUSION Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies.

BACKGROUND Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. OBJECTIVE To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. DESIGN SETTING AND PARTICIPANTS A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. RESULTS AND LIMITATIONS We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. CONCLUSION Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders). Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11) and non-responders (n = 16) to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029) gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with locally advanced rectal cancer. Moreover, our investigation added further evidence to the importance of mononuclear cells' mediated response in the neoadjuvant treatment of rectal cancer.

TRAIL/TRAIL Receptor System and Susceptibility to Multiple Sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10(-4), OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10(-5), OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.

Mutation screening of multiple genes in Spanish patients with autosomal recessive retinitis pigmentosa by targeted resequencing.

Retinitis Pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. RP is the leading cause of visual loss in individuals younger than 60 years, with a prevalence of about 1 in 4000. The molecular genetic diagnosis of autosomal recessive RP (arRP) is challenging due to the large genetic and clinical heterogeneity. Traditional methods for sequencing arRP genes are often laborious and not easily available and a screening technique that enables the rapid detection of the genetic cause would be very helpful in the clinical practice. The goal of this study was to develop and apply microarray-based resequencing technology capable of detecting both known and novel mutations on a single high-throughput platform. Hence, the coding regions and exon/intron boundaries of 16 arRP genes were resequenced using microarrays in 102 Spanish patients with clinical diagnosis of arRP. All the detected variations were confirmed by direct sequencing and potential pathogenicity was assessed by functional predictions and frequency in controls. For validation purposes 4 positive controls for variants consisting of previously identified changes were hybridized on the array. As a result of the screening, we detected 44 variants, of which 15 are very likely pathogenic detected in 14 arRP families (14%). Finally, the design of this array can easily be transformed in an equivalent diagnostic system based on targeted enrichment followed by next generation sequencing.

Hypermethylated 14-3-3-sigma and ESR1 gene promoters in serum as candidate biomarkers for the diagnosis and treatment efficacy of breast cancer metastasis

Background: Numerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of Estrogen Receptor1 (ESR1) and Stratifin (14-3-3-σ) gene promoters in disease-free and metastatic breast cancer patients. Methods: We studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR. Results: Serum levels of methylated gene promoter 14-3-3-σ significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-σ level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis. Conclusions: The relationship of 14-3-3-σ with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-σ as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Bone Mineral Density and Serum Levels of Soluble Tumor Necrosis Factors, Estradiol, and Osteoprotegerin in Postmenopausal Women with Cirrhosis after Viral Hepatitis

CONTEXT: Cirrhosis after viral hepatitis has been identified as a risk factor for osteoporosis in men. However, in postmenopausal women, most studies have evaluated the effect of primary biliary cirrhosis, but little is known about the effect of viral cirrhosis on bone mass [bone mineral density (BMD)] and bone metabolism. OBJECTIVE: Our objective was to assess the effect of viral cirrhosis on BMD and bone metabolism in postmenopausal women. DESIGN: We conducted a cross-sectional descriptive study. SETTING AND PATIENTS: We studied 84 postmenopausal female outpatients with viral cirrhosis and 96 healthy postmenopausal women from the general community. BMD was measured by dual-energy x-ray absorptiometry at lumbar spine (LS) and femoral neck (FN). RESULTS: The percentage with osteoporosis did not significantly differ between patients (LS, 43.1%; FN, 32.2%) and controls (LS, 41.2%; FN, 29.4%), and there was no difference in BMD (z-score) between groups. Serum concentrations of soluble TNF receptors, estradiol, and osteoprotegerin (OPG) were significantly higher in patients vs. controls (P < 0.001, P < 0.05, and P < 0.05, respectively). No significant difference was observed in urinary deoxypyridinoline. Serum OPG levels were positively correlated with soluble TNF receptors (r = 0.35; P < 0.02) and deoxypyridinoline (r = 0.37; P < 0.05). CONCLUSIONS: This study shows that bone mass and bone resorption rates do not differ between postmenopausal women with viral cirrhosis and healthy postmenopausal controls and suggests that viral cirrhosis does not appear to increase the risk of osteoporosis in these women. High serum estradiol and OPG concentrations may contribute to preventing the bone loss associated with viral cirrhosis in postmenopausal women.

Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

BACKGROUND We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL) and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC) and favorable outcome (LMO2, BCL6, FN1) in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.