DNA In Peripheral Blood Mononuclear Cells Research Articles

ZAP70 Methylation Is An Independent Prognostic Biomarker For Front Line Therapy Of Chronic Lymphocytic Leukemia : Results From The UK LRF CLL4 Trial

In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)).The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%.C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L<33.3%), Intermediate (C334Int 33.3-66.6%) or High (C334H >66.6%) methylation (Figure 1). [Display omitted] In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p<0.001). C334H cases were significantly enriched with good risk features such as the presence of a 13q deletion, absence of 11q deletion, absence of NOTCH1 mutation, ZAP70 negativity, CD38 negativity and IGHV mutated status (all p<0.02). However, C334H status retained significant prognostic value for PFS and OS in KM analysis when cases were stratified by each of these good risk features. Furthermore, in multivariate Cox regression analysis, C334H cases showed a significantly reduced risk of progression compared to all other cases (HR=0.67 (95%CI 0.48-0.95), p=0.023), independent of treatment received, IGHV status and 11q and 17p deletion status (Table 1).By identifying a group of patients with hypermethylation of ZAP70, who display a superior outcome with first line chemotherapy, our results confirm the prognostic value of ZAP70 methylation and show it to be superior to ZAP70 expression as measured by flow cytometry. The independent prognostic value of this, compared with established biomarkers, together with the ease and low cost of the assay, warrant its validation in other large trial cohorts. Disclosures:No relevant conflicts of interest to declare.

Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant

An infant born to a woman with human immunodeficiency virus type 1 (HIV-1) infection began receiving antiretroviral therapy (ART) 30 hours after birth owing to high-risk exposure. ART was continued when detection of HIV-1 DNA and RNA on repeat testing met the standard diagnostic criteria for infection. After therapy was discontinued (when the child was 18 months of age), levels of plasma HIV-1 RNA, proviral DNA in peripheral-blood mononuclear cells, and HIV-1 antibodies, as assessed by means of clinical assays, remained undetectable in the child through 30 months of age. This case suggests that very early ART in infants may alter the establishment and long-term persistence of HIV-1 infection.

Longitudinal analysis of HIV-1 coreceptor tropism by single and triplicate HIV-1 RNA and DNA sequencing in patients undergoing successful first-line antiretroviral therapy

ObjectivesMaraviroc has been shown to be effective in patients harbouring CCR5-tropic HIV-1. While this CCR5 antagonist has initially been used in salvage therapy, its excellent safety profile makes it ideal for antiretroviral treatment simplification strategies in patients with suppressed plasma viraemia. The aim of this study was to compare HIV-1 tropism as detected in baseline plasma RNA and peripheral blood mononuclear cell (PBMC) DNA prior to first-line therapy and to analyse tropism evolution while on successful treatment.MethodsHIV-1 tropism was determined using triplicate genotypic testing combined with geno2pheno[coreceptor] analysis at a 10% false positive rate in 42 patients. Paired pre-treatment plasma RNA and PBMC DNA and two subsequent PBMC DNA samples (the first obtained after reaching undetectable plasma HIV-1 RNA and the second after at least 2 years of suppression of plasma viraemia) were evaluated.ResultsCoreceptor tropism was completely concordant in paired pre-treatment RNA and DNA, with 26.2% of HIV-1 sequences predicted to be non-CCR5-tropic. During follow-up, coreceptor tropism switches were detected in 4 (9.5%) patients without any preferential direction. Although false positive rate discrepancies within triplicates were common, the rate of discordance of coreceptor tropism assignment among triplicate results in this mostly CCR5-tropic dataset was only 2.1%, questioning the added value of triplicate testing compared with single testing.ConclusionsHIV-1 coreceptor tropism changes during virologically successful first-line treatment are infrequent. HIV-1 DNA analysis may thus support the choice of a CCR5 antagonist in treatment switch strategies; however, maraviroc treatment outcome data are required to confirm this option.

Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group

PurposeWe have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification.ResultsThe overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/106 PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /106 PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification.ConclusionsOur results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.

HIV-1 outcompetes HIV-2 in dually infected Senegalese individuals with low CD4⁺ cell counts.

Dual infection with HIV-1 and HIV-2, which is not uncommon in West Africa, has implications for transmission, progression, and antiretroviral therapy (ART). Few studies have examined viral dynamics in this setting. Our objective was to directly compare HIV-1 and HIV-2 viral loads and to examine whether this relationship is associated with CD4⁺ cell count. This is a retrospective analysis of data from observational cohort studies. We compared HIV-1 and HIV-2 viral loads from 65 dually infected, ART-naive Senegalese individuals. Participants provided blood, oral fluid, and cervicovaginal lavage (CVL) or semen samples for virologic and immunologic testing. We assessed relationships between HIV-1 and HIV-2 levels using linear regression with generalized estimating equations to account for multiple study visits. After adjusting for CD4⁺ cell count, age, sex, and commercial sex work, HIV-1 RNA levels were significantly higher than HIV-2 levels in semen, CVL, and oral fluids. Despite similar peripheral blood mononuclear cell DNA levels among individuals with CD4⁺ cell counts above 500 cells/μl, individuals with CD4⁺ cell counts below 500 cells/μl had higher HIV-1 and lower HIV-2 DNA levels. Individuals with high CD4⁺ cell counts had higher mean HIV-1 plasma RNA viral loads than HIV-2, with HIV-1 levels significantly higher and HIV-2 levels trending toward lower mean viral loads among individuals with low CD4⁺ cell counts. Our data are consistent with the hypothesis that with disease progression, HIV-1 outcompetes HIV-2 in dually infected individuals. This finding helps explain differences in prevalence and outcomes between HIV-1, HIV-2, and HIV-dual infection.

A Dose–Response Study of Arsenic Exposure and Global Methylation of Peripheral Blood Mononuclear Cell DNA in Bangladeshi Adults

Background: Several studies employing cell culture and animal models have suggested that arsenic (As) exposure induces global DNA hypomethylation. However, As has been associated with global DNA hypermethylation in human study populations. We hypothesized that this discrepancy may reflect a nonlinear relationship between As dose and DNA methylation.Objective: The objective of this study was to examine the dose–response relationship between As and global methylation of peripheral blood mononuclear cell (PBMC) DNA in apparently healthy Bangladeshi adults chronically exposed to a wide range of As concentrations in drinking water.Methods: Global PBMC DNA methylation, plasma folate, blood S-adenosylmethionine (SAM), and concentrations of As in drinking water, blood, and urine were measured in 320 adults. DNA methylation was measured using the [3H]-methyl incorporation assay, which provides disintegration-per-minute (DPM) values that are negatively associated with global DNA methylation.Results: Water, blood, and urinary As were positively correlated with global PBMC DNA methylation (p < 0.05). In multivariable-adjusted models, 1-μg/L increases in water and urinary As were associated with 27.6-unit (95% CI: 6.3, 49.0) and 22.1-unit (95% CI: 0.5, 43.8) decreases in DPM per microgram DNA, respectively. Categorical models indicated that estimated mean levels of PBMC DNA methylation were highest in participants with the highest As exposures.Conclusions: These results suggest that As is positively associated with global methylation of PBMC DNA over a wide range of drinking water As concentrations. Further research is necessary to elucidate underlying mechanisms and physiologic implications.Citation: Niedzwiecki MM, Hall MN, Liu X, Oka J, Harper KN, Slavkovich V, Ilievski V, Levy D, van Geen A, Mey JL, Alam S, Siddique AB, Parvez F, Graziano JH, Gamble MV. 2013. A dose–response study of arsenic exposure and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults. Environ Health Perspect 121:1306–1312; http://dx.doi.org/10.1289/ehp.1206421

Effects of Raltegravir on 2-Long Terminal Repeat Circle Junctions in HIV Type 1 Viremic and Aviremic Patients

Although 2-long terminal repeat (2-LTR) circles are only a fraction of the total viral DNA in infected cells, sequence analysis of 2-LTR circles reveals critical information regarding viral DNA synthesis and the nature of actively replicating virus. It was observed that a large proportion of the 2-LTR circular molecules in the peripheral blood mononuclear cell (PBMC) DNA of infected individuals are mutated at the circle junction. The integrase inhibitor raltegravir (RAL) blocks the strand transfer step of the integration of HIV-1; as a consequence of abortive integration a significant increase of episomal 2-LTR circles is observed. Moreover, it was demonstrated that in patients treated with highly active retroviral therapy (HAART) changes in 2-LTR concentration did not affect junction sequences and flanking regions of 2-LTR. Here we evaluated whether RAL therapy could have a differential impact on the 2-LTR circle junctional sequences in patients with different virological profiles at the time of starting RAL therapy. Sequence analysis indicates that RAL acts differently in the two populations.

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

BackgroundDNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.AimTo investigate the effect of red blood cells on H2O2-induced DNA damage and repair in human peripheral blood mononuclear cells.MethodsDNA breaks were induced in peripheral blood mononuclear cells by H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by 3H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.ResultsRed blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.ConclusionRed blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.

Blood glutathione redox status and global methylation of peripheral blood mononuclear cell DNA in Bangladeshi adults

Oxidative stress and DNA methylation are metabolically linked through the relationship between one-carbon metabolism and the transsulfuration pathway, but possible modulating effects of oxidative stress on DNA methylation have not been extensively studied in humans. Enzymes involved in DNA methylation, including DNA methyltransferases and histone deacetylases, may show altered activity under oxidized cellular conditions. Additionally, in vitro studies suggest that glutathione (GSH) depletion leads to global DNA hypomethylation, possibly through the depletion of S-adenosylmethionine (SAM). We tested the hypothesis that a more oxidized blood GSH redox status is associated with decreased global peripheral blood mononuclear cell (PBMC) DNA methylation in a sample of Bangladeshi adults. Global PBMC DNA methylation and whole blood GSH, glutathione disulfide (GSSG), and SAM concentrations were measured in 320 adults. DNA methylation was measured by using the [3H]-methyl incorporation assay; values are inversely related to global DNA methylation. Whole blood GSH redox status (Eh) was calculated using the Nernst equation. We found that a more oxidized blood GSH Eh was associated with decreased global DNA methylation (B ± SE, 271 ± 103, p = 0.009). Blood SAM and blood GSH were associated with global DNA methylation, but these relationships did not achieve statistical significance. Our findings support the hypothesis that a more oxidized blood GSH redox status is associated with decreased global methylation of PBMC DNA. Furthermore, blood SAM does not appear to mediate this association. Future research should explore mechanisms through which cellular redox might influence global DNA methylation.

Abstract 3647: Dose-dependent alteration of CpG methylation in AHRR and GFI1 in mononuclear cell DNA of smokers.

Abstract About 45 million (∼19%) of U.S. adults smoke, 46 million are former smokers and &amp;gt;100 million are exposed to environmental tobacco smoke. Tobacco use is associated with many types of cancer, heart disease, stroke, and respiratory disease. While hundreds of tobacco smoke constituents cause DNA damage, many adverse outcomes are not related to DNA damage and an emerging hypothesis is that exposure-induced epigenetic effects may mediate many of these outcomes. Using epigenome-wide association, Joubert et al (Env Health Perspectives, 2012) observed Bonferroni-corrected statistically significant associations (480,000 tests, p&amp;lt;10-7) between maternal smoking and DNA methylation in cord blood at 26 CpG sites in 10 genes. We selected 21 smoking- associated CpGs in 4 genes to test as biomarkers of tobacco smoke exposure in adults. We measured methylation in peripheral blood mononuclear cell DNA of healthy adult smokers (5-70 cigarettes per day, n=43) and nonsmokers (n=19) using Illumina 450K methylation arrays. We used linear regression to assess the relationship between current smoking, pack-years, years smoked, and methylation level (log ratio beta) as the outcome. Blood from smokers and nonsmokers was also fractionated using antibody-coated magnetic beads into granulocytes, B cell, and T cell subpopulations to identify effects in specific hematopoietic lineages. Age and race were not significantly associated with methylation differences. Seven of 21 tested CpGs (AHRR, GFI1 and MYO1G) were strongly associated with both dose-dependent current smoking and years of smoking (Bonferroni, p&amp;lt;0.0023) while 13/21 displayed nominal significance (p&amp;lt;0.05). The greatest changes associated with current smoking were among CpGs located in the Ah Receptor Repressor gene (AHRR, cg05575921, delta beta = 27%, p= 1x10-10; cg23576855, delta beta=21%, p=1.96x10-6) and the GFI1 gene (four CpGs, p &amp;lt;0.005). Of the 21 CpGs tested, 17 displayed methylation differences that were directionally consistent with those observed in cord blood in relation to maternal smoking in pregnancy suggesting these CpGs may be a general hematopoietic biomarker of smoking. Surprisingly CpG methylation levels in the CYP1A1 regulatory region were not influenced by smoking, contrary to the effect in cord blood. The functional meaning of these smoking-induced alterations in CpG methylation is not yet clear but in ongoing work we are examining gene expression and cellular phenotype in the blood of smokers. Citation Format: Xuting Wang, Gary S. Pittman, Dan Su, Kelly N. Adamski, Michelle R. Campbell, Bonnie R. Joubert, Zhiqing Y. Huang, Cathrine Hoyo, Susan K. Murphy, Stephanie A. London, Douglas A. Bell. Dose-dependent alteration of CpG methylation in AHRR and GFI1 in mononuclear cell DNA of smokers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2013-3647

High ferritin and low folate increases PBMCs genomic DNA methylation in association with SHMT1–1420TT variant

Nutrient‐gene interactions within one‐carbon metabolism modulate DNA methylation, the major potentially reversible epigenetic modification in eukaryotic cells. The cytosolic serine hydroxymethyltransferase (SHMT1) regulates the metabolic balance between nucleotide synthesis and methylation in one‐carbon pathway. The SHMT1–1420T allele has been associated with a reduced enzyme activity and a decreased risk of cancer. By enhancing SHMT1expression, ferritin affects folate‐mediated one‐carbon metabolism.Aim of this study was to analyze how the interaction among ferritin, folate and SHMT1‐1420C&gt;;T polymorphism may affect peripheral blood mononuclear cells (PBMCs) DNA methylation (LC/ESI/MS method) in 537 subjects enrolled in the Verona Heart Study to identify a possible biomarker for cancer.Results showed that SHMT1‐TT carriers, under a high ferritin/low folate condition, show significantly increased PBMCs genomic DNA methylation than SHMT1‐CC subjects (P=0.01).Since cancer is usually associated with genomic hypomethylation, the increased genomic methylation in SHMT1–1420TT genotypes in presence of high ferritin/low folate, could be potentially protective for cancer risk.

Distribution of total DNA and cccDNA in serum and PBMCs may reflect the HBV immune status in HBsAg+ and HBsAg− patients coinfected or not with HIV or HCV

The potential reservoir role of serum and peripheral blood mononuclear cells (PBMCs) for total HBV DNA (tDNA) and cccDNA still remains unknown. We analyzed tDNA and cccDNA with a single sensitive and validated standardized real-time PCR method in serum and PBMCs in two populations of chronic HBV infection coinfected or not with HCV and/or HIV viruses: a retrospective cohort of 130 HBsAg-negative (HBsAg-) patients with "anti-HBc alone" or anti-HBc and anti-HBs antibodies (Ab) and a cohort of 70 HBsAg-positive patients, 16 of them being prospectively followed under treatment. Among HBsAg- patients, HBV DNA was detected in serum or PBMCs in about half of the cases with various distributions of tDNA and cccDNA: in HIV-negative patients with an "antiHBc alone" profile, tDNA was mostly detected in PBMCs suggesting a possible active role of PBMCs; although cccDNA was not detected in PBMCs in HIV-positive patients, tDNA and cccDNA were mostly observed in serum, suggesting a specific pattern of more "persistent" than "occult" infection in this population. Patients with anti-HBc and anti-HBs Ab harbored tDNA in serum or in PBMCs, regardless of their HIV or HCV status, raising the question of a viral reactivation risk during immunosupression in these patients. Among HBsAg+ patients, tDNA was detected in serum and PBMCs of 88.5% of the cases and cccDNA in 22%. Levels of tDNA in both compartments were highly correlated during treatment, suggesting a passive reservoir role for PBMCs. The respective distribution of tDNA and cccDNA in serum and PBMCs may reflect the different immune statuses of the host in HBsAg+ and HBsAg- patients. The frequency of HBV DNA in PBMCs from AgHBs- patients suggests a viral reactivation risk during immunodepression in those patients.

Global DNA Hypomethylation in Peripheral Blood Mononuclear Cells as a Biomarker of Cancer Risk

Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25-4.53) vs. 5.13 (95% CI, 5.03-5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24-4.51) vs. 5.08 (95% CI, 5.05-5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52-32.63; P = 0.013). Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status.

Demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on peripheral blood mononuclear cells in chronic hepatitis B patients

To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients. A total of 162 subjects, including 144 CHB patients and 18 healthy blood donors, were enrolled. The expression of PD-1 on PBMCs was detected by flow cytometry. And the serum HBV markers, HBV DNA load and liver function were also measured. DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli. Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined. With the PD-1 expression in normal controls (10.8% ± 4.4%) as a baseline level, the expression of PD-1 in CHB patients significantly increased. In CHB patients, the serum expression of PD-1 in PBMCs from patients with positive HBeAg (27.1% ± 18.4%) was much higher than that from those with negative HBeAg (19.6% ± 15.6%). And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase. The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601, -553, -538, -483, -463, -317 bp) were significantly correlated with PD-1 expression level (P < 0.05). The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.

Genome‐wide DNA methylation patterns in discordant sib pairs with alcohol dependence

Alcohol dependence is a complex disease caused by a confluence of environmental and genetic factors. Epigenetic mechanisms have been shown to play an important role in the pathogenesis of alcohol dependence. To determine if alterations in gene-specific methylation were associated with alcohol dependence, a genome-wide DNA methylation analysis was performed on peripheral blood mononuclear cells from alcohol-dependent patients and siblings without alcohol dependence as controls. The Illumina Infinium Human Methylation450 BeadChip was used and gene-specific methylation of DNA isolated from peripheral blood mononuclear cells was assessed. Genes ALDH1L2, GAD1, DBH and GABRP were selected to validate beadchip results by pyrosequencing. Compared to normal controls, 865 hypomethylated and 716 hypermethylated CG sites in peripheral blood mononuclear cell DNA in alcohol-dependent patients were identified. The most hypomethylated CG site is located in the promoter of SSTR4 (somatostatin receptor 4) and the most hypermethylated CG site is GABRP (gamma-aminobutyric acid A receptor). The results from beadchip analysis were consistent with that of pyrosequencing. DNA methylation might be associated with alcohol dependence. Genes SSTR4, ALDH1L2, GAD1, DBH and GABRP may participate in the biological process of alcohol dependence.

Genomic Methylation Changes Over Time in Peripheral Blood Mononuclear Cell DNA: Differences by Assay Type and Baseline Values

Lower levels of genomic DNA methylation in blood DNA has been associated with risk of different cancers and several cancer risk factors. To understand the use of genomic methylation measures as biomarkers of cancer risk, data are needed on within-individual changes over time. Using information from 77 subjects with blood collected at 2 visits on average 8 years apart, we examined whether levels of DNA methylation change with time and if so, whether selected cancer risk factors predict these changes. We measured DNA methylation levels in peripheral blood mononuclear cells (PBMC) using three assays that have been used in epidemiologic studies: (i) luminometric methylation assay (LUMA)(ii) LINE-1 by pyrosequencing, and (iii) Sat2 by MethyLight. Close to a third of all individuals had large changes over time (≥10%) in LUMA with 19.5% increasing and 13.0% decreasing. For Sat2, two-thirds of individuals had large changes with 40% increasing and 26% decreasing over time. In contrast, only 3.9% of individuals had large changes in LINE-1 over time. The degree of change in PBMC DNA methylation was statistically significantly inversely associated with methylation levels at baseline; greater decreases were observed in individuals with higher baseline values for each assay. These data, if replicated, suggest that changes in DNA methylation over time are highly associated with baseline values of the assay and vary by assay type. These findings suggest that assays that change more over time may warrant consideration for studies that measure later life exposures.

Comparing Peripheral Blood Mononuclear Cell DNA and Circulating Plasma viral RNA pol Genotypes of Subtype C HIV-1

INTRODUCTION: Drug resistance mutations (DRM) in viral RNA are important in defining to provide effective antiretroviral therapy (ART) in HIV-1 infected patients. Detection of DRM in peripheral blood mononuclear cell (PBMC) DNA is another source of information, although the clinical significance of DRMs in proviral DNA is less clear. MATERIALS AND METHODS: From 25 patients receiving ART at a center in Zimbabwe, 32 blood samples were collected. Dideoxy-sequencing of gag-pol identified subtype and resistance mutations from plasma viral RNA and proviral DNA. Drug resistance was estimated using the calibrated population resistance tool on www.hivdb.stanford.edu database. Numerical resistance scores were calculated for all antiretroviral drugs and for the subjects' reported regimen. Phylogenetic analysis as maximum likelihood was performed to determine the evolutionary distance between sequences. RESULTS: Of the 25 patients, 4 patients (2 of which had given 2 blood samples) were not known to be on ART (NA) and had exclusively wild-type virus, 17 had received Protease inhibitors (PI), 18, non-nucleoside reverse transcriptase inhibitors (NNRTI) and 19, two or more nucleoside reverse transcriptase inhibitors (NRTI). Of the 17 with history of PI, 10 had PI mutations, 5 had minor differences between mutations in RNA and DNA. Eighteen samples had NNRTI mutations, six of which demonstrated some discordance between DNA and RNA mutations. Although NRTI resistance mutations were frequently different between analyses, mutations resulted in very similar estimated phenotypes as measured by resistance scores. The numerical resistance scores from RNA and DNA for PIs differed between 2/10, for NNRTIs between 8/18, and for NRTIs between 17/32 pairs. When calculated resistance scores were collapsed, 3 pairs showed discordance between RNA and DNA for at least one PI, 6 were discordant for at least one NNRTI and 11 for at least one NRTI. Regarding phylogenetic evolutionary analysis, all RNA and DNA sequence pairs clustered closely in a maximum likelihood tree. CONCLUSION: PBMC DNA could be useful for testing drug resistance in conjunction with plasma RNA where the results of each yielded complementary information about drug resistance. Identification of DRM, archived in proviral DNA, could be used to provide for sustainable public health surveillance among subtype C infected patients.

Telomere Length Is An Independent Predictor of Outcome After Therapy in CLL: Results From the UKCLL4 Trial,

Abstract 3884▪▪This icon denotes a clinically relevant abstractTelomere length measurement has been reported to predict time to first treatment (TTFT) and overall survival from diagnosis in chronic lymphocytic leukaemia (CLL). Of note, the study by Rossi et al (Leukemia, 2009), of presentation samples from over 400 CLL patients, showed short telomere length predicted reduced TTFT, independent of established prognostic biomarkers such as disease stage, immunoglobulin heavy chain variable region (IGHV) mutation status and unfavourable genomic aberrations. However, to date no studies have been reported on the value of telomere length as a biomarker in CLL at time of first therapy.We have used the monochrome multiplex quantitative PCR (MMQPCR) method of Cawthon (Nucleic Acids Research, 2009), to determine relative average telomere length in peripheral blood mononuclear cell DNA from treatment naive CLL patients at time of enrolment into the phase III UKCLL4 trial. MMQPCR reports a telomere length value (T/S) which has been shown to correlate linearly with mean telomere terminal restriction fragment length as determined by southern blotting. Telomere length was measured for 375/777 patients selected from the trial, of the 375 cases 157, 85 and 133 were randomised to the Chlorambucil, Fludarabine or Fludarabine plus Cyclophosphamide (FC) arms respectively. Other than an increased proportion of cases from FC arm, the selected cohort did not differ from the whole trial cohort.The median T/S for all cases was 2.60 (range, 0.61–19.05). In a multiple linear regression model, only age at trial entry and IGHV homology to germline had significant predictive effect for T/S (p=0.023 and <0.0001 respectively). Whereas, Beta-2 microglobulin levels (B2M), WBC, time from diagnosis to study entry, percentage ZAP70 positive tumour cells and percentage CD38 positive tumour cells did not. T/S was significantly lower in cases with unmutated compared with mutated IGHV genes, with del 17p compared with no del 17p and with del 11q compared with no del 11q (all p<0.001) but did not significantly differ with disease stage, gender, or between those cases with or without trisomy 12.Splitting cases into two groups based on those with T/S above or below the median, short telomeres were associated with TP53 loss or mutation, del 11q and unmutated IGHV (all p<0.001) but not with gender or disease stage. Cases with short telomeres also showed significantly higher expression of ZAP70 and CD38 (both p<0.001) and of B2M (p=0.03).In univariate survival analysis, cases with short telomeres showed significantly reduced progression free survival after therapy (PFS) (median; 21.1 months v 33.3 months, p<0.001) and overall survival after therapy (OS) (median; 57.5 months v 108.7 months, p<0.001). Stratifying by IGHV mutational status; short telomeres remained associated with significantly reduced PFS and OS in both IGHV mutated cases (PFS, median; 22.1 months v 42.3 months, p=0.002 and OS, median; 75.9 months v not reached, p<0.001) and IGHV unmutated cases (PFS, median; 21.7 months v 25.7 months, p=0.049 and OS, median; 54.8 months v 81.9 months, p=0.005).Multivariate survival analysis, selected short telomere length as an independent predictor of reduced PFS (Hazard ratio (HR) 1.5; 95% CI 1.1–2.0; p=0.011) and OS (HR 1.9; 95% CI 1.3–2.8; p=0.002) in a model containing treatment arm, IGHV mutational status, age, gender, disease stage, del17p, del11q, ZAP70 expression and CD38 expression (Table 1).Table 1 –Multivariate Analysis for PFS and OS in a 236 Patient Cohort from UKCLL4 Trial.OutcomeCovariateHazard Ratio (HR)95% CI for HRSignificance (p)PFSShort telomeres1.501.10–2.050.011Treatment with FC0.360.26–0.49<0.001IGHV unmutated1.731.28–2.35<0.001Deletion 17p6.513.56–11.92<0.001Deletion 11q1.521.08–2.150.017OSShort telomeres1.881.27–2.780.002IGHV unmutated2.441.58–3.77<0.001Age1.051.03–1.07<0.001Deletion 17p6.843.63–12.89<0.001Only covariates selected as significant are listed above.These results demonstrate that telomere length is a powerful independent predictor of outcome after alkylating agent/purine analogue therapy in CLL. The simplicity and low cost of the MMQPCR assay for telomere length measurement warrants validation of these findings and comparison with other assays for telomere length in other large clinical trials. If confirmed, this prognostic biomarker has the potential for more widespread use in CLL. Disclosures:Gonzalez de Castro:The Institute of Cancer Research: Employment.

Subcutaneous 5-Azacitidine Treatment of Naturally Occurring Canine Urothelial Carcinoma: A Novel Epigenetic Approach to Human Urothelial Carcinoma Drug Development

We determined the efficacy, biological activity, pharmacokinetics and safety of the hypomethylating agent 5-azacitidine (Celgene Corp., Summit, New Jersey) in dogs with naturally occurring invasive urothelial carcinoma. We performed a preclinical phase I trial in dogs with naturally occurring invasive urothelial carcinoma to examine once daily subcutaneous administration of 5-azacitidine in 28-day cycles at doses of 0.10 to 0.30 mg/kg per day according to 2 dose schedules, including days 1 to 5 (28-day cohort) or days 1 to 5 and 15 to 19 (14-day cohort). Clinical efficacy was assessed by serial cystosonography, radiography and cystoscopy. Urinary 5-azacitidine pharmacokinetic analysis was also done. Pretreatment and posttreatment peripheral blood mononuclear cell and invasive urothelial carcinoma DNA, respectively, was analyzed for global and gene specific [CDKN2A (p14ARF)] methylation changes. Enrolled in the study were 19 dogs with naturally occurring invasive urothelial carcinoma. In the 28-day cohort the maximum tolerated dose was 0.20 mg/kg per day with higher doses resulting in grade 3 or 4 neutropenia in 4 of 6 dogs. In the 14-day cohort the maximum tolerated dose was 0.10 mg/kg per day with grade 3 or 4 neutropenia seen in 2 of 3 dogs treated at higher doses. No grade 3 or 4 nonhematological toxicity was observed during either dosing schedule. Of 18 dogs evaluable for tumor response partial remission, stable disease and progressive disease were observed in 4 (22.2%), 9 (50.0%) and 4 (22.2%), respectively. Consistent 5-azacitidine levels (205 to 857 ng/ml) were detected in urine. Pretreatment and posttreatment methylation analysis revealed no significant correlation with clinical response. Subcutaneous 5-azacitidine showed promising clinical activity in a canine invasive urothelial carcinoma model, thus meriting further development in humans with urothelial carcinoma.

Partial Retraction

In our 23 October 2009 Report, “Detection of an Infectious Retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome” (1), two of the coauthors, Silverman and Das Gupta, analyzed DNA samples from chronic fatigue syndrome (CFS) patients and healthy controls. A reexamination by Silverman and Das Gupta of the samples they used shows that some of the CFS peripheral blood mononuclear cell (PBMC) DNA preparations are contaminated with XMRV plasmid DNA (2). The following figures and table were based on the contaminated data: Figure 1, single-round PCR detection of XMRV sequences in CFS PBMC DNA samples; table S1, XMRV sequences previously attributed to CFS patients; and figure S2, the phylogenetic analysis of those sequences. Therefore, we are retracting those figures and table.