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Targeted sequencing of untreated DLBCL and TLy

Purpose: Relapsed or refractory diffuse large B-cell lymphoma (rrDLBCL) is fatal in 90% of patients, and yet little is known about its biology.Experimental Design: Using exome sequencing, we characterized the mutation profiles of 38 rrDLBCL biopsies obtained at the time of progression after immunochemotherapy. To identify genes that may be associated with relapse, we compared the mutation frequency in samples obtained at relapse to an unrelated cohort of 138 diagnostic DLBCLs and separately amplified specific mutations in their matched diagnostic samples to identify clonal expansions.Results: On the basis of a higher frequency at relapse and evidence for clonal selection, TP53, FOXO1, MLL3 (KMT2C), CCND3, NFKBIZ, and STAT6 emerged as top candidate genes implicated in therapeutic resistance. We observed individual examples of clonal expansions affecting genes whose mutations had not been previously associated with DLBCL including two regulators of NF-κB: NFKBIE and NFKBIZ. We detected mutations that may be affect sensitivity to novel therapeutics, such as MYD88 and CD79B mutations, in 31% and 23% of patients with activated B-cell–type of rrDLBCL, respectively. We also identified recurrent STAT6 mutations affecting D419 in 36% of patients with the germinal center B (GCB) cell rrDLBCL. These were associated with activated JAK/STAT signaling, increased phospho-STAT6 protein expression and increased expression of STAT6 target genes.Conclusions: This work improves our understanding of therapeutic resistance in rrDLBCL and has identified novel therapeutic opportunities especially for the high-risk patients with GCB-type rrDLBCL. Clin Cancer Res; 22(9); 2290–300. ©2015 AACR.

Supplemental methods Supplemental Figures Supplemental Figure S1. Overview of somatic copy number alterations in rrDLBCLs. Supplemental Figure S2. Consistent low coverage in the first exon of FOXO1. Supplemental Figure S3. Overview of FOXO1 mutations in rrDLBCLs. Supplemental Figure S4. VAFs corrected using purity estimates. Supplemental Figure S5. Somatic copy number alterations affecting lymphoma-related genes. Supplemental Figure S6: Recurrent deletions in NFKBIE. Supplemental Tables Supplemental Table S1. Clinical details and sample processing of 38 patients with relapsed or refractory DLBCL and TLy Supplemental Table S2. Characteristics of samples used for exome sequencing and targeted sequencing of selected genes.

DNA methylation (DNAm) is a dynamic, age-dependent, epigenetic modification that can be used to study interactions between genetic and environmental factors. Environmental exposures during critical periods of growth and development may alter DNAm patterns, leading to increased susceptibility to diseases such as asthma and allergy. One method to study the role of DNAm is the epigenetic clock – an algorithm that uses DNAm levels at select age informative Cytosine-phosphate-Guanine (CpG) dinucleotides to predict epigenetic age (EA). The difference between EA and calendar age (CA) is termed epigenetic age acceleration (EAA) and reveals information about the biological capacity of an individual. Associations between EAA and disease susceptibility have been demonstrated for a variety of age-related conditions and, more recently, phenotypes such asthma and allergic diseases which often begin in childhood and progress throughout the lifespan. In this review, we explore different epigenetic clocks and how they have been applied, particularly as related to childhood asthma and in utero and early life exposures (e.g., smoking, diet, and drugs) that result in methylation changes.

e12614 Background: Tumor infiltrating lymphocytes (TILs) have been associated with good prognosis and response to neoadjuvant chemotherapy. Several reports have shown that the heterogeneity of tumor infiltrating immune cells affects the response to chemotherapy, with for example low levels of FOXP3 expressing T cells associated with good prognosis and pathological complete response (pCR) to chemotherapy. Methods: We examined different immune cell markers on 52 pre-chemotherapy biopsy specimens obtained from triple negative breast cancer patients undergoing neo-adjuvant chemotherapy from the Q-CROC-03 trial. Slides were stained for CD8, CD3,PD-1, PDL-1, FOXP-3 and Granzyme B using multi-colour immunohistochemistry and automated cell counting of stroma and epithelial counts was conducted using the Vectra/inForm image analysis platform. We had total of 39 variables for analysis and we performed Penalized logistic regression for variable selection. Results: Nine variables were found statistically significant to predict response to chemotherapy, PD1+ stroma counts being the one with the highest probability of association with response. A tree algorithm was then used on all 9 variables to identify the best variable and threshold combination to identify patients who respond to chemotherapy. We separated our cohort in test (25% of samples n = 13) and training (75% of samples n = 39) sets for this analysis. Restricting the tree depth to 2 variables for clinical interpretability identified the combination of average counts of stromal PD1+ and average density of stromal FOXP3+ as predictors of chemo response (accuracy 0.82). Both stromal average PD1+ counts and average stromal FOXP3+ density positively correlated with the levels of TILS. Conclusions: Combining FOXP3 and PD1 protein expression in the stroma of pre-treatment biopsies of triple negative breast cancers receiving neoadjuvant chemotherapy is highly predictive of pCR.

Objective A large body of epidemiologic data strongly suggests an association between excess adiposity and coronary artery disease (CAD). Low adiponectin levels, a hormone secreted only from adipocytes, have been associated with an increased risk of CAD in observational studies. However, these associations cannot clarify whether this relationship is causal or due to a shared set of causal factors or even confounding. Genome-wide association studies have identified common variants that influence adiponectin levels, providing valuable tools to examine the genetic relationship between adiponectin and CAD. Methods Using 145 genome wide significant SNPs for adiponectin from the ADIPOGen consortium ( n = 49,891), we tested whether adiponectin-decreasing alleles influenced risk of CAD in the CARDIoGRAM consortium ( n = 85,274). Results In single-SNP analysis, 5 variants among 145 SNPs were associated with increased risk of CAD after correcting for multiple testing ( P −4 ). Using a multi-SNP genotypic risk score to test whether adiponectin levels and CAD have a shared genetic etiology, we found that adiponectin-decreasing alleles increased risk of CAD ( P = 5.4 × 10 −7 ). Conclusion These findings demonstrate that adiponectin levels and CAD have a shared allelic architecture and provide rationale to undertake a Mendelian randomization studies to understand if this relationship is causal.

The analysis of rich catalogues of genetic variation from population-based sequencing provides an opportunity to screen for functional effects. Here we report a rare variant in APOC3 (rs138326449-A, minor allele frequency ~0.25% (UK)) associated with plasma triglyceride (TG) levels (−1.43 s.d. (s.e.=0.27 per minor allele (P-value=8.0 × 10−8)) discovered in 3,202 individuals with low read-depth, whole-genome sequence. We replicate this in 12,831 participants from five additional samples of Northern and Southern European origin (−1.0 s.d. (s.e.=0.173), P-value=7.32 × 10−9). This is consistent with an effect between 0.5 and 1.5 mmol l−1 dependent on population. We show that a single predicted splice donor variant is responsible for association signals and is independent of known common variants. Analyses suggest an independent relationship between rs138326449 and high-density lipoprotein (HDL) levels. This represents one of the first examples of a rare, large effect variant identified from whole-genome sequencing at a population scale., Population-based genome sequencing provides an increasingly rich resource for the identification of low-frequency, large effect variants associated with clinically important phenotypes. Timpson et al. use UK10K data to identify a variant of the APOC3 gene strongly associated with plasma triglyceride levels.

Sixty-three patients with crescentic glomerulonephritis (cellular crescents in >50% of glomeruli) were considered for a prospective randomized trial comparing intravenous methylprednisolone, prednisone, and azathioprine with and without plasma exchange. Of 32 patients who fulfilled the inclusion criteria for this study, 16 were randomly assigned to receive drug therapy (control) and 16 to receive plasma exchange as well. The randomization was stratified for initial need of dialysis, and the presence of oliguria and sclerosis. Renal pathology was similar in the two groups of patients. There was no significant difference in the number of patients initially on dialysis who were able to discontinue it during the study (2/7 control v 3/4 plasma exchange), whereas no control but two plasma exchange-treated patients started dialysis during the study. Serum creatinine at randomization was similar in the two groups: 769 ± 486 µmol/L (8.7 + 5.5 mg/dL) in the control group versus 643 ± 275 µmol/L (7.3 ± 3.1 mg/dL) in the plasma exchange group. There was no significant difference between the two groups in mean serum creatinine, change in serum creatinine, change in reciprocal, or change in logarithm of serum creatinine at 1, 3, 6, or 12 months following randomization. Power calculation, assuming a 20% difference would be clinically relevant, was 0.94 at 12 months. There was significant morbidity in both groups; there were two deaths within 1 year of randomization, both of pulmonary infection and both in the plasma exchange group. We conclude that plasma exchange offers no additional therapeutic benefit to patients with idiopathic rapidly progressive glomerulonephritis (RPGN) who are not dialysis-dependent at presentation.

Functional single nucleotide polymorphisms within the ectoplasmic domain of the Toll-like receptor 4 (TLR4) gene have been shown to result in an endotoxin-hyporesponsive phenotype and aberrant signal transduction for bacterial agonists. TLR4 is in proximity to a genome-wide linkage peak in 9q32-33. Given the proposed function and location of TLR4, we examined the association of 2 functional variants of TLR4 in patients with ankylosing spondylitis (AS) in Newfoundland.In total, 101 AS patients and 100 ethnically matched controls were genotyped, using the Sequenom MassArray platform, for 2 functional variants in the TLR4 gene: Asp299Gly (A/G polymorphism) and Thr399Ile (C/T polymorphism).The minor allele frequency for the Asp299Gly variant (G) was significantly higher in AS cases compared to controls (7.5% vs 2.6%, respectively; OR 3.10, p = 0.037). The minor allele frequency for the Thr399Ile variant (T) for cases and controls was 7.4% vs 3.0% (OR 2.59, p = 0.071). Haplotype analysis using Haploview noted a higher proportion of GT in the cases (for GT, chi-squared p = 0.023).Given the functional role of TLR4 variants in the innate immune system, larger studies are now warranted to elucidate the association of TLR4 variants in AS.

To examine the association of single nucleotide polymorphisms (SNP) in the NFKB1 gene, as well as 2 genes in the nuclear factor (NF)-kappaB functional complex (RelA and NFKBIA), in patients with psoriatic arthritis (PsA) from Newfoundland.Patients with PsA and controls were genotyped for one 4-base insertion/deletion and 5 SNP in NFKB1, 4 SNP in RelA, and 7 SNP in NFKBIA by time-of-flight mass spectrometry, using the Sequenom platform. Chi-square analysis was used to test the single locus associations between SNP in the NF-kappaB complex and PsA. Associations between multi-locus haplotypes and case or control status were tested using the software PHASE.Two hundred and twenty-four patients with PsA (52% male) and 88 ethnically matched controls (64% male) were genotyped. No association was noted with any of the SNP tested for the single locus associations in NFKB1, RelA, and NFKBIA or with multi-locus haplotypes. In particular, the allele frequency for the NFKB1 -94delATTG was 41.7% in cases and 41.6% in the controls (p = 0.97).No association between the NFKB1 -94 ins/delATTG promoter polymorphism or with other NF-kappaB complex SNP in patients with PsA from Newfoundland was observed.

Non-invasive prediction of aluminum bone disease in hemo- and peritoneal dialysis patients. Between October 1987 and October of 1989, we conducted a prospective study to evaluate non-invasive test strategies for predicting aluminum bone disease (ABD) in a group of largely unselected dialysis patients based on their deferoxamine (DFO) test alone, or the combined results of their DFO test and intact 1-84 parathyroid hormone (PTH) levels. These test parameters were evaluated against the pathological diagnosis of ABD based on bone biopsy (“gold standard”). A total of 445 patients in three dialysis centers in Toronto were serially followed for their clinical, laboratory and risk parameters for renal osteodystrophy during the study, and 259 (142 PD and 117 HD) patients underwent a series of investigations which included the DFO test, measurement of intact 1-84 PTH levels, and an iliac crest bone biopsy. Serum aluminum ([A]) level ≥ 3700nM (or 100 µg/liter) had a positive predictive value (PPV) of 75% for ABD in our PD and 88% in our HD patients, but its sensitivity was low (10 and 37%). Delta [Al] (that is, incremental rise of serum [Al] from baseline post-DFO) was useful in predicting ABD in our PD but not HD patients. Test combination based on delta [Al] ≥ 5550nM (or 150 µg/liter) and PTH level < 20 pM (or 200 pg/ml) yielded the best PPV ≥ 95% for ABD in both PD and HD patients. This test cut-off would remain highly predictive of ABD even if the prevalence of ABD decreases to as low as 5% for the PD patients and 10% for the HD patients. However, for patients who have discontinued their aluminum gels for ≥6 months these test strategies would not be useful due to a very high frequency of false-negative cases (or low sensitivity). Thus, the main utility of these non-invasive strategies will be in those patients who continue to require treatment of aluminum gels for their phosphate control.Non-invasive prediction of aluminum bone disease in hemo- and peritoneal dialysis patients. Between October 1987 and October of 1989, we conducted a prospective study to evaluate non-invasive test strategies for predicting aluminum bone disease (ABD) in a group of largely unselected dialysis patients based on their deferoxamine (DFO) test alone, or the combined results of their DFO test and intact 1-84 parathyroid hormone (PTH) levels. These test parameters were evaluated against the pathological diagnosis of ABD based on bone biopsy (“gold standard”). A total of 445 patients in three dialysis centers in Toronto were serially followed for their clinical, laboratory and risk parameters for renal osteodystrophy during the study, and 259 (142 PD and 117 HD) patients underwent a series of investigations which included the DFO test, measurement of intact 1-84 PTH levels, and an iliac crest bone biopsy. Serum aluminum ([A]) level ≥ 3700nM (or 100 µg/liter) had a positive predictive value (PPV) of 75% for ABD in our PD and 88% in our HD patients, but its sensitivity was low (10 and 37%). Delta [Al] (that is, incremental rise of serum [Al] from baseline post-DFO) was useful in predicting ABD in our PD but not HD patients. Test combination based on delta [Al] ≥ 5550nM (or 150 µg/liter) and PTH level < 20 pM (or 200 pg/ml) yielded the best PPV ≥ 95% for ABD in both PD and HD patients. This test cut-off would remain highly predictive of ABD even if the prevalence of ABD decreases to as low as 5% for the PD patients and 10% for the HD patients. However, for patients who have discontinued their aluminum gels for ≥6 months these test strategies would not be useful due to a very high frequency of false-negative cases (or low sensitivity). Thus, the main utility of these non-invasive strategies will be in those patients who continue to require treatment of aluminum gels for their phosphate control.

In occupational cancer epidemiology, many studies are carried out without access to information on smoking and other potential confounding variables. It is unclear whether such deficiencies are likely to cause serious bias in estimates of cancer-occupation associations. An empiric investigation was carried out to determine the effect of inclusion or exclusion of three variables--smoking, ethnic group, and socioeconomic status--on estimates of odds ratios (OR) between 25 occupations and three types of cancer--lung, bladder, and stomach. Of the 75 associations studied, only one OR was distorted by more than 40% when comparing unadjusted with adjusted estimates; three were distorted by between 30% and 40%; four others by between 20% and 30%. Of the eight associations which were distorted by more than 20%, seven involved lung cancer and one involved bladder cancer; none involved stomach cancer. An additional analysis was carried out on the 25 lung cancer-occupation associations to determine whether the nature of the stratification on smoking (ie, whether crude or "precise" categories were used) gave different OR estimates. The differences in ORs induced by different parametrizations of the smoking variable were relatively small. Our results support the view that relative risks between lung cancer and occupation in excess of 1.4 are unlikely to be artifacts due to uncontrolled confounding. For bladder and stomach cancer, the corresponding cut point may be as low as 1.2. In studies of occupation and cancer, uncontrolled confounding due to smoking and social class may not be as serious a threat to the integrity of results as is sometimes feared.