Vitamin D insufficiency is associated with an increased risk of early clinical failure in follicular lymphoma.

We evaluated whether vitamin D insufficiency (VDI; 25(OH)D <20 ng/ml) was associated with adverse outcomes among follicular lymphoma (FL) patients using an observational prospective cohort study of 642 FL patients enrolled from 2002-2012. The median age at diagnosis was 60 years. At a median follow-up of 59 months, 297 patients (46%) had an event (progression, treatment failure), 78 had died and 42 (6.5%) had a lymphoma-related death. VDI was associated with inferior event-free survival (EFS) at 12 months (EFS12, odds ratio (OR)=2.05; 95% confidence interval (CI) 1.18-3.54), overall survival (OS, hazards ratio (HR)=2.35; 95%CI 1.37-4.02), and lymphoma-specific survival (LSS, HR=2.97; 95% CI 1.52-5.80) for the full cohort. Among patients treated with immunochemotherapy (IC), VDI was associated with inferior EFS12 (OR=3.00; 95% CI 1.26-7.13), OS (HR=2.86; 95% CI 1.39-5.85), and LSS (HR=2.96; 95% CI 1.29-6.79). For observed patients, VDI was associated with inferior OS (HR=2.85; 95% CI 1.20-6.76). For other therapies, VDI was associated with inferior OS (HR=3.06; 95% CI 1.01-9.24). Our work is the first to reveal an association of VDI with early clinical failure, and to demonstrate an association of VDI with adverse outcomes among patients who are observed or treated with therapies other than IC. Our findings suggest a potentially modifiable prognostic factor to address in patients with FL.

Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

Risk of non-hematologic cancer in individuals with high-count monoclonal B-cell lymphocytosis.

It is unknown whether individuals with monoclonal B-cell lymphocytosis (MBL) are at risk for adverse outcomes associated with chronic lymphocytic leukemia (CLL), such as the risk of non-hematologic cancer. We identified all locally residing individuals diagnosed with high-count MBL at Mayo Clinic between 1999 and 2009 and compared their rates of non-hematologic cancer with that of patients with CLL and two control cohorts: general medicine patients and patients who underwent clinical evaluation with flow cytometry but who had no hematologic malignancy. After excluding individuals with prior cancers, there were 107 high-count MBL cases, 132 CLL cases, 589 clinic controls and 482 flow cytometry controls. With 4.6 years median follow-up, 14 (13%) individuals with high-count MBL, 21 (4%) clinic controls (comparison MBL P<0.0001), 18 (4%) flow controls (comparison MBL P=0.0001) and 16 (12%) CLL patients (comparison MBL P=0.82) developed non-hematologic cancer. On multivariable Cox regression analysis, individuals with high-count MBL had higher risk of non-hematologic cancer compared with flow controls (hazard ratio (HR)=2.36; P=0.04) and borderline higher risk compared with clinic controls (HR=2.00; P=0.07). Patients with high-count MBL appear to be at increased risk for non-hematologic cancer, further reinforcing that high-count MBL has a distinct clinical phenotype despite low risk of progression to CLL.

Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma.

Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10(-12)) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.

Infectious complications among individuals with clinical monoclonal B-cell lymphocytosis (MBL): a cohort study of newly diagnosed cases compared to controls.

Although the risk of progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL) has been well characterized, it is unknown whether other common complications associated with CLL, such as increased risk of infection, occurs in individuals with MBL. We used the Mayo CLL database to identify cohorts of individuals with newly diagnosed MBL (n=154) or newly diagnosed CLL (n=174) who resided within 50 miles of Mayo Clinic. A cohort of 689 adult patients seen for a general medical examination who resided within 50 miles of Mayo clinic and who enrolled in a case-control study of non-Hodgkin lymphoma (NHL) was used as a comparison cohort. Hospitalization with infection was more common among individuals with MBL (25/154; 16.2%), and CLL (32/174; 18.4%) than controls (18/689; 2.6%). On pooled multivariable Cox proportional hazards analysis of all 1017 patients (controls, MBL and CLL), male sex (hazards ratio (HR)=2.3; P=0.002), major co-morbid health problems (HR=1.7, P=0.04), the presence of CLL (HR=3.2, P<0.001), treatment for progressive CLL (HR=2.4, P=0.001) and the presence of MBL (HR=3.0, P=0.001) were independently associated with risk of hospitalization for infection. These results suggest the risk of serious infection in clinical MBL is substantially greater than the risk of progression requiring treatment.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL.

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

Novel loci for major depression identified by genome-wide association study of Sequenced Treatment Alternatives to Relieve Depression and meta-analysis of three studies.

We report a genome-wide association study (GWAS) of major depressive disorder (MDD) in 1221 cases from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study and 1636 screened controls. No genome-wide evidence for association was detected. We also carried out a meta-analysis of three European-ancestry MDD GWAS data sets: STAR*D, Genetics of Recurrent Early-onset Depression and the publicly available Genetic Association Information Network-MDD data set. These data sets, totaling 3957 cases and 3428 controls, were genotyped using four different platforms (Affymetrix 6.0, 5.0 and 500 K, and Perlegen). For each of 2.4 million HapMap II single-nucleotide polymorphisms (SNPs), using genotyped data where available and imputed data otherwise, single-SNP association tests were carried out in each sample with correction for ancestry-informative principal components. The strongest evidence for association in the meta-analysis was observed for intronic SNPs in ATP6V1B2 (P=6.78 x 10⁻7), SP4 (P=7.68 x 10⁻7) and GRM7 (P=1.11 x 10⁻6). Additional exploratory analyses were carried out for a narrower phenotype (recurrent MDD with onset before age 31, N=2191 cases), and separately for males and females. Several of the best findings were supported primarily by evidence from narrow cases or from either males or females. On the basis of previous biological evidence, we consider GRM7 a strong MDD candidate gene. Larger samples will be required to determine whether any common SNPs are significantly associated with MDD.

Investigation of serotonin-related genes in antidepressant response.

In this study, we sought out to test the hypothesis that genetic factors may influence antidepressant response to fluoxetine. The investigation focused on seven candidate genes in the serotonergic pathway involved in the synthesis, transport, recognition, and degradation of serotonin. Our clinical sample consisted of 96 subjects with unipolar major depression treated with fluoxetine with response variables assessed after a 12-week trial. Patient data were also collected to investigate the pattern of drug response. Using a high-throughput single-nucleotide polymorphism (SNP) genotyping platform and capillary electrophoresis, we genotyped patients at 110 SNPs and four repeat polymorphisms located in seven candidate genes (HTR1A, HTR2A, HTR2C, MAOA, SLC6A4, TPH1, and TPH2). Statistical tests performed included single-locus and haplotype association tests, and linkage disequilibrium (LD) estimation. Little evidence of population stratification was observed in the sample with 20 random SNPs using a genomic control procedure. Our most intriguing result involved three SNPs in the TPH1 gene and one SNP in the SLC6A4 gene, which show significant single-locus association when response to fluoxetine is compared to nonresponse (P=0.02-0.04). All odds ratios indicated an increased risk of not responding to fluoxetine. In the specific response vs nonspecific and nonresponse comparison, three SNPs in the TPH2 gene (P=0.02-0.04) were positively associated and one SNP in the HTR2A gene (P=0.02) was negatively associated. When comparing specific response to nonspecific response, we found significant negative associations in three SNPs in the HTR2A gene (P=0.001-0.03) and two SNPs in the MAOA gene (P=0.03-0.05). We observed variable, although strong LD, in each gene and unexpectedly low numbers of estimated haplotypes, formed from tagged SNPs. Significant haplotype associations were found in all but the HTR1A and HTR2C genes. Although these data should be interpreted cautiously due to the small sample size, these results implicate TPH1 and SLC6A4 in general response, and HTR2A, TPH2, and MAOA in the specificity of response to fluoxetine. Intriguingly, we observe that a number of the less frequent alleles of many of the SNP markers were associated with the nonresponse and nonspecific phenotypes.

Candidate-gene association studies with pedigree data: controlling for environmental covariates.

Case-control studies provide an important epidemiological tool to evaluate candidate genes. There are many different study designs available. We focus on a more recently proposed design, which we call a multiplex case-control (MCC) design. This design compares allele frequencies between related cases, each of whom are sampled from multiplex families, and unrelated controls. Since within-family genotype correlations will exist, statistical methods will need to take this into account. Moreover, there is a need to develop methods to simultaneously control for potential confounders in the analysis. Generalized estimating equations (GEE) are one approach to analyze this type of data; however, this approach can have singularity problems when estimating the correlation matrix. To allow for modeling of other covariates, we extend our previously developed method to a more general model-based approach. Our proposed methods use the score statistic, derived from a composite likelihood. We propose three different approaches to estimate the variance of this statistic. Under random ascertainment of pedigrees, score tests have correct type I error rates; however, pedigrees are not randomly ascertained. Thus, through simulations, we test the validity and power of the score tests under different ascertainment schemes, and an illustration of our methods, applied to data from a prostate cancer study, is presented. We find that our robust score statistic has estimated type I error rates within the expected range for all situations we considered whereas the other two statistics have inflated type I error rates under nonrandom ascertainment schemes. We also find GEE to fail at least 5% of the time for each simulation configuration; at times, the failure rate reaches above 80%. In summary, our robust method may be the only current regression analysis method available for MCC data.

Confirmation of linkage of prostate cancer aggressiveness with chromosome 19q.

Regions on chromosomes 7 and 19 were recently reported to contain susceptibility loci that regulate tumor aggressiveness of prostate cancer. To confirm these findings, we analyzed genome scan data from 161 pedigrees affected with prostate cancer. Using the Gleason score as a quantitative measure of tumor aggressiveness, we regressed the squared trait difference, as well as the mean-corrected cross product, on the estimated proportion of alleles shared identical-by-descent at each marker position. Our results confirm the previous linkage results for chromosome 19q (D19S902, P<.00001). In addition, we report suggestive evidence for linkage on chromosome 4 (D4S403, P=.00012). The results of previous findings, together with our results, provide strong evidence that chromosome 19 harbors a gene for tumor aggressiveness.

The IDDM13 region containing the insulin-like growth factor binding protein-5 (IGFBP5) gene on chromosome 2q33-q36 and the genetic susceptibility to rheumatoid arthritis.

We considered that the constitutive over-expression by cultured rheumatoid arthritis (RA) fibroblast-lineage synoviocytes of genes like IGFBP5 could indicate new candidate susceptibility genes. IGFBP5 is located in a region where an insulin-dependent diabetes mellitus (IDDM) susceptibility locus, IDDM13 (2q33-q36), has been mapped. Previous evidence that non-MHC IDDM loci overlap RA susceptibility loci made IGFBP5 and its region an interesting candidate locus which was tested for linkage. Forty-nine sibships (2-4 affected siblings per sibship) with RA were genotyped with microsatellite markers covering an 11.2 cM interval in the IGFBP5/IDDM13 region. Both the two-point LOD scores and a 'nonparametric' allele-sharing analysis revealed no evidence for linkage (max LOD = 0.54, P = 0.5, respectively). Adjustments for the presence of 'shared-epitope' alleles did not significantly change the LOD scores. These results suggest that, despite the involvement of the 2q33-q36 chromosomal region in another organ-specific autoimmune disease, it is unlikely that this region harbors a RA susceptibility locus.

Case-control studies of genetic markers: power and sample size approximations for Armitage's test for trend.

The association of a candidate gene with disease can be efficiently evaluated by a case-control study in which allele frequencies are compared for diseased cases and unaffected controls. However, when the distribution of genotypes in the population deviates from Hardy-Weinberg proportions, the frequency of genotypes--rather than alleles--should be compared by the Armitage test for trend. We present formulas for power and sample size for studies that use Armitage's trend test. The formulas make no assumptions about Hardy-Weinberg equilibrium, but do assume random ascertainment of cases and controls, all of whom are independent of one another. We demonstrate the accuracy of the formulas by simulations.

Role of HPC2/ELAC2 in hereditary prostate cancer.

The HPC2/ELAC2 gene on chromosome 17p was recently identified as a candidate gene for hereditary prostate cancer (HPC). To confirm these findings, we screened 300 prostate cancer patients (2 affected members/family) from 150 families with HPC for potential germ-line mutations using conformation-sensitive gel electrophoresis, followed by direct sequence analysis. The minimum criteria for our families with HPC was the presence of 3 affected men with prostate cancer. A total of 23 variants were identified, including 13 intronic and 10 exonic changes. Of the 10 exonic changes, 1 truncating mutation was identified, a Glu216Stop nonsense mutation. This nonsense variant was found in 2 of 3 affected men in a single family. The remaining nine alterations included five missense, three silent, and one variant in the 3' untranslated region. To additionally test for potential associations of polymorphic variants and increased risk for disease, we genotyped two common polymorphisms, Ser217Leu and Ala541Thr, in 446 prostate cancer patients from 164 families with HPC and 502 population-based controls. The frequency of the Leu217 variant was similar for patients (32.3%) and controls (31.8%), as was the frequency of the Thr541 variant (5.4% among patients versus 5.2% among controls). In contrast to previous reports, we found no association of the joint effects of Leu271 and Thr541 (odds ratio, 1.04; 95% confidence interval, 0.57-1.89). Overall, our results did not reveal any association between these two common polymorphisms and the risk for HPC. The finding of a nonsense mutation in the HPC2/ELAC2 gene confirms its potential role in genetic susceptibility to prostate cancer. However, our data also suggest that germ-line mutations of the HPC2/ELAC2 are rare in HPC and that the variants Leu217 and Thr541 do not appear to influence the risk for HPC. Cumulatively, these results suggest that alterations within the HPC2/ELAC2 gene play a limited role in genetic susceptibility to HPC.

Evaluation of candidate genes in case-control studies: a statistical method to account for related subjects.

Traditional case-control studies provide a powerful and efficient method for evaluation of association between candidate genes and disease. The sampling of cases from multiplex pedigrees, rather than from a catchment area, can increase the likelihood that genetic cases are selected. However, use of all the related cases without accounting for their biological relationship can increase the type I error rate of the statistical test. To overcome this problem, we present an analysis method that is used to compare genotype frequencies between cases and controls, according to a trend in proportions as the dosage of the risk allele increases. This method uses the appropriate variance to account for the correlated family data, thus maintaining the correct type I error rate. The magnitude of the association is estimated by the odds ratio, with the variance of the odds ratio also accounting for the correlated data. Our method makes efficient use of data collected from multiplex families and should prove useful for the analysis of candidate genes among families sampled for linkage studies. An application of our method, to family data from a prostate cancer study, is presented to illustrate the method's utility.