Distinct germline genetic susceptibility profiles identified for common non-Hodgkin lymphoma subtypes.

Lymphoma risk is elevated for relatives with common non-Hodgkin lymphoma (NHL) subtypes, suggesting shared genetic susceptibility across subtypes. To evaluate the extent of mutual heritability among NHL subtypes and discover novel loci shared among subtypes, we analyzed data from eight genome-wide association studies within the InterLymph Consortium, including 10,629 cases and 9505 controls. We utilized Association analysis based on SubSETs (ASSET) to discover loci for subsets of NHL subtypes and evaluated shared heritability across the genome using Genome-wide Complex Trait Analysis (GCTA) and polygenic risk scores. We discovered 17 genome-wide significant loci (P < 5 × 10-8) for subsets of NHL subtypes, including a novel locus at 10q23.33 (HHEX) (P = 3.27 × 10-9). Most subset associations were driven primarily by only one subtype. Genome-wide genetic correlations between pairs of subtypes varied broadly from 0.20 to 0.86, suggesting substantial heterogeneity in the extent of shared heritability among subtypes. Polygenic risk score analyses of established loci for different lymphoid malignancies identified strong associations with some NHL subtypes (P < 5 × 10-8), but weak or null associations with others. Although our analyses suggest partially shared heritability and biological pathways, they reveal substantial heterogeneity among NHL subtypes with each having its own distinct germline genetic architecture.

Activation of the aryl hydrocarbon receptor and risk of lymphoma subtypes.

The aryl hydrocarbon receptor (AhR) is a transcription factor implicated in several pathways known to be relevant in lymphomagenesis. Aim of our study was to explore the link between AhR activation and risk of lymphoma subtypes. We used a Dual-Luciferase Assay® and a luminometer to detect the activation of the luciferase gene, in HepG2 cells transfected with a specific reporter systems, by a 50 ml serum aliquot of cases of diffuse large B cell lymphoma (N = 108), follicular lymphoma (N = 85), chronic lymphocytic leukemia (N = 72), multiple myeloma (N = 80), and Hodgkin lymphoma (N = 94) and 357 controls who participated in the multicentre Italian study on gene-environment interactions in lymphoma etiology (ItGxE). Risk of each lymphoma subtype associated with AhR activation was calculated with polytomous logistic regression adjusting by age, gender, and study centre. The overall prevalence of AhR activation ranged 13.9-23.6% by subtype, and it varied by study area (8-39%). Risk associated with AhR activation was moderately elevated for follicular lymphoma (OR = 1.56, 95% CI 0.86, 2.80) and chronic lymphocytic leukemia (OR = 1.56, 95% CI 0.83, 2.96). Despite our inconclusive findings about the association with risk of lymphoma subtypes, we showed that the Dual-Luciferase Assay can be reliably and easily applied in population-based studies to detect AhR activation.

A genome-wide association study by ImmunoChip reveals potential modifiers in myelodysplastic syndromes.

Because different findings suggest that an immune dysregulation plays a role in the pathogenesis of myelodysplastic syndrome (MDS), we analyzed a large cohort of patients from a homogeneous Sardinian population using ImmunoChip, a genotyping array exploring 147,954 single-nucleotide polymorphisms (SNPs) localized in genomic regions displaying some degree of association with immune-mediated diseases or pathways. The population studied included 133 cases and 3,894 controls, and a total of 153,978 autosomal markers and 971 non-autosomal markers were genotyped. After association analysis, only one variant passed the genome-wide significance threshold: rs71325459 (p = 1.16 × 10-12), which is situated on chromosome 20. The variant is in high linkage disequilibrium with rs35640778, an untested missense variant situated in the RTEL1 gene, an interesting candidate that encodes for an ATP-dependent DNA helicase implicated in telomere-length regulation, DNA repair, and maintenance of genomic stability. The second most associated signal is composed of five variants that fall slightly below the genome-wide significance threshold but point out another interesting gene candidate. These SNPs, with p values between 2.53 × 10-6 and 3.34 × 10-6, are situated in the methylene tetrahydrofolate reductase (MTHFR) gene. The most associated of these variants, rs1537514, presents an increased frequency of the derived C allele in cases, with 11.4% versus 4.4% in controls. MTHFR is the rate-limiting enzyme in the methyl cycle and genetic variations in this gene have been strongly associated with the risk of neoplastic diseases. The current understanding of the MDS biology, which is based on the hypothesis of the sequential development of multiple subclonal molecular lesions, fits very well with the demonstration of a possible role for RTEL1 and MTHFR gene polymorphisms, both of which are related to a variable risk of genomic instability.

N-acetyltransferase polymorphisms are associated with risk of lymphoma subtypes.

Genes encoding for arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) have been investigated with alternate findings in relation to risk of non-Hodgkin lymphoma (NHL). We tested functional haplotype-based NAT1 and NAT2 gene polymorphisms in relation to risk of lymphoma overall and its major B cell subtypes, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). We used allele specific primers and multiplex PCR to detect NAT1 and NAT2 haplotypes in 248 patients with incident lymphoma and 208 population controls. We inferred the NAT1 rapid and slow acetylator and the NAT2 rapid, intermediate or slow acetylator phenotype, based on published functional data on the respective genotypes. Odds ratios and 95% confidence intervals (95% CIs) for lymphoma, B-NHL, DLBCL, FL, CLL, and other B-NHL combined associated with the inferred rapid NAT1 acetylator and with the intermediate and slow NAT2 acetylator phenotypes were estimated with unconditional and polytomous logistic regression analysis, adjusting for age, gender and education. NAT1 rapid acetylators showed a 2.8-fold excess risk (95% CI 1.5-5.2) for lymphoma (all subtypes combined). Risk was highest for CLL and FL, with significant heterogeneity detected across subtypes. Risk also increased with decreasing NAT2 acetylating capacity with no heterogeneity detected across B cell lymphoma subtypes. Risks did not vary by gender. Although poor statistical power was a major limitation in our study, larger studies and pooled analyses are warranted to test whether NAT1 and NAT2 gene polymorphisms might modulate risk of specific lymphoma subtypes through the varying metabolic activity of their products. Copyright © 2015 John Wiley & Sons, Ltd.

Risk of lymphoma subtypes and dietary habits in a Mediterranean area.

BACKGROUND: Previous studies have suggested that diet might affect risk of lymphoma subtypes. We investigated risk of lymphoma and its major subtypes associated with diet in the Mediterranean island of Sardinia, Italy. METHODS: In 1998-2004, 322 incident lymphoma cases and 446 randomly selected population controls participated in a case-control study on lymphoma etiology in central-southern Sardinia. Questionnaire interviews included frequency of intake of 112 food items. Risk associated with individual dietary items and groups thereof was explored by unconditional and polytomous logistic regression analysis, adjusting by age, gender and education. RESULTS: We observed an upward trend in risk of lymphoma (all subtypes combined) and B-cell lymphoma with frequency of intake of well done grilled/roasted chicken (p for trend=0.01), and pizza (p for trend=0.047), Neither adherence to Mediterranean diet nor a frequent intake of its individual components conveyed protection. We detected heterogeneity in risk associated with several food items and groups thereof by lymphoma subtypes although we could not rule out chance as responsible for the observed direct or inverse associations. CONCLUSIONS: Adherence to a Mediterranean diet does not seem to convey protection against the development of lymphoma. The association with specific food items might vary by lymphoma subtype.

Genome-wide association study identifies five susceptibility loci for follicular lymphoma outside the HLA region.

Genome-wide association studies (GWASs) of follicular lymphoma (FL) have previously identified human leukocyte antigen (HLA) gene variants. To identify additional FL susceptibility loci, we conducted a large-scale two-stage GWAS in 4,523 case subjects and 13,344 control subjects of European ancestry. Five non-HLA loci were associated with FL risk: 11q23.3 (rs4938573, p = 5.79 × 10(-20)) near CXCR5; 11q24.3 (rs4937362, p = 6.76 × 10(-11)) near ETS1; 3q28 (rs6444305, p = 1.10 × 10(-10)) in LPP; 18q21.33 (rs17749561, p = 8.28 × 10(-10)) near BCL2; and 8q24.21 (rs13254990, p = 1.06 × 10(-8)) near PVT1. In an analysis of the HLA region, we identified four linked HLA-DRß1 multiallelic amino acids at positions 11, 13, 28, and 30 that were associated with FL risk (pomnibus = 4.20 × 10(-67) to 2.67 × 10(-70)). Additional independent signals included rs17203612 in HLA class II (odds ratio [OR(per-allele)] = 1.44; p = 4.59 × 10(-16)) and rs3130437 in HLA class I (OR(per-allele) = 1.23; p = 8.23 × 10(-9)). Our findings further expand the number of loci associated with FL and provide evidence that multiple common variants outside the HLA region make a significant contribution to FL risk.

Methylation analysis of the phosphates and tensin homologue on chromosome 10 gene (PTEN) in multiple myeloma.

BACKGROUND: Aberrant DNA methylation of promoter region CpG islands is an alternative mechanism that leads to genetic defects in the inactivation of tumor suppressor genes during myelomagenesis. The aim of this study was to examine the promoter methylation status of the phosphates and tensin homologue on chromosome 10 (PTEN) gene in a cohort of multiple myeloma patients. FINDINGS: The PTEN gene was hypermethylated in 7 out of 58 (12%) primary myeloma samples. The correlation between functional inactivation and PTEN mRNA levels was not statistically significant. The multiple myeloma subgroup with an aberrant PTEN status had a prevalence of the component IgG, Salmon Durie stage I, lower lactate dehydrogenase levels, intermediate-standard cytogenetic risk and longer overall survival with the respect to the unmethylated subgroup. CONCLUSIONS: This is the first report demonstrating the presence of PTEN promoter hypermethylation in multiple myeloma.

Patients with early-stage myelodysplastic syndromes show increased frequency of CD4+CD25+CD127(low) regulatory T cells.

Regulatory T cells (T(reg)) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed T(reg) frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25(high)+CD127(low) T(reg) than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal disorders.

Dissecting the genetic make-up of North-East Sardinia using a large set of haploid and autosomal markers.

Sardinia has been used for genetic studies because of its historical isolation, genetic homogeneity and increased prevalence of certain rare diseases. Controversy remains concerning the genetic substructure and the extent of genetic homogeneity, which has implications for the design of genome-wide association studies (GWAS). We revisited this issue by examining the genetic make-up of a sample from North-East Sardinia using a dense set of autosomal, Y chromosome and mitochondrial markers to assess the potential of the sample for GWAS and fine mapping studies. We genotyped individuals for 500K single-nucleotide polymorphisms, Y chromosome markers and sequenced the mitochondrial hypervariable (HVI-HVII) regions. We identified major haplogroups and compared these with other populations. We estimated linkage disequilibrium (LD) and haplotype diversity across autosomal markers, and compared these with other populations. Our results show that within Sardinia there is no major population substructure and thus it can be considered a genetically homogenous population. We did not find substantial differences in the extent of LD in Sardinians compared with other populations. However, we showed that at least 9% of genomic regions in Sardinians differed in LD structure, which is helpful for identifying functional variants using fine mapping. We concluded that Sardinia is a powerful setting for genetic studies including GWAS and other mapping approaches.

AIDA 0493 protocol for newly diagnosed acute promyelocytic leukemia: very long-term results and role of maintenance.

All-trans-retinoic acid (ATRA) has greatly modified the prognosis of acute promyelocytic leukemia; however, the role of maintenance in patients in molecular complete remission after consolidation treatment is still debated. From July 1993 to May 2000, 807 genetically proven newly diagnosed acute promyelocytic leukemia patients received ATRA plus idarubicin as induction, followed by 3 intensive consolidation courses. Thereafter, patients reverse-transcribed polymerase chain reaction-negative for the PML-RARA fusion gene were randomized into 4 arms: oral 6-mercaptopurine and intramuscular methotrexate (arm 1); ATRA alone (arm 2); 3 months of arm1 alternating to 15 days of arm 2 (arm 3); and no further therapy (arm 4). Starting from February 1997, randomization was limited to ATRA-containing arms only (arms 2 and 3). Complete remission was achieved in 761 of 807 (94.3%) patients, and 681 completed the consolidation program. Of these, 664 (97.5%) were evaluated for the PML-RARA fusion gene, and 586 of 646 (90.7%) who tested reverse-transcribed polymerase chain reaction-negative were randomized to maintenance. The event-free survival estimate at 12 years was 68.9% (95% confidence interval, 66.4%-71.4%), and no differences in disease-free survival at 12 years were observed among the maintenance arms.

Household contact with pets and birds and risk of lymphoma.

BACKGROUND: Contact with household pets has been suggested to be inversely associated with lymphoma risk. METHODS: We tested the hypothesis in a case-control study of lymphoma in the Sardinia region of Italy. Cases were 326 patients, first diagnosed with lymphoma in 1999-2003. Controls were 464 population controls, frequency matched to cases by age, gender, and area of residence. In person interviews included self-reported household contact with pets and birds, type of pet(s), and age at starting contact. RESULTS: Frequent contact with birds was inversely associated with lymphoma, and particularly B-cell non-Hodgkin lymphoma (odds ratio [OR] = 0.6, 95% confidence interval [95% CI]: 0.4, 0.9). Contact with chickens accounted for this inverse association, which was strongest for first contact occurring at age ≤8 years (OR = 0.4, 95% CI: 0.2, 1.0). No association was observed when first contact occurred at age 9 or older. Contact with any pets was inversely associated with risk of diffuse large B-cell lymphoma (OR = 0.4, 95% CI: 0.2, 1.0), but not other lymphoma subtypes. CONCLUSION: Our results support the hypothesis that early-life exposure to pets, birds and particularly with chickens might be associated with a reduced risk of lymphoma.

Protein tyrosine phosphatase receptor type {gamma} is a functional tumor suppressor gene specifically downregulated in chronic myeloid leukemia.

Chronic myelogenous leukemia (CML) is the most common myeloproliferative disease. Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene and a myeloid cell marker expressed by CD34(+) cells. Downregulation of PTPRG increases colony formation in the PTPRG-positive megakaryocytic cell lines MEG-01 and LAMA-84 but has no effect in the PTPRG-negative cell lines K562 and KYO-1. Its overexpression has an oncosuppressive effect in all these cell lines and is associated with myeloid differentiation and inhibition of BCR/ABL-dependent signaling. The intracellular domain of PTPRG directly interacts with BCR/ABL and CRKL, but not with signal transducers and activators of transcription 5. PTPRG is downregulated at the mRNA and protein levels in leukocytes of CML patients in both peripheral blood and bone marrow, including CD34(+) cells, and is reexpressed following molecular remission of disease. Reexpression was associated with a loss of methylation of a CpG island of PTPRG promoter occurring in 55% of the patients analyzed. In K562 cell line, the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced PTPRG expression and caused an inhibition of colony formation, partially reverted by downregulation of PTPRG expression. These findings establish, for the first time, PTPRG as a tumor suppressor gene involved in the pathogenesis of CML, suggesting its use as a potential diagnostic and therapeutic target.

Melphalan 200 mg/m(2) versus melphalan 100 mg/m(2) in newly diagnosed myeloma patients: a prospective, multicenter phase 3 study.

High-dose (200 mg/m(2), MEL200) and intermediate-dose melphalan (100 mg/m(2), MEL100) showed significant activity in myeloma. In a phase 3 study, 298 patients were randomly assigned to receive 2 autologous transplantations after conditioning with MEL200 or MEL100. Ninety-six of 149 (64%) completed MEL200 and 103 of 149 (69%) MEL100. Best response to MEL200 was: complete remission 22 of 149 (15%); partial remission 95 of 149 (64%), for an overall response rate of 79%. Best response to MEL100 was: complete remission 12 of 149 (8%); partial remission 95 of 149 (64%), for an overall response rate of 72%. Overall survival did not differ (P = .13); median progression-free survival (31.4 vs 26.2 months, P = .01), median time to progression (34.4 vs 27.0 months, P = .014) were longer in the MEL200. Treatment-related mortality was 3.1% in the MEL200 and 2.9% in the MEL100 group. Severe neutropenia and infections were marginally superior, whereas severe thrombocytopenia, mucositis, gastrointestinal adverse events, and the overall occurrence of at least 1 nonhematologic grade 3 or 4 adverse event were significantly higher in the MEL200 cohort. We conclude that MEL200 leads to longer remission duration and should be considered the standard conditioning regimen for autologous transplantation. This study was registered at www.clinicaltrials.gov as #NCT00950768.

A role of BRCA1 and BRCA2 germline mutations in breast cancer susceptibility within Sardinian population.

BACKGROUND: In recent years, numerous studies have assessed the prevalence of germline mutations in BRCA1 and BRCA2 genes in various cohorts. We here extensively investigated the prevalence and geographical distribution of BRCA1-2 mutations in the entire genetically-homogeneous Sardinian population. The occurrence of phenotypic characteristics which may be predictive for the presence of BRCA1-2 germline mutations was also evaluated. METHODS: Three hundred and forty-eight breast cancer patients presenting a familial recurrence of invasive breast or ovarian carcinoma with at least two affected family members were screened for BRCA1-2 mutations by DHPLC analysis and DNA sequencing. Association of BRCA1 and BRCA2 mutational status with clinical and pathological parameters was evaluated by Pearson's Chi-Squared test. RESULTS AND CONCLUSION: Overall, 8 BRCA1 and 5 BRCA2 deleterious mutations were detected in 35/348 (10%) families; majority (23/35;66%) of mutations was found in BRCA2 gene. The geographical distribution of BRCA1-2 mutations was related to three specific large areas of Sardinia, reflecting its ancient history: a) the Northern area, linguistically different from the rest of the island (where a BRCA2 c.8764_8765delAG mutation with founder effect was predominant); b) the Middle area, land of the ancient Sardinian population (where BRCA2 mutations are still more common than BRCA1 mutations); and c) the South-Western area, with many Phoenician and Carthaginian locations (where BRCA1 mutations are prevalent). We also found that phenotypic features such as high tumor grading and lack of expression of estrogen/progesterone receptors together with age at diagnosis and presence of ovarian cancer in the family may be predictive for the presence of BRCA1-2 germline mutations.

Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias.

Only limited data are available regarding myocardial iron overload in adult patients with transfusion dependent acquired anemias. To address this topic using MRI T2* we studied 27 consecutive chronic transfusion dependent patients with acquired anemias: (22 myelodysplastic syndrome, 5 primary myelofibrosis). Cardiac MRI T2* values obtained ranged from 5.6 to 58.7 (median value 39.8) milliseconds. Of the 24 analyzable patients, cardiac T2* correlated with transfusion burden (p=0.0002). No patient who had received less than 290 mL/kg of packed red blood cells (101 units=20 grams of iron) had a pathological cardiac T2* value (< 20 ms). All patients who had received at least 24 PRBC units showed MRI T2* detectable hepatic iron (liver T2* value

Risk of malignant lymphoma following viral hepatitis infection.

We investigated lymphoma risk following hepatitis infection in a case-control study of 274 incident lymphoma cases, defined according to the WHO classification, and 336 population controls in Sardinia, Italy. Part of our study population (198 cases and 219 controls) was included in the EPILYMPH study of Hepatitis C virus (HCV) infection in relation to non-Hodgklin's lymphoma risk. Based on questionnaire information on whether and at what age a diagnosis of hepatitis was posed by a physician, systematic anti-HCV antibodies testing in cases and controls by enzyme-linked immunoassay, and HCV-RNA assessment by PCR analyses in positive samples, we investigated more in detail whether hepatitis non-C is also associated with lymphoma risk, and whether risk varies by clinical form of hepatitis (acute or chronic infection). After adjusting by age, gender, education, and area of birth whether from the study area or elsewhere in Italy, a previous generic diagnosis of hepatitis was associated with a significantly elevated lymphoma risk [odds ratio (OR) = 1.8; 95% CI 1.1, 2.8], which was equally increased for hepatitis B (OR = 1.8; 95% CI 0.9, 3.5), for HCV positive subjects overall (OR = 2.0; 95% CI 0.8, 4.8), and for hepatitis non-B non-C (OR = 1.6; 95% CI 0.7, 3.9). Once concurrent infection from other hepatitis viruses was excluded, acute or chronic hepatitis C was the only one showing a consistent risk increase in all lymphoma subtypes, but follicular lymphoma. Some indications of an excess risk of lymphoma were observed also for acute, but not chronic forms of hepatitis B and hepatitis non-B, non C. Self-limited hepatitis C did not show an association. No significant heterogeneity in the risk of major lymphoma subtype was observed. Our results confirm a role of either acute or chronic active HCV infection in lymphomagenesis. Further studies are warranted to test the hypothesis that acute infection from other hepatitis viruses might also increase lymphoma risk.

Interleukin-1B (IL1B) and interleukin-6 (IL6) gene polymorphisms are associated with risk of chronic lymphocytic leukaemia.

Common polymorphisms in genes encoding for cytokines implicated in the inflammatory response and Th1/Th2 balance might play a role in the development and prognosis of chronic lymphocytic leukaemia (CLL). To test the hypothesis, we investigated 13 single nucleotide polymorphisms (SNPs) in nine of such genes in a population-based case-control study, conducted in the Italian region of Sardinia in 1999-2003. Forty incident CLL cases and 113 population controls were available for study. The following SNPs were selected: IL1A-889C > T, IL1RN 9589A > T, IL1B-31C > T, IL1B-511C > T, IL2-384T > G, IL6-174G > C, IL6-597G > A, IL10-1082A > G, IL10-3575T > A, TNF-308G > A, LTA- 91A > C, LTA 252A > G and CARD15 nt1007. After adjusting by age and gender, individuals homozygous for the IL1B-511T allele run a lower risk of CLL (OR = 0.1, 95% CI 0.0, 0.8, p = 0.032), while risk showed a 4.5-fold increase associated with the genotype homozygous for the IL6-174C allele (OR = 4.5; 95% CI 1.1, 19.3, p = 0.041). Individuals homozygous for the IL6-174C allele and carrying the homozygous IL1B-511C allele showed an 11-fold increase in CLL risk (OR = 11.4, 95% CI 1.9, 69.4, p = 0.008). None of the other interleukin SNPs evaluated showed any association with CLL risk. Large multicentre pooled studies are warranted, achieving the statistical power required to confirm whether IL6 and IL1B gene polymorphisms might play a role in CLL development and prognosis, as well as the null associations herein reported.