Discovery of novel predisposing coding and noncoding variants in familial Hodgkin lymphoma.

Familial aggregation of Hodgkin lymphoma (HL) has been demonstrated in large population studies, pointing to genetic predisposition to this hematological malignancy. To understand the genetic variants associated with the development of HL, we performed whole genome sequencing on 234 individuals with and without HL from 36 pedigrees that had 2 or more first-degree relatives with HL. Our pedigree selection criteria also required at least 1 affected individual aged <21 years, with the median age at diagnosis of 21.98 years (3-55 years). Family-based segregation analysis was performed for the identification of coding and noncoding variants using linkage and filtering approaches. Using our tiered variant prioritization algorithm, we identified 44 HL-risk variants in 28 pedigrees, of which 33 are coding and 11 are noncoding. The top 4 recurrent risk variants are a coding variant in KDR (rs56302315), a 5' untranslated region variant in KLHDC8B (rs387906223), a noncoding variant in an intron of PAX5 (rs147081110), and another noncoding variant in an intron of GATA3 (rs3824666). A newly identified splice variant in KDR (c.3849-2A>C) was observed for 1 pedigree, and high-confidence stop-gain variants affecting IRF7 (p.W238∗) and EEF2KMT (p.K116∗) were also observed. Multiple truncating variants in POLR1E were found in 3 independent pedigrees as well. Whereas KDR and KLHDC8B have previously been reported, PAX5, GATA3, IRF7, EEF2KMT, and POLR1E represent novel observations. Although there may be environmental factors influencing lymphomagenesis, we observed segregation of candidate germline variants likely to predispose HL in most of the pedigrees studied.

Association of polygenic risk score with the risk of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

Inherited loci have been found to be associated with risk of chronic lymphocytic leukemia (CLL). A combined polygenic risk score (PRS) of representative single nucleotide polymorphisms (SNPs) from these loci may improve risk prediction over individual SNPs. Herein, we evaluated the association of a PRS with CLL risk and its precursor, monoclonal B-cell lymphocytosis (MBL). We assessed its validity and discriminative ability in an independent sample and evaluated effect modification and confounding by family history (FH) of hematological cancers. For discovery, we pooled genotype data on 41 representative SNPs from 1499 CLL and 2459 controls from the InterLymph Consortium. For validation, we used data from 1267 controls from Mayo Clinic and 201 CLL, 95 MBL, and 144 controls with a FH of CLL from the Genetic Epidemiology of CLL Consortium. We used odds ratios (ORs) to estimate disease associations with PRS and c-statistics to assess discriminatory accuracy. In InterLymph, the continuous PRS was strongly associated with CLL risk (OR, 2.49; P = 4.4 × 10-94). We replicated these findings in the Genetic Epidemiology of CLL Consortium and Mayo controls (OR, 3.02; P = 7.8 × 10-30) and observed high discrimination (c-statistic = 0.78). When jointly modeled with FH, PRS retained its significance, along with FH status. Finally, we found a highly significant association of the continuous PRS with MBL risk (OR, 2.81; P = 9.8 × 10-16). In conclusion, our validated PRS was strongly associated with CLL risk, adding information beyond FH. The PRS provides a means of identifying those individuals at greater risk for CLL as well as those at increased risk of MBL, a condition that has potential clinical impact beyond CLL.

Parental longevity and survival among patients with multiple myeloma and monoclonal gammopathy of undetermined significance: a population-based study.

Parental longevity is associated with an increased life expectancy; results with regard to specific diseases are conflicting. There are limited data focusing on host characteristics and their effect on survival among multiple myeloma (MM) patients and individuals with monoclonal gammopathy of undetermined significance (MGUS). Therefore, our aim was to evaluate the impact of parental longevity on survival of patients with MM and MGUS. A total of 4675 patients with MM, 6812 MGUS patients and 13 398 population-based controls for MM as well as 19 110 controls for MGUS, from 1988 to 2013, were included in the study. Longevity was defined as >90 years of age. Among MM patients, parental longevity was associated with a decreased risk of death [hazard ratio (HR) = 0·92, 95% confidence interval (CI) 0·84-0·99] and the same was true for MGUS patients (HR = 0·87, 95% CI 0·78-0·96). Having one long lived parent significantly decreased the risk of death in both groups, but was not statistically significant when both parents exceeded 90 years of age. In conclusion, parental longevity decreases the risk of death for patients with MM and MGUS which may reflect the importance of the host's genetic and environmental factors in relation to survival.

Combined somatic mutation and copy number analysis in the survival of familial CLL.

Recurrent large-scale somatic copy number alterations (SCNAs), and somatic point mutations can be analysed to stratify patients with chronic lymphocytic leukaemia (CLL) into distinct prognostic groups. To investigate the relationship between SCNAs and somatic mutations, we performed whole-exome sequencing and single nucleotide polymorphism microarray analyses on 98 CLL patients from 40 families with a high burden of CLL. Overall, 69 somatic mutations in 29 CLL driver genes were detected among 45 subjects (46%), with the most frequently mutated genes being TP53 (8·2%), NOTCH1 (8·2%) and ATM (5·1%). Additionally, 142 SCNAs from 54 subjects (57%) were detected, including losses of chromosome 13q14 (28·9%), 11q (5·6%), 17p (2·1%), and gain of chromosome 12 (4·2%). We found that patients having both an adverse point mutation in a CLL driver gene and an unfavourable SCNA tended to have poorer survival (Hazard ratio [HR] = 3·17, 95% confidence interval [CI] = 0·97-10·35; P = 0·056) than patients having either a point mutation (HR = 1·34, 95%CI = 0·66-2·71; P = 0·42) or SCNAs (HR = 2·65, 95%CI = 0·77-9·13; P = 0·12). TP53 mutation carriers were associated with the poorest overall survival (HR = 4·39, 95%CI = 1·28-15·04; P = 0·018). Our study suggests that combining SCNA and mutational data could contribute to predicting outcome in familial CLL.

Germline mutations in Protection of Telomeres 1 in two families with Hodgkin lymphoma.

In a previous whole exome sequencing of patients from 41 families with Hodgkin lymphoma, we identified two families with distinct heterozygous rare coding variants in POT1 (D224N and Y36H), both in a highly conserved region of the gene. POT1 D224N mutant did not bind to a single-stranded telomere oligonucleotide in vitro suggesting the mutation perturbs POT1's ability to bind to the telomeric G-rich overhang. Human HT1080 cells expressing POT1 D224N and lymphoblastoid cells carrying Y36H both showed increased telomere length and fragility in comparison to wild type cells. This strongly suggests that mutant POT1 causes chromosome instability and may play a role in lymphomagenesis in these families.

History of autoimmune disease is associated with impaired survival in multiple myeloma and monoclonal gammopathy of undetermined significance: a population-based study.

Multiple myeloma (MM) is a plasma cell disorder preceded by monoclonal gammopathy of undetermined significance (MGUS). Incidence of MM and MGUS is higher among patients with autoimmune disease. The aim of this study was to determine whether a history of autoimmunity has an impact on survival in MM and MGUS. Using high-quality national Swedish registries, we identified 8367 patients with MM, 18,768 patients with MGUS, and 110,251 matched control subjects, and obtained information on previous autoimmune disease in patients and controls. Cox regression was used to calculate hazard ratios (HRs) for overall survival with 95 % confidence intervals (CIs). In patients with MM and a prior autoimmune disease, the risk of death was significantly increased, HR = 1.2 (95 % CI 1.2-1.3) compared to MM patients with no history of autoimmunity. In MGUS patients, a prior autoimmune disease was associated with a significantly 1.4-fold elevated risk of death (95 % CI 1.3-1.4). When analyzing different types of autoimmune diseases, a history of ulcerative colitis had a stronger impact on survival in MM than in controls. Our findings that a history of autoimmune disease has a negative impact on survival in MM and MGUS could be due to shared underlying common genetic factors, or that patients with a history of autoimmunity develop more severe cases of MM and MGUS, or cumulative comorbidity in the individual. Our results suggest that more attention should be paid to comorbidity as a prognostic factor in MGUS and MM, and underlines the need for studies aimed at tailoring therapy according to comorbidity.

Mosaic 13q14 deletions in peripheral leukocytes of non-hematologic cancer cases and healthy controls.

Loss of 13q14.3 is a chromosomal event found in ~50% of B-cell chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) cases. Surveys of somatic alterations in solid tumors have shown sporadic 13q14.3 loss in many different tumor types, but not at high frequency in any specific tumor type. In our recent survey of the single-nucleotide polymorphism (SNP) microarray data from 127 000 cancer-free or solid tumor cases, we observed mosaic 13q14.3 loss as common autosomal somatic large structural events (>2 Mb in size) in blood and buccal-derived DNA. Herein, we examined this region more closely investigating structural mosaic events <2 Mb using SNP microarray data in 46 254 non-hematologic cancer cases and 36 229 controls. We detected 60 individuals with 13q14.3 mosaic loss, 1 mosaic copy neutral uniparental disomy and 13 individuals with homozygosity. Although 13q14.3 loss size was variable, the minimally deleted region (MDR) (chr13: 49 590 000-49 983 100; GRCh36) was comparable to what is classically reported in MBL and CLL. Breakpoint analysis of the estimated boundaries reveals enrichment for genes and open chromatin. The frequency of 13q14.3 loss significantly increases with increasing age (P-value=0.028), but was not significantly different between non-hematological cancer cases and controls (0.084% versus 0.058%; P-value=0.19). These findings suggest that mosaic 13q14.3 losses accumulate with age. Individuals with detected mosaic 13q14.3 deletions may be early, undetected cases of MBL or CLL, but not necessarily all will develop MBL and CLL.

Monoclonal gammopathy of undetermined significance and risk of lymphoid and myeloid malignancies: 728 cases followed up to 30 years in Sweden.

In 728 Swedish cases of monoclonal gammopathy of undetermined significance (MGUS), followed up to 30 years (median, 10 years), we estimated the cumulative risk of hematologic disorders originating from lymphoid and myeloid lineages. Using Cox regression models, we examined associations of demographic and laboratory factors with progression and determined the discriminatory power of 3 prediction models for progression. Eighty-four MGUS cases developed a lymphoid disorder, representing a cumulative risk of 15.4%. Multiple myeloma (MM) occurred in 53 patients, and the 30-year cumulative risk was 10.6%; an ∼0.5% annual risk. Three factors were significantly associated with progression: abnormal free light-chain (FLC) ratio (<0.26 or >1.65), M-protein concentration (≥1.5 g/dL), and reduction of 1 or 2 noninvolved immunoglobulin isotype levels (immunoparesis). A prediction model with separate effects for these 3 factors and the M-protein isotype had higher discriminatory power than other models, although the differences were not statistically significant. The 30-year cumulative risk for myeloid malignancies was <2%. Our study confirms that abnormal FLC ratio and M-protein concentration >1.5 g/dL, factors previously considered by Mayo Clinic researchers, are predictors for MM progression and suggests that separate consideration of immunoparesis and the Mayo Clinic risk factors could improve identification of MGUS patients at high risk for progression.

Whole exome sequencing in families at high risk for Hodgkin lymphoma: identification of a predisposing mutation in the KDR gene.

Hodgkin lymphoma shows strong familial aggregation but no major susceptibility genes have been identified to date. The goal of this study was to identify high-penetrance variants using whole exome sequencing in 17 Hodgkin lymphoma prone families with three or more affected cases or obligate carriers (69 individuals), followed by targeted sequencing in an additional 48 smaller HL families (80 individuals). Alignment and variant calling were performed using standard methods. Dominantly segregating, rare, coding or potentially functional variants were further prioritized based on predicted deleteriousness, conservation, and potential importance in lymphoid malignancy pathways. We selected 23 genes for targeted sequencing. Only the p.A1065T variant in KDR (kinase insert domain receptor) also known as VEGFR2 (vascular endothelial growth factor receptor 2) was replicated in two independent Hodgkin lymphoma families. KDR is a type III receptor tyrosine kinase, the main mediator of vascular endothelial growth factor induced proliferation, survival, and migration. Its activity is associated with several diseases including lymphoma. Functional experiments have shown that p.A1065T, located in the activation loop, can promote constitutive autophosphorylation on tyrosine in the absence of vascular endothelial growth factor and that the kinase activity was abrogated after exposure to kinase inhibitors. A few other promising mutations were identified but appear to be "private". In conclusion, in the largest sequenced cohort of Hodgkin lymphoma families to date, we identified a causal mutation in the KDR gene. While independent validation is needed, this mutation may increase downstream tumor cell proliferation activity and might be a candidate for targeted therapy.

Whole exome sequencing reveals a C-terminal germline variant in CEBPA-associated acute myeloid leukemia: 45-year follow up of a large family.

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.

Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Common variation at 6p21.31 (BAK1) influences the risk of chronic lymphocytic leukemia.

We performed a meta-analysis of 3 genome-wide association studies to identify additional common variants influencing chronic lymphocytic leukemia (CLL) risk. The discovery phase was composed of genome-wide association study data from 1121 cases and 3745 controls. Replication analysis was performed in 861 cases and 2033 controls. We identified a novel CLL risk locus at 6p21.33 (rs210142; intronic to the BAK1 gene, BCL2 antagonist killer 1; P = 9.47 × 10(-16)). A strong relationship between risk genotype and reduced BAK1 expression was shown in lymphoblastoid cell lines. This finding provides additional support for polygenic inheritance to CLL and provides further insight into the biologic basis of disease development.

Infection in infancy and subsequent risk of developing lymphoma in children and young adults.

There is evidence that certain infections and autoimmunity predispose to the development of non-Hodgkin lymphomas (NHLs). A previous study reported that hospitalization for infections in infancy led to an increased risk of NHL. By using population-based registries in Sweden, we compared the rate of hospitalization for infections in infancy between lymphoma cases and matched controls for patients born since 1964. A history of infection was associated with a significantly increased risk of aggressive B-cell lymphomas (odds ratio 2.1, 95% confidence interval 1.11-4.04, P = .02). The specific infections involved were respiratory and intestinal. No effects were observed among cases of Hodgkin lymphoma. This association could result from the infection, its treatment, or could be a surrogate marker for underlying immune defects. Further studies are needed to determine whether this association is present among NHL occurring in older adults and if improved survival of patients with immune defects has contributed to the secular increases in incidence of NHLs.

Personal and family history of immune-related conditions increase the risk of plasma cell disorders: a population-based study.

The associations between immune-related conditions and multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) have previously been investigated with inconsistent results. In a large population-based study, we identified 19 112 patients with MM, 5403 patients with MGUS, 96 617 matched control subjects, and 262 931 first-degree relatives. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) for the association of MM and MGUS with immune-related conditions by use of logistic regression. A personal history of all infections combined was associated with a significantly increased risk of MM (OR = 1.2; 95% CI, 1.1-1.3), and a personal history of all conditions in the categories infections (OR = 1.6; 95% CI, 1.5-1.7), inflammatory conditions (OR = 1.4; 95% CI, 1.2-1.5), and autoimmune diseases (OR = 2.1; 95% CI, 1.9-2.4) was associated with a significantly increased risk of MGUS. Several specific immune-related conditions elevated the risk of MM and/or MGUS. A family history of autoimmune disease was associated with a significantly increased risk of MGUS (OR = 1.1; 95% CI, 1.00-1.2), but not MM. Our findings suggest that immune-related conditions and/or their treatment are of importance in the etiology of MGUS and possibly MM. The association of both personal and family history of autoimmune disease with MGUS indicates the potential for shared susceptibility for these conditions.

Risk of acute myeloid leukemia and myelodysplastic syndromes after multiple myeloma and its precursor disease (MGUS).

Using population-based data from Sweden, we identified all multiple myeloma (MM) patients (n = 8740) and 5652 monoclonal gammopathy of undetermined significance (MGUS) patients diagnosed between 1986 and 2005. We calculated standardized incidence rates (SIRs) for all subsequent hematologic and nonhematologic malignancies for MM patients diagnosed before/after 1995 (introduction of high-dose melphalan/autologous stem cell transplantation [HDM-ASCT]) and 2000 (introduction of immunomodulatory drugs [IMiDs]), respectively. MM patients had an 11.51-fold (95% confidence interval: 8.19-15.74) increased risk of acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS); risk was very similar before/after 1995 and 2000, respectively. MGUS patients had an 8.01-fold (5.40-11.43) increased risk of AML/MDS. Risk was confined to IgG/IgA, while no IgM MGUS patients developed AML/MDS; patients with monoclonal-protein (M-protein) concentrations > 1.5 g/dL (SIR = 11.12; 3.61-25.96) had higher risk than those < 1.5 g/dL (SIR = 4.67; 1.71-10.16). An excess risk of nonmelanoma skin cancer was observed subsequent to both MM (SIR = 2.22; 1.74-2.80) and MGUS (SIR = 3.30; 2.76-3.90). Our novel observations of an excess risk for AML/MDS following IgG/IgA (but not IgM) MGUS, and the highest risk associated with M-protein concentrations > 1.5 g/dL, support a role for nontreatment-related factors in plasma cell dyscrasias. AML/MDS risk following MM was the same before/after the introduction of HDM-ASCT. Longer follow-up is needed to characterize second tumor risks in the IMiD era.

Genome-wide association study identifies a novel susceptibility locus at 6p21.3 among familial CLL.

Prior genome-wide association (GWA) studies have identified 10 susceptibility loci for risk of chronic lymphocytic leukemia (CLL). To identify additional loci, we performed a GWA study in 407 CLL cases (of which 102 had a family history of CLL) and 296 controls. Moreover, given the strong familial risk of CLL, we further subset our GWA analysis to the CLL cases with a family history of CLL to identify loci specific to these familial CLL cases. Our top hits from these analyses were evaluated in an additional sample of 252 familial CLL cases and 965 controls. Using all available data, we identified and confirmed an independent association of 4 single-nucleotide polymorphisms (SNPs) that met genome-wide statistical significance within the IRF8 (interferon regulatory factor 8) gene (combined P values ≤ 3.37 × 10(-8)), located in the previously identified 16q24.1 locus. Subsetting to familial CLL cases, we identified and confirmed a new locus on chromosome 6p21.3 (combined P value = 6.92 × 10(-9)). This novel region harbors the HLA-DQA1 and HLA-DRB5 genes. Finally, we evaluated the 10 previously reported SNPs in the overall sample and replicated 8 of them. Our findings support the hypothesis that familial CLL cases have additional genetic variants not seen in sporadic CLL. Additional loci among familial CLL cases may be identified through larger studies.

A genome-wide association study of lung cancer identifies a region of chromosome 5p15 associated with risk for adenocarcinoma.

Three genetic loci for lung cancer risk have been identified by genome-wide association studies (GWAS), but inherited susceptibility to specific histologic types of lung cancer is not well established. We conducted a GWAS of lung cancer and its major histologic types, genotyping 515,922 single-nucleotide polymorphisms (SNPs) in 5739 lung cancer cases and 5848 controls from one population-based case-control study and three cohort studies. Results were combined with summary data from ten additional studies, for a total of 13,300 cases and 19,666 controls of European descent. Four studies also provided histology data for replication, resulting in 3333 adenocarcinomas (AD), 2589 squamous cell carcinomas (SQ), and 1418 small cell carcinomas (SC). In analyses by histology, rs2736100 (TERT), on chromosome 5p15.33, was associated with risk of adenocarcinoma (odds ratio [OR]=1.23, 95% confidence interval [CI]=1.13-1.33, p=3.02x10(-7)), but not with other histologic types (OR=1.01, p=0.84 and OR=1.00, p=0.93 for SQ and SC, respectively). This finding was confirmed in each replication study and overall meta-analysis (OR=1.24, 95% CI=1.17-1.31, p=3.74x10(-14) for AD; OR=0.99, p=0.69 and OR=0.97, p=0.48 for SQ and SC, respectively). Other previously reported association signals on 15q25 and 6p21 were also refined, but no additional loci reached genome-wide significance. In conclusion, a lung cancer GWAS identified a distinct hereditary contribution to adenocarcinoma.