BAFF-R is the primary BAFF receptor that is responsible for promoting B-cell development and survival. Malignant B-cells exploit the BAFF/BAFF receptor system and high serum BAFF levels or genetic alterations in BAFF receptors have been found in B-cell cancers. BAFF signaling impacts pro-survival pathways, however, other than NF-κB2, little is known about the specific pathways activated by individual BAFF receptors. Using a novel BAFF-R expression model we now demonstrate that activation of BAFF-R, independent of TACI and BCMA, can induce phosphorylation of Akt and GSK3β. Expression of an activated form of BAFF-R also enhanced a pro-survival gene expression pattern, including the novel BAFF-regulated gene Pin1, whose expression was PI3K-dependent. Additionally, we show that TRAF6 is essential for mediating BAFF-R-dependent activation of Akt. Together these data describe a novel role for TRAF6 in BAFF-R-specific activation of the PI3K pathway and provide evidence suggesting a new role for Pin1 in BAFF-R signaling.
BAFF-R; BAFF; lymphoma; PI3-kinase
Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. Here we perform a two-stage GWAS of 1,281 MZL cases and 7,127 controls of European ancestry and identify two independent loci near BTNL2 (rs9461741, P=3.95×10−15) and HLA-B (rs2922994, P=2.43×10−9) in the HLA region significantly associated with MZL risk. This is the first evidence that genetic variation in the major histocompatibility complex influences MZL susceptibility.
Transformation of follicular lymphoma (FL) is a critical event associated with a poor prognosis. The role of the tumor microenvironment in previous transformation studies has yielded conflicting results.
To define cell subtypes associated with transformation, we examined tissue specimens at diagnosis from patients with FL that later transformed and, using immunohistochemistry (IHC), stained for CD68, CD11c, CD21, CXCL13, FOXP3, PD-1 and CD14. Cell content and pattern of expression were evaluated. Those identified as significantly associated with time to transformation (TTT) and overall survival (OS) were further characterized by flow cytometry and multicolor IHC.
58 patients were analyzed with median TTT of 4.7 years. The pattern of PD1+ and CD14+ cells rather than the quantity of cells was predictive of clinical outcomes. On multivariate analysis including the FLIPI score, CD14+ cells localized in the follicle were associated with a shorter TTT (HR=3.0; p=0.004). PD-1+ cells with diffuse staining were associated with a shorter TTT (HR=1.9; p=0.045) and inferior OS (HR=2.5; p=0.012). Multicolor IHC and flow cytometry identified CD14+ cells as follicular dendritic cells (FDC) while PD1+ cells represented two separate populations, TFH and exhausted T-cells.
These results identify the presence of PD1+ T-cells and CD14+ FDC as independent predictors of transformation in follicular lymphoma.
CD14; PD1; follicular lymphoma; tumor microenvironment; time to transformation
Rituximab improves the outcome of patients with non-Hodgkin lymphoma, but does not completely eradicate residual B-cell populations in the microenvironment of the bone marrow and lymph nodes. Adhesion to stromal cells can protect B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance; CAM-DR). A similar mechanism of resistance to rituximab has not, to our knowledge, been described. We tested the hypothesis that the microenvironment protects malignant B-cells from rituximab-induced apoptosis, and that blocking these interactions with natalizumab, an antibody targeting VLA-4 (integrin alfa-4-beta-1/CD49d), can overcome this protection. VLA-4 is an adhesion molecule constitutively expressed on malignant B-cells and is important for pro-survival signalling in the bone marrow and lymph node microenvironment. The human bone marrow stromal cell line HS-5 was shown to strongly protect B-cell lymphoma cells from rituximab cytotoxicity, suggesting the existence of a stromal cell adhesion-mediated antibody resistance (CAM-AR) mechanism analogous to CAM-DR. Natalizumab decreased B-lymphocyte adherence to fibronectin by 75-95% and partially overcame stromal protection against rituximab and cytotoxic drugs. These pre-clinical findings suggest that the addition of stromal adhesion-disruptive drugs to rituximab-containing therapy could improve treatment efficacy.
lymphoma; stromal cells; cell adhesion-mediated drug resistance; rituximab; natalizumab
Marginal zone lymphoma (MZL) is the third most common subtype of B-cell non-Hodgkin lymphoma. Here we perform a two-stage GWAS of 1,281 MZL cases and 7,127 controls of European ancestry and identify two independent loci near BTNL2 (rs9461741, P=3.95 × 10−15) and HLA-B (rs2922994, P=2.43 × 10−9) in the HLA region significantly associated with MZL risk. This is the first evidence that genetic variation in the major histocompatibility complex influences MZL susceptibility.
Marginal zone lymphoma (MZL) is a common subtype of B-cell non-Hodgkin lymphoma. Here the authors carry out a two-stage genome-wide association study in over 8,000 Europeans and identify two new MZL risk loci at chromosome 6p, implicating the major histocompatibility complex in the disease for the first time.
While the International Prognostic Score (IPS) is the gold standard for risk-stratifying patients with classical Hodgkin lymphoma (cHL), these criteria do not accurately predict outcome. As cytokines are critically involved in driving cHL, we tested whether pretreatment serum cytokine levels could provide additional prognostic information.
Thirty cytokines were measured in pretreatment serum from 140 cHL patients and compared with 50 non-lymphoma controls. Patients were followed for event-free and overall survival, and Cox proportional hazards regression models were used to assess the association of individual cytokines and the cytokine profiles with outcome via unadjusted and IPS-adjusted hazard ratios (HR).
Twelve cytokines (EGF, FGFb, GCSF, HGF, IL-6, IL-8, IL-12, IL-2R, IP-10, MIG, TNFa and VEGF) were significantly (p<0.05) higher in cHL patients than controls; elevated levels of HGF, IL-6, IL-2R, IP-10 and MIG were all associated with poorer event-free survival (EFS). Only IL-2R (p=0.002) and IL-6 (p<0.001) were independently prognostic. Patients with increased IL-6 and IL-2R had a significantly higher risk of early relapse and death, a finding that remained significant even after IPS-based risk stratification. While elevated IL-6 and IL-2R correlated with the IPS, sCD30 and TARC levels, the 2-cytokine model remained independently predictive of prognosis.
Elevated pretreatment serum cytokines are associated with increased disease relapse and inferior survival in cHL. Thus, the pretreatment cytokine profile, particularly serum levels of IL-6 and IL-2R, may be used to identify cHL patients at high risk for early disease relapse.
Cytokines are important immune mediators of classical Hodgkin lymphoma (CHL) pathogenesis, and circulating levels at diagnosis may help predict prognosis. Germline single nucleotide polymorphisms (SNPs) in immune genes have been correlated with cytokine production and function.
We investigated whether selected germline SNPs in IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629), IL6 (rs1800795), ILRN (rs419598), INFG (rs2430561) and CCL17 (rs223828) were associated with circulating levels of related cytokines at diagnosis and progression-free survival (PFS) in CHL. Patients were from France (GELA, N = 464; median age = 32 years) and the United States (Iowa/Mayo Specialized Program Of Research Excellence [SPORE], N = 239; median age = 38 years); 22% of 346 CHL cases with EBV tumor status were positive.
There was no association with any of the SNPs with cytokine levels. Overall, there was no association of any of the SNPs with PFS. In exploratory analyses by EBV status, TNFA rs1800629 (HRAA/AG = 2.41; 95%CI, 1.17–4.94) was associated with PFS in EBV-negative GELA patients, with similar trends in the SPORE patients (HRAA/AG = 1.63; 95%CI, 0.61–4.40). In a meta-analysis of the two studies, TNFA (HRAA/AG = 2.11; 95%CI, 1.18–3.77; P = 0.01) was statistically significant, and further adjustment for the international prognostic system did not alter this result.
This study showed that germline variation in TNFA was associated with CHL prognosis for EBV-negative patients, which will require confirmation. These results support broader studies on the differential impact of genetic variation in immune genes on EBV-positive vs. EBV-negative CHL pathogenesis.
Hodgkin lymphoma; Cytokines; Polymorphism; TNFA; EBV
FCGR2A; FCGR3A; polymorphism; EBV; Hodgkin Lymphoma
Transforming growth factor beta (TGF-β) plays an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-β-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-β inhibits Tm cells, and TGF-β mediated exhaustion is associated with upregulation of CD70. We found that TGF-β upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naïve T cells. CD70 induction by TGF-β is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-β-induced exhaustion of effector Tm cells. Both TGF-β-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared to CD70− T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-β-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.
TGF-β; CD70; T-cell exhaustion; B-cell non-Hodgkin lymphoma
CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4+ and CD8+ T-cells, and skin-derived migratory dendritic cells. Together with its ligand, CXCL13, CXCR5 is involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. This study evaluated the role of common germline genetic variation in CXCR5 in the risk and prognosis of non-Hodgkin lymphoma (NHL) using a clinic-based study of 1521 controls and 2694 NHL cases including 710 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL), 586 diffuse large B-cell lymphoma (DLBCL), 588 follicular lymphoma (FL), 137 mantle cell lymphoma (MCL), 230 marginal zone lymphoma (MZL) and 158 peripheral T-cell lymphoma (PTCL). Of the ten CXCR5 tag SNPs in our study, five were associated with risk of NHL, with rs1790192 having the strongest association (OR=1.19, 95%CI 1.08–1.30; p=0.0003). This SNP was most strongly associated with the risk of FL (OR=1.44, 95%CI 1.25–1.66; p=3.1×10−7), with a lower degree of association with DLBCL (OR=1.16, 95%CI 1.01–1.33; p=0.04) and PTCL (OR=1.29, 95%CI 1.02–1.64; p=0.04) but no association with the risk of MCL or MZL. For FL patients that were observed as initial disease management, the number of minor alleles of rs1790192 was associated with better event-free survival (EFS) (HR=0.64; 95%CI 0.47–0.87; p=0.004). These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, particularly for FL.
non-Hodgkin lymphoma; SNPs; prognosis; prospective cohort; case-control
Motivation: Exome sequencing (exome-seq) data, which are typically used for calling exonic mutations, have also been utilized in detecting DNA copy number variations (CNVs). Despite the existence of several CNV detection tools, there is still a great need for a sensitive and an accurate CNV-calling algorithm with built-in QC steps, and does not require a paired reference for each sample.
Results: We developed a novel method named PatternCNV, which (i) accounts for the read coverage variations between exons while leveraging the consistencies of this variability across different samples; (ii) reduces alignment BAM files to WIG format and therefore greatly accelerates computation; (iii) incorporates multiple QC measures designed to identify outlier samples and batch effects; and (iv) provides a variety of visualization options including chromosome, gene and exon-level views of CNVs, along with a tabular summarization of the exon-level CNVs. Compared with other CNV-calling algorithms using data from a lymphoma exome-seq study, PatternCNV has higher sensitivity and specificity.
Availability and implementation: The software for PatternCNV is implemented using Perl and R, and can be used in Mac or Linux environments. Software and user manual are available at http://bioinformaticstools.mayo.edu/research/patterncnv/, and R package at https://github.com/topsoil/patternCNV/.
Supplementary data are available at Bioinformatics online.
Our genome-wide association study (GWAS) of chronic lymphocytic leukemia (CLL) identified 4 highly-correlated intronic variants within the IRF8 gene that were associated with CLL. These results were further supported by a recent meta-analysis of our GWAS with two other GWAS of CLL, supporting the IRF8 gene as a strong candidate for CLL risk.
To refine the genetic association of CLL risk, we performed Sanger sequencing of IRF8 in 94 CLL cases and 96 controls. We then performed fine-mapping by genotyping 39 variants (of which 10 were identified from sequencing) in 745 CLL cases and 1521 controls. We also assessed these associations with risk of other non-Hodgkin lymphoma (NHL) subtypes.
The strongest association with CLL risk was observed with a common SNP located within the 3’ UTR of IRF8 (rs1044873, log additive odds ratio = 0.7, P=1.81×10−6). This SNP was not associated with the other NHL subtypes (all P>0.05).
We provide evidence that rs1044873 in the IRF8 gene accounts for the initial GWAS signal for CLL risk. This association appears to be unique to CLL with little support for association with other common NHL subtypes. Future work is needed to assess functional role of IRF8 in CLL etiology.
These data provide support that a functional variant within the 3’ UTR of IRF8 may be driving the GWAS signal seen on 16q24.1 for CLL risk.
CLL; NHL; SNPs; IRF8; risk locus
A BAFF receptor mutation associated with non-Hodgkin lymphoma provides new insight into the proximal players of normal BAFF-R signaling.
The cytokine B cell activating factor (BAFF) and its receptor, BAFF receptor (BAFF-R), modulate signaling cascades critical for B cell development and survival. We identified a novel mutation in TNFRSF13C, the gene encoding human BAFF-R, that is present in both tumor and germline tissue from a subset of patients with non-Hodgkin lymphoma. This mutation encodes a His159Tyr substitution in the cytoplasmic tail of BAFF-R adjacent to the TRAF3 binding motif. Signaling through this mutant BAFF-R results in increased NF-κB1 and NF-κB2 activity and increased immunoglobulin production compared with the wild-type (WT) BAFF-R. This correlates with increased TRAF2, TRAF3, and TRAF6 recruitment to His159Tyr BAFF-R. In addition, we document a requirement for TRAF6 in WT BAFF-R signaling. Together, these data identify a novel lymphoma-associated mutation in human BAFF-R that results in NF-κB activation and reveals TRAF6 as a necessary component of normal BAFF-R signaling.
A recent meta-analysis of three genome-wide association studies of chronic lymphocytic leukaemia (CLL) identified two common variants at the 6p21.31 locus that are associated with CLL risk. To verify and further explore the association of these variants with other non-Hodgkin lymphoma (NHL) subtypes, we genotyped 1196 CLL cases, 1699 NHL cases, and 2410 controls. We found significant associations between the 6p21.31 variants and CLL risk (rs210134: P=0.01; rs210142: P=6.8×10−3). These variants also showed a trend towards association with some of the other NHL subtypes. Our results validate the prior work and support specific genetic pathways for risk among NHL subtypes.
CLL; NHL; SNPs; BAK1; risk locus
Abnormal immune function is a key factor in predisposition to non-Hodgkin lymphoma (NHL). We evaluated the association of 30 cytokines individually and as a profile with diffuse large B-cell (DLBCL) and follicular (FL) lymphomas.
We used a multiplexed assay to measure 30 cytokine concentrations in pre-treatment serum in a case-control study of 234 FL, 188 DLBCL, and 400 control participants. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) adjusted for age and sex, and polytomous regression was used to evaluate heterogeneity between FL and DLBCL. Principal components analysis (PCA) was used to assess cytokine profiles associated with FL and DLBCL.
In single cytokine modeling, we found that 12 of the 30 circulating serum cytokines were significantly (P<0.05) associated with FL and/or DLBCL after accounting for multiple testing (q<0.05). Soluble IL-2R (sIL-2R) had the strongest association with both FL (OR=6.0 for highest versus lowest tertile, 95% CI 3.8–9.5; p-trend=1.8 × 10−21) and DLBCL (OR=7.6, 95% CI 4.5–13.1; p-trend=7.2 × 10−20). IL1RA and IL-12p40 also showed similar associations for DLBCL and FL. In contrast, HGF, MIG, and MIP-1α had a stronger association with DLBCL compared to FL, and IL-6, IL-8, IL-10, IFN-γ, IP-10, and VEGF were only statistically significantly associated with DLBCL after accounting for multiple testing. However, in PCA modeling, a cytokine profile based on sIL-2R, IL-1RA, MIG, IP-10, IL-8, and IL-12p40 explained most of the variability between controls and both FL and DLBCL.
We identified some single cytokines unique to DLBCL, but overall cytokine associations were more similar than distinct for DLBCL and FL. While these data are limited by concerns of reverse causality, they do suggest cytokines and cytokine profiles that can be prioritized in future studies.
non-Hodgkin lymphoma; biomarkers; cytokines; case-control
Non-Hodgkin lymphoma (NHL) is a malignancy of lymphocytes, and there is growing evidence for a role of germline genetic variation in immune genes in NHL etiology.
To identify susceptibility immune genes, we conducted a 2-stage analysis of single nucleotide polymorphisms (SNPs) from 1,253 genes using the Immune and Inflammation Panel. In Stage 1, we genotyped 7,670 SNPs in 425 NHL cases and 465 controls, and in Stage 2 we genotyped the top 768 SNPs on an additional 584 cases and 768 controls. The association of individual SNPs with NHL risk from a log-additive model was assessed using the Odds Ratios (ORs) and 95% confidence intervals (CI).
In the pooled analysis, only the TAP2 coding SNP rs241447 (MAF=0.26; Thr655Ala) at 6p21.3 (OR=1.34, 95%CI 1.17-1.53) achieved statistical significance after accounting for multiple testing (p=3.1 × 10−5). The TAP2 SNP was strongly associated with follicular lymphoma (FL, OR=1.82, 95%CI 1.46-2.26; p=6.9 × 10−8), and was independent of other known loci (rs10484561 and rs2647012) from this region. The TAP2 SNP was also associated with diffuse large B-cell lymphoma (DLBCL, OR=1.38, 95% CI 1.08-1.77; p=0.011), but not chronic lymphocytic leukemia (OR=1.08; 95% CI 0.88-1.32). Higher TAP2 expression was associated with the risk allele in both FL and DLBCL tumors.
Genetic variation in TAP2 was associated with NHL risk overall, and FL risk in particular, and this was independent of other established loci from 6p21.3.
Genetic variation in antigen presentation of HLA class I molecules may play a role in lymphomagenesis.
genetics; non-Hodgkin lymphoma; immune function; single nucleotide polymorphisms
While standard clinical prognostic factors predict outcome in diffuse large B-cell lymphoma (DLBCL), predicting the outcome of patients might be further refined using biological factors. We tested whether serum cytokines could provide prognostic information in DLBCL patients. Thirty cytokines were measured in pre-treatment samples from newly diagnosed DLBCL patients using a multiplex ELISA. Sixty-nine patients treated with R-CHOP plus epratuzumab were used in an initial cohort and 185 patients treated with standard R-CHOP served as a subsequent validation cohort. In the initial cohort, elevated serum IL-10 (interleukin-10; HR=6.6, p=0.022), GM-CSF (granulocyte macrophage colony-stimulating factor; HR=10.8, p=0.027) and IP-10 (interferon-inducible protein-10, CXCL10; HR=3.32, p=0.015) were associated with event-free survival (EFS). An identical analysis of the subsequent validation cohort confirmed that elevated serum levels of IP-10 were strongly associated with a poor EFS (HR=2.42, p= 0.0007); and also identified IL-8 (interleukin-8; HR=3.40, p= 0.00002) and IL-2R (interleukin-2 receptor, CD25; HR=2.59, p= 0.0012) as significantly associated with prognosis. The prognostic significance of elevated IP-10 remained significant after adjustment for the International Prognostic Index (IPI; EFS – HR 1.99, p=0.009, overall survival- HR 1.93, p=0.021). Elevated pretreatment serum IP-10 levels are therefore associated with an increased likelihood of disease relapse and an inferior survival in patients with DLBCL.
IP-10; CXCL10; cytokines; diffuse large B-cell lymphoma; prognosis
The complement pathway plays a central role in innate immunity, and also functions as a regulator of the overall immune response. We evaluated whether polymorphisms in complement genes are associated with event-free survival (EFS) in follicular (FL) and diffuse large B-cell (DLBCL) lymphoma. We genotyped 167 single nucleotide polymorphisms (SNPs) from 30 complement pathway genes in a prospective cohort study of newly diagnosed FL (N=107) and DLBCL (N=82) patients enrolled at the Mayo Clinic from 2002–2005. Cox regression was used to estimate Hazard Ratios (HRs) for individual SNPs with EFS, adjusting for FLIPI or IPI and treatment. For gene-level analyses, we used a principal components based gene-level test. In gene-level analyses for FL EFS, CFH (p=0.009), CD55 (p=0.006), CFHR5 (p=0.01), C9 (p=0.02), CFHR1 (p=0.03), and CD46 (p=0.03) were significant at p<0.05, and these genes remained noteworthy after accounting for multiple testing (q<0.15). SNPs in CFH, CFHR1, and CFHR5 showed stronger associations among patients receiving any rituximab, while SNPs from CD55 and CD46 showed stronger associations among patients who were observed. For DLBCL, only CLU (p=0.001) and C7 (p=0.03) were associated with EFS, but did not remain noteworthy after accounting for multiple testing (q>0.15). Genes from the Regulators of Complement Activation (CFH, CD55, CFHR1, CFHR5, CD46) at 1q32-q32.1, along with C9, were associated with FL EFS after adjusting for clinical variables, and if replicated, these findings add further support for the role of host innate immunity in FL prognosis.
non-Hodgkin lymphoma; complement pathway; SNPs; prognosis; prospective cohort
Waldenström macroglobulinemia (WM) is a B-cell malignancy that remains incurable. Synthetic triterpenoids (ST), 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), its methyl ester derivative (CDDO-Me) and imidazolide derivative (CDDO-Im) induce cell death and inhibit growth of various malignancies and hold promise as treatment for cancer patients. We examined the therapeutic potential of these compounds in WM. All three forms of CDDO induced equal toxicity in BCWM.1 cells. In malignant B cells from WM patients, CDDO-Im induced the greatest toxicity. CDDO-Im inhibited proliferation at nanomolar concentrations and arrested the cells in G0/G1. CDDO-Im induced apoptotic cell death that was partially abolished in the presence of caspase inhibitor. CDDO-Im also inhibited survival pathways that have been shown to be important in WM. Overall, our data suggest that ST are likely to provide therapeutic efficacy for WM patients.
Waldenström macroglobulinemia; CDDO; CDDO-Im; Non-Hodgkin lymphoma; Therapy
While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (Treg) cells by enhancing expression of Foxp3 in CD4+ T cells and suppressed effector helper T (TH) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.
The cytokine IL-12 induces IFN-γ production by T and NK cells. In preclinical models, it contributes to antitumor immunity. However, in clinical testing, it has shown limited benefit in patients with any one of a variety of malignancies. Moreover, in a clinical trial testing a combination of IL-12 and rituximab in patients with follicular B cell non-Hodgkin lymphoma (FL), those treated with IL-12 showed a lower response rate, suggesting that IL-12 actually plays a detrimental role. Here, we investigated whether the failure of IL-12 treatment for FL was due to T cell exhaustion, a condition characterized by reduced T cell differentiation, proliferation, and function, which has been observed in chronic viral infection. We found that extended exposure to IL-12 induced T cell exhaustion and contributed to the poor prognosis in FL patients. Long-term exposure of freshly isolated human CD4+ T cells to IL-12 in vitro caused T cell dysfunction and induced expression of TIM-3, a T cell immunoglobulin and mucin domain protein with a known role in T cell exhaustion, via an IFN-γ–independent mechanism. TIM-3 was required for the negative effect of IL-12 on T cell function. Importantly, TIM-3 also was highly expressed on intratumoral T cells that displayed marked functional impairment. Our findings identify IL-12– and TIM-3–mediated exhaustion of T cells as a mechanism for poor clinical outcome when IL-12 is administered to FL patients.
The t(14;18)(q32;q21) is the most commonly observed chromosomal translocation in non-Hodgkin lymphoma (NHL), resulting in constitutive Bcl-2 expression and apoptosis inhibition. In addition, germline variation in both BCL2L11 (BIM) and CASP9, known regulators of apoptosis, have recently been linked to NHL risk. We conducted a comprehensive evaluation of 36 apoptosis pathway genes with risk of NHL.
We genotyped 226 single nucleotide polymorphisms (SNPs) from 36 candidate genes in a clinic-based study of 441 newly diagnosed NHL cases and 475 frequency matched controls. We used principal components analysis to assess gene-level associations, and logistic regression to assess SNP-level associations. MACH was used for imputation of SNPs in BCL2L11 and CASP9.
In gene level analyses, BCL2L11 (p=0.0019), BCLAF1 (p=0.0097), BAG5 (p=0.026) and CASP9 (p=0.0022) were associated with NHL risk after accounting for multiple testing (tail strength 0.38; 95% CI 0.05, 0.70). Two of the 5 BCL2L11 tagSNPs (rs6746608 and rs12613243), both genotyped BCLAF1 tagSNPs (rs797558 and rs703193), the single genotyped BAG5 tagSNP (rs7693), and 3 of the 7 genotyped CASP9 tagSNPs (rs6685648, rs2020902, rs2042370) were significant at p<0.05. We successfully imputed BCL2L11 and CASP9 SNPs previously linked to NHL, and replicated all 4 BCL2L11 and 2 of 3 CASP9 SNPs.
We replicated the association of BCL2L11 and CASP9 with NHL risk at the gene and SNP-level, and identified novel associations with BCLAF1 and BAG5.
Closer evaluation of germline variation of genes in the apoptosis pathway with risk of NHL and its subtypes is warranted.
Bcl-2 pathways; caspases; molecular epidemiology; non-Hodgkin lymphoma
Non-Hodgkin lymphoma (NHL) represents a diverse group of hematological
malignancies, of which follicular lymphoma (FL) is a prevalent subtype. A
previous genome-wide association study has established a marker, rs10484561 in
the human leukocyte antigen (HLA) class II region on 6p21.32 associated with
increased FL risk. Here, in a three-stage genome-wide association study,
starting with a genome-wide scan of 379 FL cases and 791 controls followed by
validation in 1,049 cases and 5,790 controls, we identified a second independent
FL–associated locus on 6p21.32, rs2647012
(ORcombined = 0.64,
Pcombined = 2×10−21)
located 962 bp away from rs10484561 (r2<0.1 in controls). After
mutual adjustment, the associations at the two SNPs remained genome-wide
significant (rs2647012:ORadjusted = 0.70,
Padjusted = 4×10−12;
rs10484561:ORadjusted = 1.64,
Padjusted = 5×10−15).
Haplotype and coalescence analyses indicated that rs2647012 arose on an
evolutionarily distinct haplotype from that of rs10484561 and tags a novel
allele with an opposite (protective) effect on FL risk. Moreover, in a follow-up
analysis of the top 6 FL–associated SNPs in 4,449 cases of other NHL
subtypes, rs10484561 was associated with risk of diffuse large B-cell lymphoma
(ORcombined = 1.36,
Pcombined = 1.4×10−7).
Our results reveal the presence of allelic heterogeneity within the HLA class II
region influencing FL susceptibility and indicate a possible shared genetic
etiology with diffuse large B-cell lymphoma. These findings suggest that the HLA
class II region plays a complex yet important role in NHL.
Earlier studies have established a marker rs10484561, in the HLA class II region
on 6p21.32, associated with increased follicular lymphoma (FL) risk. Here, in a
three-stage genome-wide association study of 1,428 FL cases and 6,581 controls,
we identified a second independent FL–associated marker on 6p21.32,
rs2647012, located 962 bp away from rs10484561. The associations at two SNPs
remained genome-wide significant after mutual adjustment. Haplotype and
coalescence analyses indicated that rs2647012 arose on an evolutionarily
distinct lineage from that of rs10484561 and tags a novel allele with an
opposite, protective effect on FL risk. Moreover, in an analysis of the top 6
FL–associated SNPs in 4,449 cases of other NHL subtypes, rs10484561 was
associated with risk of diffuse large B-cell lymphoma. Our results reveal the
presence of allelic heterogeneity at 6p21.32 in FL risk and suggest a shared
genetic etiology with the common diffuse large B-cell lymphoma subtype.