STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Mutated RAS-associating proteins and ERK activation in relapse/refractory diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling.

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

The genomic landscape of two Burkitt lymphoma cases and derived cell lines: comparison between primary and relapse samples.

Relapse occurs in 10-40% of Burkitt lymphoma (BL) patients that have completed intensive chemotherapy regimens and is typically fatal. While treatment-naive BL has been characterized, the genomic landscape of BL at the time of relapse (rBL) has never been reported. Here, we present a genomic characterization of two rBL patients. The diagnostic samples had mutations common in BL, including MYC and CCND3. Additional mutations were detected at relapse, affecting important pathways such as NFκB (IKBKB) and MEK/ERK (NRAS) signaling, glutamine metabolism (SIRT4), and RNA processing (ZFP36L2). Genes implicated in drug resistance were also mutated at relapse (TP53, BAX, ALDH3A1, APAF1, FANCI). This concurrent genomic profiling of samples obtained at diagnosis and relapse has revealed mutations not previously reported in this disease. The patient-derived cell lines will be made available and, along with their detailed genetics, will be a valuable resource to examine the role of specific mutations in therapeutic resistance.

Probabilistic modeling of cytomegalovirus infection under consensus clinical management guidelines.

BACKGROUND: Cytomegalovirus (CMV) is the most common viral pathogen after renal transplantation and remains a major therapeutic challenge with important clinical and economic implications from both direct and indirect consequences of infection. METHODS: This 5-year study modeled the relationship between CMV infection and biopsy-proven graft rejection, graft loss, or death after renal transplantation in an inception cohort using Canadian consensus guidelines for CMV management as a component of a detailed cost-analysis of viral infection. RESULTS: Probabilities of CMV viremia and syndrome/disease among 270 sequential graft recipients were 0.27 and 0.09, respectively; 91% of cases occurred in the first 6 months. Probability of CMV infection as the first event was 0.29, with a probability of subsequent biopsy-proven acute rejection (BPAR) of 0.05 (mean: 62+/-26 days, range: 32-85 days), whereas the probability of BPAR as the first event was 0.18, with a probability of subsequent CMV infection of 0.38 (mean: 63+/-31, range: 27-119 days). Probability of freedom from both CMV infection and BPAR throughout the period of observation was 0.53. Time-dependent Cox analysis showed that neither donor/recipient CMV risk stratum nor CMV infection influenced the risks of BPAR (P=0.24; P=0.74) or of graft loss or death (P=0.26; P=0.34). In contrast, BPAR significantly increased the risk of both subsequent CMV infection (hazard ratio=1.77, P=0.03) and of graft loss or death (hazard ratio=8.31, P<0.0001). CONCLUSIONS: Although current antiviral therapy seems to mitigate the reported deleterious effects of CMV infection on BPAR or graft survival, BPAR remains a significantly risk factor for both CMV infection and functional graft survival.

Probability, predictors, and prognosis of posttransplantation glomerulonephritis.

Glomerulonephritis (GN) is the leading cause of chronic kidney disease among recipients of renal transplants. Because modern immunosuppressive regimens have reduced the incidence of rejection-related graft loss, the probability and clinical significance of posttransplantation GN (PTGN) requires reevaluation. In this Canadian epidemiologic study, we monitored 2026 sequential renal transplant recipients whose original renal disease resulted from biopsy-proven GN (36%), from presumed GN (7.8%), or from disorders other than GN (56%) for 15 yr without loss to follow-up. Kaplan-Meier estimates of PTGN in the whole population were 5.5% at 5 yr, 10.1% at 10 yr, and 15.7% at 15 yr. PTGN was diagnosed in 24.3% of patients whose original renal disease resulted from biopsy-proven GN, compared with 11.8% of those with presumed GN and 10.5% of those with disorders other than GN. Biopsy-proven GN in the native kidney, male gender, younger age, and nonwhite ethnicity predicted PTGN. Current immunosuppressive regimens did not associate with a reduced frequency of PTGN. Patients who developed PTGN had significantly reduced graft survival (10.2 versus 69.7%; P < 0.0001). In summary, in the Canadian population, PTGN is a common and serious complication that causes accelerated graft failure, despite the use of modern immunosuppressive regimens.

Association between cytokine gene polymorphisms and outcomes in renal transplantation: a meta-analysis of individual patient data.

BACKGROUND: Cytokine gene polymorphisms have been associated with poor outcomes after renal transplantation such as chronic allograft nephropathy (CAN), graft rejection (GR) and graft failure (GF), but the effects of these polymorphisms are still controversial. We therefore conducted a systematic review, with individual patient data (IPD) where possible, to determine the association between cytokine polymorphisms (TGF-beta1, TNF-alpha and IL-10) and outcomes after renal transplantation. METHODS: Five investigators were willing to participate and provided IPD. The outcomes of interest were GF, GR and CAN. Subjects with at least one of these were classified as having poor outcomes. Heterogeneity of gene effects was assessed. Multiple logistic regression was applied to assess gene effects, adjusting for clinical variables such as HLA matching and age. RESULTS: One-thousand and eighty-seven subjects were included in the IPD meta-analysis. Pooled results showed no evidence of heterogeneity and indicated that the strongest variables determining poor outcomes are HLA mismatching (OR = 1.6-1.8 for >/=3 HLA-A, -B, -DR mismatches compared with those with <3 mismatches) and age (OR = 1.2-1.4 for age 45 years or more). Incremental information on risk of a poor outcome is provided by the TGF-beta1c10 polymorphism (OR = 1.5, P = 0.034, 95% CI: 1.0-2.2 for TC genotype compared to TT genotype). Haplotypes of TGF-beta1 at c10 and c25 were inferred and the C-C haplotype was a marker of a poor outcome (OR = 1.3, P = 0.177, 95% CI: 1.0-2.3). Three polymorphisms of the IL-10 gene at -1082, -819, -592 are in strong linkage disequilibrium with each other (correlation coefficients: 0.6-1) and inferred haplotypes between these three loci show some association, with ACC increasing the risk of poor events compared to GCC (OR = 1.3, P = 0.044, 95% CI: 0.9-1.6). CONCLUSION: Pooled results to date suggest possible association between both the TGF-beta1 c10 polymorphism and a 3-SNP-haplotype of IL-10 and poor outcomes in renal transplantation, but this needs to be confirmed in larger studies.

Immune response gene polymorphisms in renal transplant recipients.

BACKGROUND: T-cell activation and regulation are under genetic control and vary between individuals. However, the influence of functional immune response gene polymorphisms on transplant outcomes remains controversial. METHODS: A case-control design compared 100 white renal transplant recipients with or without acute graft rejection during the first year posttransplant and 50 normal controls. The polymorphic frequencies of the T-cell signaling genes CD45, CD40L and CTLA-4, and the cytokine genes TNF-alpha, IFN-gamma, IL-10 and TGF-beta1 were studied. The primary analysis examined rejection risk, and subsidiary analyses graft failure and patient death. RESULTS: Multivariate analysis showed no significant association between acute rejection and single nucleotide polymorphisms in CTLA-4, TGF-beta1, IL-10 or TNF-alpha genes or dinucleotide repeat polymorphisms in IFN-gamma and CD40L genes. Allele CD40L-147 was associated with reduced graft failure (P=0.004), and TGFb-25pro with increased graft failure (P=0.0007), although the latter showed a bidirectional dose effect. There was no significant association between patient death and any polymorphisms in the genes examined. The variant (G) allele of the CD45 gene was not detected in the study population. Minor differences in carriage rates observed by univariate analysis did not predict graft or patient outcome in multivariate analysis. CONCLUSION: The primary analysis demonstrated no significant association between the immune response gene polymorphisms examined and acute renal graft rejection in Caucasian patients receiving triple immunosuppression. Subsidiary analyses suggesting an influence of CD40L and TGFbeta1 genes on graft survival require independent confirmation.