Follicular lymphoma B cells exhibit heterogeneous transcriptional states with associated somatic alterations and tumor microenvironments.

Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin, which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies, combined with machine-learning approaches, we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed, proliferative, and chromatin-modifying states, with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling, we find that each state was associated with a combination of mutations in chromatin modifiers, copy-number alterations to TNFAIP3, and T follicular helper cells (Tfh) cell interactions, or primarily by a microenvironment rich in activated T cells. Altogether, these data define FL B cell transcriptional states across a large cohort of patients, contribute to our understanding of FL heterogeneity at the tumor cell level, and provide a foundation for guiding therapeutic intervention.

MicroRNA and long non-coding RNA analysis in IgM monoclonal gammopathies reveals epigenetic influence on cellular functions and oncogenesis.

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T-cell phenotype including CD57+ T follicular helper cells in the tumor microenvironment correlate with a poor outcome in follicular lymphoma.

T-lymphocytes are prevalent in the tumor microenvironment of follicular lymphoma (FL). However, the phenotype of T-cells may vary, and the prevalence of certain T-cell subsets may influence tumor biology and patient survival. We therefore analyzed a cohort of 82 FL patients using CyTOF to determine whether specific T-cell phenotypes were associated with distinct tumor microenvironments and patient outcome. We identified four immune subgroups with differing T-cell phenotypes and the prevalence of certain T-cell subsets was associated with patient survival. Patients with increased T cells with early differentiation stage tended to have a significantly better survival than patients with increased T-cells of late differentiation stage. Specifically, CD57+ TFH cells, with a late-stage differentiation phenotype, were significantly more abundant in FL patients who had early disease progression and therefore correlated with an inferior survival. Single cell analysis (CITE-seq) revealed that CD57+ TFH cells exhibited a substantially different transcriptome from CD57- TFH cells with upregulation of inflammatory pathways, evidence of immune exhaustion and susceptibility to apoptosis. Taken together, our results show that different tumor microenvironments among FL patients are associated with variable T-cell phenotypes and an increased prevalence of CD57+ TFH cells is associated with poor patient survival.

Multiomic Analysis Identifies a High-Risk Metabolic and TME Depleted Signature that Predicts Early Clinical Failure in DLBCL.

PURPOSE: 60-70% of newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients avoid events within 24 months of diagnosis (EFS24) and the remainder have poor outcomes. Recent genetic and molecular classification of DLBCL has advanced our knowledge of disease biology, yet were not designed to predict early events and guide anticipatory selection of novel therapies. To address this unmet need, we used an integrative multiomic approach to identify a signature at diagnosis that will identify DLBCL at high risk of early clinical failure. PATIENTS AND METHODS: Tumor biopsies from 444 newly diagnosed DLBCL were analyzed by WES and RNAseq. A combination of weighted gene correlation network analysis and differential gene expression analysis followed by integration with clinical and genomic data was used to identify a multiomic signature associated with high risk of early clinical failure. RESULTS: Current DLBCL classifiers are unable to discriminate cases who fail EFS24. We identified a high risk RNA signature that had a hazard ratio (HR, 18.46 [95% CI 6.51-52.31] P < .001) in a univariate model, which did not attenuate after adjustment for age, IPI and COO (HR, 20.8 [95% CI, 7.14-61.09] P < .001). Further analysis revealed the signature was associated with metabolic reprogramming and a depleted immune microenvironment. Finally, WES data was integrated into the signature and we found that inclusion of ARID1A mutations resulted in identification of 45% of cases with an early clinical failure which was validated in external DLBCL cohorts. CONCLUSION: This novel and integrative approach is the first to identify a signature at diagnosis that will identify DLBCL at high risk for early clinical failure and may have significant implications for design of therapeutic options.

Molecular classification and identification of an aggressive signature in low-grade B-cell lymphomas.

Non-follicular low-grade B-cell lymphomas (LGBCL) are biologically diverse entities that share clinical and histologic features that make definitive pathologic categorization challenging. While most patients with LGBCL have an indolent course, some experience aggressive disease, highlighting additional heterogeneity across these subtypes. To investigate the potential for shared biology across subtypes, we performed RNA sequencing and applied machine learning approaches that identified five clusters of patients that grouped independently of subtype. One cluster was characterized by inferior outcome, upregulation of cell cycle genes, and increased tumor immune cell content. Integration of whole exome sequencing identified novel LGBCL mutations and enrichment of TNFAIP3 and BCL2 alterations in the poor survival cluster. Building on this, we further refined a transcriptomic signature associated with early clinical failure in two independent cohorts. Taken together, this study identifies unique clusters of LGBCL defined by novel gene expression signatures and immune profiles associated with outcome across diagnostic subtypes.

Relationship between BCL2 mutations and follicular lymphoma outcome in the chemoimmunotherapy era.

How to identify follicular lymphoma (FL) patients with low disease burden but high risk for early progression is unclear. Building on a prior study demonstrating the early transformation of FLs with high variant allele frequency (VAF) BCL2 mutations at activation-induced cytidine deaminase (AICDA) sites, we examined 11 AICDA mutational targets, including BCL2, BCL6, PAX5, PIM1, RHOH, SOCS, and MYC, in 199 newly diagnosed grade 1 and 2 FLs. BCL2 mutations with VAF ≥20% occurred in 52% of cases. Among 97 FL patients who did not initially receive rituximab-containing therapy, nonsynonymous BCL2 mutations at VAF ≥20% were associated with increased transformation risk (HR 3.01, 95% CI 1.04-8.78, p = 0.043) and a trend toward shorter event-free survival (EFS, median 20 months with mutations versus 54 months without, p = 0.052). Other sequenced genes were less frequently mutated and did not increase the prognostic value of the panel. Across the entire population, nonsynonymous BCL2 mutations at VAF ≥20% were associated with decreased EFS (HR 1.55, 95% CI 1.02-2.35, p = 0.043 after correction for FLIPI and treatment) and decreased overall survival after median 14-year follow-up (HR 1.82, 95% CI 1.05-3.17, p = 0.034). Thus, high VAF nonsynonymous BCL2 mutations remain prognostic even in the chemoimmunotherapy era.

Characterization of immune exhaustion and suppression in the tumor microenvironment of splenic marginal zone lymphoma.

The role of the tumor microenvironment (TME) and intratumoral T cells in splenic marginal zone lymphoma (sMZL) is largely unknown. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of TFH cells varied greatly in sMZL, ICOS+ TFH cells were more abundant in sMZL than rSP, and TFH cells positively correlated with increased numbers of memory B cells. Treg cell analysis revealed that TIGIT+ Treg cells are enriched in sMZL and correlate with suppression of TH17 and TH22 cells. Intratumoral CD8+ T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data suggest that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function.

Molecular Clusters and Tumor-Immune Drivers of IgM Monoclonal Gammopathies.

PURPOSE: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. EXPERIMENTAL DESIGN: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. RESULTS: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. CONCLUSIONS: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.

Causes of death in low-grade B-cell lymphomas in the rituximab era: a prospective cohort study.

Low-grade B-cell lymphomas other than follicular and small lymphocytic lymphoma (LGBCL) account for 10% of all B-cell non-Hodgkin lymphomas. Despite improvements in survival outcomes for these patients, little is known about cause of death (COD) in the rituximab era. For a better understanding, we studied 822 newly diagnosed patients with marginal zone, lymphoplasmacytic, and unclassifiable low-grade B-cell lymphoma prospectively enrolled in the University of Iowa/Mayo Clinic Specialized Program of Research Excellence Molecular Epidemiology Resource from 2002 to 2015. COD was assigned based on medical record review using a standard protocol. At a median follow-up of 107 months, 219 (27%) patients had died. The incidence of lymphoma-related deaths when pooling across subtypes was lower than non-lymphoma-related deaths (10-year incidence, 8.0%; 95% confidence interval [CI]: 6.2-10.4 vs 13.6%; 95% CI: 11.2-16.6). The incidence of lymphoma-related deaths varied by subtype, ranging from 3.7% at 10 years in extranodal marginal zone lymphoma to 19.3% in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Patients with early progression or retreatment events, defined using event-free survival at 24 months from diagnosis, had significantly higher likelihood of lymphoma-related death compared with patients without early events (10-year estimate: 19.1% vs 5.1%, respectively; P < .001), whereas the rates for non-lymphoma-related death were comparable in patients with or without early events (10-year estimates: 11.0% vs 15.3%, respectively). In conclusion, the most common COD in LGBCLs in the first decade after diagnosis was for causes other than lymphoma. Progression or retreatment within the first 2 years of diagnosis was a strong predictor for risk of lymphoma-related death.

Phenotype, Function, and Clinical Significance of CD26+ and CD161+Tregs in Splenic Marginal Zone Lymphoma.

PURPOSE: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied. EXPERIMENTAL DESIGN: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays. RESULTS: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition. CONCLUSIONS: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease.

Vaccination History and Risk of Lymphoma and Its Major Subtypes.

BACKGROUND: Vaccinations have been hypothesized to play a role in lymphoma etiology, but there are few studies, mixed results, and limited data on lymphoma subtypes. Herein, we investigate the association of vaccinations with risk of major lymphoma subtypes. METHODS: We studied 2,461 lymphoma cases and 2,253 controls enrolled from 2002 to 2014. Participants self-reported history of vaccinations against hepatitis A, hepatitis B, yellow fever, and influenza. Polytomous logistic regression was used to estimate OR and 95% confidence intervals (CI), adjusting for potential confounders. RESULTS: After multivariable adjustment, vaccination against influenza was inversely associated with lymphoma (OR = 0.82; 95% CI, 0.66-1.02), which was stronger for last vaccination 1+ years before enrollment (OR = 0.71; 95% CI, 0.56-0.91) and for >5 influenza vaccinations (OR = 0.56; 95% CI, 0.46-0.68). Ever vaccination against hepatitis A (OR = 0.81; 95% CI, 0.66-1.00) but not hepatitis B (OR = 0.97; 95% CI, 0.81-1.18) was associated with lymphoma risk, although more recent vaccinations were inversely associated with lymphoma risk for both hepatitis A (<6 years before enrollment, OR = 0.56; 95% CI, 0.40-0.77) and hepatitis B (<9 years before enrollment, OR = 0.72; 95% CI, 0.55-0.93). Ever vaccination against yellow fever was inversely associated with risk (OR = 0.73; 95% CI, 0.55-0.96), and this did not vary by time since last vaccination. Although there was no overall statistical evidence for heterogeneity of vaccination history by lymphoma subtype, the only statistically significant inverse associations were observed for influenza and yellow fever vaccinations with diffuse large B-cell and follicular lymphoma. CONCLUSIONS: Selected vaccinations were inversely associated with lymphoma risk, with time since last vaccination relevant for some of these vaccines. IMPACT: Vaccinations against hepatitis A, hepatitis B, yellow fever, and influenza are unlikely to increase lymphoma risk.

Lack of intrafollicular memory CD4 + T cells is predictive of early clinical failure in newly diagnosed follicular lymphoma.

Despite a characteristic indolent course, a substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. Cells in the microenvironment may be a key contributor to treatment failure. We used a discovery and validation study design to identify microenvironmental determinants of early failure and then integrated these results into the FLIPI. In total, 496 newly diagnosed FL grade 1-3 A patients who were prospectively enrolled into the MER cohort from 2002 to 2012 were evaluated. Tissue microarrays were stained for CD4, CD8, FOXP3, CD32b, CD14, CD68, CD70, SIRP-α, TIM3, PD-1, and PD-L1. Early failure was defined as failing to achieve event-free survival at 24 months (EFS24) in immunochemotherapy-treated patients and EFS12 in all others. CyTOF and CODEX analysis were performed to characterize intratumoral immunophenotypes. Lack of intrafollicular CD4 expression was the only predictor of early failure that replicated with a pooled OR 2.37 (95%CI 1.48-3.79). We next developed a bio-clinical risk model (BioFLIPI), where lack of CD4 intrafollicular expression moved patients up one FLIPI risk group, adding a new fourth high-risk group. Compared with BioFLIPI score of 1, patients with a score of 2 (OR 2.17; 95% CI 1.08-4.69), 3 (OR 3.53; 95% CI 1.78-7.54), and 4 (OR 8.92; 95% CI 4.00-21.1) had increasing risk of early failure. The favorable intrafollicular CD4 T cells were identified as activated central memory T cells, whose prognostic value was independent from genetic features. In conclusion, lack of intrafollicular CD4 expression predicts early failure in FL and combined with FLIPI improves identification of high-risk patients; however, independent validation is warranted.

Expression of KLRG1 and CD127 defines distinct CD8+ subsets that differentially impact patient outcome in follicular lymphoma.

BACKGROUND: CD8+ T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied. METHODS: To characterize the phenotype of KLRG1/CD127-defined CD8+ subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL. RESULTS: We found that intratumoral CD8+ cells in FL are skewed toward effector cell subsets, particularly KLRG+CD127- and KLRG1-CD127- cells over memory cell subsets, such as KLRG1-CD127+ and KLRG1+CD127+ cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8+ T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127+KLRG1- and CD127+KLRG1+ were significantly associated with a favorable OS and EFS, while CD127-KLRG1- correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127-KLRG1+ cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8+ T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127-KLRG1+ differentiation. CONCLUSIONS: Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8+ subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8+ T cells, thereby promoting a more effective antitumor immune response.

Chronic lymphocytic leukemia B-cell-derived TNFα impairs bone marrow myelopoiesis.

TNFα is implicated in chronic lymphocytic leukemia (CLL) immunosuppression and disease progression. TNFα is constitutively produced by CLL B cells and is a negative regulator of bone marrow (BM) myelopoiesis. Here, we show that co-culture of CLL B cells with purified normal human hematopoietic stem and progenitor cells (HSPCs) directly altered protein levels of the myeloid and erythroid cell fate determinants PU.1 and GATA-2 at the single-cell level within transitional HSPC subsets, mimicking ex vivo expression patterns. Physical separation of CLL cells from control HSPCs or neutralizing TNFα abrogated upregulation of PU.1, yet restoration of GATA-2 required TNFα neutralization, suggesting both cell contact and soluble-factor-mediated regulation. We further show that CLL patient BM myeloid progenitors are diminished in frequency and function, an effect recapitulated by chronic exposure of control HSPCs to low-dose TNFα. These findings implicate CLL B-cell-derived TNFα in impaired BM myelopoiesis.

Targeting of inflammatory pathways with R2CHOP in high-risk DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma, and front line therapies have not improved overall outcomes since the advent of immunochemotherapy. By pairing DNA and gene expression data with clinical response data, we identified a high-risk subset of non-GCB DLBCL patients characterized by genomic alterations and expression signatures capable of sustaining an inflammatory environment. These mutational alterations (PIM1, SPEN, and MYD88 [L265P]) and expression signatures (NF-κB, IRF4, and JAK-STAT engagement) were associated with proliferative signaling, and were found to be enriched in patients treated with RCHOP that experienced unfavorable outcomes. However, patients with these high-risk mutations had more favorable outcomes when the immunomodulatory agent lenalidomide was added to RCHOP (R2CHOP). We are the first to report the genomic validation of a high-risk phenotype with a preferential response towards R2CHOP therapy in non-GCB DLBCL patients. These conclusions could be translated to a clinical setting to identify the ~38% of non-GCB patients that could be considered high-risk, and would benefit from alternative therapies to standard RCHOP based on personalized genomic data.

Treatment facility volume and patient outcomes in Waldenstrom macroglobulinemia.

Waldenstrom macroglobulinemia (WM) has an annual incidence of 3-3.2 cases per million-person/year. National Cancer Data Base was used to identify newly diagnosed WM cases requiring initiation of therapy and their annual facility volume was used to divide the treatment facilities into four quartiles (Qs). Cox regression was used to analyze the association between facility volume and survival, adjusted by demographics, socioeconomic, geographic, comorbidity factors and year of diagnosis. A total of 3064 patients treated in 795 facilities were included. The unadjusted median overall survival (OS) by facility volume was: Q1:6.5 years (5-year OS 55%), Q2:7 years (5-year OS 60%), Q3:8 years (5-year OS 64%), and Q4: NR (5-year OS 71%), p < 0.0001. Our results demonstrated that a volume-outcome relationship exists in WM and is an independent predictor of overall survival in addition to the established risk factors as age and disease severity.

Somatic copy number gains in MYC, BCL2, and BCL6 identifies a subset of aggressive alternative-DH/TH DLBCL patients.

Double/triple hit lymphoma (DH/TH), known as high-grade B-cell lymphoma (HGBL), is an aggressive diffuse large B cell lymphoma (DLBCL), defined as having concurrent MYC, BCL2, and/or BCL6 gene rearrangements. While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, and their contributions to rearrangements have yet to be fully elucidated. Using FISH and high-resolution CNA data, we defined the landscape of concurrent gene rearrangements and copy gains in MYC, BCL2, and BCL6, in a cohort of 479 newly diagnosed DLBCL. We also show that concurrent translocations and copy number alterations, in combinations similar to DH/TH, identify a unique subset of DLBCL, alternative DH/TH, that have survival outcomes similar to DH/TH DLBCL patients.

Increased glutathione utilization augments tumor cell proliferation in Waldenstrom Macroglobulinemia.

Metabolic reprogramming is a hallmark of cancer cells. In Waldenstrom Macroglobulinemia (WM), the infiltration of IgM-secreting lymphoplamacytic cells into the bone marrow (BM) could shift the homeostasis of proteins and metabolites towards a permissive niche for tumor growth. Here, we investigated whether alerted metabolic pathways contribute to the pathobiology of WM and whether the cytokine composition of the BM promotes such changes. Metabolomics analysis on WM patients and normal donors' serum samples revealed a total of 75 metabolites that were significantly altered between two groups. While these metabolites belonged to amino acids, glucose, glutathione and lipid metabolism pathways, the highest number of the differentially expressed metabolites belonged to glutathione metabolism. Proteomics analysis and immunohistochemical staining both confirmed the increased protein levels mediating glutathione metabolism, including GCLC, MT1X, QPCT and GPX3. Moreover, treatment with IL-6 and IL-21, cytokines that induce WM cell proliferation and IgM secretion, increased gene expression of the amino acid transporters mediating glutathione metabolism, including ASCT2, SLC7A11 and 4F2HC, indicating that cytokines in the WM BM could modulate glutathione metabolism. Glutathione synthesis inhibition using Buthionine sulphoximine (BSO) significantly reduced WM cells proliferation in vitro, accompanied with decreased NFκB-p65 and MAPK-p38 phosphorylation. Moreover, BSO treatment significantly reduced the tumor growth rate in a WM xenograft model, further highlighting the role of glutathione metabolism in promoting tumor growth and proliferation. In summary, our data highlight a central role for glutathione metabolism in WM pathobiology and indicate that intervening with the metabolic processes could be a potential therapy for WM patients.

Human Cancers Express TRAILshort, a Dominant Negative TRAIL Splice Variant, Which Impairs Immune Effector Cell Killing of Tumor Cells.

PURPOSE: TNF-related apoptosis inducing ligand (TRAIL) expression by immune cells contributes to antitumor immunity. A naturally occurring splice variant of TRAIL, called TRAILshort, antagonizes TRAIL-dependent cell killing. It is unknown whether tumor cells express TRAILshort and if it impacts antitumor immunity. EXPERIMENTAL DESIGN: We used an unbiased informatics approach to identify TRAILshort expression in primary human cancers, and validated those results with IHC and ISH. TRAILshort-specific mAbs were used to determine the effect of TRAILshort on tumor cell sensitivity to TRAIL, and to immune effector cell dependent killing of autologous primary tumors. RESULTS: As many as 40% of primary human tumors express TRAILshort by both RNA sequencing and IHC analysis. By ISH, TRAILshort expression is present in tumor cells and not bystander cells. TRAILshort inhibition enhances cancer cell lines sensitivity to TRAIL-dependent killing both in vitro and in immunodeficient xenograft mouse models. Immune effector cells isolated from patients with B-cell malignancies killed more autologous tumor cells in the presence compared with the absence of TRAILshort antibody (P < 0.05). CONCLUSIONS: These results identify TRAILshort in primary human malignancies, and suggest that TRAILshort blockade can augment the effector function of autologous immune effector cells.See related commentary by de Miguel and Pardo, p. 5546.