Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune ...dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand–induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell–inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.
Follicular lymphoma is an incurable B cell malignancy characterized by the t(14;18) translocation and mutations affecting the epigenome. Although frequent gene mutations in key signaling pathways, ...including JAK-STAT, NOTCH and NF-κB, have also been defined, the spectrum of these mutations typically overlaps with that in the closely related diffuse large B cell lymphoma (DLBCL). Using a combination of discovery exome and extended targeted sequencing, we identified recurrent somatic mutations in RRAGC uniquely enriched in patients with follicular lymphoma (17%). More than half of the mutations preferentially co-occurred with mutations in ATP6V1B2 and ATP6AP1, which encode components of the vacuolar H(+)-ATP ATPase (V-ATPase) known to be necessary for amino acid-induced activation of mTORC1. The RagC variants increased raptor binding while rendering mTORC1 signaling resistant to amino acid deprivation. The activating nature of the RRAGC mutations, their existence in the dominant clone and their stability during disease progression support their potential as an excellent candidate for therapeutic targeting.
Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier ...to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.
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•Exome sequencing in 1,001 DLBCL patients comprehensively identifies 150 driver genes•Unbiased CRISPR screen in DLBCL cell lines identifies essential oncogenes•Integrative analysis connects genomics, CRISPR hits, and clinical outcome•A genomic risk model of survival outperforms existing risk-assessment methods
An integrative analysis in 1,001 newly diagnosed DLBCL patients identifies 150 genetic drivers with functional characterization using an unbiased CRISPR screen in DLBCL cell lines and connects with clinical outcome.
The immune microenvironment is key to the pathophysiology of classical Hodgkin lymphoma (CHL). Twenty percent of patients experience failure of their initial treatment, and others receive excessively ...toxic treatment. Prognostic scores and biomarkers have yet to influence outcomes significantly. Previous biomarker studies have been limited by the extent of tissue analyzed, statistical inconsistencies, and failure to validate findings. We aimed to overcome these limitations by validating recently identified microenvironment biomarkers (CD68, FOXP3, and CD20) in a new patient cohort with a greater extent of tissue and by using rigorous statistical methodology.
Diagnostic tissue from 122 patients with CHL was microarrayed and stained, and positive cells were counted across 10 to 20 high-powered fields per patient by using an automated system. Two statistical analyses were performed: a categorical analysis with test/validation set-defined cut points and Kaplan-Meier estimated outcome measures of 5-year overall survival (OS), disease-specific survival (DSS), and freedom from first-line treatment failure (FFTF) and an independent multivariate analysis of absolute uncategorized counts.
Increased CD20 expression confers superior OS. Increased FOXP3 expression confers superior OS, and increased CD68 confers inferior FFTF and OS. FOXP3 varies independently of CD68 expression and retains significance when analyzed as a continuous variable in multivariate analysis. A simple score combining FOXP3 and CD68 discriminates three groups: FFTF 93%, 62%, and 47% (P < .001), DSS 93%, 82%, and 63% (P = .03), and OS 93%, 82%, and 59% (P = .002).
We have independently validated CD68, FOXP3, and CD20 as prognostic biomarkers in CHL, and we demonstrate, to the best of our knowledge for the first time, that combining FOXP3 and CD68 may further improve prognostic stratification.
Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells ...(PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. Methods We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes. Results Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase+ and CD68+ cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8+ , FoxP3+ , CD56+ , or CD20+ cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8+ T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8+ T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8+ T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. Conclusions Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8+ T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response.
To examine the immune microenvironment in diagnostic follicular lymphoma (FL) biopsies and evaluate its prognostic significance.
Immunohistochemistry was used to study numbers and location of cells ...staining positive for immune cell markers CD4, CD7, CD8, CD25, CD68, forkhead box protein P3 (FOXP3), T-cell intracellular antigen-1, and Granzyme B in tissue microarrays of paraffin-embedded, diagnostic lymph node biopsies taken from 59 FL patients who lived less than 5 years (short-survival group; n = 34) and more than 15 years (long-survival group; n = 25).
CD4 and FOXP3 expression were significantly different between the two groups. Samples from the long-survival group were more likely than those from the short-survival group to have CD4+ staining cells and to have FOXP3-positive cells in a perifollicular location.
This study has identified differences in immune cell composition of the diagnostic FL lymph node immune microenvironment and these have the potential for use as prognostic biomarkers in a routine histopathology setting.
Gain of function mutations in the H3K27 methyltransferase EZH2 represent a promising therapeutic target in germinal center lymphomas. In this study, we assessed the frequency and distribution of EZH2 ...mutations in a large cohort of patients with follicular lymphoma (FL) (n = 366) and performed a longitudinal analysis of mutation during the disease progression from FL to transformed FL (tFL) (n = 33). Mutations were detected at 3 recurrent mutation hot spots (Y646, A682, and A692) in 27% of FL cases with variant allele frequencies (VAF) ranging from 2% to 61%. By comparing VAF of EZH2 with other mutation targets (CREBBP, MLL2, TNFRSF14, and MEF2B), we were able to distinguish patients harboring clonal EZH2 mutation from rarer cases with subclonal mutations. Overall, the high incidence of EZH2 mutations in FL and their stability during disease progression makes FL an appropriate disease to evaluate EZH2 targeted therapy.
Key Points
Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of ...human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre-germinal center state primed to undergo class switch recombination and dissect how antibody class-dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell-mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.
An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should ...facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.
Previous studies have demonstrated the prognostic importance of the immune microenvironment in follicular lymphoma (FL). To investigate the molecular mechanisms during which tumor-infiltrating T ...cells (TILs) are altered in the FL microenvironment, we studied highly purified CD4 and CD8 TILs from lymph node biopsies at diagnosis in treatment-naive patients with FL compared with reactive tonsils and the peripheral blood of healthy donors.
Gene expression profiling of highly purified CD4 and CD8 TILs was performed on the Affymetrix platform. Diagnostic tissue microarrays from an independent patient set (n = 172) were used to verify protein expression and analyze any impact of TIL-expressed genes on outcome. Time-lapse imaging was used to assess T-cell motility.
The most upregulated genes in both CD4 and CD8 TILs were PMCH, ETV1, and TNFRSF9. PMCH is not expressed in peripheral blood T cells, but expression is highly induced on culture with FL. Both CD4 and CD8 TILs from patients with FL have significantly impaired motility compared with those of healthy TILs from reactive tonsils and this can be induced on healthy T cells by FL cells. During multivariate analysis, a model incorporating the number and location of T cells expressing PMCH, NAMPT, and ETV1 showed prognostic significance for overall survival and for time to transformation.
We showed altered gene expression in TILs in FL and demonstrated that altering the immune microenvironment in FL affects overall survival and time to transformation in this disease.
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