Project description:Human B cell lymphomas such as Follicular Lymphoma (FL) and Chronic Lymphocytic Leukemia/Small lymphocytic lymphoma (CLL) grow in lymph nodes in areas defined as malignant follicles, and proliferation centers, respectively. We used single-cell RNA-sequencing to study the tumor microenvironment of human lymph nodes involved by FL and CLL.
Project description:Comparison of gene expression profiles of follicular lymphoma vs. reactive lymph nodes. 8 cases of follicular lymphoma; 5 cases of reactive lymph nodes.
Project description:Follicular lymphoma (FL) is characterized by the t(14;18)(q32;q21), frequent numerical chromosomal alterations, and recurrent somatic mutations. However, no genetic studies are available for FL in situ (FLIS), a putative precursor lesion of FL. In this study, we analyzed cases of FLIS without manifest (m)FL, partial involvement by FL (PFL), and paired cases of FLIS and mFL to detect possible early chromosomal imbalances, as well as DNA-methylation patterns of genomic regions of selected genes. We demonstrated that paired FLIS and mFL cases are always clonally related. FLIS and PFL have no or few secondary chromosomal imbalances detectable by copy number arrays and a lower level of gene methylation as compared to mFL. Additionally, EZH2 Tyr641 mutations were detected in a subset of both FLIS and PFL cases. In conclusion, this study provides evidence that FLIS represents a FL precursor lesion of long-lived clonal B-cells carrying the t(14;18) with no or few secondary genetic changes. The earliest secondary genetic alterations detected in FLIS are the EZH2 Tyr641 mutation and low levels of CNAs, as evidence of clonal evolution. Our data suggest that there may be more than one distinct lesion driving the progression from FLIS to manifest lymphoma. DNA from cases, 20 tumor samples (10 in situ Follicular lymphoma, 4 Partial infiltration folliculary lymphoma and 6 manifest FL) were analyzed with Agilent-014693 Human Genome CGH Microarray 244A platform for copy number alterations study. For cases 9-14 paired samples of in situ follicular lymphoma and manifest follicular lymphoma were analyzed. Agilent-014693 Human Genome CGH Microarray 244A arrays were performed according to the manufacturer's directions on DNA extracted from lymph nodes that were hybridized against a normal DNA (pool) of the same gender.
Project description:Exhaustion markers are expressed by T lymphocytes in Follicular Lymphoma (FL). Through these, TIM-3 has been recently identified as a poor pronostic factor when expressed by FL CD4+ T cells. Before this study, there was no molecular characterization of unstimulated CD8+ T cells, expressing - or not - TIM-3. We used microarrays to compare the global gene expression profile between CD8+TIM-3+ T cells and CD8+TIM-3- T cells freshly sorted from follicular lymphoma biopsies. Abstract from the associated publication: Up-regulation of T-cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8+TIM-3+ T cells in lymph nodes of FL patients. When compared to their CD8+TIM-3- counterparts, CD8+TIM-3+ T cells exhibited defective cytokine production following TCR engagement. Furthermore CD8+TIM-3+ T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8+ T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8+TIM-3+ T cells was observed by phospho-flow analysis. Finally, short relapse free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3+ cells and a poor infiltrate of granzyme B+ T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8+ T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.
Project description:Gene expression profiling of biopsied human lymph node (LN) tissue comparing each patient sample against mobilised peripheral blood stem cells (PBSC), the reference channel Evaluate whether gene expression microarray can diagnose lymph node biopsies as reactive or as one of three main types of lymphoma: classical Hodgkin’s lymphoma (cHL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL).