Lenalidomide in combination with R-ESHAP in patients with relapsed or refractory diffuse large B-cell lymphoma: A phase 2 study from GELTAMO.

Diffuse large B-cell lymphoma (DLBCL) patients with relapsed or refractory (RR) disease have poor outcomes with current salvage regimens. We conducted a phase 2 trial to analyse the safety and efficacy of adding lenalidomide to R-ESHAP (LR-ESHAP) in patients with RR DLBCL. Subjects received 3 cycles of lenalidomide 10 mg/day on days 1-14 of every 21-day cycle, in combination with R-ESHAP at standard doses. Responding patients underwent autologous stem-cell transplantation (ASCT). The primary endpoint was the overall response rate (ORR) after 3 cycles. Centralized cell-of-origin (COO) classification was performed. Forty-six patients were included. The ORR after LR-ESHAP was 67% (35% of patients achieved complete remission). Patients with primary refractory disease (n = 26) had significantly worse ORR than patients with non-refractory disease (54% vs. 85%, p = 0.031). No differences in response rates according to the COO were observed. Twenty-eight patients (61%) underwent ASCT. At a median follow-up of 41 months, the estimated 3-year PFS and OS were 42% and 48%, respectively. The most common grade ≥3 adverse events were thrombocytopenia (70% of patients), neutropenia (67%) and anaemia (35%). There were no treatment-related deaths during LR-ESHAP cycles. In conclusion, LR-ESHAP is a feasible salvage regimen with promising efficacy results for patients with RR DLBCL.

Increasing genomic and epigenomic complexity in the clonal evolution from in situ to manifest t(14;18)-positive follicular lymphoma.

Follicular lymphoma (FL) is characterized besides the t(14;18)(q32;q21), by recurrent chromosomal alterations and somatic mutations. In this study, we analyzed cases of FL in situ (FLIS) without manifest FL (mFL), partial involvement by FL (PFL) and paired cases of FLIS and mFL to detect possible early chromosomal imbalances, mutations, as well as DNA-methylation patterns of genomic regions of selected genes. We demonstrate that all paired FLIS and mFL cases were clonally related, based on IGH rearrangement patterns and BCL2 breakpoint sequences. FLIS and PFL had no or few secondary chromosomal imbalances detectable by array comparative genomic hybridization (FLIS 0.8 copy number alterations (CNA)/case; PFL 2.0 CNA/case; mFL 6.3 CNA/case) and a lower level of DNA methylation of genes recurrently de novo methylated in lymphomas, as compared with mFL. EZH2 Tyr641 mutations were detected in a subset of both FLIS (2/9) and PFL (1/3) cases. In conclusion, these findings provide 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. Our data suggest that there may be more than one distinct lesion driving the progression from FLIS to manifest lymphoma.

The EMT activator ZEB1 promotes tumor growth and determines differential response to chemotherapy in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a B-cell malignancy characterized by a poor response to treatment and prognosis. Constitutive activation of different signaling pathways in subsets of MCLs, through genetic and/or nongenetic alterations, endows tumor cells with enhanced proliferation and reduced apoptosis. The canonical Wnt pathway (ß-catenin/TCF-LEF), implicated in the pathogenesis of numerous cancers, is constitutively active in half of MCLs. Here, we show that ZEB1, a transcription factor better known for promoting metastasis in carcinomas, is expressed in primary MCLs with active Wnt signaling. ZEB1 expression in MCL cells depends on Wnt, being downregulated by ß-catenin knockdown or blocking of Wnt signaling by salinomycin. Knockdown of ZEB1 reduces in vitro cell viability and proliferation in MCL cells, and, importantly, tumor growth in mouse xenograft models. ZEB1 activates proliferation-associated (HMGB2, UHRF1, CENPF, MYC, MKI67, and CCND1) and anti-apoptotic (MCL1, BCL2, and BIRC5) genes and inhibits pro-apoptotic ones (TP53, BBC3, PMAIP1, and BAX). We show that ZEB1 expression in MCL cells determines differential resistance to chemotherapy drugs and regulates transporters involved in drug influx/efflux. Downregulation of ZEB1 by salinomycin increases the sensitivity of MCL cells to the cytotoxic effect of doxorubicin, cytarabine and gemcitabine. Lastly, salinomycin and doxorubicin display a synergistic effect in established and primary MCL cells. These results identify ZEB1 in MCL where it promotes cell proliferation, enhanced tumor growth and a differential response to chemotherapy drugs. ZEB1 could thus potentially become a predictive biomarker and therapeutic target in this lymphoma.

Exome sequencing reveals novel and recurrent mutations with clinical impact in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37-99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

MicroRNA signatures in B-cell lymphomas.

Accurate lymphoma diagnosis, prognosis and therapy still require additional markers. We explore the potential relevance of microRNA (miRNA) expression in a large series that included all major B-cell non-Hodgkin lymphoma (NHL) types. The data generated were also used to identify miRNAs differentially expressed in Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) samples. A series of 147 NHL samples and 15 controls were hybridized on a human miRNA one-color platform containing probes for 470 human miRNAs. Each lymphoma type was compared against the entire set of NHLs. BL was also directly compared with DLBCL, and 43 preselected miRNAs were analyzed in a new series of routinely processed samples of 28 BLs and 43 DLBCLs using quantitative reverse transcription-polymerase chain reaction. A signature of 128 miRNAs enabled the characterization of lymphoma neoplasms, reflecting the lymphoma type, cell of origin and/or discrete oncogene alterations. Comparative analysis of BL and DLBCL yielded 19 differentially expressed miRNAs, which were confirmed in a second confirmation series of 71 paraffin-embedded samples. The set of differentially expressed miRNAs found here expands the range of potential diagnostic markers for lymphoma diagnosis, especially when differential diagnosis of BL and DLBCL is required.

E2F4 plays a key role in Burkitt lymphoma tumorigenesis.

Sporadic Burkitt lymphoma (sBL) is a rapidly growing B-cell non-Hodgkin's lymphoma whose treatment requires highly aggressive therapies that often result severely toxic. Identification of proteins whose expression or function is deregulated in sBL and play a role in its formation could facilitate development of less toxic therapies. We have previously shown that E2F1 expression is deregulated in sBL. We have now investigated the mechanisms underlying E2F1 deregulation and found that the E2F sites in its promoter fail to repress its transcriptional activity in BL cells and that the transcriptional repressor E2F4 barely interacts with these sites. We also have found that E2F4 protein levels, but not those of its mRNA, are reduced in sBL cell lines relative to immortal B-cell lines. E2F4 protein expression is also decreased in 24 of 26 sBL tumor samples from patients compared with control tissues. Our data demonstrate that enforced E2F4 expression in BL cells not only diminishes E2F1 levels, but also reduces selectively the tumorigenic properties and proliferation of BL cells, while increasing their accumulation in G(2)/M. Our results therefore point to E2F4 as a target for developing novel and less toxic treatments for sBL.

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study.

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development-namely germinal center B-cell like and activated B-cell like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver-operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1 and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

Clinical and molecular characterization of diffuse large B-cell lymphomas with 13q14.3 deletion.

BACKGROUND: Deletions at 13q14.3 are common in chronic lymphocytic leukemia and are also present in diffuse large B-cell lymphomas (DLBCL) but never in immunodeficiency-related DLBCL. To characterize DLBCL with 13q14.3 deletions, we combined genome-wide DNA profiling, gene expression and clinical data in a large DLBCL series treated with rituximab, cyclophosphamide, doxorubicine, vincristine and prednisone repeated every 21 days (R-CHOP21). PATIENTS AND METHODS: Affymetrix GeneChip Human Mapping 250K NspI and U133 plus 2.0 gene were used. MicroRNA (miRNA) expression was studied were by real-time PCR. Median follow-up of patients was 4.9 years. RESULTS: Deletions at 13q14.3, comprising DLEU2/MIR15A/MIR16, occurred in 22/166 (13%) cases. The deletion was wider, including also RB1, in 19/22 cases. Samples with del(13q14.3) had concomitant specific aberrations. No reduced MIR15A/MIR16 expression was observed, but 172 transcripts were significantly differential expressed. Among the deregulated genes, there were RB1 and FAS, both commonly deleted at genomic level. No differences in outcome were observed in patients treated with R-CHOP21. CONCLUSIONS: Cases with 13q14.3 deletions appear as group of DLBCL characterized by common genetic and biologic features. Deletions at 13q14.3 might contribute to DLBCL pathogenesis by two mechanisms: deregulating the cell cycle control mainly due RB1 loss and contributing to immune escape, due to FAS down-regulation.

Hodgkin's Lymphomas: A Tumor Recognized by Its Microenvironment.

Thomas Hodgkin's and Samuel Wilks first recognized Hodgkin disease in the first half of the 19th century. Initially described as lymphogranulomatosis, it was later recognized to be a lymphoid neoplasm derived from B cells and was classified on the basis of its histopathological features. Hodgkin lymphomas are now regarded as encompassing two clearly defined entities according to the WHO classification: nodular lymphocyte-predominant Hodgkin Lymphoma (NLPHL) and classical Hodgkin Lymphoma (CHL). This paper focuses on the current knowledge about the biological features that characterize both NLPHL and CHL, highlighting those relevant to correct pathological diagnosis and those that might be associated with patient outcome.

Identification of MNDA as a new marker for nodal marginal zone lymphoma.

Clinical and biological studies on nodal marginal zone lymphoma (NMZL) are hampered by the lack of specific diagnostic markers and the low reproducibility of this diagnosis. A comparative expression-profiling study has shown a set of markers to be differentially expressed in NMZL compared with follicular lymphoma (FL), including myeloid cell nuclear differentiation antigen (MNDA), a nuclear protein expressed by myeloid cells and a subset of B-cells. The aim of this study was to characterize the expression of MNDA in normal and reactive human tissue, and in a large series of non-Hodgkin's B-cell lymphomas, with particular emphasis on NMZL and FL. Our results showed that MNDA is expressed in normal tissue by a subset of the marginal zone B cells. They also showed MNDA expression in subgroups of chronic lymphocytic leukemia, mantle-cell lymphoma, and diffuse large B-cell lymphoma, but MNDA was especially expressed by lymphomas derived from the marginal zone, such as mucosa-associated lymphoid-tissue lymphoma, splenic marginal-zone lymphoma and NMZL. MNDA expression was rarely observed in FL, a characteristic that is of potential value in distinguishing between NMZL and FL. MNDA expression is thus a useful tool for the recognition of NMZL.