Genomic alterations important for the prognosis in patients with follicular lymphoma treated in SWOG study S0016.

Although recent advances in molecular genetics have enabled improved risk classification of follicular lymphoma (FL) using, for example, the m7-FLIPI score, the impact on treatment has been limited. We aimed to assess the prognostic significance of copy-number aberrations (CNAs) and copy-neutral loss of heterozygosity (cnLOH) identified by chromosome genomic-array testing (CGAT) at FL diagnosis using prospectively collected clinical trial specimens from 255 patients enrolled in the SWOG study S0016. The impact of genomic aberrations was assessed for early progression (progressed or died within 2 years after registration), progression-free survival (PFS), and overall survival (OS). We showed that increased genomic complexity (ie, the total number of aberration calls) was associated with poor outcome in FL. Certain chromosome arms were critical for clinical outcome. Prognostic CNAs/cnLOH were identified: whereas early progression was correlated with 2p gain (P = .007; odds ratio [OR] = 2.55 [1.29, 5.03]) and 2p cnLOH (P = .005; OR = 10.9 [2.08, 57.2]), 2p gain specifically encompassing VRK2 and FANCL predicted PFS (P = .01; hazard ratio = 1.80 [1.14, 2.68]) as well as OS (P = .005; 2.40 [1.30, 4.40]); CDKN2A/B (9p) deletion correlated with worse PFS (P = .004, 3.50 [1.51, 8.28]); whereas CREBBP (16p) (P < .001; 6.70 [2.52, 17.58]) and TP53 (17p) (P < .001; 3.90 [1.85, 8.31]) deletion predicted worse OS. An independent cohort from the m7-FLIPI study was explored, and the prognostic significance of aberration count, and TP53 and CDKN2A/B deletion were further validated. In conclusion, assessing genomic aberrations at FL diagnosis with CGAT improves risk stratification independent of known clinical parameters, and provides a framework for development of future rational targeted therapies.

Genetic drivers of oncogenic pathways in molecular subgroups of peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A /B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2 R172 mutation. CN losses were enriched in genes regulating PI3K-AKT-mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

MAPK and JAK-STAT pathways dysregulation in plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) is an aggressive B-cell lymphoma with an immunoblastic/large-cell morphology and terminal B-cell differentiation. The differential diagnosis from Burkitt lymphoma, plasma cell myeloma and some variants of diffuse large B-cell lymphoma may be challenging because of the overlapping morphological, genetic and immunophenotypic features. Furthermore, the genomic landscape in PBL is not well known. To characterize the genetic and molecular heterogeneity of these tumors, we investigated 34 cases of PBL using an integrated approach, including fluorescence in situ hybridization, targeted sequencing of 94 B-cell lymphoma-related genes, and copy-number arrays. PBL were characterized by high genetic complexity including MYC translocations (87%), gains of 1q21.1-q44, trisomy 7, 8q23.2- q24.21, 11p13-p11.2, 11q14.2-q25, 12p and 19p13.3-p13.13, losses of 1p33, 1p31.1-p22.3, 13q and 17p13.3-p11.2, and recurrent mutations of STAT3 (37%), NRAS and TP53 (33%), MYC and EP300 (19%) and CARD11, SOCS1 and TET2 (11%). Pathway enrichment analysis suggested a cooperative action between MYC alterations and MAPK (49%) and JAK-STAT (40%) signaling pathways. Of note, Epstein-Barr virus (EBV)-negative PBL cases had higher mutational and copy-number load and more frequent TP53, CARD11 and MYC mutations, whereas EBV-positive PBL tended to have more mutations affecting the JAK-STAT pathway. In conclusion, these findings further unravel the distinctive molecular heterogeneity of PBL identifying novel molecular targets and the different genetic profile of these tumors in relation to EBV infection.

Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Adult high-grade B-cell lymphoma with Burkitt lymphoma signature: genomic features and potential therapeutic targets.

The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene signature (adult-molecularly defined BL [mBL]) revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24, and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway (TCF3 and ID3) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had BCL2 translocation and mutation and elevated BCL2 mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both BCL2 and MYC translocations and/or KMT2D (MLL2) mutation. Gain/amplification of MIR17HG and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested miR-17∼92's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by copy number and mutation, leading to disruption of the p53 pathway and the BCR→PI3K or NF-κB activation, which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics.

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways. BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL.

Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma.

We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)-DLBCL compared with activated B-cell (ABC)-DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the "gold standard" GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL.

Oncogenically active MYD88 mutations in human lymphoma.

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-ß. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue.

The assignment of diffuse large B-cell lymphoma into cell-of-origin (COO) groups is becoming increasingly important with the emergence of novel therapies that have selective biological activity in germinal center B cell-like or activated B cell-like groups. The Lymphoma/Leukemia Molecular Profiling Project's Lymph2Cx assay is a parsimonious digital gene expression (NanoString)-based test for COO assignment in formalin-fixed paraffin-embedded tissue (FFPET). The 20-gene assay was trained using 51 FFPET biopsies; the locked assay was then validated using an independent cohort of 68 FFPET biopsies. Comparisons were made with COO assignment using the original COO model on matched frozen tissue. In the validation cohort, the assay was accurate, with only 1 case with definitive COO being incorrectly assigned, and robust, with >95% concordance of COO assignment between 2 independent laboratories. These qualities, along with the rapid turnaround time, make Lymph2Cx attractive for implementation in clinical trials and, ultimately, patient management.

Genome-wide copy-number analyses reveal genomic abnormalities involved in transformation of follicular lymphoma.

Follicular lymphoma (FL), the second most common type of non-Hodgkin lymphoma in the western world, is characterized by the t(14;18) translocation, which is present in up to 90% of cases. We studied 277 lymphoma samples (198 FL and 79 transformed FL [tFL]) using a single-nucleotide polymorphism array to identify the secondary chromosomal abnormalities that drive the development of FL and its transformation to diffuse large B-cell lymphoma. Common recurrent chromosomal abnormalities in FL included gains of 2, 5, 7, 6p, 8, 12, 17q, 18, 21, and X and losses on 6q and 17p. We also observed many frequent small abnormalities, including losses of 1p36.33-p36.31, 6q23.3-q24.1, and 10q23.1-q25.1 and gains of 2p16.1-p15, 8q24.13-q24.3, and 12q12-q13.13, and identified candidate genes that may be driving this selection. Recurrent abnormalities more frequent in tFL samples included gains of 3q27.3-q28 and chromosome 11 and losses of 9p21.3 and 15q. Four abnormalities, gain of X or Xp and losses of 6q23.2-24.1 or 6q13-15, predicted overall survival. Abnormalities associated with transformation of the disease likely impair immune surveillance, activate the nuclear factor-κB pathway, and deregulate p53 and B-cell transcription factors.

Gene expression signatures delineate biological and prognostic subgroups in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of neoplasms with generally poor clinical outcome. Currently 50% of PTCL cases are not classifiable: PTCL-not otherwise specified (NOS). Gene-expression profiles on 372 PTCL cases were analyzed and robust molecular classifiers and oncogenic pathways that reflect the pathobiology of tumor cells and their microenvironment were identified for major PTCL-entities, including 114 angioimmunoblastic T-cell lymphoma (AITL), 31 anaplastic lymphoma kinase (ALK)-positive and 48 ALK-negative anaplastic large cell lymphoma, 14 adult T-cell leukemia/lymphoma and 44 extranodal NK/T-cell lymphoma that were further separated into NK-cell and gdT-cell lymphomas. Thirty-seven percent of morphologically diagnosed PTCL-NOS cases were reclassified into other specific subtypes by molecular signatures. Reexamination, immunohistochemistry, and IDH2 mutation analysis in reclassified cases supported the validity of the reclassification. Two major molecular subgroups can be identified in the remaining PTCL-NOS cases characterized by high expression of either GATA3 (33%; 40/121) or TBX21 (49%; 59/121). The GATA3 subgroup was significantly associated with poor overall survival (P = .01). High expression of cytotoxic gene-signature within the TBX21 subgroup also showed poor clinical outcome (P = .05). In AITL, high expression of several signatures associated with the tumor microenvironment was significantly associated with outcome. A combined prognostic score was predictive of survival in an independent cohort (P = .004).

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

EZH2 mutations are frequent and represent an early event in follicular lymphoma.

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.

Genome-wide miRNA profiling of mantle cell lymphoma reveals a distinct subgroup with poor prognosis.

miRNA deregulation has been implicated in the pathogenesis of mantle cell lymphoma (MCL). Using a high-throughput quantitative real-time PCR platform, we performed miRNA profiling on cyclin D1-positive MCL (n = 30) and cyclin D1-negative MCL (n = 7) and compared them with small lymphocytic leukemia/lymphoma (n = 12), aggressive B-cell lymphomas (n = 138), normal B-cell subsets, and stromal cells. We identified a 19-miRNA classifier that included 6 up-regulated miRNAs and 13 down regulated miRNA that was able to distinguish MCL from other aggressive lymphomas. Some of the up-regulated miRNAs are highly expressed in naive B cells. This miRNA classifier showed consistent results in formalin-fixed paraffin-embedded tissues and was able to distinguish cyclin D1-negative MCL from other lymphomas. A 26-miRNA classifier could distinguish MCL from small lymphocytic leukemia/lymphoma, dominated by 23 up-regulated miRNAs in MCL. Unsupervised hierarchical clustering of MCL patients demonstrated a cluster characterized by high expression of miRNAs from the polycistronic miR17-92 cluster and its paralogs, miR-106a-363 and miR-106b-25, and associated with high proliferation gene signature. The other clusters showed enrichment of stroma-associated miRNAs, and also had higher expression of stroma-associated genes. Our clinical outcome analysis in the present study suggested that miRNAs can serve as prognosticators.

A new biologic prognostic model based on immunohistochemistry predicts survival in patients with diffuse large B-cell lymphoma.

Biologic factors that predict the survival of patients with a diffuse large B-cell lymphoma, such as cell of origin and stromal signatures, have been discovered by gene expression profiling. We attempted to simulate these gene expression profiling findings and create a new biologic prognostic model based on immunohistochemistry. We studied 199 patients (125 in the training set, 74 in the validation set) with de novo diffuse large B-cell lymphoma treated with rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like therapies, and immunohistochemical stains were performed on paraffin-embedded tissue microarrays. In the model, 1 point was awarded for each adverse prognostic factor: nongerminal center B cell-like subtype, SPARC (secreted protein, acidic, and rich in cysteine) < 5%, and microvascular density quartile 4. The model using these 3 biologic markers was highly predictive of overall survival and event-free survival in multivariate analysis after adjusting for the International Prognostic Index in both the training and validation sets. This new model delineates 2 groups of patients, 1 with a low biologic score (0-1) and good survival and the other with a high score (2-3) and poor survival. This new biologic prognostic model could be used with the International Prognostic Index to stratify patients for novel or risk-adapted therapies.

Genome-wide methylation analyses identify a subset of mantle cell lymphoma with a high number of methylated CpGs and aggressive clinicopathological features.

Mantle cell lymphoma (MCL) is a B-cell neoplasm with an aggressive clinical behavior characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. To clarify the potential contribution of altered DNA methylation in the development and/or progression of MCL, we performed genome-wide methylation profiling of a large cohort of primary MCL tumors (n = 132), MCL cell lines (n = 6) and normal lymphoid tissue samples (n = 31), using the Infinium HumanMethylation27 BeadChip. DNA methylation was compared to gene expression, chromosomal alterations and clinicopathological parameters. Primary MCL displayed a heterogeneous methylation pattern dominated by DNA hypomethylation when compared to normal lymphoid samples. A total of 454 hypermethylated and 875 hypomethylated genes were identified as differentially methylated in at least 10% of primary MCL. Annotation analysis of hypermethylated genes recognized WNT pathway inhibitors and several tumor suppressor genes as frequently methylated, and a substantial fraction of these genes (22%) showed a significant downregulation of their transcriptional levels. Furthermore, we identified a subset of tumors with extensive CpG methylation that had an increased proliferation signature, higher number of chromosomal alterations and poor prognosis. Our results suggest that a subset of MCL displays a dysregulation of DNA methylation characterized by the accumulation of CpG hypermethylation highly associated with increased proliferation that may influence the clinical behavior of the tumors.

Tumor-associated macrophages and survival in classic Hodgkin's lymphoma.

BACKGROUND: Despite advances in treatments for Hodgkin's lymphoma, about 20% of patients still die from progressive disease. Current prognostic models predict the outcome of treatment with imperfect accuracy, and clinically relevant biomarkers have not been established to improve on the International Prognostic Score. METHODS: Using gene-expression profiling, we analyzed 130 frozen samples obtained from patients with classic Hodgkin's lymphoma during diagnostic lymph-node biopsy to determine which cellular signatures were correlated with treatment outcome. We confirmed our findings in an independent cohort of 166 patients, using immunohistochemical analysis. RESULTS: Gene-expression profiling identified a gene signature of tumor-associated macrophages that was significantly associated with primary treatment failure (P=0.02). In an independent cohort of patients, we found that an increased number of CD68+ macrophages was correlated with a shortened progression-free survival (P=0.03) and with an increased likelihood of relapse after autologous hematopoietic stem-cell transplantation (P=0.008), resulting in shortened disease-specific survival (P=0.003). In multivariate analysis, this adverse prognostic factor outperformed the International Prognostic Score for disease-specific survival (P=0.003 vs. P=0.03). The absence of an elevated number of CD68+ cells in patients with limited-stage disease defined a subgroup of patients with a long-term disease-specific survival of 100% with the use of current treatment strategies. CONCLUSIONS: An increased number of tumor-associated macrophages was strongly associated with shortened survival in patients with classic Hodgkin's lymphoma and provides a new biomarker for risk stratification.

MicroRNA profiles of t(14;18)-negative follicular lymphoma support a late germinal center B-cell phenotype.

A total of 90% of follicular lymphomas (FLs) harbor the translocation t(14;18) leading to deregulated BCL2 expression. Conversely, 10% of FLs lack the t(14;18), and the majority of these FLs do not express BCL2. The molecular features of t(14;18)-negative FLs remain largely unknown. We performed microRNA expression analysis in 32 FL grades 1 to 3A, including 17 t(14;18)-positive FLs, 9 t(14;18)-negative FLs without BCL2 expression, and 6 t(14;18)-negative FLs with BCL2 expression. MicroRNA profiles were correlated with corresponding mRNA expression patterns, and potential targets were investigated by quantitative PCR and immunohistochemistry in an independent validation series of 83 FLs. Statistical analysis identified 17 microRNAs that were differentially expressed between t(14;18)-positive FLs and t(14;18)-negative FLs. The down-regulation of miR-16, miR-26a, miR-101, miR-29c, and miR138 in the t(14;18)-negative FL subset was associated with profound mRNA expression changes of potential target genes involving cell cycle control, apoptosis, and B-cell differentiation. miR-16 target CHEK1 showed increased expression in t(14;18)-negative FLs, whereas TCL1A expression was reduced, in line with a partial loss of the germinal center B-cell phenotype in this FL subset. In conclusion, t(14;18)-negative FL have distinct microRNA profiles that are associated with an increased proliferative capacity and a "late" germinal center B-cell phenotype.