Commentary on the WHO classification of tumors of lymphoid tissues (2008): ��Gray zone�� lymphomas overlapping with Burkitt lymphoma or classical Hodgkin lymphoma

The 2008 WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues has introduced two new categories of high-grade B-cell lymphomas: entities in which features of diffuse large B-cell lymphoma (DLBCL) overlap with Burkitt lymphoma (DLBCL/BL) or classical Hodgkin lymphoma (DLBCL/HL). The DLBCL/BL category encompasses cases that resemble Burkitt lymphoma morphologically, but have one or more immunophenotypic or molecular genetic deviations that would exclude it from the BL category; conversely, some cases have immunophenotypic and/or genetic features of BL, but display cytologic variability unacceptable for BL. Many of the cases in the DLBCL/BL category contain a translocation of MYC as well as either BCL2 or BCL6 (so-called double-hit lymphomas) and have a very aggressive clinical behavior. The DLBCL/HL category encompasses lymphomas that exhibit the morphology of classical Hodgkin lymphoma but the immunophenotype of DLBCL, or vice versa. Most DLBCL/HL cases described present as mediastinal masses, but this category is not limited to mediastinal lymphomas. These new categories acknowledge the increasing recognition of cases that display mixed features of two well-established diseases. Whether the existence of such cases reflects shortcomings of our current diagnostic armamentarium or a true disease continuum in which such hybrid or intermediate neoplasms actually exist remains to be determined.     

Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma

MicroRNAs are naturally occurring small RNA species that regulate gene expression and are frequently abnormally expressed in cancers. However, the role of microRNAs in lymphoma is poorly understood. Therefore, we undertook a comprehensive study of microRNA expression in two of the most common lymphomas: diffuse large B-cell lymphoma (DLBCL) ( n = 80) and follicular lymphoma (FCL) ( n = 18) using microarrays containing probes for 464 human microRNAs. Unsupervised cluster analysis revealed distinct expression patterns between these two lymphomas and specific microRNA signatures (including members of the miR-17–92 cluster) were derived that correctly predicted lymphoma type in >95% of cases. Furthermore, we identified microRNAs in de novo DLBCL ( n = 64) associated with germinal centre-like and non-germinal centre-like immunophenotypes, international prognostic index status and event-free survival in CHOP and rituximab (R)-CHOP treated patients. Despite the indolent nature of FCL a significant proportion of cases undergo high-grade transformation to more aggressive DLBCL. In order to see if transformation is associated with changes in microRNA expression we compared transformed DLBCL cases ( n = 16) with de novo DLBCL, as well as FCL cases that underwent subsequent transformation ( n = 7) with FCL cases that had not transformed at a median follow-up of 60 months ( n = 11). Differential expression of 12 microRNAs correctly predicted >85% of transformed versus de novo DLBCL cases; six microRNAs ( miR-223, 217, 222 , 221 and let-7i and 7b) were found which could similarly predict or transformation in FCL ( P < 0.05). These data suggest that microRNAs have potential as diagnostic and prognostic markers in these lymphomas and may be used to identify FCL patients at risk of high-grade transformation.     

Long non-coding RNA as a novel biomarker and therapeutic target in aggressive B-cell non-Hodgkin lymphoma: A systematic review

Cancer initiation and progression have been associated with dysregulated long non-coding RNA (lncRNA) expression. However, the lncRNA expression profile in aggressive B-cell non-Hodgkin lymphoma (NHL) has not been comprehensively characterized. This systematic review aims to evaluate the role of lncRNAs as a biomarker to investigate their future potential in the diagnosis, real-time measurement of response to therapy and prognosis in aggressive B-cell NHL. We searched PubMed, Web of Science, Embase and Scopus databases using the keywords “long non-coding RNA”, “Diffuse large B-cell lymphoma”, “Burkitt's lymphoma” and “Mantle cell lymphoma”. We included studies on human subjects that measured the level of lncRNAs in samples from patients with aggressive B-cell NHL. We screened 608 papers, and 51 papers were included. The most studied aggressive B-cell NHL was diffuse large B-cell lymphoma (DLBCL). At least 79 lncRNAs were involved in the pathogenesis of aggressive B-cell NHL. Targeting lncRNAs could affect cell proliferation, viability, apoptosis, migration and invasion in aggressive B-cell NHL cell lines. Dysregulation of lncRNAs had prognostic (e.g. overall survival) and diagnostic values in patients with DLBCL, Burkitt's lymphoma (BL), or mantle cell lymphoma (MCL). Furthermore, dysregulation of lncRNAs was associated with response to treatments, such as CHOP-like chemotherapy regimens, in these patients. LncRNAs could be promising biomarkers for the diagnosis, prognosis and response to therapy in patients with aggressive B-cell NHL. Additionally, lncRNAs could be potential therapeutic targets for patients with aggressive B-cell NHL like DLBCL, MCL or BL.     

Prognostic impact of C-REL expression in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) with a germinal center B-cell (GCB) phenotype is believed to confer a better prognosis than DLBCL with an activated B-cell (ABC) phenotype. Previous studies have suggested that nuclear factor-κB (NF-κB) activation plays an important role in the ABC subtype of DLBCL, whereas c-REL amplification is associated with the GCB subtype. Using immunohistochemical techniques, we examined 68 newly diagnosed de novo DLBCL cases (median follow-up 44 months, range 1 to 142 months) for the expression of c-REL, BCL-6, CD10, and MUM1/IRF4. Forty-four (65%) cases demonstrated positive c-REL nuclear expression. In this cohort of patients, the GCB phenotype was associated with a better overall survival (OS) than the non-GCB phenotype (Kaplan–Meier survival (KMS) analysis, p = 0.016, Breslow–Gehan–Wilcoxon test). In general, c-REL nuclear expression did not correlate with GCB vs. non-GCB phenotype, International Prognostic Index score, or OS. However, cases with a GCB phenotype and negative nuclear c-REL demonstrated better OS than cases with a GCB phenotype and positive nuclear c-REL (KMS analysis, p = 0.045, Breslow–Gehan–Wilcoxon test), whereas in cases with non-GCB phenotype, the expression of c-REL did not significantly impact the prognosis. These results suggest that c-REL nuclear expression may be a prognostic factor in DLBCL and it may improve patient risk stratification in combination with GCB/non-GCB phenotyping.     

Evaluation of ARG protein expression in mature B cell lymphomas compared to non-neoplastic reactive lymph node

The participation of Abl-Related Gene (ARG) is demonstrated in pathogenesis of different human malignancies. However there is no conclusive evidence on ARG expression level in mature B cell lymphomas. In this study we evaluated ARG protein expression in Follicular Lymphoma (FL), Burkitt’s Lymphoma (BL) and Diffused Large B Cell Lymphoma (DLBCL) in comparison with non-neoplastic lymph nodes. Semi-quantitative fluorescent ImmunoHistoChemistry was applied on 14, 7 and 4 patients with DLBCL, FL and BL respectively, adding to 4 normal and 4 reactive lymph nodes. The mean ratio of ARG/GAPDH expression was significantly different (p < 0.00) between lymphomas and control samples, with DLBCL having the highest ARG expression amongst all. Over expression of ARG was seen in FL and BL, with FL expressing statistically more ARG than BL. Moreover, the ARG/GAPDH expression ratio increased from DLBCL stage I towards stage VI, all showing significantly more ARG expression than FL and BL (in all cases p < 0.00).     

Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53^−/− mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53^−/− mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53^−/− mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.     

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.     

Ran GTPase-Activating Protein 1 Is a Therapeutic Target in Diffuse Large B-Cell Lymphoma

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1⁺, while reactive lymphoid hyperplasia (n = 12) was RanGAP1⁺ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.     

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.     

The alteration of lipid metabolism in burkitt lymphoma identifies a novel marker: adipophilin

Background

Recent evidence suggests that lipid pathway is altered in many human tumours. In Burkitt lymphoma this is reflected by the presence of lipid droplets which are visible in the cytoplasm of neoplastic cells in cytological preparations. These vacuoles are not identifiable in biopsy section as lipids are “lost” during tissue processing.

Methods and Results

In this study we investigated the expression of genes involved in lipid metabolism, at both RNA and protein level in Burkitt lymphoma and in other B-cell aggressive lymphoma cases. Gene expression profile indicated a significant over-expression of the adipophilin gene and marked up-regulation of other genes involved in lipid metabolism in Burkitt lymphoma. These findings were confirmed by immunohistochemistry on a series od additional histological samples: 45 out of 47 BL cases showed strong adipophilin expression, while only 3 cases of the 33 of the not-Burkitt lymphoma category showed weak adipophilin expression (p<0.05).

Conclusions

Our preliminary results suggest that lipid metabolism is altered in BL, and this leads to the accumulation of lipid vacuoles. These vacuoles may be specifically recognized by a monoclonal antibody against adipophilin, which may therefore be a useful marker for Burkitt lymphoma because of its peculiar expression pattern. Moreover this peptide might represent an interesting candidate for interventional strategies.     

MYC Protein Expression in Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System

Primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) is a rare, aggressive subtype of DLBCL, the biology of which is poorly understood. Recent studies have suggested a prognostic role of MYC protein expression in systemic DLBCL, but little is known about the frequency and significance of MYC protein expression in CNS DLBCL. Hence, we investigated MYC protein expression profiles of CNS DLBCL and assessed the relationship between MYC expression and a variety of histopathologic, immunophenotypic, genetic, and clinical features. Fifty-nine CNS DLBCL diagnosed at our institution over the past 13 years were evaluated. The majority of cases (80%) showed centroblastic morphology, and 12 (20%) displayed a perivascular pattern of infiltration. According to the Hans criteria, 41 (69%) cases had a non-germinal center B-cell and 18 (31%) had a germinal center B-cell cell-of-origin (COO) phenotype. Mean MYC protein expression was 50% (median: 50%, range: 10-80%). Forty-three cases (73%) showed MYC overexpression (≥40%), and 35 (60%) showed MYC/BCL2 coexpression. MYC overexpression was seen in the single case harboring MYC translocation and in the cases showing increased copies of MYC (27%); however, no significant difference in mean MYC expression was seen between groups harboring or lacking MYC aberrations. In our series, age was associated with a significantly increased risk of death, and the perivascular pattern of infiltration was associated with a significantly increased risk of disease progression. Neither MYC expression (with or without BCL2 coexpression) nor other variables, including COO subtype were predictive of clinical outcome. Our findings indicate that the proportion of CNS DLBCL overexpressing MYC is higher compared to systemic DLBCL, and MYC overexpression appears to be independent of genetic MYC abnormalities. Thus, MYC expression and other immunophenotypic markers used for prognostication of systemic DLBCL might not apply to CNS DLBCL due to differences in disease biology.     

MicroRNA Profiling of Primary Cutaneous Large B-Cell Lymphomas

Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs). However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs) are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) and primary cutaneous follicle center lymphoma (PCFCL) are characterized by an activated B-cell (ABC)-genotype and a germinal center B-cell (GCB)-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.     

Phosphoinositide 3-kinase/AKT/mTORC1/2 signaling determines sensitivity of Burkitt's lymphoma cells to BH3 mimetics

Burkitt's lymphoma (BL), driven by translocation and overexpression of the c-MYC gene, is an aggressive, highly proliferative lymphoma, and novel therapeutic strategies are required to overcome drug resistance following conventional treatments. The importance of the prosurvival BCL-2 family member BCL-X(L) in BL cell survival suggests that antagonistic BH3-mimetic compounds may have therapeutic potential. Here, we show that treatment of BL cell lines with ABT-737 induces caspase-3/7 activation and apoptosis with varying potency. Using selective inhibitors, we identify phosphoinositide 3-kinase (PI3K) as a proproliferative/survival pathway in BL cells and investigate the potential of combined pharmacologic inhibition of both the BCL-2 family and PI3K signaling pathway. PI3K/AKT inhibition and ABT-737 treatment induced synergistic caspase activation, augmented BL cell apoptosis, and rendered chemoresistant cells sensitive. Targeting mTORC1/2 with PP242 was also effective, either as a monotherapy or, more generally, in combination with ABT-737. The combined use of a dual specificity PI3K/mTOR inhibitor (PI 103) with ABT-737 proved highly efficacious. PI 103 treatment of BL cells was associated with an increase in BIM/MCL-1 expression ratios and loss of c-MYC expression. Furthermore, blocking c-MYC function using the inhibitor 10058-F4 also induced apoptosis synergistically with ABT-737, suggesting that maintenance of expression of BCL-2 family members and/or c-MYC by the PI3K/AKT/mTOR pathway could contribute to BL cell survival and resistance to ABT-737. The combined use of BH3 mimetics and selective mTORC1/2 inhibitors may therefore be a useful novel therapeutic approach for the treatment of B-cell malignancy, including chemoresistant lymphomas.     

A Gene Panel,Including LRP12, Is Frequently Hypermethylated in Major Types of B-Cell Lymphoma

Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt''s lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma.     

CXCR5 polymorphisms in non-Hodgkin lymphoma risk and prognosis

CXCR5 [chemokine (C-X-C motif) receptor 5; also known as Burkitt lymphoma receptor 1 (BCR1)] is expressed on mature B-cells, subsets of CD4⁺ and CD8⁺ T-cells, and skin-derived migratory dendritic cells. Together with its ligand, CXCL13, CXCR5 is involved in guiding B-cells into the B-cell zones of secondary lymphoid organs as well as T-cell migration. This study evaluated the role of common germline genetic variation in CXCR5 in the risk and prognosis of non-Hodgkin lymphoma (NHL) using a clinic-based study of 1,521 controls and 2,694 NHL cases including 710 chronic lymphocytic leukemia/small lymphocytic lymphoma, 586 diffuse large B-cell lymphoma (DLBCL), 588 follicular lymphoma (FL), 137 mantle cell lymphoma (MCL), 230 marginal zone lymphoma (MZL), and 158 peripheral T-cell lymphoma (PTCL). Of the ten CXCR5 tag SNPs in our study, five were associated with risk of NHL, with rs1790192 having the strongest association (OR 1.19, 95 % CI 1.08–1.30; p = 0.0003). This SNP was most strongly associated with the risk of FL (OR 1.44, 95 % CI 1.25–1.66; p = 3.1 × 10^?7), with a lower degree of association with DLBCL (OR 1.16, 95 % CI 1.01–1.33; p = 0.04) and PTCL (OR 1.29, 95 % CI 1.02–1.64; p = 0.04) but no association with the risk of MCL or MZL. For FL patients that were observed as initial disease management, the number of minor alleles of rs1790192 was associated with better event-free survival (HR 0.64; 95 % CI 0.47–0.87; p = 0.004). These results provide additional evidence for a role of host genetic variation in CXCR5 in lymphomagenesis, particularly for FL.