Cardioneuroablation in a Young Patient with Sinus Node Dysfunction Secondary to Cardiotoxicity in Diffuse Large B-Cell Lymphoma

Comprehensive genomic profiling of orbital and ocular adnexal lymphomas identifies frequent alterations in MYD88 and chromatin modifiers: new routes to targeted therapies

Purpose:This study aimed to investigate the effects of microRNA-222-3p on activated B cell-like-type diffuse large B-cell lymphoma cells and the regulatory relationship between microRNA-222-3p and phosphatase 2 regulatory subunit B alpha.Method:The expression of microRNA-222-3p was detected in activated B cell-like-type diffuse large B-cell lymphoma tissues and cells by quantitative reverse transcription polymerase chain reaction. The regulatory effects of microRNA-222-3p on the proliferation, invasion, and apoptosis of activated B cell-like-type diffuse large B-cell lymphoma cells were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, flow cytometry, and Transwell assay. The regulatory relationship between microRNA-222-3p and phosphatase 2 regulatory subunit B alpha was determined by luciferase reporter gene and RNA pull-down assay. In addition, the effects of microRNA-222-3p on tumor growth were further analyzed in mice.Results:MicroRNA-222-3p and phosphatase 2 regulatory subunit B alpha were significantly up- and downregulated in activated B cell-like-type diffuse large B-cell lymphoma tissues and cells, respectively. Phosphatase 2 regulatory subunit B alpha was a target of microRNA-222-3p. MicroRNA-222-3p promoted the proliferation and invasion and inhibited the apoptosis of activated B cell-like-type diffuse large B-cell lymphoma cells. Phosphatase 2 regulatory subunit B alpha reversed the tumor-promoting effects of microRNA-222-3p on activated B cell-like-type diffuse large B-cell lymphoma cells. In addition, microRNA-222-3p promoted the tumor growth in mice and downregulated phosphatase 2 regulatory subunit B alpha in tumor tissues.Conclusion:MicroRNA-222-3p promoted the proliferation and invasion and inhibited the apoptosis of activated B cell-like-type diffuse large B-cell lymphoma cells through suppressing phosphatase 2 regulatory subunit B alpha expression.

Background Orbital diffuse large B cell lymphoma(DLBCL) is a kind of malignant lymphoma with higher morbidity,but the systematic study is difficult for the fewer cases and the lack of orbital DLBCL cell line.Objective This study was to compare the differences or similarities of histopathological characteristics between orbital DLBCL and systemic DLBCL,and to discuss whether systemic DLBCL cell line is available to in vitro research of orbit DLBCL.Methods The histopathological specimens were collected from 3 orbital DLBCL patients and 15 systemic DLBCL patients in Tianjin Eye Hospital and Tianjin Tumor Hospital from 2004 to 2009.The prognosis of the patients was followed-up,and the survival curve was drew.The histopathological examination of the specimens was performed using hematoxylin and eosin staining,and the expressions of CD20,CD79α,CD45RO,CD10,BCL-6,mum-1,Ki-67 and surviving in the specimens were detected by immunochemistry.The results mentioned above were analyzed and compared between orbital DLBCL and systemic DLBCL.Results The histopathological examination showed that the centroblastic type was the primary form in both orbital DLBCL and systemic DLBCL,according with the diagnosis of DLBCL.The differences in the positive expression rate of CD20,CD79α,CD45RO,Ki-67,survivin were not statistically significant between the two types of DLBCL (P =0.167,0.442,1.000,1.000,0.442).Immunochemistry revealed that 3 orbital DLBCL patients were germinal center B-cell-like (GCB),and 2 of the patients showed the positive expression for BCL-6 and mum-1 as well as absent expression for CD10,but the other patient presented the absent expression for BCL-6,mum-1 and CD10.In 15 systemic DLBCL patients,7 were GCB type,with the positive expression for CD10 in all the 7 patients and absent expression for BCL-6 in 6 patients.In addition,in 8 of non-GCB type,3 appeared to be absent expressed for CD10 and BCL-6,and 5 were positive expressed for BCL-6 and mum-1 and absent expressed for CD10.No significant difference was found in the survival duration between the orbital DLBCL and systemic DLBCL (P =0.067).Conclusions There exists no clinically significant difference in the pathological features,the expression of tumor cell markers and prognosis in both orbital and systemic DLBCL.These two DLBCLs appear to be similar in epidemiology and clinical staging,inferring that systemic DLBCL cell line is available in the in vitro study of orbital DLBCL. Key words: Neoplasm; Orbit; Diffuse large B cell lymphoma; Pathology; Immunochemistry

Simple SummaryThere are inequalities in cancer survival between patients with or without comorbidities. The healthcare pathway (i.e., diagnostic route) of a patient is thought to explain some of these inequalities. We explore how much of the effect of comorbidity on survival of patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL) is explained by the diagnostic route (i.e., emergency diagnosis). We used mediation analysis to separate the effect of comorbidity on survival from its effect through diagnostic route. We found that, for DLBCL and FL, emergency diagnosis accounted for 24% and 16% of the inequalities in survival between comorbidity groups within 12 months since cancer diagnosis. This proportion reduced over time and was small after 5 years of follow up. Comorbidities can complicate the diagnosis and management of patients with DLBCL or FL. Our results show that greater research is needed to ensure patients with comorbidities have a timely diagnosis and will help to reduce the inequalities in cancer survival.Background: Socioeconomic inequalities in survival from non-Hodgkin lymphoma persist. Comorbidities are more prevalent amongst those in more deprived areas and are associated with diagnostic delay (emergency diagnostic route), which is also associated with poorer survival probability. We aimed to describe the effect of comorbidity on the probability of death mediated by diagnostic route (emergency vs. elective route) amongst patients with diffuse large B-cell (DLBCL) or follicular lymphoma (FL). Methods: We linked the English population-based cancer registry and hospital admission records (2005–2013) of patients aged 45–99 years. We decomposed the effect of comorbidity on survival into an indirect effect acting through diagnostic route and a direct effect not mediated by diagnostic route. Furthermore, we estimated the proportion of the comorbidity effect on survival mediated by diagnostic route. Results: For both DLBCL (n = 27,379) and FL (n = 14,043), those with any comorbidity, or living in more deprived areas, were more likely to experience diagnostic delay and poorer survival. The indirect effect of comorbidity on mortality through diagnostic route was highest at 12 months since diagnosis (DLBCL: Odds Ratio 1.10 [95% CI 1.07–1.13], FL: OR 1.09 [95% CI 1.04–1.14]). Within the first 12 months since diagnosis, emergency diagnostic route accounted for 24% (95% CI 17.5–29.5) and 16% (95% CI 6.0–25.6) of the comorbidity effect on mortality, for DLBCL and FL, respectively. Conclusion: Efforts to reduce diagnostic delay (emergency diagnosis) amongst patients with comorbidity would reduce inequalities in DLBCL and FL survival by 24% and 16%, respectively. Further public health programs and interventions are needed to reduce diagnostic delay amongst lymphoma patients with comorbidities.

The benefit of intrathecal therapy and systemic rituximab on the outcome of diffuse large B-cell lymphoma at risk of central nervous system disease is controversial. Furthermore, the effect of intrathecal treatment and rituximab in diffuse large B-cell and Burkitt lymphoma with occult leptomeningeal disease detected by flow cytometry at diagnosis is unknown. Untreated diffuse large B-cell (n=246) and Burkitt (n=80) lymphoma at clinical risk of central nervous system disease and having had pre-treatment cerebrospinal fluid were analyzed by flow cytometry and cytology. Spinal fluid involvement was detected by flow cytometry alone (occult) in 33 (13%) diffuse large B-cell and 9 (11%) Burkitt lymphoma patients, and detected by cytology in 11 (4.5%) and 5 (6%) patients, respectively. Diffuse large B-cell lymphoma with occult spinal fluid involvement had poorer survival (P=0.0001) and freedom from central nervous system relapse (P<0.0001) compared to negative cases. Burkitt lymphoma with occult spinal fluid involvement had an inferior freedom from central nervous system relapse (P=0.026) but not survival. The amount of intrathecal chemotherapy was quantitatively associated with survival in diffuse large B-cell lymphoma with (P=0.02) and without (P=0.001) occult spinal fluid involvement. However, progression of systemic disease and not control of central nervous system disease was the principal cause of treatment failure. In diffuse large B-cell lymphoma, systemic rituximab was associated with improved freedom from central nervous system relapse (P=0.003) but not with survival. Our results suggest that patients at risk of central nervous system disease should be evaluated by flow cytometry and that intrathecal prophylaxis/therapy is beneficial.

High throughput tissue microarray analysis of FHIT expression in diffuse large cell B-cell lymphoma from Saudi Arabia

Background: Chronic activation of the B-cell receptor (BCR) pathway is critical for proliferation and survival of malignant B cells, and is implicated in the pathogenesis of B-cell lymphomas. Spleen tyrosine kinase (SYK) is a central regulator of the BCR pathway. Although there are several reports of SYK inhibitory compounds in development, there appears to still be a need for a highly selective SYK inhibitor. Here we report on a novel SYK inhibitor, TAS-5567, which displays high potency with excellent kinase selectivity. In this study, we characterized the in vitro cytotoxic effect and the mechanism of action of TAS-5567 in DLBCL cells, and evaluated in vivo antitumor activity in DLBCL xenograft models. Materials and Methods: In vitro enzyme inhibition of SYK was determined by using purified SYK and the peptide substrate. Selectivity for kinases of TAS-5567 was determined in a panel of 196 kinases. To evaluate the target engagement of TAS-5567, the cellular phosphorylation of BLNK(Y84), a substrate of SYK kinase, was examined by Flow cytometry. For a growth inhibition assay, DLBCL cells were treated with TAS-5567 for 3 days, and the number of living cells was determined by measuring cellular ATP. In order to demonstrate TAS-5567’s mechanism of action in DLBCL cells, a cell cycle analysis was conducted by Flow cytometry. For evaluation of pharmacokinetics (PK), pharmacodynamics (PD), and antitumor effect of TAS-5567 in vivo, we orally dosed TAS-5567 in human DLBCL xenograft models. Results: TAS-5567 was a potent inhibitor of SYK with an IC50 value of 0.37nM, and demonstrated little activities against the other 196 kinases. In SU-DHL10 DLBCL cells, TAS-5567 potently inhibited the phosphorylation of BLNK (IC50 = 10 nM), and inhibited cell proliferation in a concentration-dependent manner (IC50 = 68 nM). Cell cycle arrest at G1 phase and apoptosis were observed in the DLBCL cells by continuous exposure to TAS-5567 at 300 nM. Also, TAS-5567 exhibited broad antiproliferation activity across various DLBCL cell lines in vitro. In vivo, TAS-5567 demonstrated favorable oral PK properties in mice, and exhibited a sustained and robust inhibition of the pharmacodynamics marker pBLNK in tumor tissues of SU-DHL10 DLBCL xenograft model. Consistent with the results of the pharmacodynamics study, TAS-5567 displayed significant antitumor effect in the SU-DHL10 xenograft models at PD effective doses with minimum body weight changes. Conclusion: TAS-5567 is an orally available, highly potent, and selective inhibitor of SYK. TAS-5567 demonstrated substantial antitumor activity in DLBCL cells both in vitro and in vivo, with an accompanying sustained and robust inhibition of SYK in the cells. These data suggest that TAS-5567 may be a promising SYK inhibitor for the treatment of DLBCL. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A273. Citation Format: Hiroki Irie, Aki Kawagishi, Keiko Ishihara, Shingo Tsuji, Yoko Nakatsuru, Jiro Kuze, Naoko Fujino, Yoshio Ogino, Hideya Komatani, Teruhiro Utsugi. TAS-5567, a highly potent and selective inhibitor of SYK kinase, demonstrated antitumor activity in diffuse large B-cell lymphoma (DLBCL) cellsin vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A273.

Role of Autoantigen Induced-B-Cell Receptor Internalization and Its Inhibition for the Biology and Survival of Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Objective To study the expression of microRNA-21（miR-21）in serum of patient with diffuse large B cell lymphoma （DLBCL） and DLBCL cell lines and validate the significance of miR-21 in early diagnosis, genotyping and prognosis estimates of DLBCL. Methods miR-21 expression were detected by fluorescent quantity polymerase chain reaction （FQ-PCR）in 9 lymphoma cell lines (OCI-Ly1，OCI-Ly3，OCI-Ly4，OCI-Ly7，OCI-Ly8，OCI-Ly10，OCI-Ly18，OCI-Ly19 and HBL), the serum from DLBCL patients (n=62) and health controls (n=50).Kaplan-Meier survival analysis was carried out during the relapse-free survival period of DLBCL patients to explore the relationship between the prognosis and microRNA expression level. Results Real time FQ-PCR result indicated that miR-21 expression was higher in DLBCL cell lines than that in normal B cells (BC).miR-21 expression in normal B cell and 9 DLBCL cell lines separately were 1.04±0.02，2.30±0.35，237.97±56.19，5.27±0.83，3.40±0.30，11.22±2.70，133.55±16.78，6.63±0.24，4.91±0.37 and 81.59±6.64.Compared with BC, the expression of miR-21 were higher in all 9 DLBCL cell lines (t=7.3，13.7，21.0，6.2，8.8，13.6，6.5，39.5，18.1；P＜0.01). miR-21 expression segregates with specific molecular subgroups of DLBCL.The expression was higher in the ABC type cell lines (OCI-Ly3, OCI-Ly10, HBL) than GCB type cell lines (OCI-Ly1, OCI-Ly4, OCI-Ly7, OCI-Ly8, OCI-Ly18, OCI-Ly19；t=11.18，P＜0.01).Consistent with the cell line models, miR-21 expression levels were higher in serum from DLBCL patients ［21.38（10.26-45.21）］ than from controls ［1.87（1.05-3.97）, U=168, P=0.000］, and the levels were higher in DLBCL cases with an ABC-type ［28.68(14.92-98.44)］ than those in GCB-type ［18.30(7.32-33.46),U=336, P=0.043］.MiR-21 expression levels were different in sera from different clinical stage DLBCL patients.The miR-21 level in serum of patients with subgroup ABC and subgroup GCB in stage Ⅰ and Ⅱ were 47.49（25.65-295.41） and 24.74（16.08-50.38） respectively and in stage Ⅲ and Ⅳ were 16.66（5.35-44.30） and 11.96（4.10-21.05） respectively.The levels were higher in DLBCL cases with Ⅰ-Ⅱ stage than those with Ⅲ-Ⅳ stage (U=62, P=0.013 in GCB type；U=53, P=0.014 in ABC type).Moreover, compare with relapse-free survival in DLBCL patients, high miR-21 expression was associated with well prognosis (U=259, P=0.035). Conclusions MiR-21 is high expression in DLBCL cell lines and DLBCL patients serum.miR-21 level in sera from DLBCL patients is associated with clinical stage, molecular subgroup and prognosis estimates.MiR-21 may serve as a new biomarker to early detection, genotyping and prognosis estimates of DLBCL.（Chin J Lab Med，2012,35：431-435） Key words: Lymphoma，large B cell，diffuse; MicroRNAs; Early detection; Tumor markers，biological

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), representing around 30% to 40% of all newly diagnosed lymphomas 1. DLBCL is clinically, morphologically and biologically a heterogeneous disease reflected in the highly variable clinical course. The 2008 World Health Organization (WHO) classification of lymphoid malignancies recognizes within the group of DLBCL, not otherwise specified (NOS) several subtypes characterized by unique clinical and pathological features including primary DLBCL of the central nervous system (CNS), primary cutaneous DLBCL, leg type, T-cell histiocyte-rich large cell lymphoma and EBV positive DLBCL of the elderly (Table 1). Nevertheless, most cases of DLBCL fall into the ‘NOS’ category. In the last 15 years our understanding of the genetic changes and biology of DLBCL has increased tremendously 2. Gene expression profiling (GEP) studies have revealed that DLBCL comprises several molecular subgroups that reflect either the stage in B cell development from which the disease originates or the activity of different biological programs 3, 4. The standard initial treatment for DLBCL is combined immunochemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) 5. While durable remissions can be achieved in the majority of cases with this combined regimen, over 30% of patients will not respond or will relapse with resistant disease. A possible explanation for such differences in therapeutic success is the considerable biological heterogeneity of DLBCL. Therefore, there is an ongoing effort to tailor therapy based on specific subtypes of DLBCL, and to identify prognostic markers like BCL2 and MYC. The diagnosis of DLBCL needs to integrate, in addition to classic morphology and immunophenotype, all the new genetic and molecular diagnostic tools. This report attempts to review distinctive pathological characteristics of DLBCL and their clinical significance. DLBCL is characterized by monoclonal rearrangement of immunoglobulin heavy (IGH) and light chain (IGκ-IGλ) genes. Analogous to most B-NHL, DLBCL derives from a mature B cell that has experienced the germinal center (GC) reaction. Based on GEP studies DLBCLs have been divided into two main subgroups based on the putative cells of origin 3, 4. GC B cell-like (GCB)-DLBCL (50% cases) exhibits a transcriptional profile that resembles that of GC B-cell with expression of genes normally detected in GC B-cells such as CD10 and the transcriptional repressor BCL6 and harboring highly mutated immunoglobulin genes with ongoing somatic hypermutations (SHM). Activated B cell-like (ABC)-DLBCL shows several features of B cell receptor (BCR) activated B-cells at a plasmablastic stage, just prior to exit the GC with up-regulation of genes required for plasma cell differentiation (IRF4/MUM1) (Figure 1). These tumors downregulate the GC-specific program, activating at the same time, the NF-κB and BCR signaling pathways 2. Consistent with their late GC origin, these tumors do not show evidence of ongoing SHM. Because not all cases of DLBCL can be characterized as GCB or ABC, a less well-characterized group comprising about 15% of the cases remain unclassifiable. Within the group of mature B cell NHL, DLBCL shows the highest degree of genomic complexity including point mutations and copy number aberrations, and less frequently chromosomal translocations and gene amplification 2. Although some of these lesions might be observed in both GCB and ABC subtypes of DLBCL, most of them are preferentially associated with one or the other subtype of DLBCL, suggesting their potential role for diagnostic, prognostic and therapeutic stratification (Figure 2). The frequent mutations found in DLBCL are thought to be the result of an aberrant function of the physiologic SHM mechanism. Accordingly, mutations in CREBBP/EP300 and MLL2 are preferentially seen in GCB-subtype. EZH2 is required for GC formation and mutations in this gene are restricted to GCB-DLBCL. Translocations resulting in deregulated BCL2 and MYC are almost restricted to GCB-DLBCL. Around 30–40% of GCB-DLBCL carry the t(14;18) translocation, whereas MYC translocations have been identified in 5–15% of DLBCL 6. In contrast, translocations involving the BCL6 locus are present in 35% of DLBCL cases, being more frequent in ABC-DLBCL (25%). Another major transformation mechanism that impairs plasma cell differentiation are mutations and deletions in PRDM1/BLIMP1 identified in 25% of ABC-DLBCL. Constitutive activation of the NF-κB transcription factor complex represents the hallmark of ABC-DLBCL 2. The underlying causes of NF-κB signaling pathway activation are diverse and include gain-of-function mutations in signal transduction components of the BCR (CD79a and CARD11) and toll-like receptor (MYD88) signaling pathways. Loss-of-function mutation and/or deletions in the NF-κB negative regulator TNFAIP3/A20 have been reported in up to 30% of ABC-DLBCL. The diagnosis of DLBCL is usually not difficult. Lymph nodes demonstrate a diffuse proliferation of large lymphoid cells that have totally or partially effaced architecture. Cytological, DLBCL can be centroblastic, immunoblastic or anaplastic in appearance 1. Centroblastic morphology is the most common variant composed of medium to large cells with round to oval vesicular nuclei and fine chromatin with two to four nuclear membrane-bound nucleoli. There is moderate amphophilic to basophilic cytoplasm. In the immunoblastic variant greater than 90% of the cells have an immunoblastic appearance with a large centrally located nucleolus and basophilic or amphophilic cytoplasm. This variant has been associated with the non-germinal center type derivation, an adverse prognosis 4 and recently as a major reservoir for MYC-IGH translocations. In most cases, however; the tumors show an admixture of centroblasts and immunoblasts. The distinction of the immunoblastic variant from the centroblastic variant is not always straightforward and has generally met poor intraobserver and interobserver reproducibility. The anaplastic variant is rather rare and is characterized by large cells with bizarre pleomorphic nuclei. These cells may resemble Hodgkin or RS cells and may show sinusoidal and/or cohesive growth pattern and even mimic undifferentiated carcinoma. The anaplastic morphology is independent of ALK expression in B-cell lymphomas. Other rare morphologic variants exist including the signet ring cell, microvillous and spindle cell variants. The neoplastic cells characteristically express pan B-cell markers including CD19, CD20, CD22, PAX5 and CD79a. Surface and/or cytoplasmic immunoglobulin (IgM > IgG > IgA) can be demonstrated in 50–75% of the cases. Other markers commonly used in the characterization of DLBCL include CD10, BCL6, BCL2 and IRF4/MUM1 1. Aberrant phenotypes are not uncommon in DLBCL and may be responsible for confusion in the diagnosis. Lack of one or more B-cell markers may occur. In 10% of the cases the tumor cells express CD5. This CD5 positive DLBCL usually represent de novo cases, and only rarely are transformed CLL cases. Expression of CD5 is associated with worse prognosis even in the rituximab era. Aberrant expression of cytoplasmic CD3 has been also documented mainly in extranodal DLBCL without the expression of other T-cell markers. DLBCL with cyclin D1 expression lack the characteristic t(11;14) translocation of MCL, have a centroblastic morphology and a post-germinal center phenotype with positivity for IRF4/MUM1. These cases should be distinguished from MCL with blastic or pleomorphic morphology. In situ hybridization for EBERs should be performed in cases with geographic necrosis and/or Reed–Stenberg-like cells to exclude the possibility of an EBV+DLBCL of the elderly. Because of the prognostic value of cell of origin and the increasing effort to tailor therapy based on molecular characteristics of DLBCL, a reliable method to identify GCB and non-GCB subtypes is needed. GEP, which is considered the gold standard to assign the molecular subtypes, is not routinely available and is not cost effective in routine diagnosis. Several studies have attempted to recapitulate the molecular subgroups (GCB vs. non-GCB) using a limited panel of antibodies available in most pathology laboratories (Figure 1). The Hans algorithm has been the most widely used in clinical trials. In this classifier three antibodies are used CD10, BCL6 and IRF4/MUM1. According to this algorithm DLBCL with CD10 expression in more than 30% cells belong to the GCB group. Cases that are CD10 negative, BCL6 positive but IRF4/MUM1 negative are also GCB subtype. Cases that are IRF4/MUM1 positive with or without expression of BCL6 are assigned to the non-GCB subtype. There have been other algorithms attempting to improve the Hans classifier. These include the use of FOXP1 and GCET1 by Choi et al., and the use of LMO2 by Natkunam et al. One study comparing different algorithms showed an 87% concordance with GEP for the Choi method and 86% for Hans scheme 7. In this same study, the Tally algorithm based on FOXP1, GCET1, CD10, IRF4/MUM1 and LMO2 antibodies was demonstrated to be the most robust. The use of BCL6 was excluded from the analysis because it was considered a problematic, poorly reproducible stain. Nevertheless, in our experience BCL6 is reliable and helpful in the routine diagnosis. Accordingly, in the report from the international DLBCL rituximab-CHOP consortium program study, an algorithm based on expression of CD10, FOXP1 and BCL6 was used, which had a 92.6% concordance with GEP 8. Although most studies find that immunohistochemical algorithms correlate with prognosis in DLBCL, everybody agrees that these algorithms are an imperfect substitution for GEP. The improvement of molecular techniques is making possible to use paraffin-embedded material with results comparable to fresh-frozen material. The recent development of a 20-gene assay using NanoString technology in paraffin-embedded tissue is a promising methodology with potential to be used as a routine method for determining cell of origin in DLBCL 9. Translocations involving the MYC oncogene are the molecular hallmark of Burkitt lymphoma (BL). Most cases involve the IGH@ gene on chromosome 14. Less commonly, the light chain genes on chromosome 2 and 22 are involved in the translocation. These translocations in BL are characteristically the sole chromosomal aberration identified 10, 11. Approximately 5 to 15% of DLBCL cases have been reported to carry MYC translocations, whereas 19–38% of cases show MYC low copy number gains 6. Most MYC rearranged DLBCL cases fall into the GCB subtype 10, 12. In contrast to BL, MYC rearrangements in DLBCL are often seen to non-IGH@ partners and with complex karyotypes 10. The prognostic significance of MYC rearrangement alone in DLBCL is controversial. Several studies have demonstrated the association of MYC rearrangement with poorer outcome in DLBCL patients treated with R-CHOP 6. However, recent studies have suggested that the impact of MYC is strongly influenced by BCL2 and that MYC alone does not have a worse prognosis 13, 14. MYC rearranged DLBCL may either arise de novo or may represent a high-grade transformation of a low-grade lymphoma, usually follicular lymphoma. In the latter case the MYC translocation is accompanied by a BCL2 rearrangement, so-called ‘double-hit’ (DH) lymphoma. Approximately 40% of all DH lymphomas represent transformed FL cases 15. However, 60–80% of MYC rearranged bona-fide de novo DLBCL are accompanied either by a BCL2 and/or a BCL6 rearrangement, representing DH or triple hit (TH) lymphomas 12, 14. Altogether DLBCL with DH/TH represent around 3–6% of all DLBCL and are generally refractory to standard chemotherapy regimens and have a poor prognosis 13, 14, 16. Importantly, not all patients with DLBCL morphology and DH/TH may have a dismal prognosis. Recently, attention has focused on the apparent importance of the MYC partner because it has been suggested that only cases with an IG/MYC translocation have an adverse prognosis. The development of FISH assays readily performed in paraffin sections and more recently a monoclonal antibody that specifically recognizes MYC protein have facilitated the recognition of MYC alterations in routine diagnosis. Break-apart FISH probes are considered to be the most sensitive assay for identifying chromosomal rearrangements. In addition, the use of MYC/IGH dual fusion probe helps to confirm the MYC partner 6. However, dual fusion probes for MYC/IGκ and MYC/IGλ are not commercially available. MYC protein expression has been proposed as a rapid and cost-effective screening tool to identify MYC rearrangements. Although some studies agree that finding >70% MYC+ cells correlates with MYC rearrangement 6, 13, a considerable number of cases with MYC rearrangement fall into the low MYC expression group (<40%) 12. A combined approach MYC-FISH and IHC seems advisable. More common than MYC translocations is MYC protein overexpression reported to occur in 25–30% of DLBCL cases 13, 14, 16. Most studies used a cut-off of 40% for MYC positivity. Of note, the negative prognostic impact of MYC protein expression is observed only in patients who simultaneously overexpressed BCL2 protein, so-called ‘double-expresser’ (DE). The cut-off for BCL2 protein overexpression varies in different studies, but most studies required at least 50% of tumor cells. MYC and BCL2 DE have been reported to occur in 19–34% of DLBCL patients, and to have a worse prognosis than patients who do not express any or only one protein 13, 14, 16, but better prognosis than DH/TH DLBCL, which have a dismal outcome. Interestingly, the DE cases appear more common in the ABC subtype, and it has been suggested that this may largely contribute to the known inferior survival of the ABC subtype 16. The morphological distinction between BL and DLBCL has been problematic for pathologists. GEP studies have shown that BL has a characteristic signature but that there are cases within the spectrum of DLBCL and aggressive B-cell lymphomas, which have a similar molecular BL signature or fell into an intermediate category 10, 11. The 2008 WHO classification recognized this problematic an added a provisional category of B cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCLU) (Figure 3) 15. BCLU is a disease of older patients presenting with nodal or extranodal disease usually in an advanced clinical stage, high lactate dehydrogenase and frequent bone marrow and CNS infiltration with a dismal prognosis. Interestingly, MYC rearrangements can be detected in 30–50% of the cases usually associated with many more chromosomal aberrations 15. The incidence of DH/TH in BCLU has been reported to be high (32%–78%).The precise morphological boundaries of this category are still not well defined. There is an ongoing discussion about how to classify aggressive lymphomas with DH/TH. One proposal is to continue classifying them based on morphology as DLBCL or BCLU or putting them together as a group of ‘DH/TH lymphomas’ regardless of the morphology of the tumor cells. The understanding of the biology of DLBCL has increased tremendously in the last 15 years. The diagnosis of DLBCL needs to integrate, in addition to standard morphology and immunohistochemistry, all available ancillary techniques. The routine use of FISH and IHC to detect MYC and BCL2 alterations/overexpression is recommended. Patients with DH and double expression of MYC and BCL2 represent poor-risk subsets in which alternative strategies should be explored 5. The distinction of GCB versus ABC-DLBCL has not yet led to differences in primary treatment. The current standard of care for most patients is R-CHOP, which has improved dramatically the outcome of DLBCL. However, for patients who fail R-CHOP, the choice of therapy is very likely to be influenced by the cell of origin 5. For example, bortezomib or BTK inhibitors have been shown to be effective in relapsed ABC-DLBCL but not in GCB subtype. On the contrary treatment with EZH2 inhibitors and BCL6 inhibitors have been shown to be effective in relapsed GCB-DLBCL. Emerging new targeted therapy will certainly influence the diagnosis and treatment of DLBCL in the near future.

From Microarray to Bedside: Targeting NF-κB for Therapy of Lymphomas

Diffuse Large B-Cell Lymphoma (DLBCL) is a clinically and biologically heterogeneous disease. The revised Classification of Lymphoproliferative diseases published in 2016 (WHO, 2016) refined the previous DLBLC subtypes and identified four categories: DLBCL not otherwise specified (NOS), other lymphomas of large B cells, high grade B-cell lymphoma, and B-cell lymphoma unclassifiable. High grade B-cell lymphomas include the entities carrying MYC, BCL2 and/or BCL6 translocations or cases with blastoid morphology without DH translocations. This classification also acknowledges the cell of origin (COO) classification, that has only a limited impact on the choice of frontline treatment for DLBCL, as most patients still receive R-CHOP chemoimmunotherapy. Attempts to improve the outcomes of specific subgroups, especially COO groups, have so far had limited success. Newer analyses have further subdivided DLBCL into genomically distinct subsets, not yet incorporated in the WHO classification, which may facilitate targeted approaches to therapy. In this review, we discuss the subgroups that are recognized by the WHO 2016 classification, review the newer genomic data, and speculate on how this could alter the treatment landscape of DLBCL in the future. We also discuss novel approaches to salvage therapy in the broad context of the heterogeneity of DLBCL.

This study aimed to investigate the performance of Pulsatilla saponin A (PsA) in diffuse large B-cell lymphoma (DLBCL) cells. Proliferation, ELISA, apoptosis, cell cycle analysis, and assays were carried out to detect the growth and apoptosis in DLBCL cells. Western blotting was used to identify the change in the protein. In cell assays, PsA significantly inhibited the growth and apoptosis in DLBCL cells. The IL-10 and TNF-α of OCI-LY10 and U2932 cells were reduced after 24 h PsA treatment. Bax, cleaved PARP, and cleaved Caspase-3 were increased, while Bcl-2 and C-Myc decreased after PsA treatment. IL-10 may regulate the expression of C-Myc protein in cells by activating the JAK2/STAT3 signaling pathway. PsA can inhibit the overexpression of p-JAK2 and p- STAT3 signaling pathways induced by IL-10 stimulants. The proliferation and apoptosis induced by PsA were confirmed in DLBCL cells. Our findings revealed that PsA may exert its antitumor effect by causing G1 arrest and apoptosis in DLBCL cells. The mechanism of PsA regulating apoptosis in DLBCL cells is probably through the JAK2/STAT3 signaling pathway in vitro.

Diffuse large B-cell lymphoma (DLBCL) is commonly treated with R-CHOP, but ~30 to 50% of the patients are poorly responsive to this strategy. Geniposide, an extract from the Gardenia jasminoides Ellis, plays antitumor roles in human gastric cancer, hepatocellular carcinoma, and oral squamous carcinoma. However, the effects of geniposide treatment on DLBCL cells, as well as its underlying mechanism, are still unknown. Here, we found that geniposide inhibited the proliferation of OCI-LY7 and OCI-LY3 cells in a dose-dependent manner. Furthermore, geniposide increased the percentage of apoptotic cells and upregulated the levels of cleaved PARP and cleaved caspase-3 in DLBCL cells. Interestingly, geniposide treatment significantly reduced the expression of the long noncoding RNA HLA complex P5 (lncRNA HCP5) in DLBCL cells. HCP5 expression was revealed to be upregulated in DLBCL tissues and cell lines. Moreover, HCP5 knockdown resulted in proliferation inhibition and apoptosis in OCI-LY7 and OCI-LY3 cells. miR-27b-3p was predicted as a potential target of HCP5 using the lnCAR web tool. Both HCP5 silencing and geniposide treatment increased the level of miR-27b-3p in DLBCL cells. Accordingly, a luciferase reporter assay identified miR-27b-3p as a direct target of HCP5. The expression of miR-27b-3p was upregulated and inversely correlated with the HCP5 level in DLBCL tissues. HCP5 knockdown reduced MET protein expression, which was subsequently rescued by miR-27b-3p silencing in DLBCL cells. Importantly, the restoration of MET partially reversed the geniposide-induced proliferation inhibition and apoptosis of DLBCL cells. In conclusion, geniposide inhibits the proliferation and induces the apoptosis of DLBCL cells at least partially by regulating the HCP5/miR-27b-3p/MET axis, indicating a potential strategy for DLBCL treatment.

Hist-O-07 - Classification of primary cutaneous large B-cell lymphomas according to cell of origin is clinically relevant