Teresa Amato

The different epidemiologic subtypes of Burkitt lymphoma share a homogenous micro RNA profile distinct from diffuse large B-cell lymphoma

Sporadic Burkitt lymphoma (sBL) can be delineated from diffuse large B-cell lymphoma (DLBCL) by a very homogeneous mRNA expression signature. However, it remained unclear whether all three BL variants—sBL, endemic BL (eBL) and human immunodeficiency virus-associated BL (HIV-BL)—represent a uniform biological entity despite their differences in geographical occurrence, association with immunodeficiency and/or incidence of Epstein–Barr virus (EBV) infection. To address this issue, we generated micro RNA (miRNA) profiles from 18 eBL, 31 sBL and 15 HIV-BL cases. In addition, we analyzed the miRNA expression of 86 DLBCL to determine whether miRNA profiles recapitulate the molecular differences between BL and DLBCL evidenced by mRNA profiling. A signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was obtained that differentiated BL from DLBCL. The miRNA profiles of sBL and eBL displayed only six differentially expressed miRNAs, whereas HIV and EBV infection had no impact on the miRNA profile of BL. In conclusion, miRNA profiling confirms that BL and DLBCL represent distinct lymphoma categories and demonstrates that the three BL variants are representatives of the same biological entity with only marginal miRNA expression differences between eBL and sBL.

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2‐like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B‐ and T‐cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B‐ and T‐cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T‐lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed–Sternberg cells of classical Hodgkins lymphoma also showed strong nuclear staining. Diffuse large B‐cell, Burkitts lymphomas, and peripheral T‐cell lymphomas, among T‐cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B‐cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkins lymphomas, Burkitts lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated. Copyright

Distinct Viral and Mutational Spectrum of Endemic Burkitt Lymphoma

Endemic Burkitt lymphoma (eBL) is primarily found in children in equatorial regions and represents the first historical example of a virus-associated human malignancy. Although Epstein-Barr virus (EBV) infection and MYC translocations are hallmarks of the disease, it is unclear whether other factors may contribute to its development. We performed RNA-Seq on 20 eBL cases from Uganda and showed that the mutational and viral landscape of eBL is more complex than previously reported. First, we found the presence of other herpesviridae family members in 8 cases (40%), in particular human herpesvirus 5 and human herpesvirus 8 and confirmed their presence by immunohistochemistry in the adjacent non-neoplastic tissue. Second, we identified a distinct latency program in EBV involving lytic genes in association with TCF3 activity. Third, by comparing the eBL mutational landscape with published data on sporadic Burkitt lymphoma (sBL), we detected lower frequencies of mutations in MYC, ID3, TCF3 and TP53, and a higher frequency of mutation in ARID1A in eBL samples. Recurrent mutations in two genes not previously associated with eBL were identified in 20% of tumors: RHOA and cyclin F (CCNF). We also observed that polyviral samples showed lower numbers of somatic mutations in common altered genes in comparison to sBL specimens, suggesting dual mechanisms of transformation, mutation versus virus driven in sBL and eBL respectively.

Selective influences in the expressed immunoglobulin heavy and light chain gene repertoire in hairy cell leukemia

Hairy cell leukemia is a rare, chronic B-cell neoplasm characterized by leukemic hairy cells. This immunogenetic analysis of the expressed immunoglobulin heavy ad light chain gene repertoire suggests that immunoglobulin gene selection may play an important role in the pathognesis of this neoplasm. Background We previously reported ongoing mutational and isotype switch events in the immunoglobulin (Ig) heavy chain (H) locus in hairy cell leukemia. Those analyses raised questions on the incidence and type of selective influences occurring on the tumor B-cell receptor of hairy cell leukemia. Design and Methods To further investigate this issue, we examined the full IGH and κ and λ light chains (IGκ and IGλ) variable and constant region transcripts expressed in a large cohort of patients with hairy cell leukemia (n=88). Results Multiple IgH isotypes were expressed in 46/56 (82%) cases of hairy cell leukemia. Comparison of tumor with normal B-cell repertoires revealed preferential usage of IGHV3-21, IGHV3-30 and IGHV3-33 in hairy cell leukemia (p=0.001, p=0.003 and p=0.001, respectively). Light chain analysis demonstrated preferential Igl use with an inverted IGk:IGl ratio (0.7:1) and universal usage of IGLJ3. Analysis of LCDR3 junctions revealed highly homologous motifs in 40% of IGL. Parallel analysis of IGH and IGL showed selective pairing of IGHV3-21/30/33 segments to specific LCDR3-J3 subsets (p=0.008). Of 40 cases of hairy cell leukemia, 38 had mutated IGHV and/or IGK/LV, with variations in 13/13 cloned cases, while two had 100% unmutated IGHV and IGK/LV. Conclusions Overall, biased IGV usage, preference for Igλ with universal IGLJ3 usage and a high incidence of LCDR3 homologous motifs suggest selective influences on the B-cell receptor of hairy cell leukemia. Ongoing mutations and isotype switching suggest that influences occur on the tumor B-cell receptor at ectopic sites.

IRTA1+ monocytoid B cells in reactive lymphadenitis show a unique topographic distribution and immunophenotype and a peculiar usage and mutational pattern of IgVH genes

The origin and function of monocytoid B cells (MBCs) are poorly understood. Taking advantage of their strong expression of IRTA1 (a receptor that is also associated with MALT marginal zone B cells), we have comprehensively analysed MBCs in 25 cases of lymphadenitis of different aetiologies, shedding new light on the topographical distribution, immunophenotype and IgVH gene usage and mutational profile of this B cell subset. IRTA1+ MBCs, although predominantly located in the subcapsular and intermediary sinuses, were also observed scattered within germinal centres (GCs) in all lymphadenitis cases examined. The molecular characterization of IgVH genes revealed that IRTA1+ MBCs residing in different areas of the lymph node (subcapsular sinus, intermediary sinuses and GCs) can be clonally related (with intraclonal variation), and that those located in GCs are consistently more mutated and selected for expression of a functional antigen receptor than those located in the sinuses. Moreover, by contrast, IRTA1+ MBCs in GCs express the memory B cell marker CD27. Finally, in toxoplasmic lymphadenitis, the IRTA1+ MBC population shows a highly preferential usage of the VH genes 3‐7 and 3‐30 (without any obvious peculiarity in their CDR3s), possibly suggesting that a superantigen expressed by Toxoplasma gondii may be involved in the early activation of this B cell subset. Copyright

Complete molecular remission induced by concomitant cladribine--rituximab treatment in a case of multi-resistant hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare B-cell neoplasm associated with a good outcome after treatment with single-agent purine-analogs (PAs) Pentostatin or Cladribine [1]. However, responses are not universal and the refractory patients often respond poorly to subsequent treatments [2,3]. Additionally, relapsefree survival curves do not reach a plateau in responders due to a significant proportion of patients who relapse and who require re-treatment [2,3]. Quality of response is important and patients with complete remission (CR) have a longer diseasefree survival [2,3]. Thus, it becomes relevant to seek strategies to improve the type of response in HCL. Early studies in refractory/relapsed HCL demonstrated some efficacy of Rituximab given weekly for 4 weeks [4,5], and regimens extended to 8 weeks increased efficacy with high CR rates [6]. Rituximab in combination with Cladribine produced higher levels of response. In two independent studies, intravenous Cladribine followed by 4 – 8 weekly doses of Rituximab determined CRs with minimal residual disease (MRD) eradication in 490% of patients [7,8]. While the data indicated high efficacy, the best timing for Cladribine administration has not been investigated. We evaluated toxicity and efficacy of Cladribine and Rituximab given ‘‘concomitantly’’ in a refractory/relapsed 62-year-male HCL patient. The patient was diagnosed HCL 15 years ago and had received eight separate treatment-lines with either Interferona or Pentostatin or Cladribine or Rituximab. None of them determined bone marrow remission, and normalization of peripheral blood counts was transient. Even the Rituximab regimen (375 mg/m, 4 weekly doses) was ineffective and two subsequent treatment-lines with Pentostatin and weekly Cladribine produced no response. ‘‘Concomitant’’ treatment consisted of Cladribine given 0.1 mg/kg/day intravenously over 2 h daily for five consecutive days starting 1 day after the first of eight Rituximab weekly doses (375 mg/m/dose). Clinical and haematological features pre-, during and after Cladribine – Rituximab therapy are described in Table I and the immunophenotype of hairy cells performed prior to treatment showed no atypical features (CD19þve, CD11cþve, CD25þve, CD103þve, FMC7þve and CD20þve at high intensity). MRD was determined in flow cytometry and by polymerase-chain-reaction (PCR) as described in the legend to Table I. Hematological or non-hematological toxicity or infections were not documented during treatment or follow-up. Remarkably, evaluation of response performed 3 and 7 months after treatment completion demonstrated a CR with total eradication of MRD. Ten months after treatment termination, the patient had no complications and complete blood cell counts were normal.

Clonality Analysis of Immunoglobulin Gene Rearrangement by Next-Generation Sequencing in Endemic Burkitt Lymphoma Suggests Antigen Drive Activation of BCR as Opposed to Sporadic Burkitt Lymphoma

Objectives: Recent studies using next-generation sequencing (NGS) analysis disclosed the importance of the intrinsic activation of the B-cell receptor (BCR) pathway in the pathogenesis of sporadic Burkitt lymphoma (sBL) due to mutations of TCF3/ID3 genes. Since no definitive data are available on the genetic landscape of endemic Burkitt (eBL), we first assessed the mutation frequency of TCF3/ID3 in eBL compared with sBL and subsequently the somatic hypermutation status of the BCR to answer whether an extrinsic activation of BCR signaling could also be demonstrated in Burkitt lymphoma. Methods: We assessed the mutations of TCF3/ID3 by RNAseq and the BCR status by NGS analysis of the immunoglobulin genes (IGs). Results: We detected mutations of TCF3/ID3 in about 30% of the eBL cases. This rate is significantly lower than that detected in sBL (64%). The NGS analysis of IGs revealed intraclonal diversity, suggesting an active targeted somatic hypermutation process in eBL compared with sBL. Conclusions: These findings support the view that the antigenic pressure plays a key role in the pathogenetic pathways of eBL, which may be partially distinct from those driving sBL development.

Plasmablastic transformation of a pre-existing plasmacytoma: a possible role for reactivation of Epstein Barr virus infection

Background: Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal myeloid neoplasm of early childhood associated with mutations in Ras pathway genes (PTPN11, KRAS, NRAS, CBL and NF1). Elevated fetal hemoglobin (HbF) levels and monosomy 7 are frequently observed. Stem cell transplantation is the only available curative treatment option but only provides an event-free survival of about 50%. Aims: Gain insight in the molecular networks involved in JMML pathogenesis based on mRNA, microRNA and long non-coding RNA transcriptome analysis of JMML samples. Methods: Expression of 27958 mRNA probes and 23042 lncRNA probes was assessed in diagnostic bone marrow or peripheral blood mononuclear cells of 63 JMML patients and 5 healthy donors, using a custom designed Agilent array. In addition, cDNA of 768 microRNAs was pre-amplified and quantified using miRNA specific Taqman probes. Results: Unsupervised clustering of an initial cohort of 14 patients generated two subgroups with let-7e and RNA-binding protein LIN28B amongst the most significantly differentially expressed genes. In the final cohort, relative higher LIN28B expression was observed in 35 of 63 cases (55.6%) and was defined as the average of the healthy donors plus three standard deviations. Univariable Cox regression showed that logarithmic LIN28B expression as a dichotomous variable can predict overall survival (p=0.035, exp(B) = 4.227, CI(95%) = 1.108 – 16.125). Patients with higher LIN28B mRNA levels experience a significant worse overall survival (Kaplan-Meier plot, p=0.022). HbF and platelet count were also significant prognostic factors, as described previously (p=0.023 and 0.027 respectively). There was no association between LIN28B expression and Ras pathway mutation status. We observed the strongest miRNA anti-correlation between LIN28B and five let-7 family members (d, b, g, e and a), and the second highest positive mRNA correlation between LIN28B and HMGA2. Recently, it was shown that the LIN28B – let-7 – HMGA2 axis determines higher self-renewal of fetal hematopoietic stem cells (Copley, 2013). This indicates that LIN28B confers augmented self- renewal to leukemic hematopoietic stem cells in JMML and – since this is an early childhood disease – this is potentially already initiated during embryogenesis. JMML patients frequently show elevated HbF levels at diagnosis. A positive correlation was found between LIN28B expression and HbF levels (rs=0.64, p<0.001, N = 41). Interestingly, our gene expression profiling data showed that both probes corresponding to HBG1 (encoding the human gamma globin chain) and HBBP1 (encoding a lncRNA-affiliated hemoglobin beta pseudogene) were strongly correlated with LIN28B expression in our patient series. This emphasizes the central role for LIN28B in the fetal (leukemic) hematopoietic stem cell system. Strikingly, patients with monosomy 7 (n=7/56) never displayed increased LIN28B expression (Chi-square p = 0.0017), suggesting the presence of a LIN28B activating transcription factor on chromosome 7. We identified MNX1 (HLXB9) as a possible activator of LIN28B based on a very strong correlation and siRNA knockdown.

Lack of allelic exclusion by secondary rearrangements of tumour B-cell receptor light chains in hairy cell leukaemia

Analyses of the tumour immunoglobulin (Ig) gene (IG) heavy (H) and light chains show heterogeneity of mutational status, but reveal common features of ongoing IGH isotype‐switching with multiple IGH isotype expression and preference of IG lambda (IGL) light chain with selective use of IGLJ3. Phenotypic and immunogenetic analyses were performed in a series of 105 HCL patients to estimate prevalence of multiple IG light chain expression by the tumour cells. By phenotype, 3/105 HCL (2.9%) expressed double tumour‐related Ig kappa (K) and L light chain proteins. By immunogenetic analysis, functional mutated double IGKI/IGKII, IGKI/IGLI and IGLI/IGLII transcripts were cloned and sequenced in 3/71 (4.2%) HCL. These latter three HCL expressed multiple IGH isotypes with mutated IGHVDJ rearrangements at the time of AID transcript expression. Most interestingly, the three cases had reinduced RAG1 transcript. In the double IGL expresser, single‐cell analysis documented co‐expression of the tumour‐related IGLs in 5/6 cells (83%). In the IGK/IGL co‐expresser, evidence of surface IgK/IgL isotype proteins confirmed functionality of the tumour‐derived transcripts. The evidence of double light chain expression in single HCs and the new observation of RAG re‐induction suggest ongoing selective influences on the BCR that may promote or maintain the HCL clone in the periphery. Copyright

The cell of origin of Burkitt lymphoma: germinal centre or not germinal centre?

Sir: We read with interest the ‘Accepted article’ in Histopathology of Park and colleagues that reports novel insight into the early histopathogenesis of immunodeficiency-associated Burkitt lymphoma (BL), suggesting a possible relationship with monocytoid B cell and speculating on the BL cell of origin. In addition, the paper raises the question of the polymicrobial nature of BL, as cytomegalovirus (CMV) was also detected. We wish to point out, however, that previous published papers demonstrated that all BL subtypes are definitely related to germinal centre (GC) B lymphocytes. Interestingly, follicular colonization or follicular growth can be observed occasionally in BL samples (Figure 1). Conversely, it has been hypothesized that endemic and human immunodeficiency virus (HIV)-related BL cases, which are associated commonly with Epstein–Barr virus (EBV) infection, may derive from a later developmental stage of B cells than sporadic BL, i.e. post-germinal centre/ memory B cells. This is in line with the fact that in healthy carriers, EBV resides in memory immunoglobulin (Ig)M-positive B cells that re-enter the GC reaction following antigen stimulation. In fact, activation of the B cell receptor by malaria infection and/or other pathogens induces the clonal expansion of memory B cells that express Epstein– Barr nuclear antigen 1 (EBNA-1) (latency I) and activation-induced cytidine deaminase (AID) when they divide. EBNA-1 expression in this subset of cells allows viral DNA to replicate and may thus result in the up-regulation of hsa-miR-127 and in the shift to the characteristic GC phenotype and reentry into the GC reaction. The finding of clusters of BL cells intermingled with monocytoid B cells may well thus represent the first in-vivo picture of the origin of BL from this IgM-positive memory B cell subset during clonal expansion under the pressure of antigens (Figure 2). The MYC/IgH translocations may thus occur during clonal expansion of activated memory B cells or when they re-enter the GC, being the active form of AID expressed regardless of the B cell stage. Park et al. raise the possibility of a direct role of CMV in promoting BL by directly infecting B cells. However, so far no evidence of such a phenomenon is present in the literature, and the data presented by the authors are insufficient to draw this conclusion. In fact, CMV was identified only in the original specimen but not in the subsequent BL. Therefore, it is unlikely that the virus had been integrated into B cells. In addition, a recent study on endemic BL (eBL) detected the presence of CMV only in bystander cells, raising the possibility that CMV infection, along with other microbes, may contribute to the chronic antigenic stimulation in which eBL occurs and may also induce the EBV lytic cycle through B cell reactivation and spreading of EBV infection out of its natural niche of memory B cells.