Chronic lymphocytic leukemia (CLL) cells are considered to be derived from antigen-exposed B cells. To further explore the antigen-driven selection behind the leukemogenesis of CLL, we performed immunoglobulin (Ig) specificity screening of 7 CLL cell lines and 23 primary CLL clones from patient peripheral blood. We also included a recombinant monovalent monoclonal antibody (mAb) belonging to a subset of CLL cases with identical or semiidentical heavy chain complementarity determining region 3 (HCDR3) of the IGHV3-21 gene rearrangement. We found CLL mAb specificities against vimentin, filamin B, cofilin-1, proline-rich acidic protein 1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae polysaccarides. These molecules are functionally associated with microbial infection and/or apoptotic cell removal. An antigen-driven selection would therefore imply that CLL B cell precursors are involved in the elimination and scavenging of pathogens and apoptotic cells, which could trigger the development of the disease. The limited in vitro survival of CLL cells makes Epstein-Barr virus (EBV) immortalization of CLL cells a useful experimental model for studies on antibody-specificity screening. Considering the intricate procedure of EBV transformation of CLL cells and the many false cell lines used worldwide, we also wanted to characterize and evaluate the authentic origin of several previously established CLL cell lines and their normal lymphoblastoid counterparts. Three of the CLL cell lines tested were truly authentic (I83-E95, CLL-HG3 and CII), two had features of a biclonal Ig expression (232B4 and WaC3CD5+), one was only tentatively verified (PGA-1), whereas one cell line could not be verified (EHEB) due to lack of original patient cells for comparison. Two of the presumed normal lymphoblastoid cell lines tested were shown to be a neoplastic CLL clone. This study emphasizes the importance of proper cell line authentication and we will continue to verify additional cell lines not yet proven authentic. In conclusion, we provide evidence for natural Ab production by CLL cells and suggest that these cells might be derived from B cell precursors involved in the innate immunity and, thus, providing a first-line-defence against pathogens and in elimination of apoptotic cells.

In recent years, great progress has been achieved towards identifying novel biomarkers in lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), at the genomic, transcriptomic and epigenomic level for accurate risk-stratification and prediction of treatment response. In paper I, we validated the prognostic relevance of a recently proposed RNA-based marker in CLL, UGT2B17, and analyzed its expression levels in 253 early-stage patients. Besides confirming its prognostic impact in multivariate analysis, we could identify 30% of IGHV-mutated CLL (M-CLL) cases with high expression and poor outcome, which otherwise lacked any other poor-prognostic marker. In paper II, we investigated the prognostic impact of a previously reported 5 CpG signature that divides CLL patients into three clinico-biological subgroups, namely naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL) and intermediate CLL (i-CLL), in 135 CLL patients using pyrosequencing. We validated the signature as an independent marker in multivariate analysis and further reported that subset #2 cases were predominantly classified as i-CLL, although displaying a similar outcome as n-CLL. In paper III, we investigated the methylation status and expression level of miR26A1 in both CLL (n=70) and MCL (n=65) cohorts. High miR26A1 methylation was associated with IGHV-unmutated (U-CLL) and shorter overall survival (OS) in CLL, while it was uniformly hypermethylated in MCL. Furthermore, overexpression of miR26A1 resulted in significant downregulation of EZH2 that in turn led to increased apoptosis. In paper IV, we performed DNA methylation profiling in 176 CLL cases assigned to one of 8 major stereotyped subsets (#1-8) in relation to non-subset CLL (n=325) and different normal B-cell subpopulations. Principal component analysis of subset vs. non-subset CLL revealed that U-CLL and M-CLL subsets generally clustered with n-CLL and m-CLL, respectively, indicating common cellular origins. In contrast, subset #2 emerged as the first defined member of the i-CLL subgroup, which in turn alludes to a distinct cellular origin for subset #2 and i-CLL patients. Altogether, this thesis confirms the prognostic significance of RNA and epigenetic-based markers in CLL, provides insight into the mechanism of miRNA deregulation in lymphoid malignancies and further unravels the DNA methylation landscape in stereotyped subsets of CLL.

La leucémie lymphoïde chronique (LLC) est une hémopathie maligne caractérisée par l’accumulation, dans le sang et les organes lymphoïdes secondaires, de lymphocytes B matures résistants à l’apoptose. Le microenvironnement tumoral de la LLC au sein des organes lymphoïdes secondaires, et notamment les « Nurse-Like Cells » (NLCs), joue un rôle majeur dans la promotion de la survie et de la prolifération des cellules leucémiques. Au cours de cette étude, la surexpression du récepteur de la neurotensine NTSR2, un récepteur couplé aux protéines G, a été caractérisée. Il est activé de façon constitutive dans les cellules leucémiques circulantes, et son activation dépend de son interaction avec le récepteur à activité tyrosine kinase TrkB suite à la liaison de son ligand, le BDNF, tous deux également surexprimés. L’activation de NTSR2-TrkB par le BDNF entraine une signalisation de survie par les voies Src et Akt, aboutissant à la surexpression des protéines anti-apoptotiques Bcl-2 et Bcl-XL. L’inhibition du récepteur NTSR2 dans ces cellules fait diminuer leur viabilité. En présence des NLCs, les expressions de NTSR2, TrkB, BDNF, et de la sortiline, protéine de transport des neurotrophines et des récepteurs Trk, est accentuée. Les NLCs produisent elle-même du BDNF, activent la voie de signalisation Src, et leur rôle protecteur des cellules leucémiques est BDNF-dépendant. L’ensemble de ces travaux met en évidence un rôle capital de NTSR2-TrkB-BDNF dans la signalisation de survie des cellules leucémiques circulantes et au sein du microenvironnement tumoral de la LLC, et pourrait ainsi constituer une nouvelle cible thérapeutique potentielle. / Chronic lymphocytic leukemia (CLL) is a malignant hemopathy characterized by the accumulation of apoptosis resistant mature B lymphocytes in peripheral blood and secondary lymphoid organs. In these secondary lymphoid organs, the tumor microenvironment, notably Nurse-like Cells (NLCs), plays a major role in leukemic cells survival and proliferation promotion. In this study, an overexpression of neurotensin receptor NTSR2, a G-protein coupled receptor, was identified. NTSR2 is constitutively activated in circulating leukemic cells and its activation depends on its interaction with tyrosine kinase activity receptor TrkB upon binding of its ligand, BDNF. Activation of NTSR2-TrkB by BDNF induces survival signaling by Src and Akt pathways, and in term anti-apoptotic proteins Bcl-2 and Bcl-XL overexpression. Inhibition of NTSR2 in those cells impacts their viability. In the presence of NLCs, expressions of NTSR2, TrkB, BDNF, and sortilin, a neurotrophin and Trk receptor transport regulator, are enhanced. NLCs produce BDNF, stimulate Src activation, and their protective role on leukemic cells is BDNF-dependent. Taken together, this study highlights a key role of NTSR2-TrkB-BDNF in leukemic cells survival signaling, both in the circulation or in the tumor microenvironment, and might thus constitute a potential new therapeutic target.

Multiple myeloma is characterized by the malignant proliferation of clonal plasma cells producing monoclonal paraproteins, leading to multi-organ damage. On the other hand monoclonal B-cell lymphocytosis (MBCL) is characterized by the malignant proliferation of clonal B-lymphocytes, with potential to develop into chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). CLL/SLL can result in visceromegaly, anemia, thrombocytopenia, fevers, night sweats and unintentional weight loss. Literature review demonstrates these two malignant clonal bone marrow disorders are most frequently seen independently in patients; however, we report one rare diagnostic challenge where both clonal disorders were identified in a single patient concurrently. A 64-year-old man initially presented with worsening back pain. Thoracic spine x-ray revealed a T11 compression fracture, confirmed by magnetic resonance imaging. Complete blood count revealed a white blood cell count of 7.3 K/uL with 54% lymphocyte predominance and peripheral smear demonstrated a population of small lymphocytes with round nuclei and an atypical chromatin pattern suggestive of CLL/MBCL. Flow cytometry revealed a monoclonal B-cell CD5 positive, CD23 positive, CD10 negative population with an absolute count of 1.6 K/uL. Due to the instability and pain associated with the spinal fracture, patient had kyphoplasty performed and intraoperative bone biopsies were taken from both T11 and T12 vertebrae. Interestingly each bone biopsy revealed involvement by both a kappa-light chain restricted plasma cell neoplasm, ranging from 15% to 30% cellularity, as well as a CD5-positive B-cell lymphocyte population. It suggested two concurrent but pathologically distinct pathologies including plasma cell myeloma and a separate B-cell lymphoproliferative disorder with immunophenotypic features suggestive of CLL/MBCL. Bone marrow biopsy was performed for definitive evaluation and confirmed multiple myeloma with 15-20% kappa-restricted plasma cells identified, and also confirmed concurrent MBCL with CD5 and CD23-positive, kappa-restricted B-cells identified on bone marrow flow cytometry. Adding an additional layer of complexity, bone marrow molecular genetics revealed presence of a MYD88 mutation, raising concern for possible lymphoplasmacytic lymphoma (LPL). However, secondary pathologic review ruled out LPL, as the immunophenotypic pattern of the clonal B-cells was not consistent with that of LPL, and although the MYD88 mutation is predominantly seen in LPL, it has also been seen in a small percentage of CLL/SLL cases and exceedingly rarely described in MM as well. Serum protein electrophoresis with immunofixation, serum quantitative immunoglobulins and serum quantitative free light chain assay revealed findings consistent with IgG kappa multiple myeloma and systemic CT imaging was negative for any lymphadenopathy, confirming MBCL. Patient was started on first-line multiple myeloma systemic therapy for transplant eligible patients and has demonstrated an excellent response to treatment thus far. This patient case serves to demonstrate the importance of maintaining a broad differential when approaching hematological problems; It also underlines the necessity for a complete diagnostic evaluation to identify rare clinical conundrums such as with our patient, allowing for proper and timely treatment. While we use “Occam’s razor” to explain multiple problems with a single unifying diagnosis the rare possibility of divergent diagnosis is to be always entertained.

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Yes / In this paper, a pyramidal horn antenna loaded with unit cell of metamaterial is proposed, designed and realized for L-band that including terrestrial digital audio broadcasting TDAB, GPS and GSM. The proposed antenna operates in the frequency range from 1.722 GHz to 1.931 GHz. The metamaterial is fabricated on a printed circuit board as Capacitive Loaded Loop (CLL). The work aims to exhibit the advantage of metamaterial loaded inside the horn antenna in terms of the gain enhancement of the radiation pattern and the resonant frequency shift towards lower frequency. The retrieval technique used show that the constitutive parameters of the unit cell as CLL have a zero index metamaterial (ZIM) from 1.34 GHz to 1.49 GHz and a near zero index of refraction from 1.495 GHz to 2 GHz, which is within the operating frequency of the horn antenna. The achieved results show that the total gain is improved over the frequency range. The simulation and the measurement are in good agreement.

Incomplete biological concepts in lymphoid neoplasms still dictate to a large extent the limited availability of efficient targeted treatments, which entertains the mostly unsatisfactory clinical outcomes. Aberrant expression of the embryonal and lymphatic TCL1 family of oncogenes, i.e., the paradigmatic TCL1A, but also TML1 or MTCP1, is causally implicated in T- and B-lymphocyte transformation. TCL1A also carries prognostic information in these particular T-cell and B-cell tumors. More recently, the TCL1A oncogene has been observed also in epithelial tumors as part of oncofetal stemness signatures. Although the concepts on the modes of TCL1A dysregulation in lymphatic neoplasms and solid tumors are still incomplete, there are recent advances in defining the mechanisms of its (de)regulation. This review presents a comprehensive overview of TCL1A expression in tumors and the current understanding of its (dys)regulation via genomic aberrations, epigenetic modifications, or deregulation of TCL1A-targeting micro RNAs. We also summarize triggers that act through such transcriptional and translational regulation, i.e., altered signals by the tumor microenvironment. A refined mechanistic understanding of these modes of dysregulations together with improved concepts of TCL1A-associated malignant transformation can benefit future approaches to specifically interfere in TCL1A-initiated or -driven tumorigenesis.

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