Tasoula Touloumenidou

Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein–Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

Toll-like receptor signaling pathway in chronic lymphocytic leukemia: distinct gene expression profiles of potential pathogenic significance in specific subsets of patients

Background Signaling through the B-cell receptor appears to be a major contributor to the pathogenesis of chronic lymphocytic leukemia. Toll-like receptors bridge the innate and adaptive immune responses by acting as co-stimulatory signals for B cells. The available data on the expression of Toll-like receptors in chronic lymphocytic leukemia are limited and derive from small series of patients. Design and Methods We profiled the expression of genes associated with Toll-like receptor signaling pathways in 192 cases of chronic lymphocytic leukemia and explored potential associations with molecular features of the clonotypic B-cell receptors. Results Chronic lymphocytic leukemia cells express all Toll-like receptors expressed by normal activated B cells, with high expression of TLR7 and CD180, intermediate expression of TLR1, TLR6, TLR10 and low expression of TLR2 and TLR9. The vast majority of adaptors, effectors and members of the NFKB, JNK/p38, NF/IL6 and IRF pathways are intermediately-to-highly expressed, while inhibitors of Toll-like receptor activity are generally low-to-undetectable, indicating that the Toll-like receptor-signaling framework is competent in chronic lymphocytic leukemia. Significant differences were identified for selected genes between cases carrying mutated or unmutated IGHV genes or assigned to different subsets with stereotyped B-cell receptors. The differentially expressed molecules include receptors, NFκB/MAPK signaling molecules and final targets of the cascade. Conclusions The observed variations are suggestive of distinctive activation patterns of the Toll-like receptor signaling pathway in subgroups of cases of chronic lymphocytic leukemia defined by the molecular features of B-cell receptors. Additionally, they indicate that different or concomitant signals acting through receptors other than the B-cell receptor can affect the behavior of the malignant clone.

A certified plasmid reference material for the standardisation of BCR–ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

ATM mutations in major stereotyped subsets of chronic lymphocytic leukemia: enrichment in subset #2 is associated with markedly short telomeres

ATM mutations in major stereotyped subsets of chronic lymphocytic leukemia: enrichment in subset 2 is associated with markedly short telomeres, ATM mutations in major stereotyped subsets of chronic lymphocytic leukemia: enrichment in subset 2 is associated with markedly short telomeres, ATM mutations in major stereotyped subsets of chronic lymphocytic leukemia: enrichment in subset 2 is associated with markedly short telomeres.

Tp53 gene p72R polymorphism in chronic lymphocytic leukemia: incidence and clinical significance amongst cases with unmutated immunoglobulin receptors

Chronic lymphocytic leukemia (CLL) displays extreme clinical heterogeneity likely reflecting the underlying biological heterogeneity. For this reason, intense research efforts have addressed progno...

In vitro evidence of complement activation in patients with sickle cell disease

Sickle cell disease (SCD) remains a devastating and painful condition leading to significant morbidity and mortality in the era of hydroxycarbamide.[1][1] Current understanding of disease pathophysiology has focused not only on the interaction between sickle red blood cells and neutrophils,

Sequential transient novel chromosomal translocations in a patient with chronic myelogenous leukemia in complete cytogenetic remission after therapy with imatinib mesylate.

Imatinib mesylate (IM) is the first tyrosine kinase inhibitor (TKI) introduced for the treatment of chronic myelogenous leukemia (CML) with remarkable high rates of complete hematologic and cytogenetic response and an acceptable safety and toxicity profile. Clonal aberrations (CAs) in Philadelphia (Ph)-chromosome negative metaphases have been reported in patients with CML after treatment with IM, with a varying frequency between series (1.6e20.6%) (1e3). Most CAs are numerical aberrations, whereas structural aberrations, in particular, balanced translocations are much less frequent (4e8). In our single-center series of 142 CML cases treated with TKIs, with amedian follow-up of 72months (range, 4e135), 10 cases (7%) developed CAs in Ph-chromosome negative cells; all cases except one carried numerical CAs. The remaining case that we describe here concerns a CML patient treated with IM who sequentially developed two novel chromosomal translocations in Ph-chromosome negative metaphases. The present case concerns a 35-year-old male diagnosed with Ph-chromosome positive CML in chronic phase in March 1993. He was treated with hydroxyurea and alpha-interferon for 12 months and achieved complete hematological remission, however, with no cytogenetic response. He was then referred for autologous hematopoietic stem cell transplantation (AHSCT), which was performed in September 1994, after stem cell mobilization with one course of the ICE protocol (idarubicin, cytarabine, etoposide, and granulocyte colony-stimulating factor) and conditioning with busulfan and cyclophosphamide. At þ3 months after AHSCT, the patient attained major cytogenetic response (MCyR) and was given maintenance therapy with alpha-interferon. At þ6 months, he attained complete cytogenetic response (CCyR) lasting 3 years; thereafter, repeated classic cytogenetic studies were positive for the t(9;22)(q34;q11), ranging from 3.3e42% of all metaphases. In January 2002, the patient started IM at 400 mg/day, leading to the attainment of CCyR within 3 months. At þ12 months after the initiation of IM, quantitative realtime PCR (RQ-PCR) revealed a 3-log reduction in BCR-ABL1 chimeric transcript levels demonstrating major molecular response (MMR). In August 2007, at þ67 months on IM, classic cytogenetic analysis identified loss of the Y chromosome in