Minimal residual disease is an independent predictor for 10-year survival in CLL

Minimal residual disease (MRD) negativity, defined as <1 chronic lymphocytic leukemia (CLL) cell detectable per 10 000 leukocytes, has been shown to independently predict for clinical outcome in patients receiving combination chemoimmunotherapy in the frontline setting. However, the long-term prognostic value of MRD status in other therapeutic settings remains unclear. Here, we retrospectively analyzed, with up to 18 years follow-up, all patients at our institution who achieved at least a partial response (PR) with various therapies between 1996 and 2007, and received a bone marrow MRD assessment at the end of treatment according to the international harmonized approach. MRD negativity correlated with both progression-free survival (PFS) and overall survival (OS) independent of the type and line of treatment, as well as known prognostic factors including adverse cytogenetics. The greatest impact of achieving MRD negativity was seen in patients receiving frontline treatment, with 10-year PFS of 65% vs 10% and 10-year OS of 70% vs 30% for MRD-negative vs MRD-positive patients, respectively. Our results demonstrate the long-term benefit of achieving MRD negativity, regardless of the therapeutic setting and treatment modality, and support its use as a prognostic marker for long-term PFS and as a potential therapeutic goal in CLL

Results of the randomized phase IIB ADMIRE trial of FCR with or without mitoxantrone in previously untreated CLL

ADMIRE was a multi-center, randomized-controlled, open, phase IIB superiority trial in previously untreated Chronic Lymphocytic Leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). Initial evidence from non-randomized Phase II trials suggested that the addition of mitoxantrone to FCR (FCM-R) improved remission rates. 215 patients were recruited to assess the primary endpoint of complete remission (CR) rates according to IWCLL criteria. Secondary endpoints were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity and safety. At final analysis, CR rates were 69.8% FCR vs 69.3% FCM-R [adjusted odds ratio (OR): 0.97; 95%CI: (0.53-1.79), P=0.932]. MRD-negativity rates were 59.3% FCR vs 50.5% FCM-R [adjusted OR: 0.70; 95% CI: (0.39-1.26), P=0.231]. During treatment, 60.0% (n=129) of participants received G-CSF as secondary prophylaxis for neutropenia, a lower proportion on FCR compared with FCM-R (56.1 vs 63.9%). The toxicity of both regimens was acceptable. There are no significant differences between the treatment groups for PFS and OS. The trial demonstrated that the addition of mitoxantrone to FCR did not increase the depth of response. Oral FCR was well tolerated and resulted in impressive responses in terms of CR rates and MRD negativity compared to historical series with intravenous chemotherapy

Combined Patterns of IGHV Repertoire and Cytogenetic/Molecular Alterations in Monoclonal B Lymphocytosis versus Chronic Lymphocytic Leukemia

Background:Chronic lymphocytic leukemia (CLL)-like monoclonal B lymphocytosis (MBL) with (MBLhi) or without (MBLlo) absolute B-lymphocytosis precedes most CLL cases,the specific determinants for malignant progression remaining unknown.Methodology/Principal Findings:For this purpose, simultaneous iFISH and molecular analysis of well-established cytogenetic alterations of chromosomes 11, 12, 13, 14 and 17 together with the pattern of rearrangement of the IGHV genes were performed in CLL-like cells from MBL and CLL cases. Our results based on 78 CLL-like MBL and 117 CLL clones from 166 subjects living in the same geographical area, show the existence of three major groups of clones with distinct but partially overlapping patterns of IGHV gene usage, IGHV mutational status and cytogenetic alterations. These included a group enriched in MBLloclones expressing specific IGHV subgroups (e.g. VH3-23) with no or isolated good-prognosis cytogenetic alterations, a second group which mainly consisted of clinical MBLhiand advanced stage CLL with a skewed but different CLL-associated IGHV gene repertoire (e.g. VH1-69), frequently associated with complex karyotypes and poor-prognosis cytogenetic alterations, and a third group of clones with intermediate features, with prevalence of mutated IGHV genes, and higher numbers of del(13q)+clonal B-cells.Conclusions/Significance:These findings suggest that the specific IGHV repertoire and IGHV mutational status of CLL-like B-cell clones may modulate the type of cytogenetic alterations acquired, their rate of acquisition and/or potentially also their clinical consequences. Further long-term follow-up studies investigating the IGHV gene repertoire of MBLloclones in distinct geographic areas and microenvironments are required to confirm our findings and shed light on the potential role of some antigen-binding BCR specificities contributing to clonal evolution

High Mitochondrial DNA Stability in B-Cell Chronic Lymphocytic Leukemia

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) leads to progressive accumulation of lymphocytes in the blood, bone marrow, and lymphatic tissues. Previous findings have suggested that the mtDNA could play an important role in CLL. METHODOLOGY/PRINCIPAL FINDINGS: The mitochondrial DNA (mtDNA) control-region was analyzed in lymphocyte cell DNA extracts and compared with their granulocyte counterpart extract of 146 patients suffering from B-Cell CLL; B-CLL (all recruited from the Basque country). Major efforts were undertaken to rule out methodological artefacts that would render a high false positive rate for mtDNA instabilities and thus lead to erroneous interpretation of sequence instabilities. Only twenty instabilities were finally confirmed, most of them affecting the homopolymeric stretch located in the second hypervariable segment (HVS-II) around position 310, which is well known to constitute an extreme mutational hotspot of length polymorphism, as these mutations are frequently observed in the general human population. A critical revision of the findings in previous studies indicates a lack of proper methodological standards, which eventually led to an overinterpretation of the role of the mtDNA in CLL tumorigenesis. CONCLUSIONS/SIGNIFICANCE: Our results suggest that mtDNA instability is not the primary causal factor in B-CLL. A secondary role of mtDNA mutations cannot be fully ruled out under the hypothesis that the progressive accumulation of mtDNA instabilities could finally contribute to the tumoral process. Recommendations are given that would help to minimize erroneous interpretation of sequencing results in mtDNA studies in tumorigenesis

Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact

Vision and visual history in elite-/near-elite level cricketers and rugby-league players

Background: The importance of optimal and/or superior vision for participation in high-level sport remains the subject of considerable clinical research interest. Here we examine the vision and visual history of elite/near-elite cricketers and rugby-league players. Methods: Stereoacuity (TNO), colour vision, and distance (with/without pinhole) and near visual acuity (VA) were measured in two cricket squads (elite/international-level, female, n=16; near-elite, male, n=23) and one professional rugby-league squad (male, n=20). Refractive error was determined, and details of any correction worn and visual history were recorded. Results: Overall, 63% had their last eye-examination within 2 years. However, some had not had an eye examination for 5 years, or had never had one (near-elite-cricketers: 30%; rugby-league players: 15%; elite-cricketers: 6%). Comparing our results for all participants to published data for young, optimally-corrected, non-sporting adults, distance VA was ~1 line of letters worse than expected. Adopting α=0.01, the deficit in distance-VA deficit was significant, but only for elite-cricketers (p0.02 for all comparisons). On average, stereoacuity was better than in young adults, but only in elite-cricketers (p<0.001; p=0.03, near-elite-cricketers; p=0.47, rugby-league -players). On-field visual issues were present in 27% of participants, and mostly (in 75% of cases) comprised uncorrected ametropia. Some cricketers (near-elite: 17.4%; elite: 38%) wore refractive correction during play but no rugby-league player did. Some individuals with prescribed correction choose not to wear it when playing. Conclusion: Aside from near stereoacuity in elite-cricketers, these basic visual abilities were not better than equivalent, published data for optimally-corrected adults. 20-25% exhibited sub-optimal vision, suggesting that the clearest possible vision might not be critical for participation at the highest levels in the sports of cricket or rugby-league. Although vision could be improved in a sizeable proportion of our sample, the impact of correcting these, mostly subtle, refractive anomalies on playing performance is unknown

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

Most consensus leukemia lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies

MHC class I–associated phosphopeptides are the targets of memory-like immunity in Leukemia

Deregulation of signaling pathways involving phosphorylation is a hallmark of malignant transformation. Degradation of phosphoproteins generates cancer-specific phosphopeptides that are associated with MHC-I and II molecules and recognized by T-cells. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8 T-cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and HLA-matched primary leukemia cells ex vivo. Healthy individuals showed surprisingly high levels of CD8 T-cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients, which correlated with clinical outcome, and was restored following allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T-cell adoptive transfer immunotherapies.

The 5th edition of the World Health Organization classification of haematolymphoid tumours: lymphoid neoplasms

We herein present an overview of the upcoming 5th edition of the World Health Organization Classification of Haematolymphoid Tumours focussing on lymphoid neoplasms. Myeloid and histiocytic neoplasms will be presented in a separate accompanying article. Besides listing the entities of the classification, we highlight and explain changes from the revised 4th edition. These include reorganization of entities by a hierarchical system as is adopted throughout the 5th edition of the WHO classification of tumours of all organ systems, modification of nomenclature for some entities, revision of diagnostic criteria or subtypes, deletion of certain entities, and introduction of new entities, as well as inclusion of tumour-like lesions, mesenchymal lesions specific to lymph node and spleen, and germline predisposition syndromes associated with the lymphoid neoplasms