EuroFlow: Resetting leukemia and lymphoma immunophenotyping. Basis for companion diagnostics and personalized medicine

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License.-- Editorial.We are grateful to Dr Jean-Luc Sanne of the European Commission for his support and monitoring of the EuroFlow project.Peer Reviewe

Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45-/EpCAM+), germ cell tumors (CD56+/CD45-/NG2+/CD10+) and eventually also hemangiopericytomas (CD45-/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.This work has been partially supported by the following grants: EO was partially supported by grant from CNPq- Brazilian National Research Council (Brasília, Brazil). MF was partially supported by a grant from FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil) and now she is partially supported by a grant from CAPES/Ministério da Educação (Brasília, Brazil). VB was partially supported by FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil). ESC was partially supported by grant from CNPq- Brazilian National Research Council (Brasília, Brazil) and FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil).Peer Reviewe

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by atp

AbstractThe incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis

Changes in adult rat liver mitochondrial populations at different energy states analyzed by flow cytometry

AbstractThe present work studies the changes in green fluorescence intensity after Rh-123 staining of the low (LFP) and the high fluorescence populations (HFP) in isolated mitochondria from rat liver. The results show that the HFP represents a mitochondrial compartment less sensitive to changes in energy states. In addition, it is concluded that the use of Rh-123 to monitor changes in mitochondrial membrane potential should be undertaken with caution because, under certain circumstances, there is no correlation between the Rh-123 intensity of fluorescence due to its uptake by mitochondria and previously reported changes in the mitochondrial membrane potential

KIT mutation analysis in mast cell neoplasms: Recommendations of the European Competence Network on Mastocytosis

PMCID: PMC4522520.-- et al.Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals.M. Arock is supported by Fondation de France; P. Dubreuil is supported by La Ligue Nationale Contre le Cancer (équipe labellisée) and INCa; A. Garcia-Montero and A. Orfao are Supported by grants from the Instituto de Salud Carlos III, Ministry of Economy and Competitivity, Madrid, Spain (grant numbers RD12/0036/0048 and PI11/02399, FEDER) and from Fundacion Ramon Areces, Madrid, Spain (grant number CIVP16A1806); D.D. Metcalfe is supported in part by the Division of Intramural Research, NIAID; P. Valent is supported by Austrian Science Funds (FWF) Project SFB F4611 and SFB F4704-B20.Peer Reviewe

Common Infectious Agents and Monoclonal B-Cell Lymphocytosis: A Cross-Sectional Epidemiological Study among Healthy Adults

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- Primary Health Care Group of Salamanca for the Study of MBL: et al.[Background]: Risk factors associated with monoclonal B-cell lymphocytosis (MBL), a potential precursor of chronic lymphocytic leukaemia (CLL), remain unknown. [Methods]: Using a cross-sectional study design, we investigated demographic, medical and behavioural risk factors associated with MBL. >Low-count> MBL (cases) were defined as individuals with very low median absolute count of clonal B-cells, identified from screening of healthy individuals and the remainder classified as controls. 452 individuals completed a questionnaire with their general practitioner, both blind to the MBL status of the subject. Odds ratios (OR) and 95% confidence interval (CI) for MBL were estimated by means of unconditional logistic regression adjusted for confounding factors. [Results]: MBL were detected in 72/452 subjects (16%). Increasing age was strongly associated with MBL (P-trend<0.001). MBL was significantly less common among individuals vaccinated against pneumococcal or influenza (OR 0.49, 95% confidence interval (CI): 0.25 to 0.95; P-value = 0.03 and OR: 0.52, 95% CI: 0.29 to 0.93, P-value = 0.03, respectively). Albeit based on small numbers, cases were more likely to report infectious diseases among their children, respiratory disease among their siblings and personal history of pneumonia and meningitis. No other distinguishing epidemiological features were identified except for family history of cancer and an inverse relationship with diabetes treatment. All associations described above were retained after restricting the analysis to CLL-like MBL. [Conclusion]: Overall, these findings suggest that exposure to infectious agents leading to serious clinical manifestations in the patient or its surroundings may trigger immune events leading to MBL. This exploratory study provides initial insights and directions for future research related to MBL, a potential precursor of chronic lymphocytic leukaemia. Further work is warranted to confirm these findings.This work was financially supported by the following grants: Red Temática de Investigación Cooperativa en Cáncer del Instituto de Salud Carlos III - FONDOS FEDER (RD06/0020/0035, 2006-RET2039-O, CIBERESP 06/02/0073); FIS PI06/0824-FEDER, PS09/02430-FEDER and PI11/01810-FEDER, from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain; GRS206/A/08 from the Gerencia Regional de Salud de Castilla y León and Ayuda al Grupo GR37 de Excelencia de Castilla y León, Consejería de Educación, and SAN/1778/2009, Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain; and Agència de Gestió d’Ajuts Universitaris i de Recerca Generalitat de Catalunya (AGAUR 2009SGR1465). AR-C is supported by a grant from Fundación Científica de la Asociación Española contra el Cáncer.Peer Reviewe

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry

PMCID: PMC4488901.-- et al.[Purpose]: According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1- positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. [Experimental Design]: We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24MCLand 13MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. [Results]: We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed betweenMCLandMALD1and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. [Conclusion]: We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment.This work has been supported, in part, by grants from Instituto de Salud Carlos III RD07/0020/2004, RD09/0076/00036, RD12/0036/0044, (RTICC, FEDER), Generalitat de Catalunya 2009/SGR541, and the “Xarxa de Bancs de Tumors” sponsored by Pla Director d'Oncologia de Catalunya (XBTC).Peer Reviewe

Desarrollo y evaluación de KIT NAPPA Array para su uso como plataforma de secreening de fármacos

Comunicaciones a congreso

Early diagnostic of B cell chronic leukemia by novel biomarkers

Comunicaciones a congreso

Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

This is an open access paper.-- et al.The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating laboratories. The second aimed to resolve outstanding technical issues and develop a consensus approach to analysis of plasma cells. The primary clinical applications identified were: differential diagnosis of neoplastic plasma cell disorders from reactive plasmacytosis; identifying risk of progression in patients with MGUS and detecting minimal residual disease. A range of technical recommendations were identified, including: 1) CD38, CD138 and CD45 should all be included in at least one tube for plasma cell identification and enumeration. The primary gate should be based on CD38 vs. CD138 expression; 2) after treatment, clonality assessment is only likely to be informative when combined with immunophenotype to detect abnormal cells. Flow cytometry is suitable for demonstrating a stringent complete remission; 3) for detection of abnormal plasma cells, a minimal panel should include CD19 and CD56. A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples, particularly normal plasma cells in MRD negative cases.The meetings were sponsored by the European Myeloma.Peer Reviewe