Optimization of CSF biological investigations for CNS lymphoma diagnosis.

Diagnosis of lymphoma leptomeningeal dissemination is challenging and relies on a wide array of methods. So far, no consensus biological guidelines are available. This increases the chance of intra- and interpractice variations, despite the shared concern to perform the minimum amount of tests while preserving clinically relevant results.We evaluated a training cohort of 371 cerebrospinal fluid (CSF) samples from patients with putative lymphomatous central nervous system (CNS) localization using conventional cytology (CC), flow cytometry (FCM), molecular clonality assesment by PCR and cytokine quantification (CQ). This led us to propose a biological algorithm, which was then verified on a validation cohort of 197 samples. The samples were classified according to the clinical context and the results of each technique were compared. Using all four techniques was not useful for exclusion diagnosis of CNS lymphoma (CNSL), but they proved complementary for cases with suspected CNSL. This was particularly true for CQ in primary CNSL. Overall, diagnosis can be obtained with a two-step approach. The first step comprises CC and FCM, as results are available quickly and FCM is a sensitive method. Both PCR and CQ can be postponed and performed in a second step, depending on the results from the first step and the clinical context.The proposed algorithm missed none of the CNSL samples of the validation cohort. Moreover, applying this algorithm would have spared 30% of PCR tests and 20% of CQ over a one-year period, without compromising clinical management.

A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection.

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.

Establishment and characterization of a new human erythroleukemic cell line, ERY-1.

The growth factor-independent erythroleukemic cell line ERY-1 was established from the peripheral blood of a 87-year-old woman with chronic myeloid leukemia (CML) in the acute phase. Immunophenotyping showed that fresh leukemic cells were positive for CD13, CD33, CD36 and CD235a (glycophorin A), a phenotype compatible with that of erythroblastic cells. Cytogenetic and fluorescence in situ hybridization (FISH) analysis demonstrated classical t(9;22)(q34;q11) chromosomic translocation associated with a duplication of the BCR-ABL fusion gene. Other cytogenetic abnormalities were detected in all analyzed mitosis, the most frequent being a trisomy of chromosome 8. The established ERY-1 cell line retains these immunophenotypic and cytogenetic features, and light and electron microscopy confirmed the relatively mature erythroblastic phenotype of the cells. In addition, ERY-1 cell line expressed beta-globin mRNA and a non-phosphorylable form of the erythropoietin receptor, even in presence of erythropoietin. Of note, the proliferation of ERY-1 cells was inhibited by TGFbeta1 or STI-571 (Gleevec), without significant induction of further differentiation. In conclusion, ERY-1 is a new growth factor-independent human erythroleukemic cell line with a relatively mature phenotype that may be useful to study the molecular events involved in erythroblastic differentiation.

Novel flow-cytometric analysis based on BCD5+ subpopulations for the evaluation of minimal residual disease in chronic lymphocytic leukaemia.

We describe a new flow-cytometric analysis using quadruple labelling with anti-CD19, CD20, CD5, CD79b monoclonal antibodies and sequential gating. We determined a novel criteria defined by BCD5+CD79b-/low/total BCD5+ cells ratio (BCD5+R), and compared it with the previous definition of phenotypic remission, based on CD19+CD5+ coexpression, and with complementarity-determining region 3 polymerase chain reaction (CDR3 PCR) and clonotypic PCR (cPCR). A series of 54 peripheral blood samples from 21 chronic lymphocytic leukaemia (CLL) patients in complete haematological remission and a series of 16 from normal volunteers were analysed. In normal controls, the BCD5+R was always < 0.2. The sensitivity of the BCD5+R was 1 x 10-4vs 5 x 10-2 for CDR3 PCR and 1 x 10-5 for cPCR. Among the 54 CLL samples, 35 had a BCD5+R < 0.2 and showed polyclonal CDR3 PCR, whereas the cPCR was positive in 12 out of 20 tested. In the remaining 19 samples, BCD5+R was > 0.2, CDR3 PCR was monoclonal in 16 out of 19 and cPCR positive in 14 out 14 tested, including one out of three samples with polyclonal CDR3 amplification. Even though cPCR remains the most sensitive method to evaluate MRD, this new, sensitive and specific flow cytometric parameter, the BCD5+R, is more suitable than CDR3 PCR for routine clinical MRD assessment in CLL.