Minimal residual disease detection in Tunisian B-acute lymphoblastic leukemia based on immunoglobulin gene rearrangements.

IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.

Minimal residual disease detection in Tunisian B-acute lymphoblastic leukemia based on immunoglobulin gene rearrangements

IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.

Chronic natural killer lymphoproliferative disorders: characteristics of an international cohort of 70 patients.

BACKGROUND: The 2008 World Health Organization (WHO) classification distinguishes three entities among the large granular lymphocytic leukemia (LGL leukemia): T-cell LGL leukemia (T-LGL leukemia), aggressive natural killer (NK) cell leukemia, and chronic NK lymphoproliferative disorders (LPD), the later considered as a provisional entity. Only a few and small cohorts of chronic NK LPD have been published. PATIENTS AND METHODS: We report here clinicobiological features collected retrospectively from 70 cases of chronic NK LPD, and compared with those of T-LGL leukemia. RESULTS: There were no statistical differences between chronic NK LPD and T-LGL leukemia concerning median age [61 years (range 23-82 years)], organomegaly (26%), associated autoimmune diseases (24%), and associated hematological malignancies (11%). Patients with chronic NK LPD were significantly less symptomatic (49% versus 18%, P < 0.001) and the association with rheumatoid arthritis was more rarely observed (7% versus 17%, P = 0.03). The neutropenia (<0.5 × 10(9)/l) was less severe in chronic NK LPD (33% versus 61%, P < 0.001) without difference in the rate of recurrent infections. STAT3 mutation was detected in 12% of the cohort, which is lower than the frequency observed in T-LGL leukemia. Thirty-seven percent of the patients required specific therapy. Good results were obtained with cyclophosphamide. Overall and complete response rates were, respectively, 69% and 56%. Overall survival was 94% at 5 years. CONCLUSION: This study suggests very high similarities between chronic NK LPD and T-LGL leukemias. Since chronic NK LPD is still a provisional entity, our findings should be helpful when considering further revisions of the WHO classification.

High level of soluble programmed cell death ligand 1 in blood impacts overall survival in aggressive diffuse large B-Cell lymphoma: results from a French multicenter clinical trial.

The dosage of soluble programmed cell death ligand 1 (sPD-L1) protein in the blood of adults with cancer has never been performed in a prospective patient cohort. We evaluated the clinical impact of sPD-L1 level measured at the time of diagnosis for newly diagnosed diffuse large B-cell lymphoma (DLBCL). Soluble PD-L1 was measured in the plasma of 288 patients enrolled in a multicenter, randomized phase III trial that compared R-high-dose chemotherapy with R-CHOP. The median follow-up was 41.4 months. A cutoff of 1.52 ng/ml of PD-L1 level was determined and related to overall survival (OS). Patients with elevated sPD-L1 experienced a poorer prognosis with a 3-year OS of 76% versus 89% (P<0.001). Considering clinical characteristics, the multivariate analysis retained this biomarker besides bone marrow involvement and abnormal lymphocyte-monocyte score as independently related to poor outcome. sPD-L1 was detectable in the plasma and not in the serum, found elevated in patients at diagnosis compared with healthy subjects and its level dropped back to normal value after CR. The intention-to-treat analysis showed that elevated sPD-L1 was associated with a poorer prognosis for patients randomized within the R-CHOP arm (P<0.001). Plasma PD-L1 protein is a potent predicting biomarker in DLBCL and may indicate usefulness of alternative therapeutic strategies using PD-1 axis inhibitors.

IL-6 supports the generation of human long-lived plasma cells in combination with either APRIL or stromal cell-soluble factors.

The recent understanding of plasma cell (PC) biology has been obtained mainly from murine models. The current concept is that plasmablasts home to the BM and further differentiate into long-lived PCs (LLPCs). These LLPCs survive for months in contact with a complex niche comprising stromal cells (SCs) and hematopoietic cells, both producing recruitment and survival factors. Using a multi-step culture system, we show here the possibility to differentiate human memory B cells into LLPCs surviving for at least 4 months in vitro and producing immunoglobulins continuously. A remarkable feature is that IL-6 is mandatory to generate LLPCs in vitro together with either APRIL or soluble factors produced by SCs, unrelated to APRIL/BAFF, SDF-1, or IGF-1. These LLPCs are out of the cell cycle, express highly PC transcription factors and surface markers. This model shows a remarkable robustness of human LLPCs, which can survive and produce highly immunoglobulins for months in vitro without the contact with niche cells, providing the presence of a minimal cocktail of growth factors and nutrients. This model should be useful to understand further normal PC biology and its deregulation in premalignant or malignant PC disorders.

Evaluation of CytoDiff™ on cord blood WBC differential.

INTRODUCTION: An umbilical cord blood bank was recently opened in our institution as an alternative source of hematopoietic stem cells. Before inclusion of a cord blood in an international register, a WBC with differential is requested, among others. Currently, the reference method is the microscopic manual count, and we sought to evaluate the routine flow cytometric method (CytoDiff™) as an alternative. METHODS: A total of 161 cord bloods were analyzed between November 2010 and February 2011. WBC differentials were determined for each sample, by (i) the cell counter (DxH800), (ii) a manual review, and (iii) the flow cytometry using the CytoDiff™ antibody cocktail. RESULTS: Correlation coefficients between flow cytometry and microscopic count were satisfying for neutrophils, lymphocytes, and immature granulocytes and acceptable for eosinophils. On the other hand, we found lower correlation coefficient for basophils and monocytes. Monocytes' correlation was better when comparing flow cytometry with cell counter. CONCLUSION: The flow cytometric approach is suitable to realize cord blood WBC differential and allows for the identification of additional cell subsets.

Flow cytometry and IG/TCR quantitative PCR for minimal residual disease quantitation in acute lymphoblastic leukemia: a French multicenter prospective study on behalf of the FRALLE, EORTC and GRAALL.

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and CD40L signaling.

Interleukin-15 (IL-15) has been extensively studied for its role in the survival and proliferation of NK and T cells through a unique mechanism of trans-presentation by producer cells. Conversely, whereas activated B cells have been described as IL-15-responding cells, the cellular and molecular context sustaining this effect remains unexplored. In this study, we found that, whereas human B cells could not respond to soluble IL-15, monocytes and lymphoid tissue-derived macrophages but not stromal cells efficiently trans-present IL-15 to normal B cells and cooperate with T-cell-derived CD40L to promote IL-15-dependent B-cell proliferation. Furthermore, CD40L signaling triggers a Src-independent upregulation of STAT5 expression and favors a Src-dependent phosphorylation of STAT5 in response to IL-15. In follicular lymphoma (FL), immunohistochemical studies reported a strong relationship between malignant B cells, infiltrating macrophages and T cells. We show here an overexpression of IL-15 in purified tumor-associated macrophages, and STAT5A in purified tumor B cells. Moreover, FL B cells respond to IL-15 trans-presented by monocytes/macrophages, in particular, in the presence of CD40L-mediated signaling. This cooperation between IL-15 and CD40L reinforces the importance of tumor microenvironment and unravels a mechanism of FL growth that should be considered if using IL-15 as a drug in this disease.

Characterization of intratumoral follicular helper T cells in follicular lymphoma: role in the survival of malignant B cells.

Accumulating evidences indicate that the cellular and molecular microenvironment of follicular lymphoma (FL) has a key role in both lymphomagenesis and patient outcome. Malignant FL B cells are found admixed to specific stromal and immune cell subsets, in particular CD4(pos) T cells displaying phenotypic features of follicular helper T cells (T(FH)). The goal of our study was to functionally characterize intratumoral CD4(pos) T cells. We showed that CXCR5(hi)ICOS(hi)CD4(pos) T cells sorted from FL biopsies comprise at least two separate cell populations with distinct genetic and functional features: (i) CD25(pos) follicular regulatory T cells (T(FR)), and (ii) CD25(neg) T(FH) displaying a FL-B cell supportive activity without regulatory functions. Furthermore, despite their strong similarities with tonsil-derived T(FH), purified FL-derived T(FH) displayed a specific gene expression profile including an overexpression of several genes potentially involved directly or indirectly in lymphomagenesis, in particular TNF, LTA, IL4 or CD40LG. Interestingly, we further demonstrated that these two last signals efficiently rescued malignant B cells from spontaneous and rituximab-induced apoptosis. Altogether, our study demonstrates that tumor-infiltrating CD4(pos) T cells are more heterogeneous than previously presumed, and underlines for the first time the crucial role of T(FH) in the complex set of cellular interactions within FL microenvironment.

Follicular lymphoma cell niche: identification of a preeminent IL-4-dependent T(FH)-B cell axis.

Follicular lymphoma (FL) B cells contract tight connections with their microenvironment, which governs the pathogenesis and progression of the disease. Indeed, specific immune response gene signatures, obtained from whole biopsy samples, have been associated with patient survival. In this study, we performed gene expression profiling of purified B cell and non-B cell compartments obtained from FL and reactive lymph nodes. We identified 677 non-redundant genes defining the FL interface and involving 26 FL-specific functional networks. This approach highlighted an interleukin-4 (IL-4)-centered pathway associated with an activation of signal transducer and activator of transcription 6 (STAT6), which favors overexpression of IL-4-target genes. In addition, FL microenvironment was characterized by a strong enrichment in follicular helper T cells (T(FH)), as demonstrated through transcriptomic and flow cytometry analyses. The majority of phospho-STAT6(pos) B cells were located at the vicinity of cells expressing the programmed death 1 (PD-1) T(FH) marker. Moreover, purified FL-derived T(FH), expressed IL4 at very high levels compared with purified tonsil-derived T(FH) or non-T(FH) microenvironment. Altogether, our study demonstrated that tumor-infiltrating T(FH) specifically express functional IL-4 in FL, creating an IL-4-dependent T(FH)-B cell axis. This cross talk could sustain FL pathogenesis and represent a new potential therapeutic target.

[Hemophagocytic syndrome and toxoplasmic primo-infection]. / Syndrome d'activation macrophagique et primo-infection toxoplasmique.

Hemophagocytic syndrome (HPS) is a clinical entity that combines the clinical, biological and histological symptoms. The physiopathological mechanism involves the interaction between T lymphocytes/NK cells and macrophages, at the origin of an uncontrolled activation of the macrophages. The consequence is a hemophagocytosis extending to numerous organs, preferentially bone marrow. Clinical symptoms include cytopenia, fever unresponsive to antibiotics and multiple organ dysfunctions. Infections, lymphoproliferative disorders, cancers, systemic diseases are the most prevalent triggers or etiologies of HPS. Because of its high risk of mortality, HPS constitutes a diagnostic and therapeutic urgency. The search for an aetiology, in particular by serological testing, is essential because it conditions the treatment and thus the evolution of the disease. We report here the case of a 12 years-old boy presenting a HPS secondary to a toxoplasmic primo-infection. The objective of this work is to present the step of the biological diagnosis of HPS. Moreover, this observation allows the study of a very rare clinical presentation of toxoplasmic primo-infection, in an immunocompetant patient.

Clinical spectrum of gammadelta+ T cell LGL leukemia: analysis of 20 cases.

We report on the clinico-biological characteristics of 20 cases of gammadelta T cell large granular lymphocyte (LGL) leukemia. All the data were compared to that of 196 cases with alphabeta T cell subtype, which represents the majority of T cell LGL leukemias. Clinical findings were quite similar in the two groups regarding age, sex ratio, recurrent infections, and association with auto-immune diseases especially rheumatoid arthritis. Gammadelta LGL predominantly expressed a CD3+/CD4-/CD8+/CD16+/CD57+ phenotype, in 50% of cases. Clinical outcome was favorable for these patients with overall survival of 85% at 3 years. Fifty percent of gammadelta patients required treatment and the response to therapy was estimated at 55%. gammadelta and alphabeta T cell LGL leukemia harbor a very similar clinico-biological behavior and represent part of an antigen-driven T cell lymphoproliferation.

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT >> 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.

Human CD34-positive hematopoietic stem cells constitute targets for carcinogenic polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are major carcinogenic environmental contaminants known to exert bone marrow toxicity and to induce leukemias, suggesting that these chemicals target hematopoietic stem cells. To investigate this hypothesis, we studied the effects of PAHs on cell proliferation and differentiation in human hematopoietic CD34+ cell cultures. Benzo(a)pyrene (BP), a prototypical PAH, was shown to markedly impair CD34+ cell expansion and to inhibit CD34+ cell differentiation into various hematological cell lineages, including erythroid, granulomacrophagic, and megakaryocytic lineages. This was associated with the induction of a caspase- and mitochondrion-related apoptosis process. CD34+ progenitor cells were found to exhibit functional expression of the aryl hydrocarbon receptor (AhR), and the use of the pure AhR antagonist 3'-methoxy-4'-nitroflavone partially counteracted the deleterious effects of BP in CD34+ cell cultures, underlining the involvement of AhR in BP toxicity. Additional events such as CYP1A1/1B1-dependent PAH metabolism and adduct formation were also required since 1) 2,3,7,8-tetrachlorodibenzo-p-dioxin, a very potent ligand of the AhR that is poorly metabolized and therefore does not generate reactive metabolites in contrast to PAHs, failed to affect CD34+ cell expansion; 2) the CYP1A1/1B1 inhibitor alpha-naphthoflavone blocked both BP adduct formation and BP toxicity; and 3) benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide, a highly reactive BP metabolite, exerted a marked toxicity toward CD34+ cell cultures. Overall, these data indicate that human hematopoietic CD34+ cells can bioactivate chemical carcinogens such as PAHs and, in this way, constitute targets for such carcinogenic environmental contaminants.

Relationship between expression of genes involved in cell cycle control and apoptosis in diffuse large B cell lymphoma: a preferential survivin-cyclin B link.

Accumulating evidence suggests that lack of balance between proliferation and apoptosis may lead to clonal expansion and cancer emergence. In diffuse large B cell lymphoma (DLBCL), survivin expression by tumor cells has been recently described as a poor prognostic marker. We assessed the relationship between survivin gene up-regulation and several other factors involved in either cell cycle or apoptosis control. The expression of 34 genes from 27 cases of DLBCL with typical IPI factor-related poor prognostic outcome was analyzed by RNase protection assay. Using non-neoplastic tissues and low grade lymphomas as control, survivin expression was high in 80% of the cases without significant relation to patient overall survival (P = 0.64). However, the expression of several genes encoding for cell cycle inhibitors, cyclins, Bcl-2 or IAP family factors was significantly associated with the survivin up-regulation. Gene expression profiling showed that both survivin and cyclin B expression can define two subgroups of DLBCL: the previously described germinal center-like and activated B-like lymphomas, determined by protein expression analysis. We also identified a preferential survivin-cyclin B relationship (P = 0.017), suggesting that cyclin B over-expression, when linked to survivin over-expression in aggressive forms of lymphoma, might demonstrate a specific G2/M transition promotion.

c-myc box II mutations in Burkitt's lymphoma-derived alleles reduce cell-transformation activity and lower response to broad apoptotic stimuli.

In addition to c-myc rearrangement, over 50% of Burkitt's lymphoma cases present clustered mutations in exon 2, where many of the functional activities of c-Myc protein are based. This report describes the functional consequences induced by tumour-derived c-myc mutations located in c-myc box II. Two mutated alleles were studied, focusing on the P138C mutation, and compared to wild-type c-myc. The c-Myc transformation, transactivation and apoptosis activities were explored based on cells over-expressing c-Myc. While the transcriptional activation activity was not affected, our experiments exploring the anchorage-independent growth capacity of c-Myc-transfected Rat1a cells showed that c-Myc box II mutants were less potent than wild-type c-Myc in promoting cell transformation. Considering the possibility that these mutations could be interfering with the ability of c-Myc to promote apoptosis, we tested c-Myc-transfected Rat1a fibroblasts under several conditions: serum deprivation-, staurosporine- and TNFalpha-induced cell death. Interestingly, the mutated alleles were characterized by an overall decrease in ability to mediate apoptosis. Our study indicates that point mutations located in c-Myc box II can decrease the ability of the protein to promote both transformation and apoptosis without modifying its transactivating activity.