Lymphocyte expansion after unrelated cord blood allogeneic stem cell transplantation in adults.

Limited information is available regarding the incidence and features of lymphocyte expansions after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Large granular lymphocytes (LGL) expansions have been reported after bone marrow or peripheral blood, but not after unrelated cord blood (UCB) allo-HSCT, associated with indolent clinical courses and favorable outcomes. Here, we considered 85 recipients of UCB allo-HSCT to more broadly define the impact of lymphocytosis, not limited to LGL. Sustained lymphocytosis was observed in 21 (25%) patients at a median onset of 12.6 months and with a median duration of 12 months. Immunophenotypic analysis showed predominantly CD8+ T and/or polyclonal B-cell expansions. Three patients only had monoclonal T-cell expansion. CMV reactivation was significantly more frequent in the group of patients with lymphocytosis (76% vs 28%, P=0.0001), but was not associated with survival. Conversely, 2-year disease-free survival and overall survival were significantly higher for lymphocytosis patients (85% vs 55%, P=0.01 and 85% vs 63%, P=0.03, respectively). In conclusion, expansion of T or B lymphocytes after UCB allo-HSCT in adults is not a rare event. Although occurring relatively late after transplant, this feature is predictive of a better outcome for the patients.

Flow cytometry in hematological nonmalignant disorders.

Multiparameter flow cytometry (MFC) has become an integral part of the diagnosis and classification of hematological malignancies. However, several nonmalignant or premalignant disorders may benefit from this technology in hematology laboratories. This review provides information on the normal immunophenotypic characteristics of peripheral blood leukocyte subsets and their modifications in several clinical conditions. The usefulness of MFC and the specific markers that can be investigated in hyperlymphocytosis, infection, hypereosinophilia, paroxysmal nocturnal hemoglobinuria, and large granular lymphocyte disorders is described. Mention is also made of the developments of MFC for analyses of red blood cells or platelets.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10-4 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10-4 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10-4 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.

Autocrine insulin-like growth factor 1 and stem cell factor but not interleukin 6 support self-renewal of human myeloma cells.

In this study, we have identified the growth factors supporting myeloma self-renewal in eight myeloma cell lines. All cell lines able to form self-colonies displayed constitutive P-AKT and P-ERK1,2 but not P-STAT3 and did not express CD45, suggesting the presence of an insulin-like growth factor 1 (IGF1) loop. We showed that a blocking anti-insulin-like growth factor 1 receptor (IGF1R) monoclonal antibody (mAb) inhibited colony formation in correlation with IGF1R expression and decreased P-AKT. Imatinib or a blocking anti-stem cell factor (SCF) mAb also inhibited colony formation of two cell lines expressing C-KIT and SCF, and decreased P-AKT. Moreover, the PI3K/AKT pathway inhibitor wortmannin inhibited colony formation. Blocking interleukin (IL)6R did not inhibit colony formation in good agreement with a lack of constitutive P-STAT3. We showed that primary cells frequently co-expressed IGF1R/IGF1 but not C-KIT/SCF or IL6R/IL6, suggesting that in vivo autonomous growth could be possible via IGF1R. Despite their similar role in clonogenic growth and shared signaling pathway, IGF1R and C-KIT had opposite prognostic values, suggesting that they were surrogate markers. Indeed, we showed that both C-KIT and IGF1R prognostic values were not independent of MMSET expression. This study highlights the autocrine role of IGF1 in myeloma cells and reinforces the interest in targeting IGF1R in IGFR1(+) CD45(+/-) patients, such as MMSET(+) patients.

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.

Selection of Epstein-Barr virus specific cytotoxic T lymphocytes can be performed with B lymphoblastoid cell lines created in serum-free media.

Epstein-Barr Virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) are currently used for numerous applications in cellular immunology. Where protocols destined for clinical application are concerned, the final choice of assay is made according to a risk/benefit ratio analysis. In this balance the use of xenogenic or allogenic serum has always been a major concern, as it carries both an infectious and an immunological risk. So far, it is unknown whether serum can be omitted from the entire BLCL selection procedure. In addition, as BLCL have been described as heterogeneous, serum deprivation may affect their antigen-presenting capacity. In the present study, BLCL were generated in the absence or presence of fetal calf serum (referred to as BLCL0 or BLCL(FCS), respectively). Next, in order to assess the antigen-presenting capacity of these cells, we compared the ability of BLCL0 and BLCL(FCS) cells to stimulate the EBV-specific repertoire of the corresponding donor's peripheral blood mononuclear cells in vitro. Our results showed that addition of serum was not essential for BLCL infection and culture, and that as far as we could determine, BLCL0 cells were as effective as BLCL(FCS) in reactivating the EBV-specific T-cell repertoire in vitro. Notably, FCS-specific T-lymphocytes can be detected among the BLCL(FCS)-specific CD4+-CTL. Not only was this latter observation unexpected for an EBV-seropositive donor, but it implied that the BLCL had captured and processed the corresponding FCS-derived solubles antigens; taken together our results emphasized the interest of the possibility to generate BLCL0, both for research and for clinical applications.

Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival.

We and others have shown that Mcl-1 was essential for the survival of human myeloma cells in vitro. Furthermore, this antiapoptotic protein is upregulated by interleukin-6, which plays a critical role in multiple myeloma (MM). For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC), that is, myeloma cells from 51 patients with MM and 21 human myeloma cell lines (HMCL) using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. In total, 52% of patients with MM at diagnosis (P=0.017) and 81% at relapse (P=0.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only HMCL but not reactive plasmacytoses have abnormal Mcl-1 expression, although both PC expansions share similar high proliferation rates. Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. Finally, the level of Mcl-1 expression is related to disease severity, the highest values at diagnosis being associated with the shortest event-free survival (P=0.002). In conclusion, Mcl-1, which has been shown to be essential for the survival of human myeloma cells in vitro, is overexpressed in vivo in MM in relation with relapse and shorter survival. Mcl-1 represents a potential therapeutical target in MM.

Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups in multiple myeloma.

Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients. The method was very specific and sensitive for the detection of hyperdiploidy. Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%). Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.

A one-step RT-PCR and a flow cytometry method as two specific tools for direct evaluation of human herpesvirus-6 replication.

In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The first method consisted of a one-step reverse transcription PCR amplifying a part of the late alternatively spliced U100 gene which encode the gp 82-105 viral glycoprotein. Two extraction methods and two RT-PCR kits were evaluated, leading to the selection of TaKaRa mRNA selective PCR kit. The second procedure consisted in a flow cytometry method to analyze the expression of two late viral HHV-6 antigens using 7C7 and 10G6 monoclonal antibodies. Four fixation permeabilization procedures were compared and the preparation of cells with paraformaldehyde (PFA) 4% was found to be optimal. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This kinetic study confirmed that Mabs recognized late antigens and demonstrate that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms are detectable earlier in the cycle.

Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.

R115777, a nonpeptidomimetic farnesyl transferase inhibitor has recently demonstrated a significant antileukemic activity in vivo in acute myeloid leukemia. Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of long-lived plasma cells within the bone marrow. In the present study, we have investigated the effect of the R115777 on growth and survival of myeloma cells. We have found that R115777 induced (1) a significant and dose-dependent growth inhibition of the three myeloma cell lines tested; and (2) a significant and time-dependent apoptosis. R115777 also induced apoptosis in the bone marrow mononuclear cell population of four MM patients, being almost restricted to the malignant plasma cells. Finally, we have investigated the effect of the R115777 in the Ras/MAPK and JAK/STAT pathways which are implicated in survival and/or proliferation in MM. The phosphorylation of both STAT3 and ERK1/2 induced by IL-6 was totally blocked at 15 microM of R115777 and partially blocked when R115777 was used at 10 and 5 microM. The induction of apoptosis by R115777 in myeloma cells and its implication in the regulation of JAK/STAT signalling suggest that R115777 might be an interesting therapeutical approach in MM.

Use of pathology-specific peripheral blood CD34 thresholds to predict leukapheresis CD34 content with optimal accuracy: a bicentric analysis of 299 leukaphereses.

CD34+ cell counts in peripheral blood (PB) and corresponding numbers of CD34+ cells and colony-forming units-granulocyte/macrophage (CFU-GM) in 299 leukapheresis products of 209 patients undergoing PB progenitor cell (PBPC) mobilization for autologous transplantation in two different centers were analyzed and compared according to diagnosis: non-Hodgkin lymphoma (NHL, 94 leukaphereses), multiple myeloma (MM, 75), Hodgkin's disease (HD, 37), solid tumors (35), and chronic myeloid leukemia (CML, 32). Without separating disease entities, correlations between PB CD34+ cell counts and leukapheresis content of CD34+ cells (r>0.83, P<0.01) and CFU-GM (r>0.81, P<0.01) were excellent. In both centers, a PB CD34 threshold ensuring a leukapheresis yield > 10(6) CD34/kg was determined. This threshold was higher in center 1 than in center 2, and its predictive accuracy (91.4%, i.e., prediction correct 91.4% of the time) was significantly lower than in center 2 (98.4%, P=0.02). When data were analyzed by pathology, PB CD34+ cell counts and leukapheresis content of CD34+ cells and CFU-GM remained well correlated, and in both centers PB CD34 thresholds predictive of a yield > 10(6) CD34/kg per leukapheresis could be determined for each pathology. For most patients, pathology-specific PB CD34 thresholds could be obtained directly from the equation of the PB CD34/leukapheresis CD34 correlation curve; they varied depending on both pathology and center (range: 7-20 x 10(6) CD34/l). Pathology-specific thresholds predicted a leukapheresis yield > or = 10(6) CD34/kg accurately 100% of the time for MM patients in center 2 and HD and solid tumor patients of both centers, resulting in overall rates of accurate prediction of sufficient graft CD34 content of 96.6% in center 1 and 98.9% in center 2.

Reactive plasmacytoses in multiple myeloma during hematopoietic recovery with G- or GM-CSF.

We report on three cases of reactive plasmacytoses (RP) in the course of multiple myeloma (MM). The three patients achieved complete remission following high dose melphalan and peripheral blood stem cell transplantation. These transient plasmacytoses had all the characteristics of RP, i.e. expansion of highly proliferative polyclonal plasma cells (PC) with a normal phenotype and genotype and corresponding to expansion of both PC progenitors (plasmablasts) and PC precursors (early plasma cells). These cells were easily distinguished from malignant PC of the corresponding patients evaluated at diagnosis, especially by their phenotypic and genotypic features.

IgG-secreting lymphoplasmacytoid leukaemia: a B-cell disorder with extensively mutated VH genes undergoing Ig isotype-switching frequently associated with trisomy 12.

We investigated 16 patients with elevated serum monoclonal IgG and a leukaemic B-cell lymphocytic disorder different from multiple myeloma. Their clinical history was that of a non-aggressive disease with dominant splenomegaly and long survival. Whereas abnormal blood and bone marrow cells were predominantly small lymphocytes with a few lymphoplasmacytoid cells, histopathological features included a lymphoplasmacytic infiltrate in eight cases. Most frequently, abnormal blood cells displayed a CD19+CD5-CD23+/- immunophenotype different from that of chronic lymphocytic leukaemia, except in two cases with a CD19+CD5+CD23+ phenotype. Interestingly, a coexistent serum monoclonal IgM and/or surface IgMG+ with identical light chain was identified in 10 patients, whereas in the remaining six patients only IgG expression was determined. VH gene analysis was performed in eight patients to investigate the clonal origins of tumour cells. All cases utilized the VH3 family, with evidence of extensive somatic mutations and intraclonal homogeneity in all cases. VH gene analysis indicated a clonal relationship between cells expressing IgM and IgG, with one case being biclonal. Cytogenetic evaluation showed a high incidence of trisomy 12 (60%) and 13q14 deletion (40%). In conclusion, we have described an unusual subset of low-grade lymphoma with high-serum IgG and frequent lymphoplasmacytoid features in which tumour cells derive from post-follicular memory B cells undergoing isotype switching with some cases arrested at both the IgM and IgG stage and others as IgG-positive cells only.