ALBERT

Description: Reduced-intensity conditioning regimens (RIC) had become a classical strategy of allogeneic hematopoietic stem cell transplantation (HSCT) and many patients are now transplanted with unrelated donor. The aim of this restrospective study was to evaluate the impact of HLA mismatches between donor (D) and recipient (R) at the allelic level on survival after RIC. We analyzed 103 patients registered in France from Jan 1999 to Dec 2003 with a median age of 46 years (18–67). All patients had hematologic malignancies: AL (n=35), MM (n=18), CLL (n=5), NHL (n=11), HD (n=9), CML (n=12), MDS (n=9), and MPS (n=4). 39% of the patients were in an advanced phase of the disease at time of HSCT. Anti-thymocytes globulins (ATG) were part of the conditioning regimen for 77% of patients. The main source of stem cells was PBSC (n=65). Seventy-one D/R pairs (69%) were 10/10 HLA match at the allelic level. Mismatches concerned 5, 6, 15, 2 and 7 D/R pairs for HLA-A, -B, -C, -DRB1 and -DQB1, respectively. The results showed that 96% of patients engrafted. Acute GVHD grade II to IV and grade III/IV occurred in 46% and 19% of patients, respectively. The risk of developing cGvHD was 45% at 2 years. Overall survival (OS) was 42% at five years. Among the 47 patients alive, the median disease free survival (DFS) was 28 months. Among non-HLA parameters studied, the only factor associated with a good OS was the diagnosis of lymphoid disease (HD or NHL or CLL) (p=0.003). Recipient age

Description: Background :AZA improves overall survival (OS) in higher risk MDS, but only 50-60% of the patients respond, and median OS with AZA is only 20-24 months. As OS improvement is obtained at modest response rates, OS rather than response should probably remain the primary endpoint for all combinations with AZA, requiring large phase III trials with significant follow up. On the other hand, combinations that do not increase response will likely not improve OS. We therefore tested, based on a "pick the winner" approach, AZA combinations with the HDAC inhibitor VPA, LEN or IDA to identify, based on response, the most promising combination with AZA in higher risk MDS, that could be subsequently compared with AZA alone in a larger phase III study. Methods : AZA-PLUS (#NCT01342692)was an adaptive two-stage phase II trial based on Jung design (Stat Med.2008;27:568) that randomly assigned higher-risk MDS, low blast count AML (20-30%) and CMML to: AZA (75 mg/m2/d d1-7 of 28-day cycles); AZA plus LEN (10 mg/d on d1-14); AZA plus VPA( 50 mg/kg/d on d1-7; 35 mg/kg/d in patients〉 60y) or AZA plus IDA (10 mg/m2on d1 for the first 9 cycles). The primary end point was response rate (RR, including CR, PR, marrow CR, based on IWG 2006) of the combination arms vs AZA alone. Given a 30% RR with AZA alone, we considered that a ≥45% RR would make combination(s) promising. Controlling for type I and type II errors at 0.15 and 0.20, 40 patients per arm were to be enrolled at each stage. Any experimental arms with RR lower than those observed in the AZA arm at the first stage should be stopped. At the second stage, any arm with 〉 6 more responses than AZA alone should be selected for further testing. Secondary endpoint were ORR (RR+ stable disease with HI (HI) and OS. Results : After inclusion of 40 pts/arm (first stage) all experimental arms had at least the same number of responses as the control arm and were continued in second stage. Overall, 322 pts were enrolled from 06/2011 to 07/2017: 81, 80, 80, 81 in the AZA, AZA+VPA, AZA+LEN and AZA+IDA arms, respectively. Baseline characteristics were well-balanced across arms. Median age was 74.6 y, 213 pts were male, IPSS was INT-2 in 54% and High in 46%. IPSS Karyotype was fav, int and poor in 40%, 26% and 34%, respectively. Pts received a median of 7 cycles and median follow-up was 15.1 months. Prevalence of trial discontinuation due to adverse events was 32%, 29%, 28% and 31% in the AZA , AZA+VPA , AZA+LEN and AZA+IDA arms, respectively (p=0.95). Rates of hospitalization during the first 6 cycles were 38%, 44.7% , 55.1%, 59.7% in the AZA, AZA +VPA, AZA+LEN and AZA+IDA arms, respectively (p=0.028), suggesting increased myelosuppression in the experimental arms, especially in the LEN and IDA arm. In the control arm, 29 responses (CR+PR+mCR) after 6 cycles were observed, with 29, 25 and 29 responses observed in AZA+VPA , AZA+LEN and AZA+IDA arms, respectively. Thus, no combination demonstrated benefit over AZA. The RR was estimated at 34.8% (18.6% CR, 3.1% PR, and 13.0% mCR) and the ORR after 6 cycles was 40.4%. The RR after 6 cycles (35.8% for AZA, 36.2% for AZA+VPA, 31.2% for AZA+LEN, and 35.8% for AZA+IDA) and the ORR after 6 cycles (41.9% for AZA; 41.2% for AZA+VPA, 40.0% for AZA+LEN and 38.3% for AZA+IDA) were close across study arms. By multivariate analysis, factors associated with better ORR were higher Hb level (p=0.05), low fibrinogen (p=0.008) and low LDH (p=0.01). 17 (5%) pts were bridged to allogeneic SCT: 6 on AZA, 5 on AZA+VPA, none in the AZA+LEN arm and 6 on AZA+IDA arm (p=0.03). At the reference date of July 2018, median EFS was 16.6 months for in AZA, 14.5 months for in AZA+VPA, 15.1 months for in AZA+LEN and 13.2 months for in AZA+IDA (p=0.74) (Fig A). Multivariable Cox model selected Hb level (p=0.02), presence of circulating blasts (p

Description: Introduction: Even though the diagnosis criteria of chronic myelomonocytic leukemia (CMML) have been recently revised by the World Health Organization (WHO), recognition of this disease may be challenging. This myelodysplastic/myeloproliferative neoplasm can also be diagnosed by a relative accumulation of classical monocytes (cMO, CD14++CD16-) ≥94% of total peripheral blood monocytes and a decrease of intermediate (iMO, CD14++CD16+) and non-classical monocytes (ncMO, CD14lowCD16+) percentages measured by flow cytometry (Selimoglu-Buet, 2015; Talati, 2017; Patnaik, 2017; Hudson, 2018). However, inflammatory diseases concomitant to CMML or inflammatory state in CMML patients can provoke an increase of the iMO percentage leading to a decrease of the relative cMO percentage below the 94% threshold (Selimoglu-Buet, 2017). In these cases, the decrease of the relative ncMO percentage persists, hence it might be a useful diagnostic criterion relevant for CMML diagnostic. Since accurate delineation of the iMO and ncMO populations remains debated, the use of a ncMO specific marker, such as slan (6-sulfo LacNac), could be of interest. Objective:We aimed to assess the clinical utility of the slan marker in peripheral monocytosis exploration and CMML diagnosis, especially in inflammatory state. Methods: From November 2017 to July 2018, whole blood samples collected on EDTA or peripheral blood mononuclear cells (PBMC) were stained with the following antibodies as previously described: anti-CD45, CD2, CD56, CD24, CD14, CD16 (purchased either from Beckman-Coulter or Becton-Dickinson) and anti-slan (Miltenyi Biotec). Sample analysis was performed either with a Navios (BC) or a Fortessa (BD) cytometer. Fifty-four controls (19 young healthy blood donors and 35 age-matched healthy donors), 13 patients with reactive monocytosis and 37 patients newly diagnosed with a CMML were enrolled in this study. Results: Firstly, we analyzed the expression of the slan marker in the different circulating mature populations from control whole blood samples. We found that slan is only expressed in monocytes and not in neutrophils or lymphocytes (Figure 1A), especially not in NK cells. Among the three monocyte subpopulations, the expression of slan is restricted to ncMO with 98.9%±0.7% of slan-positive cells gathering within this subpopulation (Figure 1B). However, only 49.1%±12.3% of the ncMO are slan positive, corresponding to ncMo cells with the weakest expression of CD14 (Figure 1C, compare red population to blue one). Yet, both slan-positive and slan-negative ncMO subpopulations displayed similar morphological features after cell-sorting and MGG staining (Figure 1D). Next, we assessed slan expression within the ncMO subpopulation in comparison with relative cMO percentage in healthy donors, patients presenting a reactive monocytosis or a CMML. Thirty-two out of the 37 CMML patients displayed cMO percentage above 94% as expected (Figure 1E). A significant decrease of slan-positive ncMO percentage was observed in CMML patients compared to healthy donors and patients with reactive monocytosis (Figure 1G). All the five patients whose cMO percentage was below the threshold (Figure 1E, blue triangles amongst red ones) displayed the well-recognized "bulbous" aspect (Figure 1F), with an increase of the iMO leading to the decrease of the relative cMO percentage. Interestingly, these patients that couldn't be diagnosed as CMML using the relative accumulation of cMO displayed a low percentage of slan-positive ncMO (Figure 1G, blue triangles amongst red ones). Eventually, we established a Receiver Operator Curve (ROC) and obtained a 1.4% cut-off value of slan-positive ncMo with an area under the ROC curve (AUC) of 0.999 (Figure 1H). The use of the relative slan-positive ncMo percentage led to an improvement of the sensitivity of the CMML flow cytometry assay compared to the relative cMO% (100% vs 86%), as all the false negatives were retrieved. Conclusion: Here, we describe a new parameter for CMML diagnosis, namely the decrease of the relative slan-positive ncMo percentage below 1.4%. This criterion, associated to the relative cMO quantification, may be useful, especially when CMML patient displays an inflammatory state. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 508 Background: Contrary to the “5q syndrome”, prognosis of MDS with del 5q with increased BM blast % and/or additional cytogenetic abnormalities (abn) and of AML with del 5q (isolated or complex) is poor. Furthermore, del 5q is present in 40–50% of higher risk MDS and AML with complex karyotype. Those patients respond poorly to IC with 20–30% CR, of short duration (Estey, Hematologica 2000) and to azacytidine (AZA) (Mufti, ASH 2009, abstr n° 1755). Lenalidomide (LEN) yields hematological but also frequently cytogenetic CR in lower risk MDS with del 5q. In a recent phase II study of LEN in higher risk MDS or AML with del 5q, 28% responded, some with cytogenetic responses, but with significant myelosuppression (Ades, Blood 2008). This prompted us to combine IC and REV in this patient population. Methods: This still ongoing phase I/II study (Clinical trial.gov n° NCT00885508) combines induction DNR (45 mg/m2/d d1-3, escalated to 60 mg/m2/d d1-3) + AraC (200mg/m2/d d1-7) to LEN (10 mg/d d1-21)and G-CSF (from day 8 to end of aplasia) in IPSS int 2 or high MDS or AML with del 5q (isolated or not). Responders, ie CR, CRi or marrow CR according to AML criteria (Cheson JCO, 2003) receive 6 consolidation courses of DNR (45 mg/m2 d1), AraC (120 mg/m2/d×5) and LEN 10 mg/d d1-15, followed by maintenance LEN (14 days/month) until progression. After the first cohort at DNR 45 mg/m2/d d1-3 (n=31) proved safe, escalation to DNR 60 mg/m2/d during induction and consolidation courses was made (n=17). Results: Between Feb 2009 and April 2010, 48 pts from 11 centres were included: 22 Female, 26 Male, median age was 65 years (range 30–79), 36 (75%) AML and 12 (25%) RAEB-2 (WHO classification). 38/48 (79%) pts had complex Karyotype (median number of 6 abn (range 2–13) in addition to del 5q), 5 pts had isolated del 5q and 5 pts had del 5q+1 abn. 11 (23%) pts had therapy related disease. At inclusion, ECOG was 0 in 14 pts, 1 in 21 pts, 2 in 4 pts and 3 in 1 pt (8 missing data). Median baseline WBC, platelets and Hb level were 2.85 G/l (0.7-100), 45 G/l (11-213) and 8.7 g/dl (7.2-11.5) respectively (resp). 31 pts were included in the fist cohort (DNR 45 mg/m2) and 17 were included in the second cohort (DNR 60 mg). All but 7 pts received the planned schedule of LEN (21 days). Overall, 5 pts had early death related to severe sepsis in 3, multiorgan failure in 1 and myocardial infarction in 1 pt. Median duration of hospitalisation during induction treatment was 30.5 days (range 19–43). In responders, median ti me to ANC〉1G/l and platelet 〉50G/l was 23 days (range 1–36) and 22 days (range 9–38), 19 d and 19 d, 24d and 24d in the whole cohort, in DNR45 cohort and in DNR60 cohort resp (p=0.1). Median number of RBC and platelet units transfused during induction treatment was 9 (4-16) and 7 (3-21), resp. 24 pts (50%) achieved CR, 1 achieved CRi (2%), 1 PR (2%) and 3 pts (6%) normalized PB count without marrow response, leading to an ORR of 29/48 (60%). Of the 17 pts with hematological CR/CRi evaluable for cytogenetic response, 8 (Including 4 complex karyotype, 3 isolated del 5q and 1 del5q+1 abn) achieved cytogenetic CR, 5 cytogenetic PR and 4 had no cytogenetic response. Eight of 12 (66%) MDS achieved CR/CRi compared to 17/36 (47%) AML (p=0.32). Four of 5 (80%), 4/5 (80%) and 17/38 (45%) pts with isolated del5q, del5q+1 abn and complex carytotype, resp, achieved CR/CRi (p=0.134). 17 of 31 (55%) pts in the DNR45 cohort achieved CR, vs 8/17 (47%) pts in the DNR60 cohort (p=0.76). Sex, ECOG status, Age, WBC count, Hb level, Platelet count had no effet on CR achievement. Among the 25 pts in CR, 14 relapsed and 2 died in CR (1 during consolidations and 1 after SCT). 1y DFS was 26.5% and significantly shorter in females (p=0.03) while other factors did not influence DFS. Estimated 2y OS was 30% and WHO diagnosis, Sex, Karyotype, and ECOG had no impact on OS. Conclusion: Intensive chemotherapy (IC) and LEN can be combined in higher risk MDS and AML with del 5q without unexpected additive myelosuppression or extra hematological DLT. In this cohort of elderly pts with very poor cytogenetics, The CR rate was 50%, higher than generally reported with CT alone in similar pts. DFS remained however short, suggesting that induction or consolidation therapy should be improved. Based on the better efficacy of DNR 90 mg/m2/d (Lowenberg et al, and Fernandez H et al, NEJM, 2009) in the chemotherapy of elderly AML, and the adequate tolerance in our DNR 60 cohort, we will increase the DNR dose to 90 mg/m2/d in a next patient cohort. Disclosures: Off Label Use: Lenalidomide in the treatment of AML and High risk MDS. Fenaux:CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Honoraria, Research Funding.

Description: Hydroxyurea is the standard therapy of chronic myelomonocytic leukemia (CMML) presenting with advanced myeloproliferative and/or myelodysplastic features. Response to hypomethylating agents has been reported in heterogeneous series of CMML. We conducted a phase 2 trial of decitabine (DAC) in 39 patients with advanced CMML defined according to a previous trial. Median number of DAC cycles was 10 (range, 1-24). Overall response rate was 38% with 4 complete responses (10%), 8 marrow responses (21%), and 3 stable diseases with hematologic improvement (8%). Eighteen patients (46%) demonstrated stable disease without hematologic improvement, and 6 (15%) progressed to acute leukemia. With a median follow-up of 23 months, overall survival was 48% at 2 years. Mutations in ASXL1, TET2, AML1, NRAS, KRAS, CBL, FLT3, and janus kinase 2 (JAK2) genes, and hypermethylation of the promoter of the tumor suppressor gene TIF1γ, did not predict response or survival on DAC therapy. Lower CJUN and CMYB gene expression levels independently predicted improved overall survival. This trial confirmed DAC efficacy in approximately 40% of CMML patients with advanced myeloproliferative or myelodysplastic features and suggested that CJUN and CMYB expression could be potential biomarkers in this setting. This trial is registered at EudraCT (eudract.ema.europa.eu) as #2008-000470-21 and www.clinicaltrials.gov as #NCT01098084.

Description: Introduction: The IMDSFlow working group reported recently the usefulness of immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes (MDS) (Westers, 2017). This multicenter study revealed that analysis of CD36 and CD71 expression on nucleated erythroid cells as coefficient of variation (CV), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between MDS and non-clonal cytopenias. These four parameters were analyzed on bone marrow samples after red blood cell lysis (RBC) procedure. Since this latter could also remove some nucleated erythroid cells, it was proposed to use a nuclear dye in a whole no-lysis bone marrow strategy for analysis of CD36 and CD71 expression (Mathis, 2013). So far, no study has compared the four ELN dyserythropoiesis parameters after or without lysis procedure. Objective:We aimed to evaluate whether no-lysis procedure can lead to a better assessment of dyserythropoiesis by flow cytometry compared to lysis procedure by preserving erythroblast cells. Methods: One hundred patients referred to our laboratory for bone marrow investigations between November 2017 and June 2018 were included in this prospective study. Nineteen patients were used as control samples without cytopenia whereas 81 presented at least one cytopenia. Complete blood count parameters as well as morphological and cytogenetic analyses were used to classify these patients as 25 MDS or 56 non-MDS, referred thereafter as pathological controls. Bone marrow specimens were collected on EDTA and were processed within 4h following aspiration. A similar amount of each sample was stained in parallel with the same antibody panel (CD36, CD71, CD117 and CD45), yet according to two different protocols, one with CyTrak orange without lysis procedure and one with RBC lysis using VersaLyse (Beckman-Coulter). Data were acquired using a Navios cytometer and analyzed using the Kaluza software (BC). Geometrical means of fluorescence (GMFI) of CD71 as well as CD36 and CD71 coefficients of variation and the percentage of CD117+ erythroid progenitors were collected in order to calculate the ELN dyserythropoiesis score as previously described (see Figure 1 A-B for erythroblast selection). Results: Firstly, we compared the percentages of erythroblasts obtained with the no-lysis and the RBC lysis strategies as reported to the total nucleated cells analyzed (corresponding to the CD45 positive cells in addition to erythroblasts selected as CD71 and CD36 positive cells). Surprisingly, we found that the lysis protocol led to a higher percentage of erythroblasts than the no-lysis protocol (19.0±11.8% vs 15.4±10.8%; p

Description: Background : Most non-del 5q lower risk MDS patients (pts) are first treated with ESA, with about 50% (generally transient) responses, and second line treatments (TX) including hypomethylating agent (HMA), Lenalidomide (LEN) and investigational drugs are then often proposed, but their effect on overall survival (OS) is unknown. In a previous work on 253 such pts, we found worse OS with early failure to ESA, i.e. primary resistance (RES) or relapse (REL) 〈 6 months after ESA onset (Kelaidi, Leukemia, 2013), but only few pts had received, after ESA failure, TX other than RBC transfusions. In the present study, we gathered non-del 5q lower risk MDS treated with ESA from several EU MDS cooperative groups, and analyzed their outcome after ESA failure, and the effect of second line TX on survival. Methods : 1611 IPSS low and int-1 (lower risk) non del 5q MDS pts included in the French (GFM), Italian (FISM), Spanish (GESMD), Greek, Düsseldorf and Munich registries between 1997 and 2014, and treated by ESA were studied. Survival was assessed from failure of ESA (i.e. from primary failure evaluated after 12 to 24 weeks of ESA treatment, or from relapse after a response). Progression at ESA failure was defined upon progression to a higher IPSS-R class at ESA failure as compared with ESA onset. Results : At ESA onset, the 1611 pts were reclassified by IPSS-R in 16% very low, 54% low, 13% int, 6% high, 1% very high and 10% ND. HI-E (using IWG 2006 criteria) to ESA treatment was 66.9%, and the median duration of response was 15 months. The cohort of 1038 pts with ESA failure included 521 RES and 517 REL. Median OS was 4.2 years in REL and 3.7 years in RES pts (p=0.56), and no significant difference was seen, even after restricting the analysis to very low and low IPSS-R pts (p=0.81), or when analyzing "early" vs "late" failures, with cut-off points at 6 or 12 months, as we previously reported (Kelaidi, Leukemia, 2013). 336 (32%) pts received second line treatment (TX2) other than RBC transfusions, including HMA in 88 pts, LEN in 169 pts, and other TX (OT) in 79 pts (including 11 chemotherapy, 17 thalidomide, 11 immunosuppressors (ATG, cyclosporine), or investigational drugs), with response rates of 46%, 39% and 33% respectively (p=0.4). 87 pts had a third line TX (mostly a new drug, but also 7 pts who received HMA after LEN, and 33 pts LEN after HMA). Pts treated with LEN as TX2 were younger (median age 70 vs 75 for BSC, and 70 for HMA p

Description: The immunophenotypic characterization of Waldenstrom’s macroglobulinemia (WM) is still unclear, despite being an essential tool in the diagnosis of hematological malignancies. We retrospectively reviewed the immunophenotypic profile of 63 cases of WM showing monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow infiltration, and of 26 cases of other chronic B-cell lymphoproliferative disorders having monoclonal IgM in the serum (LPD-M), including marginal zone (n=16), mantle cell (n=8) and follicular (n=2) lymphomas. The median age at diagnosis was 64[46–92] years. Immunophenotypic analysis was performed by flow cytometry between January 1998 and July 2007, using four or six monoclonal antibody combinations. In WM and LPD-M groups, all patients showed a monoclonal tumoral B-cell population, detected and studied in blood (21 and 23 patients, respectively), blood and bone marrow (19 and 2 patients, respectively) or bone marrow samples (23 and 1 patient, respectively). Patients with a Matutes score 〉 3 were excluded. Neoplastic cells, in all cases, expressed a monoclonal immunoglobulin light chain (kappa for 70% WM and 73% LPD-M, lambda for 30% WM and 27% LPD-M). The intensity of expression of the monoclonal light chain was particularly heterogeneous in both groups: high, normal or low expression in 43%, 27% or 30% of WM, and in 52%, 33% or 15% of LPD-M, respectively. Pan-B antigens (CD20, CD19, CD79b) were positive for at least 97% of patients. The results obtained with other antigens in WM compared to LPD-M were: CD10 = 10 versus 7%, CD23 = 33 versus 56%, CD5 = 14 versus 26%, FMC7 = 76 versus 89%, CD38 = 56 versus 41%, CD25 = 86 versus 84%, CD43 = 12 versus 16%, and CD11c = 10 versus 36%. The intensity of expression of these antigens was heterogeneous in both groups. Among the antigens only tested in the WM group, CD1c and CD27 were positive for 70% of patients, IgM and IgD for 95% of patients, and CD103 as well as CD117 were negative in all cases. Considering all (blood and/or bone marrow) WM samples, plasma cells (CD38/CD138 positive cells) were found at low levels (less than 2.5% of B-cells) for 46% of WM. Comparing blood versus bone marrow WM samples, no differences were found for all previous antigens except the higher frequency of low counts of plasma cells in bone marrow (observed in 71% of WM) versus blood samples (28% of WM). Among the 10 WM patients tested for ZAP-70 expression, nine were negative and one showed a low intensity expression. In conclusion, our results show that the immunophenotypic analysis usually performed with standard antigens in WM overlaps with other B-cell lymphoproliferative disorders. Studies involving the expression of new antigens and/or other biological approaches are required to identify the WM among the B-cell malignancies and are ongoing in our group.

Description: The immunophenotypic characterization is an essential tool in the diagnosis of hematological malignancies but the immunophenotypic features in Waldenstrom’s macroglobulinemia (WM) remain not clearly defined. We studied 96 cases of WM diagnosed by monoclonal IgM in the serum and morphological lymphoplasmacytic bone marrow infiltration, and we compared results to 33 cases of other chronic B-cell lymphoproliferative disorders (LPD), including marginal zone (MZL)(n=23), mantle cell (MCL)(n=8) and follicular (FL)(n=2) lymphomas. Patients with a Matutes score 〉3 (chronic lymphocytic leukemia) and with pathognomonic immunophenotype (hairy cell leukemia) were excluded. Immunophenotypic analysis was performed by flow cytometry using six-colour staining (FACS Canto II, Becton Dickinson). In WM and LPD groups, a monoclonal B-cell population was identified in blood (31 and 28 patients, respectively), blood and bone marrow (28 and 4 patients) or bone marrow samples (23 and 1 patients). Overall, 61% of WM patients showed a monoclonal B-cell population in blood. Neoplastic cells of WM and LPD patients with blood and/or bone marrow involvement expressed a monoclonal immunoglobulin light chain kappa (in 70% and 73% of cases respectively) or lambda (30% and 27%). The intensity of expression of the light chain was heterogeneous in both groups (high, normal or low expression in 43%, 27% or 30% of WM, and in 52%, 33% or 15% of LPD, respectively). All pan-B antigens (CD20, CD19, CD79b) were positive for at least 97% of patients. Results obtained with other antigens in WM compared to LPD were: CD10 = 10% vs 7% of patients, CD23 = 33% vs 56%, CD5 = 14% vs 26%, FMC7 = 76% vs 89%, CD38 = 56% vs 41%, CD25 = 86% vs 84%, CD43 = 12% vs 16%, and CD11c = 10% vs 36%. The intensity of expression of these antigens was heterogeneous in both groups. Among the antigens only tested in the WM group, CD1c and CD27 were positive for 70% of patients, IgM and IgD for 95% of patients, and CD103 as well as CD117 were negative in all cases. No difference was found between blood and bone marrow for all previous antigens. Plasma cells (CD38/CD138 positive cells) were found at low levels (less than 2.5% of B-cells) for 46% of WM in blood and/or bone marrow samples. Among the 10 WM patients tested for ZAP-70 expression, 9 were negative and 1 showed a low intensity expression. These results confirm that the immunophenotypic analysis usually performed with standard antigens does not allow defining a typical profile of WM. In order to tentatively identify the WM among the B-cell malignancies, we studied the expression of molecules known to be involved in B-cell development or in costimulatory pathways of antigenic activation, namely CD69, CD83, CD80 and CD86. We first analyzed blood samples of 24 WM patients showing a peripheral monoclonal B-cell population. CD80 was positive (〉 20% of B-cells) in all cases and CD83, CD69 and CD86 were always negative. Among these WM patients, 13 were also studied for the bone marrow phenotype. No difference was found between blood and bone marrow phenotype in 11/13 WM cases. We then studied 11 LPD with blood tumoral involvement (MZL(n=7), MCL(n=2) and FL(n=2)). In these LPD, CD69 and CD83 were always negative and, in most cases (9/11 patients), CD80 and CD86 were also negative. Interestingly, CD80 was found positive in 2 patients with MZL, but the CD80 positivity was always associated to the CD86 positivity. Altogether, these data suggest that the inclusion of CD80 and CD86 in the panel of cytometric analysis allow to discriminate WM from other B-LPD with peripheral blood involvement.