Search

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of hematologic diseases. World Health Organization classification divides MPNs into different categories based on the presence of an underlying genetic abnormality. The JAK2 V617F somatic mutation is observed in polycythemia vera, primary myelofibrosis and essential thrombocythemia (ET), the BCR-ABL1 fusion gene is observed in chronic myeloid leukemia (CML). JAK2 V617F mutation and BCR-ABL1 translocation have been considered mutually exclusive. We report a case of a 76-year-old woman who initially presented with ET which progressed to CML, reverting to the ET phenotype once the treatment with tyrosine kinase inhibitors was initiated. JAK2 V617F somatic mutation was the first molecular abnormality found, followed by the appearance of the BCR-ABL1 rearrangement, with the uncommon e1a2 BCR-ABL1 transcript isoform, described very rarely in CML. We believe that this might be a “dual” disease, very rarely recognized and described.

We analyzed the clinical features and prognostic factors for transformation of immunoglobulin M–related disorders (IgM-RDs) to malignant lymphoproliferative disease (MLD) in 83 patients with IgM-RDs. We studied 19 patients with type I cryoglobulinemias, 56 patients with type II cryoglobulinemias, 5 patients with peripheral neuropathies (PNs), and 3 patients with idiopathic thrombocytopenic purpuras. Fourteen patients with cryoglobulinemias presented with mild to moderate hepatomegaly with or without splenomegaly. Fourteen patients with type II cryoglobulinemias had arthralgias and/or vascular purpura (12 receiving corticosteroids), and 7 presented with PN. These latter patients and those with PNs without cryoglobulinemia were treated with steroids, cyclophosphamide, or polychemotherapy with/without plasma-exchange. Cumulative probability of evolution to MLD at 5 years was 15% (95% CI; 5%-25%). At a median of 62 months (12-195 months), 8 cases of IgM-RDs (8.4%) evolved to overt Waldenström's macroglobulinemia (n = 6), 1 case to non-Hodgkin's lymphoma, and 1 case to B-cell chronic lymphocytic leukemia. At univariate analysis, male sex (P= 0.02), IgM level = 3 g/dL (P< 0.0001), detectable Bence Jones proteinuria (P= 0.0005), lymphocytosis (P= 0.049), and high erythrocyte sedimentation rate (P= 0.003) significantly correlated with the evolution risk. Age, blood cell counts, ß2-microglobulin level, degree of marrow lymphoplasmacytic infiltration, type of cryoglobulinemia, and hepatitis C virus positivity did not correlate with transformation. Although IgM-RDs represent a distinct clinical entity frequently requiring treatment in view of the IgM-related symptoms, their evolution probability and prognostic factors for malignant transformation seem to widely overlap those described for asymptomatic IgM monoclonal gammopathies.

We evaluated the risk of transformation of asymptomatic immunoglobulin (Ig) M monoclonal gammopathy (aIgM-MG) into symptomatic lymphoproliferative disease in 384 patients, in subgroups of patients with IgM MG of undetermined significance (MGUS) and smoldering Waldenström's macroglobulinemia (sWM). One hundred seventy-two patients with aIgM-MG with bone marrow (BM) histopathology and immunophenotyping were available for analysis. After a median of 45 months (range, 12-233 months), 45 cases of aIgM-MG (11.7%) evolved into lymphoproliferative disease, as follows: symptomatic WM (n = 41), non-Hodgkin's lymphoma (NHL; n = 2), IgM multiple myeloma (IgM-MM; n = 1), and primary amyloidosis (n = 1). Cumulative transformation probability at 5 and 10 years was 8% and 29%, respectively. The parameters significantly correlated with evolution were, at univariate analysis, BM lymphoplasmacytic infiltration, high erythrocyte sedimentation rate, hemoglobin (Hb) level, IgM size, and peripheral lymphocytosis; at multivariate analysis, the parameters were IgM size and peripheral lymphocytosis, with Hb level associated with a trend toward higher progression risk. Of the 138 cases of IgM-MGUS, 14 (10.1%) evolved (13 WM, 1 IgM-MM) after a median of 75 months (range, 12-117 months); of the 34 cases of sWM, 13 (38.2%) progressed to WM after 55 months (range, 13-154 months). In patients with IgM-MGUS, event-free survival at 5 and 10 years was 95% and 83%, respectively, and in patients with sWM, 77% and 42%, respectively (P= 0.0001). Among patients with aIgM-MG, those at high risk of evolution are patients with sWM, a distinct entity with clear BM evidence of NHL.

Factors predicting evolution to symptomatic disease were investigated in 27 patients with smouldering Waldenstrom's macroglobulinemia (SWM) among 172 patients with Waldenstom's macroglobulinemia (WM), selected on the basis of the following criteria: (1) IgM paraprotein 3 g/dL, and/or (2) bone marrow (BM) lymphoplasmacytoid (LPC) infiltration = 30%, and/or (3) diffuse infiltration pattern on BM biopsy, and (4) no treatment requirement for at least 12 months. Cumulative probability of survival was calculated by means of Kaplan-Meier. The Mantel and Haenszel test and multivariate Cox model were used to identify possible predictors for evolution. At a median follow-up of 79 months (range, 14 to 204), 11 patients (40.7%) showed progression to symptomatic disease, with the median interval from diagnosis being 46 months (range, 12 to 154). Event-free survival (EFS) at 5 and 10 years was 65% (95% confidence interval [CI], 45% to 85%) and 53% (95% CI, 31% to 75%), respectively. At multivariate analysis, paraprotein 3 g/dL (hazard ratio [HR], 15.1; 95% CI, 3.01 to 75.64; P< .0009) and hemoglobin = 12.5 g/dL (HR, 3.75; 95% CI, 1.05 to 13.34; P< .042) independently predicted transformation into symptomatic disease (P< .0006). Neither BM findings nor other laboratory parameter was associated with overt WM development. In conclusion, in SWM monoclonal IgM levels 3 g/dL and hemoglobin levels = 12.5 gr/dL are likely to predict SWM transformation into active disease requiring treatment. Semin Oncol 30:231-235. © 2003 Elsevier Inc. All rights reserved.

The natural history of asymptomatic IgM monoclonal gammopathies (MG) and variables predicting evolution to symptomatic lymphoproliferative disorders were investigated in 452 patients diagnosed from 1975 to 2001. Univariate and multivariate Cox models were used to identify possible predictors of disease progression. At a median follow-up of 49 months (range, 12 to 233), 41 cases (9.1%) evolved to symptomatic Waldenstrom's macroglobulinemia (n = 36), non-Hodgkin's lymphoma (n = 2), B-cell chronic lymphocytic leukemia (n = 1), IgM multiple myeloma (n = 1), and primary amyloidosis (n = 1); the median interval from diagnosis was 53 months (range, 12 to 154). The cumulative probabilities of transformation into a symptomatic lymphoproliferative disease at 5 and 10 years were 8% (95% confidence interval [CI], 6% to 12%) and 21% (95% CI, 16% to 29%), respectively. At univariate analysis, monoclonal component size and hemoglobin level as continuous parameters, lymphocytosis (4 × 109/L), bone marrow lymphoplasmacytoid infiltration (10%), erythrocyte sedimentation rate (40 mm/h), and detectable Bence Jones proteinuria were significantly related with evolution probability. At multivariate analysis, paraprotein level (P< .0001), hemoglobin level (P< .05), and lymphocytosis (P< .0001) independently predicted malignant evolution (P< .0001). In conclusion, patients with asymptomatic IgM-MG showing hematological features predictive of progression should be carefully monitored in view of an early treatment of the disease. Semin Oncol 30:172-177. © 2003 Elsevier Inc. All rights reserved.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with aggressive clinical course and poor prognosis. Diagnosis is based on detection of CD4+ CD56+, CD123high, TCL-1+, and blood dendritic cell antigen-2/CD303+ blasts, together with the absence of lineage specific antigens on tumour cells. In this report we present a case of BPDCN presenting with extramedullary and bone marrow involvement, extensively studied by flow cytometry and immunohistochemistry, who achieved complete remission after acute lymphoblastic leukemia like chemotherapy and allogeneic hematopoietic stem cell transplantation.

bone marrow blast count is a crucial parameter to determinate the prognostic score according to IPSS in myelodysplastic syndromes. However even among the major experts the agreement about detection of a blast cells by optical microscopy is less than 90% (Mufti et al: Haematologica 2008). The aim of this study is to evaluate prognostic significance of immunocytometric count of medullar blasts compared to cytological count.in a retrospective analysis of 104 patients with a minimum follow-up of three years, IPSS was calculated replacing cytological count of blasts with the flow cytometry one. Blasts were expressed as percentages of total cellularity, and identified by the combination of CD45/SSC with/without CD33, CD34 and CD117 antigens. The monoclonal antibodies used were CD34 FITC, CD117 PE, CD45 PerCp, CD33 APC. Acquisition of information on 1×105 stained cells corresponding to the whole bone marrow cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Survival of “low + intermediate-1 risk” (“IPSS1+2”) patients was compared to “intermediate-2 + high risk” ones (“IPSS3+4”) using Kaplan Meier method followed by logrank test. The comparison of ability among all blast count models to distinguish the probability of survival of the two groups (“IPSS 1+2” vs “IPSS3+4”) was based on χ2 value obtained by the logrank test.considering IPSS obtained from any of the immunocytometric markers considered (CD33, CD34, CD117 and CD45dim/SSC), “IPSS1+2” group had always an extremely better survival than “IPSS 3+4” group with a level of statistical significance similar to survival study of “standard IPSS”. Of all markers, CD45dim/SSC was the most capable to distinguish survival of “IPSS1+2” group versus “IPSS3+4” group (χ2 = 42. 5). Among others, CD117 and CD33 had a better discriminating power than cytological count (respectively χ2 = 38, 1 and χ2 = 22, 3 versus χ2 18, 2); only CD34 was worse than cytological count (χ2 =14, 2).the immunocytometric markers considered are demonstrated to be even better than cytological count as parameter in IPSS score. Their use has to be encouraged, especially where there is little experience in optical microscopy.No relevant conflicts of interest to declare.

In the absence of serum hepatitis B (HB) surface antigen (HBsAg) and clinical evidence of HB, an occult HB virus (HBV) infection (OBI) is defined as the existence of HBV DNA in the serum, in the peripheral blood mononuclear cells (PBMC), and/or in the liver. Most frequently, OBI follows the resolution of acute hepatitis and continues indefinitely after HBsAg clearance and liver function improvement. Usually, this form of infection, termed secondary OBI (sOBI), is associated with the presence of serum anti-HB core antibodies (anti-HBc). Up to 20% of individuals with OBI do not react for either anti-HBc or any other serological indicator of HBV exposure. Consistent with the woodchuck model, this form of infection, termed primary OBI (pOBI), is characterized by the presence of DNA in both serum and PBMC, but not in the liver. Although reactivation of sOBI has been reported in a high proportion of hemopoietic stem cell transplant (HSCT) recipients, pOBI reactivation risk has not yet been fully investigated in the transplant setting. We followed up 9 long term surviving HSCT recipients with pOBI, in order to define its clinical significance in immune suppressed patients.A total of 75 consecutive patients with hematological disease, receiving autologous or allogeneic HSCT between April 2006 and March 2007, were included in a database and retrospectively examined. As a prerequisite for inclusion in the study, each of the following was needed: (i) negative serological indicators of HBV exposure and absence of clinical signs/symptoms of hepatitis at the time of HSCT, (ii) positive HBV DNA, as detected by polymerase chain reaction (PCR), in both serum and PBMC samples drawn within 6 months before transplant, and (iii) availability of stocked frozen serum/PBMC samples collected after HSCT from each patient. Following these criteria, 9 patients (7 males, 2 females) were selected for the analysis. Five patients (1 Hodgkin disease, 3 non-Hodgkin lymphomas, and 1 B-cell chronic lymphocytic leukemia) received autologous HSCT, and 4 patients (1 acute myeloid leukemia, 1 multiple myeloma, 1 myelodysplastic syndrome, and 1 acute lymphoblastic leukemia) received allogeneic HSCT. Median age at transplant was 49 (range: 33–63) years. No patient received lamivudine prophylaxis. Serum and PBMC samples collected after HSCT were analyzed for HBV serology and DNA detection. The latter was performed by in-house PCR amplification by nested primers (detection limit 50 copies/ml) from highly conserved S region encompassing a-determinant of HBV. Direct sequencing of all HBV DNA amplified products was used to confirm the specificity of the reaction.In 8 cases, one serum/PBMC sample per patient was available, at a median of 27 (range: 21–34) months after HSCT; in 1 case, three serum/PBMC samples were available, at 17, 22, and 28 months after HSCT, respectively. During follow-up, positive HBV DNA was found, both in the serum and in the PBMC. All patients were confirmed to be infected with HBV subgenotype D1, with mutations known to impair HBsAg antigenicity. Three patients were confirmed to be further infected with HBV subgenotype A2. So far, 41 to 52 months after transplant (median: 46 months), neither patients showed seroconversion nor developed clinical hepatitis, suggesting pOBI persistence.Although the virus persisting in pOBI remains biologically competent, it is likely neither to reach liver-pathogenic doses nor to induce “classical” HBV reactivation during PBMC-restricted replication. However, prospective studies with large sample size and long-term follow-up are warranted, to define the actual clinical relevance of HBV occult infection. Indeed, OBI with low viremia could induce DNA damage, cellular demise, immune hypo-responsiveness, and oxidative stress in peripheral blood lymphocytes.No relevant conflicts of interest to declare.

obtained by the two methods were matched with RCO. A combined method assessment (CMA) was also tested, accounting as negative those samples not assessed as frankly positive by at least one method.Thirty-six percent of BCF resulted RCO-positive. By applying each method to RCO, concordant results between FCM and CM were observed in 69 cases (75%). Fifty-six cases (61%) were double true negative (TN), 11 cases (12%) were double true positive (TP), and 2 cases (2%) were double false negative (FN). The latter were BALF samples from patients with primary pulmonary lymphomatoid granulomathosis in which FCM revealed a prominent inflammatory background of CD3+ T-cells; clonality could not be assessed due to the small number of B-cells. Mismatched results between FCM and CM were observed in 23 cases (25%). FCM gave TP results in 19 cases (20.5%) with FN or uncertain CM, TN results in 3 cases (3%) with uncertain CM, and FN results in 1 case (1%) with TP CM. This was a pleural effusion in which, in the presence of anaplastic cell features, primary effusion lymphoma was definitively diagnosed by immunocytochemical staining for ORF73/latent nuclear antigen-1 of HHV-8 on B-cell marker negative neoplastic cells. By applying the CMA to RCO, 59 TN results (64%), 31 TP results (33.5%) and 2 (double) FN results (2%) were obtained. Overall, 100% specificity and positive predictive value were detected for each method. FCM/CMA sensitivity was significantly higher than CM sensitivity (p<.0001). The highest sensitivity for each method was displayed in the analysis of samples from B/T-cell precursor lymphoma/leukemia. FCM sensitivity was significantly higher than that of CM in the evaluation of effusions (p<.005) and of samples from B/T-cell differentiated lymphoma (p<.0001). CMA sensitivity was the highest in the latter, and reached 100% in the former. In the BALF setting, FCM/CMA displayed 75% sensitivity, while CM being non-diagnostic in each of RCO-positive samples.In conclusion, clinical follow-up demonstrates FCM to retain significantly higher accuracy than CM in detecting hematologic malignancy contamination in both effusions and BALF, especially in the subset of differentiated lymphomas. Combining methods improves diagnostic performance almost completely by means of FCM.No relevant conflicts of interest to declare.

Introduction: Although blast-rich specimens immunophenotype studies in myelodysplastc syndromes (MDS) could associate bone marrow (BM) blast expression of CD7 and/or CD117 antigens with poor outcome (Ogata et al., Blood 2002), the prognostic role of markers of myeloid cell immaturity and committment in not enriched BM samples is largely unexplored. Patients and Methods: The expression of CD33, CD34 and CD117 antigens in not enriched BM samples of 50 newly diagnosed MDS was compared with both BM blast WHO category and IPSS score. Immunophenotyping was carried out by using the panel of quadruple monoclonal antibodies CD34/CD117/CD45/CD33, conjugated with the fluorochromes FITC, PE, PerCP, APC, respectively. Acquisition of information on 1x105 stained cells corresponding to the whole BM cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Multiple group comparisons were made using non parametric ANOVA for BM blasts; general linear model with Wald’s test and Kruskal-Wallis (KW) test to confirm significance was used for IPSS. Results: According to IPSS, 5 (10%) low risk, 27 (54%) intermediate risk-1, 14 (28%) intermediate risk −2 and 4 (8%) high risk pts were identified, respectively. The expression of CD33, CD34 and CD117 significantly correlated with both blast WHO category and IPSS, as shown in the Table 1. Interestingly, by analyzing the subset of 30 pts with BM blasts <5%, a correlation was found between IPSS and the expression of both CD33 (p=0.0097) and CD34 (p=0.0299); CD117 expression increased in pts with higher IPSS, without, however, reaching statistical significance. Conclusions: Both markers of myeloid cell committment (i.e., CD33 and CD34) and markers of myeloid cell immaturity (i.e., CD117) correlate with BM blast WHO category and IPSS score. Expression of CD33 and CD34 may have prognostic significance independently from blast percentage. The evaluation of CD33, CD34 and CD117 expression on not enriched BM samples may represent a simple tool for the prognostic assessment of MDS patients, in addition to previously established methods. Tab. 1: Correlation of CD33, CD34, CD117 expression with IPSS and percentage of BM blast in 50 MDS pts at diagnosis

Introduction: Flow cytometry is well known to detect malignant cells in peripheral blood and bone marrow of patients with hematologic malignancies. However, its role in evaluating non-hematic body fluid contamination by tumor cells is largely unexplored. Patients and Methods: Data detected by flow cytometry in non-hematic body fluid samples drawn between 2002 and 2007 from patients with hematologic neoplasms were retrospectively compared with morphological findings obtained from cytospin slides. Immunophenotyping was carried out by using disease-specific multicolor panels of quadruple monoclonal antibodies, conjugated with the fluorochromes FITC, PE, PerCP, and APC, respectively. Acquisition of information on 1x104 to 1x105 stained cells depending on the whole sample cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José, CA, USA). Results: Fourty-five samples (bronchoalveolar fluid, n=5; ascites, n=2; hydrocele, n=2; pleural effusion, n=8; and cerebrospinal fluid, n=28) from 32 patients were available for comparison. Diagnoses were as follows: chronic myelomonocitic leukemia (n=1), acute promyelocytic leukaemia (n=2), acute myelomonocytic leukaemia (n=1), B-chronic lymphocitic leukemia (n=4), follicular lymphoma (n=1), acute lymphoblastic leukaemia (n=6), lymphoblastic lymphoma (n=1), high grade non-Hodgkin’s lymphoma (NHL), Burkitt-like (n=3), diffuse large B-cell NHL (n=6), peripheral T-cell NHL (n=3), and NHL, unspecified (n=4). Flow cytometry detected neoplastic cells in 24 cases. Of these cases, only 17 were positive also by morphology. In 7 cases, in which tumor cells were detected by flow cytometry but not by morphology, clinical data confirmed the presence of the disease. Flow cytometry did not show neoplastic cells in 21 cases. Of these cases, only 18 were negative also by morphology. In the remaining 3, the suggestion of diffuse large B-cell NHL contamination by morphology was not confirmed by flow cytometry, demonstrating T-reactive lymphocytes that were clearly negative for disease-specific markers. Conclusions: Our data suggest that flow cytometry is a useful tool complementary to morphology for the screening of non-hematic body fluid contamination in patients with hematologic neoplasms.

Introduction : The International Prognostic Scoring System (IPSS) better defines the outcome and transformation risk of myelodysplastic syndromes (MDS) than FAB classification. The latter was previously found to identify different patterns of peripheral blood (PB) CD34+ cells in MDS. Whether a correlation exists between PB CD34+ cells and IPSS classification of MDS is undetermined. Patients and Methods : PB CD34+ cells detected at the diagnosis of 29 MDS [according to WHO, 8 refractory anemias (RA), 7 refractory cytopenias with bi-/tri-lineage dysplasia, 5 RA with excess of blasts (RAEB)-I, 5 RAEB-II, 4 mixed MDS/myeloproliferative disorders] were compared with IPSS and its determinants [karyotype class, bone marrow (BM) blasts percentage, number of cytopenias]. Karyotype classes were as follows: good (GK): normal, isolated 5q-, 20q-, -Y; poor (PK): more than 2 anomalies, chromosome 7 abnormalities; intermediate (IK): other. Multiple group comparisons were made using Kruskal-Wallis one way analysis of variance. Continuous and categorical variables were compared by means of the Mann-Witney U test and the Fisher exact test, respectively. Results: According to IPSS, 10 (34.5%) low risk (LR), 10 (34.5%) intermediate risk (IR)-1, 8 (28%) IR-2 and 1 (3%) high risk (HR) patients (pts) were identified, respectively. Nineteen (66%), 6 (21%), and 4 (19%) patients presented GK, IK and PK, respectively. Chromosome 7 and chromosome 17 anomalies were reported in 14% and 10% of patients, respectively. Each 5q- and -Y occurred in 7% of patients. Neutrophil counts less than 1.8 x 10(9)/L, hemoglobin less than 10 g/dL, and platelet counts less than 100 x 10(9)/L were detected in 9 (31%), 19 (66%), and 14 (48%) pts, respectively. Less than 5%, 5 to 10%, and 11 to 20% BM blasts were observed in 18 (62%), 6 (21%) and 5 (17%) patients, respectively. Median PB CD34+ cells were 10.4/mcL (range, 2.7–66.4). Proportion of LR/IR-1 patients with 10 or more CD34+ cells/mcL was significantly lower than that of IR-2/HR pts (35% vs 100%, p=0.001). In the latter, median PB CD34+ cells (25/mcL, range 10–66.4) were significantly higher than those detected in LR (5.6/mcL, range 2.7–10.4) and IR-1 (14/mcL, range 3.1–37.3) patients, respectively (p=0.0003 and =0.045, respectively). Median PB CD34+ cells in the GK group (4.7/mcL, range 2.7–15) were significantly lower than those in the IK (30/mcL, range 4.6–56) and PK (36/mcL, range 13–66.4) groups, respectively (p=0.03 and =0.01, respectively). Similarly, median PB CD34+ cells were significantly lower in patients with less than 5% BM blasts than in patients with 11–20% BM blasts (4.9/mcL vs 25.2/mcL, p=0.036), and in patients with 0–1 cytopenias than in patients with 2–3 cytopenias (6.2/mcL vs 16.8/mcL, P=0.023). Conclusions: By dividing MDS according to IPSS, definite patterns of PB CD34+ cells can be identified. Intermediate and unfavourable karyotype, previously correlated with reduced BM CD34+ cell apoptosis, are also associated with high PB CD34+ cell counts. A prognostic impact of PB CD34+ cell assessment in newly diagnosed MDS is suggested.

Introduction: The International Prognostic Scoring System (IPSS) better defines the outcome and transformation risk of myelodysplastic syndromes (MDS) than FAB classification. The latter was previously found to identify different patterns of peripheral blood (PB) CD34+cells in MDS. Whether a correlation exists between PB CD34+cells and IPSS classification of MDS is undetermined.

Chronic myelomonocytic leukemia (CMML) is a heterogeneous hematological malignancy, which shows features of both myelodysplastic and myeloproliferative disorders. It has poor prognosis due to the lack of effective treatment and very little is known about its pathogenesis. A point mutation in the RAS genes is detected in 20 to 35% of patients with CMML, with higher percentage among patients with proliferative variant of the disease (MP-CMML, WBC > 13 x 109/L). The expression of Bcl-2 family proteins, which are key regulators of the mitochondrial-mediated pathway of apoptosis, have been reported as being altered in MDS and other hematological disorders. However, the role of these proteins has not been systematically investigated in CMML. In this study, we evaluated by Western Blotting the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in peripheral blood mononuclear cells (PBMNC) from 21 CMML patients and 8 age-matched healthy controls. We also investigated the presence of point mutations within codons 12, 13, and 61 of N- and K-RAS by sequencing analysis, and assessed in vitro colony growth of PBMNC with and without growth factors (GFs) [SCF, GM-CSF, IL-3, Epo]. Our series included 10 males and 11 females, with a median age of 74 years. Median WBC value was 9.2 x 109/L (range 3.2 to 17.2), while differential showed median monocyte values of 27.5% and 2.0 x 109/L. Six out of the 21 patients had MP-CMML. All patients but one had normal karyotype. A point mutation within N-RAS (G12D) was detected in two patients, both with MP-CMML. Spontaneous growth was observed both in normal controls and in CMML (median 19 and 14 CFU-GM/105 MNCs, respectively), with a higher number of CFU-GM in the MP-CMML than in the dysplastic subgroup (MD-CMML) (46 vs 12). After addition of GFs, we observed an increase of colony number in all groups. Interestingly, in vitro colony growth was remarkably higher in the two patients with mutant N-RAS than in patients with wild type RAS, especially after the addition of GFs (200 vs 57, p <0.05). The expression of Bax was higher in CMML than in the control group (3.81 vs 1.39, p = 0.07), whereas Bcl-2 was lower (0.88 vs 1.41, p = .16), even though these differences were not statistically significant. No differences were observed between subgroups of patients with MD- and MP-CMML. When the Bcl-2/Bax ratio was calculated, we observed lower values in CMML than in normal controls (0.27 vs 1.08, p <0.01). In the two patients with mutated RAS, Bcl-2 expression was significantly higher than in patients with wt-RAS (2.07 vs 0.75, p <0.05). Noteworthy, Bcl-2 expression in patients with mutant RAS was also higher than in the control group. In conclusion, our data suggest a deregulation of mechanisms controlling apoptosis in CMML, with no significant differences between MD and MP variants. The finding of higher Bcl-2 expression in patients with mutant RAS suggests a possible cooperation of this anti-apoptotic protein with RAS-activated intracellular pathways, warranting further confirmation in larger CMML series. In agreement with previous reports, we observed higher in vitro spontaneous growth in MP-CMML than in MD-CMML. Hypersensitivity to GFs (possibly GM-CSF) in cells from patients with mutant RAS is suggested and merits further investigation.