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The GIMEMA LAL1509 protocol, designed for adult (≥18-60 years) de novo Ph+ acute lymphoblastic leukemia patients, was based on a dasatinib plus steroids induction - with central nervous system prophylaxis - followed by dasatinib alone in patients in complete molecular response or chemotherapy and/or allogeneic transplantation in patients not reaching a complete molecular response. Sixty patients (median age 41.9 years) were enrolled: 33 were p190+, 18 p210+ and 9 p190/p210+. At the end of induction (day +85), 58 patients (97%) achieved a complete hematologic remission. No deaths in induction were recorded. Eleven patients (18.3%) obtained a complete molecular response. Among non-complete molecular responders (n=47), 22 underwent an allogeneic transplant. Seventeen hematologic relapses occurred (median 7 months, range 3-40.1), 13 during consolidation and 4 post-transplant. ABL1 mutations (5 T315I, 3 V299L, 1 E281K and 1 G254E) were found in 10/13 relapsed cases. With a median follow-up of 57.4 months (range: 4.2-75.6), overall survival and disease-free survival are 56.3% and 47.2%. A better diseasefree survival was observed in patients who obtained a molecular response at day +85 compared to cases who did not. The presence of additional copy number aberrations - IKZF1 plus CDKN2A/B and/or PAX5 deletions - was the most important unfavorable prognostic factor on overall and disease-free survival (p=0.005 and p=0.0008). This study shows that in adult Ph+ ALL long-term survivals can be achieved with a total-therapy strategy based on a chemo-free induction and, in complete molecular responders, also without further systemic chemotherapy. Finally, the screening of additional copy number aberrations should be included in the diagnostic work-up. EudraCT 2010-019119-39.

Extramedullary disease is relatively frequent in multiple myeloma, but our knowledge on the subject is limited and mainly relies on small case series or single center experiences. Little is known regarding the role of new drugs in this setting. We performed a meta-analysis of eight trials focused on the description of extramedullary disease characteristics, clinical outcome, and response to new drugs. A total of 2,332 newly diagnosed myeloma patients have been included; 267 (11.4%) had extramedullary disease, defined as paraosseous in 243 (10.4%), extramedullary plasmocytoma in 12 (0.5%), and not classified in 12 (0.5%) patients. Median progression-free survival was 25.3 months and 25.2 in extramedullary disease and non-extramedullary disease patients, respectively. In multivariate analysis the presence of extramedullary disease did not impact on progression-free survival (hazard ratio 1.15, P =0.06), while other known prognostic factors retained their significance. Patients treated with immunomodulatory drugs, mainly lenalidomide, or proteasome inhibitors had similar progression-free survival and progression-free survival-2 regardless of extramedullary disease presence. Median overall survival was 63.5 months and 79.9 months ( P =0.01) in extramedullary and non-extramedullary disease patients, respectively, and in multivariate analysis the presence of extramedullary disease was associated with a reduced overall survival (hazard ratio 1.41, P <0.001), in line with other prognostic factors. With the limits of the use of low sensitivity imaging techniques, that lead to an underestimation of extramedullary disease, we conclude that in patients treated with new drugs the detrimental effect of extramedullary disease at diagnosis is limited, that lenalidomide is effective as are proteasome inhibitors, and that these patients tend to acquire a more aggressive disease in later stages. (EUDRACT2005-004714-32, NCT01063179 NCT00551928, NCT01091831, NCT01093196, NCT01190787, NCT01346787, NCT01857115)., (Copyright© 2020 Ferrata Storti Foundation.)

Background and Objectives: The outcome of adult patients with acute lymphoblastic leukemia (ALL) is discouraging, only about 30% of them becoming long-term survivors. A small fraction of patients are resistant to the first line treatment, while most patients relapse within two years of achieving complete remission (CR). No standard treatment exists for refractory or relapsed patients. The GIMEMA group designed a phase II trial for adult ALL patients with refractory or relapsed disease., Design and Methods: Patients aged >15 years with primary refractory or relapsed ALL were eligible for this study. The salvage strategy included a single high dose of idarubicin combined with high dose cytarabine, followed by consolidation therapy leading to a stem cell transplant procedure according to donor availability., Results: From 1998 to 2002, 135 patients were enrolled. Seventy-five patients (55%) achieved CR, including 12 Philadelphia-positive cases; 44 patients had persistent leukemia and 16 died during reinduction. Fifty patients received a stem cell transplant: 19 from an HL-A identical sibling, 16 from an unrelated donor, 7 from a haploidentical relative, 2 received cord blood, and 6 had an autotransplant. The median disease-free and overall survival were both short (5.0 and 6.4 months, respectively); however, after a median follow-up of 40 months, 13 patients are alive, 10 of whom are free of disease (9 transplanted), while 3 are alive with leukemia., Interpretation and Conclusions: The treatment induced CR in a high percentage of poor prognosis patients, thus rendering a transplant procedure feasible in most of them. However, significant rates of transplant-related mortality and post-transplant relapse encourage the search for more effective and less toxic conditioning regimens.

Background and Objective: CD34+ hematopoietic progenitor cells (HPCs) constitute a heterogeneous population both in size and in immunological features. Lack of CD38, HLA-DR and lineage committed antigens as well as the co-expression of Thy-1 (CDw90) and c-kit receptor (CD117), are able to identify the so-called stem cells. A flow cytometric study was carried out to investigate the co-expression of Thy-1 and c-kit receptors, both members of Ig superfamily adhesion molecules, involved in cell to cell and cell to stroma interactions, on bone marrow (BM), mobilized peripheral blood (PB) and human umbilical cord blood (HUCB) CD34+ HPCs., Design and Methods: Lysed whole blood from 15 BM, 25 mobilized PB and 25 HUCB samples were used to perform a five-dimensional flow cytometric evaluation of both CDw90 and CD117 on CD34+ cells., Results: Few CD34+ cells co-expressed Thy-1 antigen in all three compartments (BM: 11.2 +/- 7.2%; PB: 6.2 +/- 3.6%; HUCB: 6 +/- 2.9%; BM vs PB < 0.04; BM vs HUCB < 0.008; PB vs HUCB ns). c-kit receptor was detected on the majority of CD34+ HPCs, particularly in HUCB (HUCB: 80.7 +/- 8.2%; BM: 72.3 +/- 13.1%; PB: 64.2 +/- 17%; HUCB vs BM < 0.03; HUCB vs PB < 0.0001; BM vs PB ns). CD34+Thy-1+ and CD34+c-kit+ HPCs generally displayed HLA-DR antigen, as expression of early cell commitment. However, the most immature CD34+Thy-1+HLA-DR- (HUCB: 1 +/- 0.6%; BM: 0.4 +/- 03%; PB: 0.7 +/- 0.5%; HUCB vs BM < 0.0001; BM vs PB < 0.04; HUCB vs PB ns) and CD34+c-kit+HLA-DR- HPCs (HUCB: 6.5 +/- 4.4%; BM: 6.3 +/- 4.8%; PB: 2.2 +/- 1.8%; HUCB vs BM ns; BM vs PB < 0.0001; HUCB vs PB < 0.0001) were mainly detected in HUCB. Finally, the greatest percentage of CD34+Thy-1+c-kit+ cells was found in BM (6.9 +/- 4.1%) followed by leukapheretic samples (4.4% +/- 2.7) and then by HUCB (3.7 +/- 1.2%; BM vs PB ns; BM vs HUCB < 0.001; HUCB vs PB ns)., Interpretation and Conclusions: Since the blood release or HPCs is probably due to a perturbation of the adhesive interactions between these cells and the marrow stroma, the different pattern of Thy-1 and c-kit receptor expression on CD34+ HPCs found in the three hemopoietic compartments evaluated can lead to new knowledge about the mobilization kinetics in which the Ig superfamily adhesion molecules are involved.

Background and Objective: The CD117 molecule is an antigen more frequently found on early normal and leukemic hematopoietic cells, but its correlation with the FAB subtypes and with other lineage and stage associated antigens is still not well established. In this study we investigated the surface expression of CD117 antigen in 135 patients with acute leukemia in relationship to de novo or secondary origin of AML, subtypes of FAB classification, expression of other antigens such as CD34, HLA-DR, CD15, CD14, CD45RA, CD45RO, CD11b, CD11c, CD4, CD7, mixed antigen co-expression (LyAg + AML and MyAg + ALL) and features of leukemic mass., Design and Methods: The CD117 antigen expression (clone 95C3) was determined by flow cytometry in a series of 135 patients with acute leukemia at diagnosis consecutively observed during the years 1995-1997: 82 AML (including 51 cases of de novo AML, 22 cases of AML following myelodysplastic syndromes (MDS), 9 cases of myeloid blastic crisis of chronic myeloid leukemia (BC-CML) and 53 ALL. All cases were stratified in CD117+ and CD117- groups and the differences were analyzed by using appropriate statistical analyses., Results: CD117 antigen was found over 10% in 74% of AML without significant differences of positivity between AML after MDS or BC-CML and de novo AML. We did not note a significant correlation between FAB classification and CD117 which was expressed in 100% of M1 and M7 cases, in 80% of M0 cases, in 75% of M2 cases, in 70% of M3 cases and in 82% of M4 cases. Instead, in M5 subtype CD117 was strictly restricted to earlier stages: ten of the eleven M5b (91%) cases completely lacked CD117 antigen expression, whereas 100% of M5a cases were positive. The results of Pearson's coefficient showed: 1) a significant inverse relationship between CD117 and CD15, CD4 and CD14 (only in M5 subtypes) and CD11b, CD11c and CD45RO (in all cases); 2) a significant direct correlation between CD117 and CD34 and CD45RA (in all cases); and 3) an independent expression between CD117 and CD15 associated with a low correlation between CD117 and HLA-DR antigen (only in non-monocytic cases). In ALL, whether of B or T lineages, surface expression of CD117 was never observed., Interpretation and Conclusions: We conclude that the CD117 antigen shows a high specificity for AML, independently upon FAB classification, and represents a reliable marker in characterizing the differentiative degree of the monocytic blasts.

Background and Objective: One of the most important potential advantages in the use of human umbilical cord blood (HUCB) for hematopoietic reconstitution after myeloablative therapy seems to be the lower occurrence of acute graft-versus-host-disease (GvHD) in recipients after allogeneic transplantation. Since mature T cells play an important role in GvHD pathogenesis, we tried to verify whether a different immunophenotypic pattern exists between HUCB and peripheral blood (PB) T cells., Design and Methods: An immunophenotypic study on 40 HUCB and 40 PB samples from healthy adult volunteers was performed, by means of flow cytometry using a large panel of monoclonal antibodies in double labeling., Results: The absolute lymphocyte count was greater in HUCB (5233 +/- 1808 microL) than in adult PB (1941 +/- 378 microL). Significant differences in percentage were found between cord and adult T-cells, respectively (CD3+: 59.9 +/- 12 vs 74.9 +/- 4.6%), CD3- CD16+ and/or CD56+ natural killer (NK) cells (23.8 +/- 10.1 vs 10.8 +/- 5.3%) and CD3+CD16+ and/or CD56+ cytotoxic T lymphocyte subset (0.3 +/- 0.3 vs 10.7 +/- 4.1%). There was no difference in CD4/CD8 ratio (1.7 +/- 0.5 vs 1.6 +/- 0.2%) between the two groups. In absolute terms, HUCB contained an higher number of all lymphocyte subsets, with the exception of CD3+CD16+ and/or CD56+ T lymphocyte subpopulation, CD3+CD25+ and CD3+HLADR+ activated T-cells. CD38, a marker of activation and immaturity, was present on virtually all cord T cells and on approximately half of the adult T cells. The large majority of HUCB T cells co-expressed CD45RA naive antigen (CD4+CD45RA+: 87.6 +/- 5.2%, CD8+ CD45RA+: 93.5 +/- 7.8%; CD4+CD45RO+: 12.3 +/- 5.2%; CD8+CD45RO+: 6.4 +/- 7.8%) whereas in adult PB T cells an higher number of CD45RO+ memory cells was detected (CD4+CD45RA+: 44.8 +/- 9.6%; CD8+CD45RA: 71.5% +/- 8.1%; CD4+CD45RO+: 55.2 +/- 9.6%; CD8+ CD45RO+: 28.5 +/- 8.1%). Finally, less than 14% of lymphocytes were shown to belong to B lineage in both sources, while, in absolute terms, they were more represented in HUCB with respect to adult PB., Interpretation and Conclusions: In the present study we found significant difference between HUCB and adult PB lymphocytes in their immunophenotypic profile. In particular HUCB showed T lymphocytes that appeared to be phenotypically immature. Indeed, as a likely consequence of poor antigenic experience during pregnancy, the majority of HUCB cells were naive, expressing the RA isoform of the CD45 molecule. These findings could justify the previously reported reduced cord blood lymphocyte alloreactivity when allogeneic transplantation is performed and require further functional studies in order to confirm the impairment of HUCB immune system response to alloantigens.

Background and Objective: Although less specific than sCD23, sCD54 levels have clinico-prognostic relevance in B-cell chronic lymphocytic leukemia (CLL). Since serological markers are now emerging as potentially important in CLL, we tried to verify whether sCD54 might complement clinical stages., Methods: Serum levels of sCD54 were determined at the time of diagnosis in 115 previously untreated CLL patients. Results were correlated with clinicobiological parameters as well as with survival., Results: Life-expectancy was significantly shorter in patients with higher serum levels of sCD54 (p < 0.001); however, in a Cox's multivariate survival analysis, the only variables which entered the regression model at a significant level were bone marrow (BM) histology (p = 0.03) and lymphocyte doubling time (LDT) (p = 0.04). Interestingly, when LDT was excluded from analysis the only significant variables were clinical stages (p < 0.05) and sCD54 (p < 0.05). These results suggest that sCD54 and LDT give similar prognostic information., Interpretation and Conclusions: In CLL, sCD54 is a reliable prognostic parameter whose value is independent of clinical stages. When investigated in relation to clinical outcome, serum levels of sCD54 were able to predict progression to a more advanced clinical stage. On the basis of these data, an integrated clinico-biological classification which separates intermediate risk into two prognostic subgroups is proposed.

Background and Objective: The prognostic significance of CD34 antigen expression in acute lymphoblastic leukemias (ALL), especially in adult patients, is still not well established. In the present report we analyzed a series of biological and clinical findings from 128 ALL patients in order to evaluate the possible clinical significance of this marker., Methods: The clinical and biological significance of CD34 expression, an early marker of hemopoietic cells, was analyzed by flow cytometry in a series of 128 patients affected by ALL, including 78 adults and 50 children under 15 years old., Results: Overall, 68.7% of patients showed significant ( > 10%) CD34 expression. There was no difference between CD34+ and CD34- ALL with respect to age, sex, FAB morphology, hepatosplenomegaly, Plt count, Hb level, DNA index, P-170 expression. CD34+ ALL displayed a significantly lower frequency of extramedullary involvement, a lower LDH level and lower WBC count, lower proliferative activity (as evaluated by the Ki67 monoclonal antibody) than CD34- ALL. CD34 expression was also associated with early phenotypes in both B- and T-ALL, co-expression of myeloid antigens, and the presence of the Ph1 chromosome. Due to a different distribution of prognostic factors investigated, DFS and OS were both significantly better in CD34+ than in CD34- childhood ALL, whereas no statistical difference was found in adults. Multivariate analyses confirmed these data in children., Interpretation and Conclusions: Expression of the CD34 antigen is a positive prognostic factor in childhood ALL. In adult ALL the presence of this marker on leukemic cell does not seem to influence the clinical outcome of these patients.

Background: Human umbilical cord blood (HUCB) is a possible alternative to bone marrow (BM) and mobilized peripheral blood (PB) for transplantation of hematopoietic progenitors. The aim of this study was to evaluate the phenotypic profile of CD34+ progenitors present in HUCB., Materials and Methods: A flow cytometric analysis was performed on 20 HUCB samples, using a large panel of monoclonal antibodies recognizing different lineage or activation antigens, in double labeling with CD34., Results: A toal of 13,897 +/- 2,529 cells/microL, 0.84 +/- 0.83% of which were CD34+, was found. The large majority of CD34+ cells were committed toward initial myeloid differentiation (CD33+, CD13+) and expressed the transferrin receptor (CD71). A substantial proportion of these cells (about 40%) co-expressed CD45RA and CD117, while a very small number displayed markers of advanced myeloid commitment, such as CD14, CD15 and CD41 (less than 2%), or those of lymphoid differentiation: CD2, CD5, CD7, CD10 and CD19 (less than 6%). About 11% of HUCB CD34+ cells were primitive progenitors, as suggested by the absence of HLA-DR and CD38 on their surface., Conclusions: As previously observed in BM and mobilized PB, the phenotype of HUCB CD34+ cells is quite heterogeneous. In particular, HUCB contains subpopulations of both early and committed hematopoietic progenitors which may represent a valid source for transplantation.

Background: Hematopoietic progenitor cells (HPC), identified by expression of the CD34 surface antigen, show morphological and phenotypic heterogeneity in bone marrow (BM) and peripheral blood (PB)., Methods: CD34+ HPC subpopulations present in 18 PB leukaphereses after high-dose chemotherapy in cancer patients and in 11 BM samples from patients with stage IA lymphoma were characterized. In order to identify CD34+ HPC subsets within these two compartments, the expression of lineage- or activation-associated antigens and the c-kit gene product (CD117) was studied by flow cytometry, using a large panel of monoclonal antibodies (MoAb) in double labelling., Results: We observed a higher proportion of CD34+/CD13+ and CD34+/CD33+ cells (myeloid commitment) in harvested leukapheresis products than in BM. On the contrary, a higher percentage of CD34+/CD10+ and CD34+/CD19+ cells (B-lymphoid commitment) was found in BM. The percentage of the most immature subset of CD34+ HPC (CD38- and HLA-DR-) was also higher in BM than in mobilized PB. No differences in proportions were found with respect to the expression of CD14, CD15, CD45RA (myeloid commitment), CD2, CD5, CD7 (T-lymphoid commitment), CD117, CD71 and CD45RO antigens. In terms of absolute values, however, significantly higher amounts of CD34+ HPC co-expressing CD13, CD33, CD5, CD7, CD71, CD117, CD45RA, CD45RO were detected in leukaphereses than in BM. The absolute number of immature HPC (CD34+/CD38- and CD34+/HLA-Dr-) was also significantly increased in mobilized PB., Conclusions: Our data confirm the heterogeneous phenotypic profile of HPC, thus supporting the hypothesis that different CD34+ subpopulations may have clinical relevance on the rapidity and long-term durability of engraftment in patients who undergo high-dose chemotherapy followed by rescue with HPC. We also demonstrated that mobilized PB is a particularly rich source of both primitive and committed HPC, more than BM.

Background: A series of immunological abnormalities has been described in patients with beta-thalassemia. The aim of this study was to investigate whether the measurement of serum levels of selected cytokines and soluble molecules (deriving from cell membrane antigens) involved in the immune response could be useful for a better definition of such alterations., Patients and Methods: Serum levels of interleukin-2 (IL-2), IL-6, tumor necrosis factor (TNF), soluble (s) CD4, sCD8, sCD23 and sCD25 were measured using immunoenzymatic assays in 45 transfusion-dependent patients affected by beta-thalassemia major and correlated to conventional immunological indexes, such as peripheral lymphocyte subpopulations and circulating immunoglobulins., Results: Patients with beta-thalassemia major showed increased TNF, sCD8, sCD23 and sCD25 and lower sCD4 values compared to normal controls. IL-2 and IL-6 were found to be undetectable or within the normal range in all patients. Splenectomized patients presented lower levels of sCD8 and sCD23 than those observed in unsplenectomized ones. A series of correlations involving TNF, sCD8, sCD23, sCD25, serum immunoglobulins and some lymphocyte subpopulations was observed. In addition, serum markers of immune activation (TNF, sCD23, sCD25) correlated directly with the annual blood transfusion requirement. Despite this series of immunological anomalies, no patient had a history of repeated infectious episodes., Conclusions: Polytransfused beta-thalassemic patients are characterized by a partial functional immunodeficiency determined by increased activity of CD8+ suppressor/cytotoxic lymphocytes and possibly reduced activity of the CD4+ helper/inducer subset. B-lymphocytes also appear highly activated. The allo-antigenic stimulation of transfusions seems to play a major role in the determination of these defects; however, this functional immunological imbalance does not seem to have any clinical relevance.

We describe the case of an infant with immune thrombocytopenia whose bone marrow showed an increased percentage of CD10/TdT-positive lymphoid cells that resembled the onset of an acute lymphoproliferative disorder. Genotypic analysis of bone marrow, however, failed to reveal the malignant origin of these B cell precursors. After 8 months of follow-up, the child is alive and well, and shows a chronic form of ITP. Although a relation between this B cell proliferation and the onset of ITP cannot be excluded, it is important to consider this atypical pattern as a benign hematologic condition.

Background: The use of monoclonal antibodies has been recently proposed as a more sensitive technique than conventional cytochemistry in recognizing myeloperoxidase (MPO) in leukemic cells., Methods: In order to verify the diagnostic relevance of such an approach, the immunocytochemical detection of MPO by means of the APAAP technique and a monoclonal antibody directed against an antigenic determinant of MPO (MPO7) was performed in 10 patients with undifferentiated or biphenotypic (myeloid and lymphoid) acute leukemia. All the cases were negative for conventional cytochemical staining of MPO., Results: Positive cells (15 to 65%) were found in three patients. Some clinical data and biomolecular studies of T-cell receptor and immunoglobulin genes, available in five patients, indirectly confirmed these findings., Conclusions: Although some technical and biological aspects remain to be better characterized, immunocytochemical methods which utilize monoclonal antibodies against MPO may represent a simple and reliable tool for improving the diagnostic capacity to identify undifferentiated myeloid blasts in difficult cases of acute leukemia.