Margherita Bonferroni

Interleukin 2 does not promote the in vitro and in vivo proliferation and growth of human acute leukaemia cells of myeloid and lymphoid origin

The effect of recombinant interleukin 2 (IL2) on the in vitro and in vivo proliferation and growth of human acute leukaemia cells of both myeloid and lymphoid origin was investigated. In none of the 25 primary samples tested could a continuously in vitro growing cell line be obtained by adding IL2 to the culture medium. Although IL2 induced a proliferative signal in three of the 31 acute leukaemias analysed, the overall 3H‐thymidine uptake of the neoplastic cells was significantly reduced (P<0.05) in the presence of IL2. The unlikelihood of an important proliferative signal triggered by IL2 was confirmed in a semisolid clonogenic assay, which failed to document an increased colony growth in the 26 samples studied. Furthermore, using a colorimetric assay as a test for cell proliferation and survival, in seven of the 11 fresh acute leukaemia samples tested a 22–40% reduction in viability was observed in the presence of IL2, while in the remaining four, IL2 was ineffective. In order to investigate the effect of IL2 in an in vivo setting, an experimental model in heavily immunosuppressed nu/nu mice was established. In no case did IL2 promote the in vivo proliferation and growth of human myeloid and lymphoid acute leukaemia cells injected in the mice. On the contrary, with seven of the eight leukaemic cell lines which gave rise spontaneously to leukaemic masses, this could be prevented when the mice received locally 300 U of IL2 three times daily for 90 d. IL2 also blocked the growth in vivo of three fresh acute leukaemia samples (two myeloid and one lymphoid). Co‐culture experiments using leukaemic cell lines and increasing numbers of normal lymphocytes suggest that the inhibitory effect of IL2 is probably exerted via an indirect mechanism. These findings, coupled to the well‐documented ability of IL2 to generate lymphokine activated killer cells cytolytic against leukaemic blasts, further point to the potential role of immunotherapy with IL2 in the management of patients with haematological malignancies.

Treatment of hairy-cell leukaemia with α-interferon (α-IFN)

Twenty-three patients with hairy-cell leukaemia (HCL), six of whom were previously splenectomized, were treated with α-interferon (α-IFN) 3 MU per day for 3–6 months and then with 3 MU three times per week for at least 3 further months. Seven patients (two splenectomized) showed a complete response (CR), 11 patients achieved a partial response (PR) and the remaining five experienced only a minor response (MR). All seven patients who achieved a CR are still in CR after 10–21 months from the onset of the disease. Among the 11 PRs, five showed an increase in the number of circulating hairy cells during the follow-up; they were re-started on α-IFN and an improvement of the haematological values was again obtained. One patient who achieved only a MR died after 1 month therapy because of severe infection. Following treatment with α-IFN, the improvement or normalization of the peripheral blood counts was paralleled by an improvement of the immunologic surface markers, as determined by monoclonal antibodies, and by an improvement of the response to PHA and of the natural killer activity. These findings, coupled to the mild drug-related toxicity observed, confirm that treatment with α-IFN represents a safe and effective therapeutic approach for both splenectomized and non-splenectomized HCL patients.

Lymphokine-activated killer (LAK) cells inhibit the clonogenic growth of human leukemic stem cells

The effect of lymphokine‐activated killer (LAK) cells on the in vitro clonogenic capacity of acute myeloid leukemia (AML) blasts was investigated in a semisolid medium assay. The leukemic clonogenic capacity of 11 AML cases, selected on the basis of their ability to grow in vitro, was highly reduced following overnight preincubation with LAK effectors. The degree of colony inhibition, which ranged between 66% and 98% (mean 83.8% ± 11.4 SD), was quantitatively greater than by 51Cr release, which gave rise to lytic values between 5% and 65% (mean 43.2% ± 19.2 SD). The demonstration that the clonogenic inhibition was still induced following a shorter pre‐incubation period (4 hours) suggests that the effect is unlikely to be due only to the generation of cytotoxic activity during the incubation time. The possibility that LAK cells may be employed in the management of residual disease is strengthened by the evidence that the clonogenic potential of samples containing as few as 20% and 14.3% leukemic cells could be almost completely abolished by LAK effectors. These findings further point the possible role of adoptive immunotherapy with interleukin 2/LAK cells in the treatment of patients with acute leukemia.

Prognostic relevance of cytometric quantitative assessment in patients with myelodysplastic syndromes.

Objectives: Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. Methods: Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia‐free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (<3% vs. ≥3%), CD117, and CD11b−/CD66b− (<5% vs. ≥5%); myeloid maturation was analyzed by the expression of CD11b+/CD66b++ (<15% vs. ≥15%) and CD11b+/CD66b+ (<25% vs. ≥25%). Results: In univariate analysis, the expression of immaturity markers (CD34+, CD117+, and CD11b−/CD66b−) was associated with shorter LFS and OS (P < 0.0001); higher expression of differentiation markers (CD11b+/CD66b++ and CD11b+/CD66b+) was associated with longer LFS (P < 0.0001 and P = 0.0002, respectively) and OS (P < 0.0001). In multivariate analysis, expression of CD34+ (P = 0.007), CD117+ (P = 0.013), and CD11b+/CD66b++ (P = 0.023) retained independent prognostic value for OS, while only the expression of CD34+ was a prognostic factor for LFS (P = 0.0003). Two different risk groups were defined according to the presence of 0–1 or ≥2 of these factors with significant different LFS and OS (P < 0.0001). This score showed prognostic value in predicting survival even in subanalysis according to IPSS and WHO subgroups. Conclusions: Flow cytometric analysis in MDS may provide meaningful prognostic information. Blast percentage expressed as CD117+ or CD34+ cells and the quantitative assessment of myeloid maturation showed prognostic value for survival.

Peripheral Blood CD34+ Percentage at Hematological Recovery after Chemotherapy Is a Good Early Predictor of Harvest: A Single-Center Experience

Several algorithms for early prediction of poor-mobilizing patients after chemotherapy and granulocyte colony-stimulating factor administration have been proposed. They generally define peripheral blood cut-off levels of CD34+ cells at a fixed day after starting chemotherapy, mostly with cyclophosphamide. To define an algorithm for early addition of plerixafor regardless of the chemotherapy regimen used, we retrospectively analyzed 280 chemomobilization attempts in 236 patients treated at our institution between 2002 and 2012. In multivariate analysis, CD34+ absolute count and CD34+ percentage upon total leukocyte count at day 1 (defined as the first day in which leukocytes reached a value > 1 × 10(9)/L) were the only factors able to predict a total harvest ≥ 2 × 10(6) CD34+/kg. In patients with day 1 CD34+ lower than 20/μL, the CD34+ percentage was a more reliable predictor of stem cell harvest in the following days than CD34+ absolute count. Upon definition of the best CD34+ cut-off value for identification of poor-mobilizing patients, an algorithm was set up to guide plerixafor administration. It was prospectively validated in 20 patients in 2013 with encouraging results in terms of low incidences of both mobilization failure and plerixafor use. Large prospective trials that define the most cost-effective strategy for just-in-time rescue plerixafor are warranted.

Lymphokine Activated Killer (LAK) Cells Inhibit the in vitro Growth of Myeloid and Erythroid Progenitor Cells Via the Release of Tumor Necrosis Factor-Alpha†

Adoptive immunotherapy with lymphokine-activated killer (LAK) cells induced by Interleukin 2 (IL2) has provided a new and promising strategy for the treatment of cancer patients. The clinical observation of variable degrees of cytopenia(s) in patients with solid tumors treated with LAK cells suggests that IL2-activated effector cells may play a role on the normal progenitor cell compartment. We therefore carried out in vitro studies designed to assess the influence of LAK cells on normal hemopoiesis. LAK cells from different individuals were cultured with enriched normal peripheral blood and bone marrow colony-forming cells at effector: target ratios ranging between 1:1 and 10:1. Both LAK effectors generated from peripheral blood mononuclear non-adherent cells (PBL-LAK) and from sheep erythrocyte rosette-forming cells (RFC-LAK) suppressed in a reproducible manner the growth of CFU-GM and CFU-E from both peripheral blood and bone marrow. This inhibitory effect is dose-dependent, appears to be smaller with LAK cells generated from RFC rather than from unfractionated PBL and is less evident on the early erythroid compartment. In general, the effect is more pronounced when LAK cells are pre-incubated for 18 hours with the target cell populations prior to seeding. Both autologous and allogeneic PBL-LAK inhibit colony formation. The mechanism of killing implicates a release of soluble factor(s) after close cell-to-cell interaction, as only cell-free supernatants produced after pre-incubation of PBL-LAK cells with hemopoietic progenitors give rise to inhibitory effects. The evidence that during this incubation time high levels of Tumor Necrosis Factor-alpha (TNF) are released and that the use of an anti-TNF antibody completely abolishes the inhibitory effect suggests that TNF plays a part in the LAK cell-induced inhibitory activity.

Erythroid response during iron chelation therapy in a cohort of patients affected by hematologic malignancies and aplastic anemia with transfusion requirement and iron overload: a FISM Italian multicenter retrospective study

Emanuela Messa, Lucia Biale, Anna Castiglione, Monia Lunghi, Margherita Bonferroni, Flavia Salvi, Bernardino Allione, Dario Ferrero, Chiara Calabrese, Marco De Gobbi, Paolo Nicoli, Daniela Gioia, Alessandro Levis, Giuseppe Saglio and Daniela Cilloni Department of Internal Medicine, ASL To5-Turin, Italy; AOU Citt a della Salute e della Scienza di Torino – Presidio Ospedaliero Molinette – SC Banca del Sangue e del plasma, Turin, Italy; Unit of Cancer Epidemiology and CPO Piedmont, S. Giovanni Battista Hospital, Torino, Italy; Division of Haematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern Piedmont, Italy, Novara, Italy; Department of Hematology, Ospedale S. Croce e Carle, Cuneo, Italy; Department of Hematology, AO SS Antonio e Biagio e C. Arrigo, Alessandria, Italy; Division of Hematology, AOU Citt a della Salute e della Scienza di Torino, Torino, Italy; Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; FISM Registry, SS Antonio e Biagio e C. Arrigo Hospital, Alessandria, Tuscany, Italy; Department of Clinical and Biological Sciences, Division of Hematology, University of Turin, Turin, Italy

Effects of erythropoiesis-stimulating agents on overall survival of IPSS low/INT-1 risk, transfusion independent myelodysplastic syndrome patients: a cohort study

Clinical guidelines recommend the use of erythropoietin-stimulating agents (ESA) in anemic patients with lower risk myelodysplastic syndrome (MDS)[1][1]–[4][2] and two registration trials for ESA have just been completed.[5][3],[6][4] We conducted a retrospective study in MDS patients selected by

O-020 Erythropoietin alpha therapy in 1110 lower-risk MDS patients: A real life survey from the network of regional Italian MDS Registries

Background: Erythroid stimulating agents (ESAs) are a common treatment for lower risk anaemic MDS patients (pts). The response rate varies from 20 to 60% according to the different published studies and ESAs are recommended in International Guidelines. Many clinical and biological parameters have been proposed as predictors of response. Little is known about ESAs therapy in real life. Purpose:We performed an analysis based on the network of regional Italian MDS Registries in order to assess which group of patients could benefit more of ESAs treatment. Materials and Methods: From 2487 pts enrolled in the Registry from 1999 to November 2012, we selected a group of 1411 low or intermediate-1 cases with follow-up data available. No differences in leukemic evolution rate have been observed by ESAs treatment (p=0,279). Subsequently, in order to study the effect of ESAs on survival, we excluded the 92 pts who experienced a leukemic evolution and the cases whose evolution was influenced by lenalidomide and/or azacytidine treatments. Cases treated with ESAs other than erythropoietin alpha (EPOa) were excluded because of the low number of observations and/or incomplete information and/or inhomogeneous dosages. Finally, we obtained a data base of 1110 pts treated by EPOa or observation only. We evaluated Overall Survival (OS) according to EPOa treatment considering the degree of anaemia at baseline. Results: The 1110 pts were subdivided according to Hb level as follows: 123 (11%) with Hb 10 g/dL. Three hundred and fifty six pts were treated by EPOa. The difference in OS by EPOa treatment in the whole cohort was not statistically significant. However, when considering patients not yet transfusion dependent with Hb levels ranging from 8 g/dL to 10 g/dL, there was a clear statistically significant difference in OS: median time 216 months in EPOa treated group vs 99 months in non treated pts (p=0,002). The influence of EPOa on OS in pts with Hb 10 g/dL did not reached a significant level. Additionally, no statistically significant difference was observed in deaths for thrombotic events. Conclusions: Our real life experience confirms that EPOa treatment is safe inMDS patients and it is particularly useful in prolonging OS of the selected group of lower risk pts with mild anaemia not yet transfusion dependent.