Costanza Bosi

Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myeloid leukemia (AML). The progression of myelodysplastic syndromes (MDSs) to AML is thought to be associated with abrogation of apoptotic control mechanisms. However, little is known about signal transduction pathways which may be involved in enhanced survival of MDS cells. In this report, we have performed immunocytochemical and flow cytometric analysis to evaluate the levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt) staining in 90% of the cases (n=22) diagnosed as high-risk MDS, whereas mononuclear cells from normal bone marrow or low-risk MDS patients showed low or absent Ser473 p-Akt staining. Furthermore, all high-risk MDS patients also demonstrated high expression of the Class I PI3K p110δ catalytic subunit and a decreased expression of PTEN. Taken together, our results suggest that Akt activation might be one of the factors contributing to the decreased apoptosis rate observed in patients with high-risk MDS.

Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS

Lipid signaling pathways are involved in cell growth, differentiation, and apoptosis, and could have a role in the progression of myelodysplastic syndromes (MDS) into acute myeloid leukemia (AML). Indeed, recent studies showed that phosphoinositide-phospholipase (PI-PL)Cbeta1 mono-allelic deletion correlates with a higher risk of AML evolution. Also, a single patient treated with azacitidine, a DNA methyltransferase inhibitor currently used in MDS, displayed a direct correlation between PI-PLCbeta1 gene expression and drug responsiveness. Consequently, we hypothesized that PI-PLCbeta1 could be a target for demethylating therapy. First, we analyzed the structure of PI-PLCbeta1 gene promoter, then quantified the degree of PI-PLCbeta1 promoter methylation and gene expression in MDS patients at baseline and during azacitidine administration. Indeed, PI-PLCbeta1 mRNA increased in responder patients, along with a reduction of PI-PLCbeta1 promoter methylation. Also, the molecular response correlated to and anticipated the clinical outcome, thus suggesting that PI-PLCbeta1 gene reactivation could predict azacitidine responsiveness. Our results demonstrate not only that PI-PLCbeta1 promoter is hypermethylated in high-risk MDS patients, but also that the amount of PI-PLCbeta1 mRNA could predict the clinical response to azacitidine, therefore indicating a promising new therapeutic approach.

The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation.

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is important for both cell growth and survival. In particular, an impaired regulation of the Akt/mTOR axis has been strongly implicated in mechanisms related to neoplastic transformation, through enhancement of cell proliferation and survival. Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by a high risk of evolution into acute myelogenous leukemia (AML). The pathogenesis of the MDS evolution into AML is still unclear, although some recent studies indicate that aberrant activation of survival signaling pathways could be involved. In this investigation, done by means of immunofluorescent staining, we report an activation of the Akt/mTOR pathway in high-risk MDS patients. Interestingly, not only mTOR was activated but also its downstream targets, 4E-binding protein 1 and p70 ribosomal S6 kinase. Treatment with the selective mTOR inhibitor, rapamycin, significantly increased apoptotic cell death of CD33(+) (but not CD33(-)) cells from high-risk MDS patients. Rapamycin was ineffective in cells from healthy donors or low-risk MDS. Moreover, incubation of high-risk MDS patient CD34(+) cells with rapamycin decreased the in vitro clonogenic capability of these cells. In contrast, the phosphoinositide 3-kinase inhibitor, LY294002, did not significantly affect the clonogenic activity of high-risk MDS cells. Taken together, our results indicate that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients. Therefore, this signaling network could become an interesting therapeutic target for treating more advanced MDS cases.

Phosphoinositide-Phospholipase C β1 Mono-Allelic Deletion Is Associated With Myelodysplastic Syndromes Evolution Into Acute Myeloid Leukemia

PURPOSE To evaluate the association between the presence of phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) mono-allelic deletion with the clinical outcome of myelodysplastic syndromes (MDS) patients. METHODS PI-PLCbeta1, PI-PLCbeta4, and PI-PLCgamma1 cytogenetic investigations were performed on 80 newly diagnosed MDS patients (18 low risk, 26 intermediate 1, 18 intermediate 2, 18 high risk) comparing the results with the clinical outcome of the patients. Moreover, fluorescent in situ hybridization results were validated by real-time polymerase chain reaction (PCR). Finally, PI-PLCbeta1 gene and protein expression were assessed by both real-time PCR and immunocytochemical experiments. RESULTS Collectively, 35 (43.75%) of 80 of the MDS patients showed a specific mono-allelic deletion of PI-PLCbeta1. Kaplan-Meier analysis revealed a significant association (P < .0001) between the PI-PLCbeta1 mono-allelic deletion and a higher risk of evolution into acute myeloid leukemia (AML), since 23 of 35 MDS patients (65.7%) bearing the PI-PLCbeta1 mono-allelic deletion evolved into AML. Even in multivariate analysis, the PI-PLCbeta1 mono-allelic deletion retained a higher significance, with a P < .001, as a prognostic factor of evolution into AML (odds ratio [OR] 1.83; 95% CI, 2.26 to 17.24; P = .00045). Finally, PI-PLCbeta1 deletion was related to an altered gene and protein expression. CONCLUSION PI-PLCbeta1 mono-allelic deletion is associated with a worse clinical outcome in MDS patients, hinting at the identification of a new group at higher risk of AML evolution and representing a reliable prognostic tool. Moreover, targeting PI-PLCbeta1 pathways might emerge as a new therapeutic strategy for MDS.

PI-PLCbeta-1 and activated Akt levels are linked to azacitidine responsiveness in high-risk myelodysplastic syndromes.

PI-PLC β -1 and activated Akt levels are linked to azacitidine responsiveness in high-risk myelodysplastic syndromes

Anti-leukemic and anti-GVHD effects of campath-1H in acute lymphoblastic leukemia relapsed after stem-cell transplantation

Despite aggressive approaches, including second transplant, donor lymphocyte infusion and several new agents, the prognosis of acute lymphoid leukemia (ALL) patients relapsing after stem-cell transplantation (SCT) remains poor. Monoclonal-antibodies (moAb) could provide a useful tool in this setting. In particular, anti-CD52 moAb is useful in lymphoid malignancies. We thus treated as compassionate with campath-1H 3 ALL patients relapsed after SCT. In 2 cases we observed a reduction of peripheral blood and/or bone marrow blasts. In 1 case a GVHD grade reduction was observed. Larger trials are required in order to define the role of campath-1H in ALL.

PKR is activated in MDS patients and its subcellular localization depends on disease severity.

PKR is activated in MDS patients and its subcellular localization depends on disease severity

An increased expression of PI-PLCβ1 is associated with myeloid differentiation and a longer response to azacitidine in myelodysplastic syndromes

This study tested the hypothesis that PI‐PLCβ1 is associated with myeloid differentiation and that its expression could be useful for predicting the response of MDS patients to azacitidine, as the clinical effect of epigenetic treatments is often detectable only after several cycles of therapy. To this end, PI‐PLCβ1 was quantified on 70 MDS patients (IPSS risk: 13 Low, 20 Int‐1, 31 Int‐2, 6 High) at baseline and during the first 3 cycles of azacitidine. Results were then compared with the hematologic response, as assessed after the sixth cycle of azacitidine therapy. Overall, 60 patients completed 6 cycles of azacitidine, and for them, a clinical and molecular evaluation was possible: 37 of these patients (62%) showed a specific increase of PI‐PLCβ1 mRNA within the first 3 cycles, which was associated with a longer duration of response and with an increased myeloid differentiation, as evidenced by PI‐PLCγ2 induction and the recruitment of specific myeloid‐associated transcription factors to the PI‐PLCβ1 promoter during azacitidine response. Moreover, the increase of cyclin D3 gene expression throughout all of the therapy showed that PI‐PLCβ1‐dependent signaling is indeed activated in azacitidine responder patients. Taken together, our results show that PI‐PLCβ1 quantification in MDS predicts the response to azacitidine and is associated with an increased myeloid differentiation.

Safety and efficacy of granulocyte colony-stimulating factor biosimilars in engraftment after autologous stem cell transplantation for haematological malignancies: a 4-year, single institute experience with different conditioning regimens.

BACKGROUND Filgrastim biosimilars have recently been introduced into clinical practice. To date biosimilars have demonstrated comparable efficacy and safety as the originator in chemotherapy-induced neutropenia. Published experience in engraftment after autologous stem cell transplantation (ASCT) is limited and concerns relatively few patients. MATERIALS AND METHODS With the aim of assessing the efficacy and the safety of filgrastim biosimilars in post-ASCT bone marrow recovery, we conducted a single institution, retrospective study in 56 lymphoma and myeloma patients who received filgrastim biosimilars (Tevagrastim(®) and Zarzio(®)) at standard doses from day 5. We compared our results with recently published data on the originator. A cost analysis of each biosimilar was performed. RESULTS Neutrophil counts recovered in 55 patients. The median number of filgrastim biosimilar vials injected was seven per patient. The median time to neutrophil and platelet recovery was 10 and 12 days, respectively. Twenty-six patients had febrile neutropenia, in half of whom the agent involved was identified. In the cost analysis, the use of Tevagrastim(®) and Zarzio(®) was associated with cost reductions of 56% and of 86%, respectively. DISCUSSION Despite differences in CD34+ cell counts and time of starting filgrastim, our results in terms of time to engraftment and median number of vials injected are similar to published data. Comparing our results by single conditioning regimen to recent literature data, the time to engraftment and duration of hospitalisation were equivalent. Significant differences were observed in the incidence of febrile neutropenia, perhaps due to different preventive and prophylactic protocols for infections. Although prospective studies should be performed to confirm our results, filgrastim biosimilars were found to be effective and safe in engraftment after ASCT.

Baseline factors associated with response to ruxolitinib: an independent study on 408 patients with myelofibrosis

In patients with Myelofibrosis (MF) treated with ruxolitinib (RUX), the response is unpredictable at therapy start. We retrospectively evaluated the impact of clinical/laboratory factors on responses in 408 patients treated with RUX according to prescribing obligations in 18 Italian Hematology Centers. At 6 months, 114 out of 327 (34.9%) evaluable patients achieved a spleen response. By multivariable Cox proportional hazard regression model, pre-treatment factors negatively correlating with spleen response were: high/intermediate-2 IPSS risk (p=0.024), large splenomegaly (p=0.017), transfusion dependency (p=0.022), platelet count <200×109/l (p=0.028), and a time-interval between MF diagnosis and RUX start >2 years (p=0.048). Also, patients treated with higher (≥10 mg BID) average RUX doses in the first 12 weeks achieved higher response rates (p=0.019). After adjustment for IPSS risk, patients in spleen response at 6 months showed only a trend for better survival compared to non-responders. At 6 months, symptoms response was achieved by 85.5% of 344 evaluable patients; only a higher (>20) Total Symptom Score significantly correlated with lower probability of response (p<0.001). Increased disease severity, a delay in RUX start and titrated doses <10 mg BID were associated with patients achievinglower response rates. An early treatment and higher RUX doses may achieve better therapeutic results.In patients with Myelofibrosis (MF) treated with ruxolitinib (RUX), the response is unpredictable at therapy start. We retrospectively evaluated the impact of clinical/laboratory factors on responses in 408 patients treated with RUX according to prescribing obligations in 18 Italian Hematology Centers. At 6 months, 114 out of 327 (34.9%) evaluable patients achieved a spleen response. By multivariable Cox proportional hazard regression model, pre-treatment factors negatively correlating with spleen response were: high/intermediate-2 IPSS risk (p=0.024), large splenomegaly (p=0.017), transfusion dependency (p=0.022), platelet count <200x109/l (p=0.028), and a time-interval between MF diagnosis and RUX start >2 years (p=0.048). Also, patients treated with higher (≥10 mg BID) average RUX doses in the first 12 weeks achieved higher response rates (p=0.019). After adjustment for IPSS risk, patients in spleen response at 6 months showed only a trend for better survival compared to non-responders. At 6 months, symptoms response was achieved by 85.5% of 344 evaluable patients; only a higher (>20) Total Symptom Score significantly correlated with lower probability of response (p<0.001). Increased disease severity, a delay in RUX start and titrated doses <10 mg BID were associated with patients achievinglower response rates. An early treatment and higher RUX doses may achieve better therapeutic results.