Comparison between azacitidine and decitabine as front-line therapy in elderly acute myeloid leukemia patients not eligible for intensive chemotherapy.

We compared the efficacy of azacitidine (AZA) and decitabine (DEC) in elderly patients with untreated AML, diagnosed according to WHO criteria. In the two groups, we evaluated complete remission (CR), overall survival (OS) and disease free survival (DFS). The AZA and DEC groups included 139 and 186 patients, respectively. To minimize the effects of treatment selection bias, adjustments were made using the propensity-score matching method, which yielded 136 patient pairs. In the AZA and DEC cohort, median age was 75 years in both, (IQR, 71-78 and 71-77), median WBCc at treatment onset 2.5 × 109/L (IQR, 1.6-5.8) and 2.9 × 109/L (IQR, 1.5-8.1), median bone marrow (BM) blast count 30% (IQR, 24-41%) and 49% (IQR, 30-67%), 59 (43%) and 63 (46%) patients had a secondary AML, respectively. Karyotype was evaluable in 115 and 120 patients: 80 (59%) and 87 (64%) had intermediate-risk, 35 (26%) and 33 (24%) an adverse risk karyotype, respectively. Median number of cycles delivered was 6 (IQR, 3.0-11.0) and 4 (IQR, 2.0-9.0), CR rate was 24% vs 29%, median OS and 2-year OS rates 11.3 (95% CI 9.5-13.8) vs 12.0 (95% CI 7.1-16.5) months and 20% vs 24%, respectively. No differences in CR and OS were found within the following subgroup: intermediate- and adverse-risk cytogenetic, frequency of WBCc at treatment ≥ 5 × 10^9 L and < 5 × 10^9/L, de novo and secondary AML, BM blast count < and ≥ 30%. Median DFS for AZA and DEC treated patients was 9.2 vs 12 months, respectively. Our analysis indicates similar outcomes with AZA compared to DEC.

The emerging role of measurable residual disease detection in AML in morphologic remission.

Despite the increasing knowledge of the genomic landscape of acute myeloid leukemia (AML), prediction merely based on genetics fails to anticipate outcome, presumably due to the heterogeneous composition of the leukemic clone determining complex interactions between different genetic abnormalities. Therefore, the introduction of a post-treatment biomarker exploring the quality of response to therapy such as assessment of measurable (previously minimal) residual disease (MRD) may lead to refinements of the prognostic assessment in AML. In this view, the European LeukemiaNet has recently endorsed the achievement of a MRD negative morphologic complete remission as a purpose the treatment. Techniques like multiparametric flow cytometry and reverse transcriptase-quantitative polymerase chain reaction have reached a level of sensitivity and specificity that make them ready for introduction in clinical practice. In the present review, we will give an update on the efforts in harmonization and/or standardization of MRD assessment in AML, focusing on the newest acquisitions in the clinical applications of MRD, and considering issues like relationship of MRD with leukemic stem cells or MRD assessment in peripheral blood.

Fungal infections of the central nervous system and paranasal sinuses in onco-haematologic patients. Epidemiological study reporting the diagnostic-therapeutic approach and outcome in 89 cases.

Invasive fungal infections (IFI) of the Central Nervous System (IFI-CNS) and Paranasal Sinuses (IFI-PS) are rare, life-threatening infections in haematologic patients, and their management remains a challenge despite the availability of new diagnostic techniques and novel antifungal agents. In addition, analyses of large cohorts of patients focusing on these rare IFI are still lacking. Between January 2010 and December 2016, 89 consecutive cases of Proven (53) or Probable (36) IFI-CNS (71/89) and IFI-PS (18/89) were collected in 34 haematological centres. The median age was 40 years (range 5-79); acute leukaemia was the most common underlying disease (69%) and 29% of cases received a previous allogeneic stem cell transplant. Aspergillus spp. were the most common pathogens (69%), followed by mucormycetes (22%), Cryptococcus spp. (4%) and Fusarium spp. (2%). The lung was the primary focus of fungal infection (48% of cases). The nervous system biopsy was performed in 10% of IFI-CNS, and a sinus biopsy was performed in 56% of IFI-PS (P = 0.03). The Galactomannan test on cerebrospinal fluid has been performed in 42% of IFI-CNS (30/71), and it was positive in 67%. Eighty-four pts received a first-line antifungal therapy with Amphotericine B in 58% of cases, Voriconazole in 31% and both in 11%. Moreover, 58% of patients received 2 or more lines of therapy and 38% were treated with a combination of 2 or more antifungal drugs. The median duration of antifungal therapy was 60 days (range 5-835). A surgical intervention was performed in 26% of cases but only 10% of IFI-CNS underwent neurosurgical intervention. The overall response rate to antifungal therapy (complete or partial response) was 57%, and 1-year overall survival was 32% without significant differences between IFI-CNS and IFI-PS. The overall mortality was 69% but the IFI attributable mortality was 33%. Mortality of IFI-CNS/PS remains high but, compared to previous historical data, it seems to be reduced probably due to the availability of newer antifungal drugs. The results arising from this large contemporary cohort of cases may allow a more effective diagnostic and therapeutic management of these very rare IFI complications in haematologic patients.

Venetoclax: Bcl-2 inhibition for the treatment of chronic lymphocytic leukemia.

Venetoclax (ABT-199) is a small-molecule selective oral inhibitor of the antiapoptotic protein Bcl-2 that promotes programmed cell death of chronic lymphocytic leukemia (CLL) cells regulating the release of proapoptotic factors, such as Smac/Diablo, apoptosis-inducing factor (AIF) and cytochrome c. In April 2016, the U.S. Food and Drug Administration (FDA) granted accelerated approval to venetoclax for patients diagnosed with CLL with 17p deletion, as detected by an FDA-approved test, who have received at least one prior therapy. This review will focus on the mechanism of action, preclinical studies and clinical development of venetoclax both as a monotherapy and in combination with other drugs for CLL in the current milieu of therapy dominated by novel tyrosine kinase inhibitors such as ibrutinib and idelalisib.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4ß1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

Minimal residual disease negativity in elderly patients with acute myeloid leukemia may indicate different postremission strategies than in younger patients.

In the present analysis, we evaluated whether in elderly acute myeloid leukemia (AML) patients (>60 years), minimal residual disease (MRD) assessed by flow cytometry may have a role in guiding choice of postremission strategies. We analyzed 149 young and 61 elderly adults who achieved morphological CR after induction course of EORTC/GIMEMA protocols. Elderly patients reached a postconsolidation MRD negative status less frequently than younger ones (11 vs 28 %, p = 0.009). MRD negativity resulted in a longer 5-year disease-free survival (DFS) both in elderly (57 vs 13 %, p = 0.0197) and in younger patients (56 vs 31 %, p = 0.0017). Accordingly, 5-year cumulative incidence of relapse (CIR) of both elderly (83 vs 42 %, p = 0.045) and younger patients (59 vs 24 % p = NS) who were MRD positive doubled that of MRD negative ones. Nevertheless, CIR of MRD negative elderly patients was twofold higher than that of younger MRD negative ones (42 vs 24 %, p = NS). In conclusion, elderly patients in whom chemotherapy yields a MRD negative CR have duration of DFS and rate of CIR significantly better than those who remain MRD positive. Nonetheless, the high CIR rate observed in the elderly suggests that MRD negativity might have different therapeutic implications in this population than in the younger counterpart.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells.

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d(+)CD38(+) and CD49d(+)CD38(-) primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d(+)CD38(-) CLL-derived cell line Mec-1. Results indicate that CD49d(+)CD38(+) cells adhered more efficiently onto CD49d-specific substrates than CD49d(+)CD38(-) cells (P < 0.001). Upon adhesion, CD49d(+)CD38(+) cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d(+)CD38(-) cells (P = 0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d(+)CD38(+) cells were more resistant to serum-deprivation-induced (P < 0.001) and spontaneous (P = 0.03) apoptosis than the CD49d(+)CD38(-) counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

Circulating regulatory T cells in "clinical" monoclonal B-cell lymphocytosis.

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with "clinical" MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to "clinical" MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.

Deregulation of the mitochondrial apoptotic machinery and development of molecular targeted drugs in acute myeloid leukemia.

Apoptosis plays a key role in the control of rapidly renewing tissues, such as the hematopoietic system and leukemia cells invariably have abnormalities in one or more apoptotic pathways, determining a survival advantage of these cells and the development of drug resistance. These defects are also frequently associated with a low rate of response to standard chemotherapy and with a poor survival in acute myeloid leukemia (AML). The major form of apoptosis proceeds through the mitochondrial pathway, with the mitochondrial outer membrane permeabilization, leading to the release of proteins normally found in the space between the inner and outer mitochondrial membranes (cytochrome C, AIF and others). Higher levels of anti-apoptosis proteins bcl-2, bcl-x(L), Mcl-1 block permeabilization of the membrane and are reported in AML patients presenting a poor outcome. On the contrary, activated pro-apoptotic bax or bad proteins allow this permeabilization and are correlated to a good prognosis in AML. Defects in the mitochondrial pathway induce multidrug-resistance and confer important prognostic information in AML. High ratios of bcl-2 to bax protein confer a poor prognosis with decreased rates of complete remission and overall survival. The prognostic information from the ratio of the proteins is greater than bcl-2 levels alone. Recently, we confirmed the impressive impact of the bax/bcl-2 ratio, determined by flow cytometry, on AML prognosis (complete remission and overall survival) in 255 AML patients. Bcl-2 down regulation might lower the apoptotic threshold of leukemic cells and, through this mechanism, favor response to chemotherapy. Phase II studies of oblimersen (antisense Bcl-2), cytarabine and daunorubicin or oblimersen plus gentuzumab, a cytotoxic antibody directed against CD33+ cells in relapsed AMLs, showed promising results. Defects in apoptosome proteins, such as APAF-1, are frequent in AML and treatment with 5-aza-2'-deoxycytidine, a specific inhibitor of DNA methylation, restored APAF-1 expression in leukemic cells. In conclusion, targeted therapies that are designed to induce apoptosis in leukemic cells, are the most promising anti-leukemia strategies. The elucidation of the apoptotic machinery and of its defects in AML lays the basis for developing new drugs able to trigger apoptosis and overcome therapy resistance.

The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia.

UNLABELLED: We assessed by multiparametric flow cytometry the levels of minimal residual disease (MRD) in 100 adult patients with acute myelogenous leukemia (AML) achieving complete remission after intensive chemotherapy. The aim of the study was to determine the optimal threshold, in terms of residual leukemic cells, and the time point of choice, that is, post-induction (post-Ind) or post-consolidation (post-Cons), able to better predict outcome. By applying the maximally selected log-rank statistics, the threshold discriminating MRD- from MRD+ cases was set at 3.5 x 10(-4) residual leukemic cells, a level that allowed the identification of distinct subgroups of patients, both at post-Ind and post-Cons time points. Post-Cons MRD- patients had a superior outcome in terms of relapse rate, overall survival (OS) and relapse-free survival (RFS) (P<0.001, for all comparisons), regardless of the MRD status after induction. In particular, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. Multivariate analysis, including karyotype, age, MDR1 phenotype, post-Ind and post-Cons MRD levels, indicated that the post-Cons MRD status independently affected relapse rate, OS and RFS (P<0.001, for all comparisons). IN CONCLUSION: (1) the threshold of 3.5 x 10(-4) is valid in discriminating risk categories in adult AML and (2) post-Cons MRD assessment is critical to predict disease outcome.

Pretransplant minimal residual disease level predicts clinical outcome in patients with acute myeloid leukemia receiving high-dose chemotherapy and autologous stem cell transplantation.

A total of 31 adult patients with AML entered in the EORTC/GIMEMA AML-10 trial, who received autologous stem cell transplantation (ASCT) after induction and consolidation chemotherapy, were prospectively evaluated for minimal residual disease (MRD) by multidimensional flow cytometry (MFC). Using a cutoff level of 3.5 x 10(-4) leukemic cells pre-ASCT, 12 patients (39%) were stratified to MRD high-risk group and 19 (61%) into MRD low-risk group. During follow-up, all patients who were in the high-risk group relapsed at a median time of 7 months; in the low-risk group, five patients relapsed at a median time of 11 months and 14 remained in remission for 56 (range 7-80) months (P=0.00004). Longitudinal MFC determinations post-ASCT showed increased MRD levels in three of the five patients who underwent subsequent relapse, while disease recurrence was unpredicted in the remaining two cases. The pre-ASCT MRD status was the factor most strongly associated with relapse risk in the multivariate analysis (P=0.0014). We conclude that: (1) pre-ASCT MRD status predicts successful outcome in patients receiving ASCT; (2) high-dose chemotherapy conditioning regimen followed by ASCT has no impact on the unfavorable prognostic value of high pre-ASCT MRD level; and (3) sequential MRD monitoring post-ASCT may allow the prediction of impending relapse.

Clinical significance of CD38 expression in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) follows heterogeneous clinical courses, and several biological parameters need to be added to the current clinical staging systems to predict which patients will experience an indolent or an aggressive outcome. This study analyzed CD38 expression by flow cytometry and soluble APO1/Fas (sAPO1/Fas), Bcl-2 (sBcl-2), and CD23 (sCD23) proteins by immunoenzymatic methods to evaluate their effect on the clinical course of 168 unselected B-CLL patients. Intermediate/high risk modified Rai stages were characterized by a higher CD38(+) B-cell number (P =.0002) and higher sCD23 levels (P <.0001). Moreover, CD38(+) B-cell percentages were significantly and directly associated both with beta(2)-microglobulin and sCD23 concentrations (P <.0001 and P =.002, respectively). Both a higher tumor burden (lymphadenopathy/splenomegaly) and a lymphocyte doubling time less than 12 months were significantly associated with higher CD38(+) percentages (P <.0001 and P =.0001, respectively). With regard to clinical outcome, progression-free survival was significantly longer (75% versus 37% at 5 years; P =.00006) in patients with lower CD38(+) B-cell percentages. Furthermore, the risk of partial or no response to fludarabine increased with increasing CD38 expression (P =.003), and a shorter overall survival (50% versus 92% at 8 years; P <.00001) characterized patients with more than 30% CD38(+) B-cell number. The predictive value of CD38 expression was maintained among the patients within the Rai intermediate risk group and was confirmed in multivariate analysis. Thus, the percentage of CD38(+) B cells appears to be an accurate predictor of clinical outcome and therefore could be used to indicate when more novel chemotherapeutic approaches are needed.

Femoral catheters: safety and efficacy in peripheral blood stem cell collection.

Central venous access is necessary in patients candidate for peripheral blood stem cell (PBSC) collection. We report our experience with a dual lumen femoral catheter (Gamcath, 11 french), initially designed for hemodialysis. We studied 147 patients and performed 488 collections after mobilization with either G-CSF alone or chemotherapy + G-CSF, when the white blood cell count exceeded 1 x 10(9)/L, or when a measurable population of CD34+ cells (20/microL) was detected in peripheral blood. All patients received systemic anticoagulation with a low weight heparin and ultrasound examination was performed after the removal of the catheter. Seven patients developed thrombosis (4.7%), ten experienced hematomas at the site of catheter placement (6.8%) despite prophylactic platelet transfusions, while only one patient (0.6%) had a catheter-related infection. In conclusion, the short-term use of large bore femoral catheters in setting up PBSC collection seems to be associated with minimal risk of infection and low thrombotic incidence.