Roberto Massimo Lemoli

The addition of plerixafor is safe and allows adequate PBSC collection in multiple myeloma and lymphoma patients poor mobilizers after chemotherapy and G-CSF

We report 13 multiple myeloma (MM) or lymphoma patients who were failing PBSC mobilization after disease-specific chemotherapy and granulocyte-CSF (G-CSF), and received plerixafor to successfully collect PBSCs. Patients were considered poor mobilizers when the concentration of PB CD34+ cells was always lower than 10 cells/μL, during the recovery phase after chemotherapy and/or were predicted to have inadequate PBSC collection to proceed to autologous transplantation. Plerixafor (0.24 mg/kg) was administered subcutaneously for up to three consecutive days, while continuing G-CSF, 10–11 h before the planned leukapheresis. Plerixafor administration was safe and no significant adverse events were recorded. We observed a 4.7 median fold-increase in the number of circulating CD34+ cells after plerixafor as compared with baseline CD34+ cell concentration (from a median of 6.2 (range 1–12) to 21.5 (range 9–88) cells/μL). All patients collected >2 × 106 CD34+ cells/kg in 1–3 leukaphereses. In all, 5/13 patients have already undergone autograft with plerixafor-mobilized PBSCs, showing a rapid and durable hematological recovery. Our results suggest that the pre-emptive addition of plerixafor to G-CSF after chemotherapy is safe and may allow the rescue of lymphoma and MM patients, who need autologous transplantation but are failing PBSC mobilization.

Larger Size of Donor Alloreactive NK Cell Repertoire Correlates with Better Response to NK Cell Immunotherapy in Elderly Acute Myeloid Leukemia Patients

Purpose: In acute myeloid leukemia (AML), alloreactive natural killer (NK) cells are crucial mediators of immune responses after haploidentical stem cell transplantation. Allogeneic NK cell infusions have been adoptively transferred with promising clinical results. We aimed at determining whether the composition of NK graft in terms of frequency of alloreactive NK cells influence the clinical response in a group of elderly AML patients undergoing NK immunotherapy. Experimental Design: Seventeen AML patients, in first complete remission (CR; median age 64 years, range 53–73) received NK cells from haploidentical KIR-ligand–mismatched donors after fludarabine/cyclophosphamide chemotherapy, followed by IL2. To correlate donor NK cell activity with clinical response, donor NK cells were assessed before and after infusion. Results: Toxicity was moderate, although 1 patient died due to bacterial pneumonia and was censored for clinical follow-up. With a median follow-up of 22.5 months (range, 6–68 months), 9 of 16 evaluable patients (0.56) are alive disease-free, whereas 7 of 16 (0.44) relapsed with a median time to relapse of 9 months (range, 3–51 months). All patients treated with molecular disease achieved molecular CR. A significantly higher number of donor alloreactive NK cell clones was observed in responders over nonresponders. The infusion of higher number of alloreactive NK cells was associated with prolonged disease-free survival (0.81 vs. 0.14, respectively; P = 0.03). Conclusions: Infusion of purified NK cells is feasible in elderly AML patients as post-CR consolidation strategy. The clinical efficacy of adoptively transferred haploidentical NK cells may be improved by infusing high numbers of alloreactive NK cells. Clin Cancer Res; 22(8); 1914–21. ©2016 AACR. See related commentary by Muntasell and López-Botet, p. 1831

Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells.

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.

Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn’s disease patients in a mouse model of colitis

IntroductionDue to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution.MethodsColitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 106 cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy.ResultsWe found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of inflammatory mediators. Finally, Nile Red staining of MSCs is very efficient, stable and does not impair their vitality and function. Nile Red-labelling was clearly detected in the colitic area of adCD-MSCs injected mice and it was significantly brighter in the colon sections of mice that had received adCD-MSCs/hCBPL.ConclusionsIn summary, with this study we propose a novel and promising adCD-MSC/hCBPL-based therapy for refractory IBDs.

High feasibility and antileukemic efficacy of fludarabine, cytarabine, and idarubicin (FLAI) induction followed by risk-oriented consolidation: A critical review of a 10-year, single-center experience in younger, non M3 AML patients.

About 105 consecutive acute myeloid leukemia (AML) patients treated with the same induction‐consolidation program between 2004 and 2013 were retrospectively analyzed. Median age was 47 years. The first induction course included fludarabine (Flu) and high‐dose cytarabine (Ara‐C) plus idarubicin (Ida), with or without gemtuzumab‐ozogamicin (GO) 3 mg/m2 (FLAI‐5). Patients achieving complete remission (CR) received a second course without fludarabine but with higher dose of idarubicin. Patients not achieving CR received an intensified second course. Patients not scheduled for early allogeneic bone marrow transplantation (HSCT) where planned to receive at least two courses of consolidation therapy with Ara‐C. Our double induction strategy significantly differs from described fludarabine‐containing regimens, as patients achieving CR receive a second course without fludarabine, to avoid excess toxicity, and Ara‐C consolidation is administrated at the reduced cumulative dose of 8 g/m2 per cycle. Toxicity is a major concern in fludarabine containing induction, including the recent Medical Research Council AML15 fludarabine, cytarabine, idaraubicin and G‐CSF (FLAG‐Ida) arm, and, despite higher anti‐leukemic efficacy, only a minority of patients is able to complete the full planned program. In this article, we show that our therapeutic program is generally well tolerated, as most patients were able to receive subsequent therapy at full dose and in a timely manner, with a 30‐day mortality of 4.8%. The omission of fludarabine in the second course did not reduce efficacy, as a CR rate of 83% was achieved and 3‐year disease‐free survival and overall survival (OS) were 49.6% and 50.9%, respectively. Our experience shows that FLAI‐5/Ara‐C + Ida double induction followed by risk‐oriented consolidation therapy can result in good overall outcome with acceptable toxicity. Am. J. Hematol. 91:755–762, 2016.

BU/melphalan and auto-SCT in AML patients in first CR: a ‘Gruppo Italiano Trapianto di Midollo Osseo (GITMO)’ retrospective study

AML patients (total 129; median age =50 years; range 16–72) in first CR received BU and melphalan (BU/Mel) as conditioning regimen before auto-SCT. In all, 82 patients (63.6%) received PBSCs and 47 patients (36.4%) received BM cells. The distribution of cytogenetic categories was conventionally defined as favorable (15.5%), intermediate (60.1%) and unfavorable (24.3%). With a median follow-up of 31 months, the 8-year projected OS and disease-free survival (DFS) was 62 and 56% for the whole population, respectively. The relapse rate was 46% and the non-relapse mortality was 4.65%. Although PBSC transplantation led to a faster hematological recovery than BM transplantation, in univariate analysis the stem cell source, cytogenetics and different BU formulations did not significantly affect OS and DFS, whereas age and the number of post-remission chemotherapy cycles did have a significant effect on the clinical outcome. Multivariate analysis identified age <55 years as the only important independent predictor for OS and DFS. Our data suggest that BU/Mel, being associated with a low toxicity profile (mainly mucositis) and mortality, is an effective conditioning regimen even for high-risk AML patients in first CR undergoing auto-SCT.

Extracellular ATP induces apoptosis through P2X7R activation in acute myeloid leukemia cells but not in normal hematopoietic stem cells

Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2×7R is the most consistently expressed by tumors. P2×7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2×7R. Here, we show that P2×7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2×7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo. Overall, our results demonstrate that P2×7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.

Dual NAMPT and BTK Targeting Leads to Synergistic Killing of Waldenström Macroglobulinemia Cells Regardless of MYD88 and CXCR4 Somatic Mutation Status.

Purpose: Nicotinamide phosphoribosyltransferase (Nampt) regulates intracellular NAD+ pool and is highly expressed in a number of malignancies. FK866, a selective inhibitor of Nampt, depletes intracellular NAD+ levels, thereby blocking cellular metabolism and triggering sensitization to other drugs and cell death. Here we characterized the antitumor effects of Nampt inhibition in Waldenström macroglobulinemia. Experimental Design: We investigated Nampt role in MW cells using both mRNA and protein expression analyses. We have also used loss-of-function approaches to investigate the growth and survival effects of Nampt on MW cells and further tested the anti-MW activity of dual Nampt and BTK inhibition in vitro and in vivo. Results: We found that Waldenström macroglobulinemia cells exhibit high levels of Nampt compared with normal B cells. Loss of function studies suggested a potential oncogenic role of Nampt in Waldenström macroglobulinemia cells, and BTK-inhibitor ibrutinib and FK866 resulted in a significant and synergistic anti-Waldenström macroglobulinemia cell death, regardless of MYD88 and CXCR4 mutational status. Cell death was associated with: (i) activation of caspase-3, PARP and downregulation of Mcl-1, (ii) enhanced intracellular ATP and NAD+ depletion, (iii) inhibition of NF-κB signaling, and (iv) inhibition of multiple prosurvival signaling pathways. In a murine xenograft Waldenström macroglobulinemia model, low-dose combination FK866 and ibrutinib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Conclusions: Our results show intracellular NAD+ level as crucial for proliferation and survival of Waldenström macroglobulinemia cells, and provides the mechanistic preclinical rationale for targeting Nampt, either alone or with Ibrutinib, to overcome drug resistance and improve patient outcome in Waldenström macroglobulinemia. Clin Cancer Res; 22(24); 6099–109. ©2016 AACR.

Combined assessment of WT1 and BAALC gene expression at diagnosis may improve leukemia-free survival prediction in patients with myelodysplastic syndromes

Several genes with relevant pathogenetic and prognostic value have been identified in patients with myelodysplastic syndromes. Overexpression of WT1 at diagnosis has been associated with increased progression to acute myeloid leukemia and reduced leukemia free survival. Conversely, few data are available on the prognostic value of BAALC gene overexpression in AML and MDS patients. We evaluated the prognostic value of the combined expression of WT1 and BAALC genes at diagnosis in 86 MDS patients who had been stratified according to IPSS and R-IPSS scoring systems. Our results suggest that in the whole group of patients, low levels of both WT1 and BAALC were associated with a favorable outcome (3-year LFS 74.5%, median not reached), whereas patients presenting high expression levels of both genes had the worst prognosis (3-year LFS 0%, median 12 months, p<0.001). More specifically, molecular profiling was especially useful for intermediate 1 and intermediate-2/high risk groups. This study suggests that evaluating WT1 and BAALC gene expression at diagnosis may improve standard risk stratification and possibly refine the therapeutic approach for MDS patients.

A blastic plasmacytoid dendritic cell neoplasm-like phenotype identifies a subgroup of npm1-mutated acute myeloid leukemia patients with worse prognosis

To the Editor: As widely reported, isolated NPM1 mutations display a positive prognostic value in acute myeloid leukemia (AML) since they are associated with high complete response rate to chemotherapy and with reduced relapse risk, especially if a molecular complete remission (mCR) is achieved. However, a minority of NPM-mut AML patients do not achieve hematological or mCR or display early relapse, irrespectively of FLT3 status. Despite its recognition as a distinct WHO 2016 entity, NPM-mut AML displays indeed a certain degree of clinical and biological heterogeneity. Morphologic spectrum is wide and can involve all the FAB subtypes, with the exception of M3, with blasts frequently showing monocytic differentiation and cup-like nuclei. Even immunophenotype (IF) is not univocal; NPM-mut cells are usually CD34 negative, CD33 and CD13 positive and a “myeloid” or “monocytic” IF can be usually distinguished. No prognostic relevance has been associated to morphological and immunophenotypic features so far. We retrospectively evaluated 38 consecutive young, de novo NPM-mut AML patients diagnosed in our institution between 2006 and 2014 and treated with a fludarabine, high dose cytarabine and idarubicin (FLAI) based induction. Multicolor cytofluorimetric analysis was routinely performed on bone marrow samples obtained at diagnosis, to define lineage according to WHO 2016, and to identify the leukemia associated phenotypes for minimal residual disease (MRD) monitoring. MRD assessment was performed in all patients with both IF and RQ-PCR for NPM1 expression levels quantification, after induction and each of the consolidation courses. In our experience, a greater than 3.5 logarithmic reduction of NPM1 expression after FLAI induction identified patients with the best probability to achieve mCR and best long term outcome. The retrospective review of leukemic immunophenotypes at diagnosis allowed us to identify three different subgroups of patients; 16/38 displayed a myeloid IF [CD33/CD13/CD38/CD117/MPO (1)]; 7/38 a monocytic IF [CD33/CD64/cyLys/CD11b/CD15 (1) with 3/7 patients CD131]; the third group included 10 patients who displayed both myeloid, and monocytic features [CD33/CD13/CD38/CD117/MPO/ CD64/cLys/CD11b/CD15 (1)]. Five patients could not be assigned to any of those groups. FLT3-ITD mutation was detected in 16/38 (42%) patients. Its incidence was significantly higher in the monocytic group, however this did not translate in a worse outcome (data not shown). No statistically significant differences in relapse free survival (RFS) and overall survival (OS) were detectable among the three IF groups. The expression of CD34 did not negatively affect RFS and OS. Interestingly, searching for recurrent aberrant antigen combinations, we identified six patients with [CD56/CD123/CD4 (1)] coexpression; in other seven patients only two of these three markers were present. Since these markers represent part of the typical blastic plasmacytoid dendritic cell neoplasm (BPDCN) IF, we named this phenotype “BPDCNlike”. BPDCN-like IF was equally distributed among the previously described IF subgroups. Three out of the 6 BPDCN-like patients displayed concomitant FLT3 ITD mutation and all patients had normal karyotype. None of these BPDCN-like patients displayed clinical, morphological and biological features generally associated with BPDCN. Overall, the outcome of BPDCN-like patients was poorer compared to those not expressing this antigen combination. Specifically, five out of six BPDCN-like patients achieved CR after induction (83%) but only one patient achieved mCR. Allogeneic stem cell transplantation (HSCT) was scheduled for refractory patients and for those not achieving mCR, with three patients being transplanted. Three out of five patients not obtaining mCR could not be transplanted due to a sudden unresponsive disease relapse. A complete overview of BPDCN-like patients’ features at diagnosis, response to treatment, and long-term outcome is provided in Table 1. Three year RFS was 28 and 72%, respectively, for patient with or without BPDCN-like phenotype (P< .05), whereas 3-year OS was 0 and 63%, respectively (P<0.05). Furthermore, a trend towards an inferior OS was observed even in the seven patients presenting only two of three BPDCN markers. Although the negative impact of each of these antigens has already been described, to the best of our knowledge this is the first report on the prognostic impact of CD123, CD56, and CD4 coexpression in NPM mut AML. CD123 is strongly expressed by plasmacytoid dendritic cells and by their pathological counterpart in BPDCN and it is widely expressed in hematological malignancies. It is also expressed on physiological CD341 hemopoietic progenitors and on leukemic AML stem cells (LSC). The number of CD1231 LSC has been shown to be predictive of clinical outcome. Interestingly, a negative prognostic impact of CD123 expression in NPM-mut AML has already been