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Suppl. Fig.1. Nampt staining on normal Lymph Nodes; Suppl. Fig. 2. Nampt over-expression in BCMW.1 cells. Suppl. Fig. 3. Dose-response effect curves of FK866; Suppl. Fig. 4. Expression levels of Nampt in WM cells after BTK inhibition (GSE65816); Suppl. Fig. 5. MYD88 L265P and MYD88 WT expressing cells treated with FK866, ibrutinib, or both. Suppl. Fig. 6. MYD88 L265P (BCMW.1 and MWCL-1) and MYD88 WT ( MEC.1 and RL) expressing cells were treated with an inhibitor of Nampt (FK866), BTK (ibrutinib), or both. Suppl. Fig. 7. Effects of co-treatment on primary tumor cells. Suppl. Fig. 8. Densitometric analysis of WB represented in Figure 3B. Suppl. Fig. 9. Rescue experiments with exogenous NAD. Suppl. Fig. 10. Effects of co-treatments on animals body weight.

Suppl. figure legends

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.

We studied pre-transplant minimal residual disease (MRD) in 224 patients (median age 44 years; range 17-65) with acute myeloid leukemia (AML) undergoing allogeneic stem cell transplant (HSCT) in complete remission. MRD was evaluated on marrow samples using multicolor flow cytometry (MFC) and assessment of WT1 gene expression. Both methods showed a strong prognostic value and their combination allowed the identification of 3 groups of patients with different risk of relapse. In multivariate analysis, combined MRD was the only predictor of cumulative incidence of relapse, regardless of donor type, conditioning regimen, first or second CR at HSCT, HSCT year and ELN risk group. Multivariate regression model showed that only negative combined MRD status (p

Metabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. ʟ-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a poor prognosis myeloid malignancy characterized by an atypical phenotype (CD123+, CD56+, and CD4+). We reported that BPDCN-like phenotype (CD123+ and either CD56+ or CD4+ or both) confers poor prognosis to acute myeloblastic leukemia (AML) patients with mutated NPM1. Here, we evaluated the incidence and the prognostic relevance of BPDCN-like phenotype in cytogenetically normal AML (CN-AML) patients. From 2006 to 2016, 83 young (age60 yrs), consecutive, CN-AML patients underwent intensive treatment. Fifteen patients (18%) showed a BPDCN-like phenotype with no difference between

Background Erythropoiesis‐stimulating agents effectively improve the hemoglobin levels in a fraction of anemic patients with myelodysplastic syndromes (MDS). Higher doses (HD) of recombinant human erythropoietin (rhEPO) have been proposed to overcome suboptimal response rates observed in MDS patients treated with lower “standard doses” (SD) of rhEPO. However, a direct comparison between the different doses of rhEPO is lacking. Methods A cohort of 104 MDS patients treated with HD was retrospectively compared to 208 patients treated with SD in a propensity score‐matched analysis to evaluate hematological improvement‐erythroid (HI‐E) rate induced by the different doses of rhEPO. The impact of rhEPO doses on survival and progression to leukemia was also investigated. Results Overall HI‐E rate was 52.6%. No difference was observed between different rhEPO doses (P = .28) in matched cohorts; in a subgroup analysis, transfusion‐dependent patients and patients with higher IPSS‐R score obtained a higher HI‐E rate with HD, although without significant impact on overall survival (OS). Achievement of HI‐E resulted in superior OS. At univariate analysis, a higher HI‐E rate was observed in transfusion‐independent patients (P, When compared in a propensity‐score matched analysis, standard doses of rhEPO showed similar effects to those of higher doses in anemic MDS patients. Achievement of favorable response with hematologic improvement in this clinical scenario is possible by reducing drug doses and costs.

Cisplatin-containing salvage regimens followed by autologous hematopoietic stem cell (HSC) transplantation are the current standard of care for relapsed or refractory (R/R) lymphomas. We retrospectively analyzed efficacy and stem cell mobilizing activity of oxaliplatin, cytarabine, dexamethasone and rituximab (R-DHAOx) in 53 R/R diffuse large B cell lymphomas (DLBCL) treated in our center (median lines 2, range 2-5; median age 59, range 22-79). Hematological toxicity was manageable and no patients experienced renal impairment. After 2 courses the overall response rate was 60% (CR 49%, PR 11%). Median overall survival (OS) was 30.53 months (95% CI 11.5-49.55), 3-year OS 40.5%. Twenty-two eligible patients collected HSC and transplantation was performed in 21/22 patients (95%), after a median of 52 days from last cycle. Our results suggest that in DLBCL R-DHAOx has an excellent stem cell mobilizing capability, response rate comparable to cisplatin-containing regimens and good toxicity profile.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for “priming” MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

The myelodysplastic syndromes are a group of diseases characterized by impairment in hemopoiesis and variable tendency to progress to acute myeloid leukemia. Recent data regarding the MDS pathogenesis highlight alterations in several compartments of DNA replication, cell cycle control, and gene expression machinery. As previously reported, miRNA families are involved in MDS pathogenesis. Also, different miRNAs have been characterized in AML and MDS and allow the identification of prognostic risk categories independent from the revised international prognostic scoring system (R-IPSS). Herein we report the results and the possible scenarios regarding pathogenesis and disease progression secondary to the evidence of considerable gene network derangement following miRNA evaluation in a cohort of patients.

BACKGROUND Azacitidine (AZA) is the standard treatment for myelodysplastic syndromes (MDS); however, many patients prematurely stop therapy and have a dismal outcome. METHODS The authors analyzed outcomes after AZA treatment for 402 MDS patients consecutively enrolled in the Italian MDS Registry of the Fondazione Italiana Sindromi Mielodisplastiche, and they evaluated the North American MDS Consortium scoring system in a clinical practice setting. RESULTS At treatment discontinuation, 20.3% of the patients were still responding to AZA, 35.4% of the cases had primary resistance, and 44.3% developed adaptive resistance. Overall survival (OS) was better for patients who discontinued treatment while in response because of planned allogeneic hematopoietic stem cell transplantation (HSCT; median OS, not reached) in comparison with patients with primary resistance (median OS, 4 months) or adaptive resistance (median OS, 5 months) or patients responsive but noncompliant/intolerant to AZA (median OS, 4 months; P = .004). After AZA discontinuation, 309 patients (77%) received best supportive care (BSC), 60 (15%) received active treatments, and 33 (8%) received HSCT. HSCT was associated with a significant survival advantage, regardless of the response to AZA. The North American MDS Consortium scoring system was evaluable in 278 of the 402 cases: patients at high risk had worse OS than patients at low risk (3 and 7 months, respectively; P < .001). The score was predictive of survival both in patients receiving BSC (median OS, 2 months for high-risk patients vs 5 months for low-risk patients) and in patients being actively treated (median OS, 8 months for high-risk patients vs 16 months for low-risk patients; P < .001), including transplant patients. CONCLUSIONS Real-life data confirm that this prognostic scoring system for MDS patients failing a hypomethylating agent seems to be a useful tool for optimal prognostic stratification and for choosing a second-line treatment after AZA discontinuation.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

NPM1 and FLT3 mutational status assessment is recommended in acute myeloid leukemia (AML) at diagnosis by European Leukemia Net (ELN) risk stratification. The presence of NPM1 mutation (NPM1-mut) partially overcomes the negative prognostic impact of FLT3-ITD, which is also modulated by FLT3-ITD/wild-type allelic ratio. Targeted drugs are available for FLT3-mutated AML but no data are available so far on the efficacy of intensified front-line regimens in overcoming the negative prognostic role of FLT3-ITD mutation. We investigated the efficacy of an intensive fludarabine, high dose cytarabine (ARA-C) and Idarubicin (IDA) induction regimen (FLAI) as frontline treatment for fit, de novo AML patients according to NPM1 and FLT3-ITD mutational status. One-hundred and forty-nine consecutive AML patients, treated in 3 Hematology Italian centers from January 2008 to January 2018, were included in this analysis. Twenty nine patients had isolated FLT3-ITD, 59 concomitant FLT3-ITD and NPM1-mut and 61 isolated NPM1-mut. Median age was 51 yrs (range: 18-65). All patients received FLAI induction (fludarabine 30 mg/sqm and ARA-C 2g/sqm on days 1 to 5 plus IDA 10 mg/sqm on days 1-3-5). For patients achieving CR fludarabine was omitted on II induction and IDA dose was increased to 12 mg/sqm. Before ELN 2017 risk stratification was adopted, patients with isolated FLT3-ITD mutation were scheduled to receive allogeneic bone marrow transplantation (allo-BMT) in first CR regardless of allelic burden. For patients with AML with NPM1 mutation and concomitant FLT3-ITD indication to allo-BMT in 1st complete remission was mainly based on minimal residual disease (MRD) status. MRD was evaluated on marrow samples using multicolor flow-cytometry (MFC) or NPM1 expression levels. Negative MFC-MRD was defined by the presence of less than 25 clustered leukemic cells/105 total events (threshold of 0.025%, Minetto et. al, BJH 2019). NPM1-mut (NPM1-A, B and D) was measured using Muta Quant Kit Ipsogen from Qiagen. FLT3-ITD allelic burden was available in31/64 of FLT3-ITD patients. Overall, 60-days mortality was 4.7%. After first induction cycles, 129 patients achieved CR (86.6%). Thirty-five/129 (25.5%) CR patients underwent BMT in first CR. After a median follow up of 68 months, 3-year overall survival (OS) was 58.6% (median not reached). In univariate analysis OS duration was affected by NPM1, FLT3 mutational status and ELN risk score. However, NPM1-mutated patient was not negatively affected by the presence of FLT3-ITD, regardless of allelic burden. This observation was even more evident in patients younger than 55 yrs (3 yy OS 64% and 68% for NPM1-mutated with or w/o FLT3-ITD, respectively (p=n.s, Figure 1), regardless of FLT3-ITD allelic burden. ELN 2017 high risk patients displayed the worst prognosis (3-year OS 35.2%). Multivariate analysis showed that NPM1 mutation was the strongest predictor of survival. In order to assess the impact of allo-BMT in 1st CR we performed a Landmark Analysis including patients alive and in CR at day 90. Interestingly, performing allo-BMT in 1st CR did not impact OS in the whole cohort of patients and irrespectively of NPM1 and FLT3 mutational status. The only subgroup who benefit from allo-BMT in first CR was high risk ELN2017 (p Despite the potential bias due to the retrospective nature of the analysis, our data indicate that intensive fludarabine-high dose cytarabine-based induction exerts a strong anti-leukemic efficacy in younger AML patients carrying NPM1 mutation irrespectively of FLT3 mutational status. Our data potentially question the role of BMT in first CR in this setting. Figure Disclosures Bocchia: CELGENE: Honoraria; Incyte: Honoraria.

Background: Minimal residual disease (MRD) assessment retains high prognostic value in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy. Widely available MRD techniques include multicolour flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan- leukemic marker WT1. However, most of the data on the prognostic value of MRD come from trials including younger patients treated with conventional 3+7 regimen. AML arising from a previous myelodisplastic syndrome (s-AML) and therapy-related AML (t-AML) are usually under-represented in trial involving younger patients and are unlikely to respond to conventional induction. Few data are therefore available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients, particularly in the context of alternative induction regimens. Aims: We evaluated MRD in a cohort of elderly s-AML or t-AML patients receiving induction therapy either with a fludarabine-containing regimen or CPX-351, in order to compare the probability of achieving MRD negativity, to disclose the prognostic value of MRD in this setting and to define the best time-points for MRD assessments. Methods: A total of 136 elderly (>60 year, median age 67, range 60-75) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=35) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was retrospectively analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels.All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria. Results: After induction, CR was achieved in 83 patients (61%). CR rate was 28/35 in patients treated with CPX-351 (80%), significantly higher when compared to patients receiving FLAI (55/101, 54.5%, p Moreover, MRD showed significant prognostic value in terms of Overall Survival in all treatment group (2-year OS of 74 and 36% in patients with or without residual MFC MRD after induction, respectively, p Conclusion: In conclusion MRD assessment retains a strong prognostic value and may help to identify patients with suboptimal response to treatment. The higher rate of MRD negativity observed with CPX-351 may be related with a more efficient anti-leukemic activity of the drug in this particular setting. The evaluation of MRD with both MFC and WT1-based assessment lead to superimposable conclusions and allowed us to obtain MRD data from virtually all AML patients treated in the selected time period. Disclosures No relevant conflicts of interest to declare.

Background: The presence of peripheral blasts (PB) is a negative prognostic factor in patients (pts) with primary and secondary myelofibrosis (PMF/SMF) and PB ≥4% was associated with a particularly unfavorable prognosis (Masarova L et al, Cancer 2020). Ruxolitinib (RUX) is the JAK1/2 inhibitor most used for treatment of MF-related splenomegaly and symptoms. Its role has not been assessed in correlation with PB. Aims: To evaluate the impact of PB percentage on RUX efficacy and prognosis Methods: After IRB approval, the "RUX-MF" retrospective study collected 804 RUX-treated chronic-phase (CP, defined as PB Comparisons of quantitative variables between the 3 groups were carried out by Kruskal-Wallis and Dunn's tests while association between categorical variables was tested by the χ2 test. Variables significantly associated to RUX stop/leukemic transformation (LT)/overall survival (OS) in univariate analysis (Log-rank test) were considered for multivariable analyses, carried out using the Cox regression model, with evaluation of the model's performance in terms of goodness of fit. Results: Pts were categorized according to PB at RUX start: PB-0 (no PB; n. 487, 61.3%), PB-5 (PB 1-5%; n. 283, 35.8%), and PB-9 (PB 6-9%; n. 24, 2.9%). Pts characteristics at RUX start were: median age 68.1y (24-89); males 58.1%; PMF 52.5%; JAK2, CALR and MPL mutated: 80.5%, 13.1% and 2% (4.4% triple negative), high DIPSS: 7.6%; PLT25 x10 9/l: 10.8%/16.4%; spleen length ≥10 cm: 47.8%, TSS ≥20: 60.6%; ≥1/≥2 high-risk mutation (HMR): 69/18 out of 167 evaluable (41.3%/10.8%); fibrosis grade ≥2: 77.9%; starting/cumulative RUX dose ≥15 mg BID: 61.4%/52.6%. Higher PB count was associated to high DIPSS risk (PB-0: 1.9%, PB-5: 16.2%, PB-9: 21.7%, p At 3 and 6 mos, 26.9% and 30.4% of evaluable pts achieved a SR, while 59.7% and 68.1% were in SyR, respectively. At 3 mos, SR (PB-0: 31.8%, PB-5: 20.6%, PB-9: 10%, p=0.001) and SyR (PB-0: 62.9%, PB-5: 55.5%, PB-9: 36.8%, p=0.02) were less frequently achieved by PB-5 and PB-9 pts compared to PB-0 pts. This association remained significant for SR at 6 mos (PB-0: 35%, PB-5: 23.9%, PB-9: 14.3%, p=0.006) and for both SR (p=0.003) and SyR (p=0.01) at any time. After a median RUX exposure of 1.5 y (0.1-8.9), 491 (61.8%) pts stopped RUX, 110 (13.9%) had a LT and 365 (46%) died. In univariate analysis, at 2y PB-9 pts had higher rates of RUX stop (73.9% vs 40.8% and 34.3% in PB-5 and PB-0 pts, log-rank p In multivariable analysis, PB-9 pts confirmed their association with: 1) RUX stop (HR 3.74, 95%CI 1.51-3.70, p20 (HR 1.66, 95%CI 1.05-2.61, p=0.03), and HMR≥2 (HR 2.69, 95%CI 1.26-4.47, p=0.007); 2) LT (HR 3.71, 95%CI 1.71-8.04, p Conclusions: CP-MF pts with PB>5% have a worse response to RUX and a worse outcome. Personalized approaches beyond RUX monotherapy may be useful in this context. Further clinical trials evaluating combination strategies and new drugs are required. Figure 1 Figure 1. Disclosures Palandri: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; AOP: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Membership on an entity's Board of Directors or advisory committees; CTI: Consultancy. Abruzzese: Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Iurlo: Bristol Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Speakers Bureau; Incyte: Speakers Bureau. Bonifacio: Bristol Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Cuneo: AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Pane: AbbVie; Amgen; Novartis: Other: Travel, accommodation, expenses; Novartis Pharma SAS;: Research Funding; AbbVie; Amgen; Novartis, GSK , Incyte: Consultancy; AbbVie; Amgen; Novartis, GSK, Incyte: Speakers Bureau. Lemoli: Celgene: Other: Support for attending meetings and/or travel; Jazz, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Daiichi Sankyo, Servier: Honoraria, Speakers Bureau. Cavo: Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Breccia: Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Bristol Myers Squibb/Celgene: Honoraria; Abbvie: Honoraria.

Genomic instability plays a pathological role in various malignancies, including acute myeloid leukemia (AML), and thus represents a potential therapeutic target. Recent studies demonstrate that SIRT6, a NAD + -dependent nuclear deacetylase, functions as genome-guardian by preserving DNA integrity in different tumor cells. Here, we demonstrate that also CD34 + blasts from AML patients show ongoing DNA damage and SIRT6 overexpression. Indeed, we identified a poor-prognostic subset of patients, with widespread instability, which relies on SIRT6 to compensate for DNA-replication stress. As a result, SIRT6 depletion compromises the ability of leukemia cells to repair DNA double-strand breaks that, in turn, increases their sensitivity to daunorubicin and Ara-C, both in vitro and in vivo In contrast, low SIRT6 levels observed in normal CD34 + hematopoietic progenitors explain their weaker sensitivity to genotoxic stress. Intriguingly, we have identified DNA-PKcs and CtIP deacetylation as crucial for SIRT6-mediated DNA repair. Together, our data suggest that inactivation of SIRT6 in leukemia cells leads to disruption of DNA-repair mechanisms, genomic instability and aggressive AML. This synthetic lethal approach, enhancing DNA damage while concomitantly blocking repair responses, provides the rationale for the clinical evaluation of SIRT6 modulators in the treatment of leukemia.

Tyrosine kinases have been implicated in promoting tumorigenesis of several human cancers. Exploiting these vulnerabilities has been shown to be an effective anti-tumor strategy as demonstrated for example by the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, for treatment of various blood cancers. Here, we characterize a new multiple kinase inhibitor, ARQ531, and evaluate its mechanism of action in preclinical models of acute myeloid leukemia. Treatment with ARQ531, by producing global signaling pathway deregulation, resulted in impaired cell cycle progression and survival in a large panel of leukemia cell lines and patient-derived tumor cells, regardless of the specific genetic background and/or the presence of bone marrow stromal cells. RNA-seq analysis revealed that ARQ531 constrained tumor cell proliferation and survival through Bruton's tyrosine kinase and transcriptional program dysregulation, with proteasome-mediated MYB degradation and depletion of short-lived proteins that are crucial for tumor growth and survival, including ERK, MYC and MCL1. Finally, ARQ531 treatment was effective in a patient-derived leukemia mouse model with significant impairment of tumor progression and survival, at tolerated doses. These data justify the clinical development of ARQ531 as a promising targeted agent for the treatment of patients with acute myeloid leukemia.

Acute myeloid leukemia with biallelic mutation of CEBPA (CEBPA-dm AML) is a distinct good prognosis entity recognized by WHO 2016 classification. However, testing for CEBPA mutation is challenging, due to the intrinsic characteristics of the mutation itself. Indeed, molecular analysis cannot be performed with NGS technique and requires Sanger sequencing. The association of recurrent mutations or translocations with specific immunophenotypic patterns has been already reported in other AML subtypes. The aim of this study was the development of a specific cytofluorimetric score (CEBPA-dm score), in order to distinguish patients who are unlikely to harbor the mutation. To this end, the correlation of CEBPA-dm score with the presence of the mutation was analyzed in 50 consecutive AML patients with normal karyotype and without NPM1 mutation (that is mutually exclusive with CEBPA mutation). One point each was assigned for expression of HLA DR, CD7, CD13, CD15, CD33, CD34 and one point for lack of expression of CD14. OS was not influenced by sex, age and CEBPA-dm score. Multivariate OS analysis showed that CEBPA-dm (p 0.02) and FLT3-ITD (p 0.01) were the strongest independent predictors of OS. With a high negative predictive value (100%), CEBPA-dm score6 was able to identify patients who are unlikely to have the mutation. Therefore, the application of this simple score might optimize the use of expensive and time-consuming diagnostic and prognostic assessment in the baseline work up of AML patients.

Specific myeloid-related and inositide-specific gene mutations can be linked to myelodysplastic syndromes (MDS) pathogenesis and therapy. Here, 44 higher-risk MDS patients were treated with azacitidine and lenalidomide and mutations analyses were performed at baseline and during the therapy. Results were then correlated to clinical outcome, overall survival (OS), leukemia-free-survival (LFS) and response to therapy. Collectively, 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases): 17 patients showed a durable response, 9 patients early lost response and 8 patients never responded. The most frequently mutated genes were ASXL1, TET2, RUNX1, and SRSF2. All patients early losing response, as well as cases never responding, acquired the same 3 point mutations during therapy, affecting respectively PIK3CD (D133E), AKT3 (D280G), and PLCG2 (Q548R) genes, that regulate cell proliferation and differentiation. Moreover, Kaplan–Meier analyses revealed that this mutated cluster was significantly associated with a shorter OS, LFS, and duration of response. All in all, a common mutated cluster affecting 3 inositide-specific genes is significantly associated with loss of response to azacitidine and lenalidomide therapy in higher risk MDS. Further studies are warranted to confirm these data and to further analyze the functional role of this 3-gene cluster.

Introduction. The association of Azacitidine (AZA) and Lenalidomide (LEN), either administered concurrently or sequentially, has proven effective in Myelodysplastic Syndromes (MDS), however the optimum dose and schedule remains unknown. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in higher-risk MDS pts. Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the IWG criteria (Cheson, 2006). Moreover, the aim of this analysisis is to enucleate the clinical and biological features of pts who showed long-lasting (≥ 20 cycles) responses. Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS with IPSS risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles. For responder pts the same treatment was continued until disease progression or unacceptable toxicity. Results. From March 2013, 44 pts (27 males), median age: 72 (48-83 yrs) were enrolled, from 13 hematologic Centers. 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. Treatment was given for a median of 8.5 (1-68) cycles; in ARM 1: 9 (1-68) cycles; in ARM 2: 8 (1-63) cycles, respectively. Median follow-up: 15 (2-77) months. 10/44 pts (22.7%) did not complete at least 6 cycles of treatment for causes other than disease progression, and were not considered evaluable for response. Among the 34/44 pts (77.3%) evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. Intention-to-treat (ITT) analysis: ORR: 59.1%. First response was observed after a median of 2 (1-8) cycles. The Best Response achieved was: CR: 8 pts (23.5%) (ITT: 18.1%), PR: 1 pt (2.9%) (ITT: 2.2%), mCR: 3 pts (8.8%) (ITT: 6.8%), HI: 8 pts (23.5%) (ITT: 18.1%), mCR+HI: 6 pts (17.6%) (ITT: 13.6%). Median duration of hematologic response: 10.5 months. 37 pts (84.1%) died , and 20 pts (45.4%) showed progression to AML. Grade >2 non haematological toxicity: 54.5%. Median OS: 15 months. OS was significantly longer in responder pts as compared to the other pts (28 vs 7 months, p2 non haematological toxicity (ARM 1: 66.7%; ARM 2: 43.5%), AML incidence (ARM 1: 33.3%; ARM 2: 56.5%; p=0.2150) and OS (ARM 1: 14 months; ARM 2: 16 months). However, among responder pts, sequential treatment showed a longer clinical benefit, as compared to combined treatment. Responder pts of ARM 2 showed a significantly longer median duration of response (18 vs 6 months, p=0.0481), a longer median duration of therapy (28 vs 10 months, p=0.0870; 20 vs 10 cycles, p=0.1181), more long-lasting (≥ 20 cycles) responses (34.8% vs 9.5%, p=0.1017) and a longer OS (35 vs 26 months, p=0.3868), as compared to responder pts of ARM 1. Overall, 10/44 long-responder pts (22.7%) received ≥ 20 cycles; 5/10 pts (50%) achieved CR. IPSS risk: Intermediate-2 (8 pts); High (2 pts); IPSS-R risk: Intermediate (2 pts); High (6 pts); Very High (2 pts); IPSS cytogenetic risk: Good (5 pts); Intermediate (3 pts); Poor (2 pts); IPSS-R cytogenetic risk: Good (5 pts); Intermediate (4 pts); Very Poor (1 pt); 4/6 patients with altered karyotype achieved cytogenetic remission; it is noteworthy that the only 3 pts of the entire series who showed no gene mutations at baseline are included in this subset of long-responders pts, while 5/10 pts showed at baseline ≥ 1 prognostically unfavorable gene mutations (none with TP53 mutations), with variable VAFs during treatment. Moreover all long-responder pts showed a common gene mutation on SOD2 gene, and mutations on PLCG2 gene. Conclusions. Our results confirm the efficacy of both AZA+LEN treatment regimens in higher-risk MDS pts, in terms of ORR and OS, although sequential treatment was associated with a longer clinical benefit among responder pts. A subset of pts (22,7 %) with less unfavorable cytogenetic and molecular characteristics showed a long-lasting response to treatment. Disclosures Finelli: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Crugnola:BMS: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Rigolin:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Jannsen, BMS, Celgene, Sanofi, GlaxoSmithKline, Takeda, Amgen, Oncopeptides, AbbVie, Karyopharm, Adaptive: Consultancy, Honoraria.

In recent years, dermatologic manifestations in oncohematologic patients have become more common. The aim of our study was to determine the incidence and heterogeneity of skin manifestations in patients followed at our Hematology Department. This observational monocentrical study was conducted on 60 patients. We divided the observed conditions in exanthematous, purpuric, vesicular-bullous, papulonodular, urticarial, and eczematous manifestations. Moreover, all lesions were classified according to pathogenesis, in (a) specific skin manifestations, caused by neoplastic skin infiltration; (b) immune-mediated manifestations, based on immunological mechanisms; and (c) skin lesions due to immunodeficiency. Altogether, 73 clinical manifestations were reported. Specific manifestations (a) were detected in 15.1% of the cases, mainly with papulonodular appearance. Immune-mediated manifestations (b) were found in 37 cases (50.7%), particularly with eczematous or exanthematous appearance, and leukemia was the malignancy most frequently reported in these patients. Eventually, lesions due to immunosuppression (c) were reported in 34.2% of the cases. They were represented by infections and cutaneous malignancies, and usually manifested with papulonodular lesions. Skin lesions in oncohematologic patients are a common event. A multidisciplinary approach based on the collaboration between the hematologist and the dermatologist is crucial to achieve a proper diagnosis, and correctly manage these manifestations.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) are a very rare group of diseases included by WHO 2016 classification among the myeloid neoplasms and usually display an aggressive course with dismal outcome. BPDCN are characterized by a recurrent phenotype (CD45low/CD34-/CD56+/CD4+/CD123+), in the absence of other lineage differentiation markers. Our group recently reported that a subset of patients diagnosed with AML with NPM1-mutation carrying co-expression of CD123, CD56 and CD4, a "BPDCN-like" phenotype, showed poor prognosis. The aim of the present study was to evaluate the incidence and the prognostic impact of BPDCN-like phenotype in a wider cohort of cytogenetically normal AML patients, irrespectively of NPM1-mutational status. Methods We retrospectively evaluated a cohort of 83 younger (age In all patient, 4 (until 2012) or 8 color immunophenotypic analysis was performed at diagnosis by analysing erythrocyte-lysed whole BM blasts to identify relevant antigen aberration patterns and the pathological leukemia phenotype for future minimal residual disease assessment. We defined a BPDCN-like signature the positivity of at least two among CD56/CD4/CD123 antigens and we evaluated the prognostic impact and the correlation with biological, molecular and cytogenetic features. Results Fifteen patients (18%) showed a BPDCN-like signature. Neither the presence of NPM1-mutation nor FLT3-ITD or biallelic CEBPA mutation showed a significant correlation with BPDCN-like signature. BPDCN-like patients had significantly higher WBC count at diagnosis (p Among analyzed variables, only the presence of NPM1-mutation correlated with complete response (CR) probability. With a median follow-up of 63 months, 3-year Overall Survival (OS) was 52% in the whole cohort (median 38 months). Patient with NPM1 mutation or biallelic CEBPA mutation had a better outcome (p However, as we previously reported, in the subgroup of 30 patients with NPM-1 mut AML, the presence of BPDCN-like features conferred a poor prognosis (3-year OS 25% vs 77% for BPDCN positive and negative NPM-1 mut patients, respectively, p On the contrary in the 38 NPM-1 wt patients a trend towards better outcome was observed in BPDCN-like patient, unless not statistically significant (3-year OS 71% vs 35% for BPDCN-like positive and negative NPM-wt patients, respectively, p=0.156). No difference in MRD clearance probability was observed in this subgroup. Conclusions Our extended analysis confirms that a peculiar BPDCN-like immunophenotype among NPM-1 mut AML patients is associated with significantly worse outcome in this otherwise favorable subset of AML. Patient with BPDCN-like features showed high level of minimal residual disease after induction, suggesting an intrinsic chemo-resistance. Interestingly, this observation was strictly restricted to NPM-1 mutated AML, suggesting that peculiar alterations in this distinct entity contribute to the prognostic impact of BPDCN-like features in this setting. The biological explanation of this finding is not clear and further gene-expression profiling studies are ongoing in order to explain our findings. Figure. Figure. Disclosures Gobbi: Pfister: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Purpose To investigate the progression-free survival (PFS) of patients with advanced Hodgkin lymphoma (HL) after a risk-adapted treatment strategy that was based on a positive positron emission tomography scan performed after two doxorubicin, vinblastine, vincristine, and dacarbazine (ABVD) cycles (PET2). Patients and Methods Patients with advanced-stage (IIB to IVB) HL were consecutively enrolled. After two ABVD cycles, PET2 was performed and centrally reviewed according to the Deauville five-point scale. Patients with a positive PET2 were randomly assigned to four cycles of escalated bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPP) followed by four cycles of standard BEACOPP with or without rituximab. Patients with a negative PET2 continued ABVD, and those with a large nodal mass at diagnosis (≥ 5 cm) in complete remission with a negative PET at the end of chemotherapy were randomly assigned to radiotherapy or no further treatment. The primary end point was 3-year PFS. Results Of 782 enrolled patients, 150 (19%) had a positive and 630 (81%) a negative PET2. The 3-year PFS of all patients was 82%. The 3-year PFS of those with a positive and negative PET2 was 60% and 87%, respectively ( P < .001). The 3-year PFS of patients with a positive PET2 assigned to BEACOPP with or without rituximab was 63% versus 57% ( P = .53). In 296 patients with both interim and post-ABVD–negative PET who had a large nodal mass at diagnosis, radiotherapy was randomly added after chemotherapy without a significant PFS improvement (97% v 93%, respectively; P = .29). The 3-year overall survival of all 782 patients was 97% (99% and 89% for PET2 negative and positive, respectively). Conclusion The PET-driven switch from ABVD to escalated BEACOPP is feasible and effective in high-risk patients with advanced-stage HL.