ALBERT

Description: The primary end point of the study was the successful mobilization of a target cell dose of 2 x 106 CD34+ cells/kg in lymphoma patients receiving ifosfamide, epirubicin and etoposide (IEV) chemotherapy and a fixed dose (6 mg) of pegfilgrastim given as single subcutaneous injection. An open-label phase II study including 25 relapsed or refractory patients (Hodgkin’s disease=4; aggressive non-Hodgkin’s lymphoma=21) was conducted to evaluate the efficacy of pegfilgrastim, in combination with salvage chemotherapy, mobilizing CD34+ stem cells into peripheral blood. Following chemotherapy, all patients had grade 4 neutropenia with a median duration of 1.5 days (1–3). Pegfilgrastim treatment was well tolerated and only 2/25 patients required pain-control medication. CD34+ cells were mobilized in all patients. The median (range) peak value of peripheral blood CD34+cells after IEV chemotherapy and pegfilgrastim was 141/microL (12.8–386) and occurred almost invariably on day +14 (13–16). Twenty three/25 patients underwent a single apheresis to collect a median of 8.7 CD34+cells/Kg (1.8–17.3). Twenty four/25 patients (96%) reached the target cell dose of 2 x 106 CD34+ cells/kg. High concentrations of circulating CD34+ cells (〉 50/microL) were observed for several days after the achievement of the peak value. All patients have been transplanted with pegfilgrastim-mobilized CD34+ cells and all of them showed a rapid and sustained engraftment after high-dose chemotherapy. Our results show that pegfilgrastim as adjunct to chemotherapy is a predictable and highly effective mobilization regimen in lymphoma patients

Description: Background: Several studies have suggested that genetic variability related with single nucleotide polymorphisms (SNPs) of the BER system, DNA synthesis and folate-metabolizing pathway genes could modulate DNA repair capacity. Moreover, these genes are supposed to be related to cancer risk. However, the prognostic impact of the association of individual and/or combined genetic variants in patients with myelodysplastic syndromes (MDS) remains undetermined. Methods: We genotyped 113 MDS patients, 54 with IPSS low/int-1 receiving only best supportive care (BSC group) and 59 with IPSS int-2/high treated with azacitidine (AZA-group), for the following polymorphisms: XRCC1 194 and 399, APE1 148, XRCC3 241, TS5'-UTR (2R/3R and G/C) and 3'-UTR (6bp+/6bp-), MTHFR 677 and 1298. Genomic DNA was analyzed by High Resolution Melting assay and restriction digests of PCR products. Overall survival (OS) was calculated using the Kaplan-Meier estimate probabilities, and differences between survival curves were analyzed by the log-rank test. Multivariate analyses were performed using the Cox method. Results: For all the target genes, the distribution of genotypes was consistent with the Hardy-Weinberg equilibrium. Among the baseline characteristics analyzed (age, sex, diagnosis according to WHO, hemoglobin) there was no statistically significant difference in the genotype distribution of studied polymorphisms. In the BSC group, the variants XRCC1 399 GG [Hazard ratio (HR)=7.07; p=0.02], -6/-6 of TS3'-UTR (HR=4.65; p=0.05), 2R/3G, 3C/3G, 3G/3G of TS5'-UTR (HR=11.44; p=0.02) and TT of MTHFR 677 (HR=67.12; p

Description: Background. Nilotinib 300 mg BID was approved as frontline treatment in chronic phase chronic myeloid leukemia (CP-CML) patients and allowed to reach deep molecular responses in a shorter median time with reduction of progression rate. Nilotinib is associated to a specific safety profile, with metabolic side effect as the most common events and increased probability of cardiovascular disorders. Aim. Aim of our study is to prospectively assess metabolic changes and cardiovascular safety during treatment with nilotinib, in a single arm multicentric Italian GIMEMA trial (0811), testing the drug as frontline treatment with the primary endpoint to obtain MR4 at 24 months. All metabolic changes were classified according to CTC grade. Lipidic changes were assessed according to American Association of Clinical endocrinologist criteria of 2012 and glucose abnormalities according to American diabetologist association (ADA). Results. One hundred and thirty patients were enrolled in 33 different centers: median age 50.5 years (range 18-85), 64.6% male. Mean body mass index (BMI) was 25.3, with 40% of patients being overweight/obese according to WHO classification. At last contact, 100 patients were still in treatment, the majority with full dose (86%). According to ADA criteria 47%, 10%, 4.6% and 6% of patients experienced grade 1 (101-125 mg/dl), grade 2 (126-150 mg/dl), grade 3 (151-200 mg/dl) and grade 4 (〉200 mg/dl), increased fasting glucose, respectively. As compared to baseline, a significant variation was observed after 1 year (p8), respectively according to ADA criteria. AACE criteria identified a significant reduction of triglycerides (p

Description: In multiple myeloma (MM) adoptive immunotherapy through allogeneic donor lymphocyte infusions results in a well-documented graft versus myeloma effect, but it is associated with high incidence of graft versus host disease. The idiotype (Id) expressed by MM cells can be regarded as a tumor-specific antigen and it has been used for immunotherapy. To enhance antitumor immunity and reduce alloreactivity, allogeneic T cells were activated against tumor-derived Id and then purified according to IFN-γ production. Total peripheral blood mononuclear cells from healthy donors were incubated with autologous monocyte-derived dendritic cells and pulsed with patient-derived Id protein. Cells were maintained in serum-free medium and supplemented during the priming phase with IL-7 and IL-12. Subsequently, the T-cell culture was restimulated every 7 days with pulsed DCs in the presence of low doses of IL-2. After 2 or 3 stimulations, the percentage of IFN-γ-producing T cells was as high as 5–10%, whereas that observed in presence of not stimulated T-cells was undetectable. Based on their IFN-γ production, T cells were isolated by using a commercial immunomagnetic IFN-γ capture assay. The purity of enriched IFN-γ-producing T cells ranged between 60 and 90% as evaluated by flow cytometry. The yield was 60% of pre-selection IFN-γ positive T cells and cell viability after selection was 80%. Functionally, IFN-γ purified T cells showed better Id-specific proliferative response when compared both to not-stimulated T cells and to stimulated but not purified T cells. IFN-γ purified T cells were shown to be memory CD45RO+ T cells expressing HLA-DR, CD25, CD95 at high level, whereas the negative fraction was mainly composed of naive, poorly activated T cells. T-cell receptor spectratyping analysis demonstrated that IFN-γ positive T cells were represented by a pauciclonal cell population as compared to policlonal T cells obtained before cell culture. Moreover, addition of not-stimulated T cells resulted in a significant reduction of allogeneic CD34+-derived colony-forming capacity. Conversely, the absolute number of allogeneic total colony-forming units-cells was not affected by IFN-γ purified T cells.These data demonstrate that Id-specific T cells may be generated from healthy donors and significantly enriched on the basis of their IFN-γ production. Id-specific IFN-γ - purified T cells have better anti-Id response and reduced alloreactivity than unselected T cells.

Description: Tyrosine-kinase inhibitors (TKIs)have completely changed the expected survival of chronic myeloid leukemia (CML) patients which is now approaching that of the general population: a relevant proportion of CML patients are currently elderly or very elderly. Very elderly patients represent generally a small proportion in published experiences. Older CML patients imatinib treated, as it happens in the general population, receive other drug treatments for associated chronic illnesses. Our aim is to assess if and which classes of concomitant drugs have an impact on cytogenetic response in chronic phase (CP)-CML very elderly (age 〉75 years) patients. Two hundred and twelve very elderly CP-CML patients, imatinib treated at 33 italian hematological institutions have been retrospectively evaluated. Median age at diagnosis was 78.5 years (range 75.0-93.0); 111 (52.4%) were male. Sixty-two (29.2%) were Sokal high risk. Sixty-seven (31.8%) were treated with reduced dose imatinib (400 mg/day. Concomitant drugs were 1-2 in 73 (34.4%) patients, 3-4 in 59 (27.8%), and 〉5 in 64 (30.2%); 16 (7.6%) did not assume any concomitant drug. Drugs more frequently used were antiplatelets, assumed by 104 (49.1%) patients, followed by diuretics in 91 (42.9%) patients, proton pump inhibitors (PPIs) in 86 (40.6%), ACE inhibitors in 55 (25.9%), beta blockers in 44 (20.7%), angiotensin II receptors blockers (ARB) in 41 (19.3%), calcium channel blockers in 34 (16%), statins in 25 (11.8%), and alpha blockers in 11 (5.2%). Univariate logistic regression models were computed to assess the association between cytogenetic response after 6 or 12 months of imatinib treatment and number of concomitant drugs or selected drug classes. Statistical analyses were done using JMP 11.1 (SAS Institute Inc., Cary, NC, USA). Complete cytogenetic response (CCyR) was obtained in 124 (58.8%) patients, of whom 70 (33%) within 6 months. Consequently, we focused our study on the impact of number and types of drugs on CCyR rate, which represents the primary therapeutic endpoint in the elderly. Cytogenetic response distribution according to concomitant drugs is reported in table 1. We did not find any significant correlation between number of concomitant drugs, single classes of antihypertensive drugs, antiplatelets, PPIs or statins and CCyR rate at 6 or 12 months. Even though few pharmacokinetic interactions are reported between imatinib and some of medications we considered, this does not seem to have an impact on cytogenetic response rate in our cohort. Indeed, our results confirm the well-known safety and efficacy of imatinib also in very elderly CML patients. Table 1. Cytogenetic response according to concomitant drugs Drug classes Cytogenetic response CCyR 12 months No CCyR Antiplatelets (n=104) 38 (36.5%) 31 (29.8%) 11 (10.6%) 24 (23.1%) Diuretics (n=91) 32 (35.2%) 21 (23.1%) 13 (14.3%) 25 (27.4%) Proton pump inhibitors (n=86) 30 (34.9%) 22 (25.6%) 13 (15.1%) 21 (24.4%) ACE inhibitors (n=55) 19 (34.6%) 11 (20%) 12 (21.8%) 13 (23.6%) Beta blockers (n=44) 18 (40.9%) 11 (25%) 3 (6.8%) 12 (27.3%) Angiotensin II receptor blockers (n=41) 19 (46.3%) 11 (26.8%) 5 (12.3%) 6 (14.6%) Calcium channel blockers (n=34) 10 (29.4%) 7 (20.6%) 6 (17.7%) 11 (32.3%) Statins (n=25) 9 (36%) 7 (28%) 2 (8%) 7 (28%) Alpha blockers (n=11) 4 (36.4%) / 1 (9.1%) 6 (54.5%) Disclosures Castagnetti: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Tiribelli:Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Rosti:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau.

Description: Background: The most important drawback of clinical trials of high-dose therapy (HDT) followed by autologous stem cell transplant (ASCT) in lymphomas is the high heterogeneity of histological entities. Therefore, the statistical power is reduced, and data are not conclusive. We previously demonstrated the safety of a new conditioning regimen with bendamustine, etoposide, cytarabine, and melphalan (BeEAM) prior to ASCT in resistant/relapsed lymphoma patients. This combination of drugs was able to induce a high CR rate in a population that did not have an opportunity of being cured with other therapies. However, that study enrolled both Hodgkin and non-Hodgkin lymphoma patients. Aims: We designed a phase II study to evaluate the efficacy of the BeEAM conditioning in resistant/relapsed diffuse large B-cell non-Hodgkin lymphoma (DLBCL) patients. Patients and methods: The study was registered at European Union Drug Regulating Authorities Clinical Trials (EudraCT) N. 2011-001246-14. Until now, 61 patients (median age 54 years, range 19-69) with resistant/relapsed DLBCL were enrolled. The primary end-point of the study is to evaluate the 1-year complete remission rate. Results: Briefly, 46/61 patients had advanced stage disease (III-IV); 20 were primary refractory and 41 had relapsed after a median number of 2 lines of therapy (range: 1-3). Twenty-one patients had 1 or more relevant comorbidities (range: 1- 5). 30 patients were in II or subsequent CR after salvage therapy, whereas 27 were in PR and 4 had stable or progressive disease. A median number of 5.72x106 CD34+/kg cells (range 2.21-10.60) collected from peripheral blood was reinfused to patients. All patients engrafted, with a median time to ANC〉0.5x109/l of 10 days. Median times to achieve a platelet count 〉20x109/l and 〉50x109/l were 12 and 17 days respectively. Twenty-two out of 61 patients presented a fever of unknown origin (36%), whereas 24 patients (39%) presented a clinically documented infection. All patients received G-CSF after transplant for a median time of 8 days (range: 8-13). One patient died due to an incomplete hematological recovery after transplant, producing an overall transplant related mortality of 2.7%. Fifty-seven patients are evaluable for response: 48/57 (84%) obtained a CR, 3/57 (5%) a PR, whereas 6/57 (11%) did not respond to therapy. After a median follow-up of 10.5 months after transplant (range 3-37), 6/57 (11%) patients were refractory, 12/57 (21%) relapsed and 39/57 (68%) are still alive, in continuous CR. Conclusion: Our clinical trial was designed to closely resemble real-world treatment for these patients. During the study, we transplanted a similar proportion of the patients that would have received ASCT in a real-world scenario. While we cannot make sound comparisons without head-to-head trials, results from previous studies using HDT regimens in DLCBL have not been as encouraging as ours. Accordingly, our data preliminary provide the evidence that the Benda-BEAM regimen is safe and has promising high efficacy in resistant-relapsed aggressive DLBCL patients. Acknowledgments: The study was supported in part by AIL Pesaro Onlus. Mundipharma Italy is grateful acknowledged for providing Bendamustine free of charge. Disclosures Patriarca: Janssen-Cilag, Celgene, Merck Sharp & Dohme: Honoraria. Zinzani:Gilead: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; J&J: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Older (age ≥60 years) patients with acute myeloid leukemia (AML) have poor outcomes and intensive induction chemotherapy is frequently unsuitable. Thus, new safe and effective therapies are urgently needed. Tosedostat, a new, orally bioavailable inhibitor of members of the M1 and M17 classes of aminopeptidases, was proven to be effective in both de novo and relapsed AML. We hypothesize that the addition of tosedostat to cytarabine may improve the response rate and remission duration over what is expected with chemotherapy or tosedostat alone. Methods: This was a phase II, prospective, multicenter study, designed according to Fleming's method. Fixing the lowest acceptable rate as 10% and the successful rate as 25%, with a significance level alpha=0.05 and a power 1-beta =0.80, the sample size was estimated in 33 patients. Thirty-three patients (median age 75 years) received Tosedostat 120 milligrams orally once daily until disease progression, coupled with intermittent low-dose cytarabine given subcutaneously at 20 milligrams twice/day for 10 days. Courses of cytarabine were repeated every 4 weeks in the absence of disease progression or unacceptable toxicity, up to 8 cycles. Global gene expression profiling (GEP, Affymetrix Human Gene 2.0 Array) was performed on purified AML blasts of 29 patients from peripheral blood or bone marrow at diagnosis before treatment initiation. Unsupervised clustering was generated using a hierarchical algorithm based on the average-linkage method. Principal component analysis (PCA) and supervised gene expression analysis was performed by using GeneSpring GX 12.0 (Agilent, USA). Results: The characteristic of enrolled patients are listed in table 1 Induction-period mortality was 12%, with 4 deaths occurring in aplasia. According to intent-to-treat, the CR rate was 48.5% (16/33 patients); 2 additional patients obtained a partial response, for an overall response rate of 54.6%. In addition, 4/33 patients remained in stable disease for a median time of 9 months (range: 4-14). Seven patients did not respond and died with progressive disease after having received a median of 2 cycles of cytarabine and 45 days of tosedostat. In responding patients, the median time to best response was 74 days (range 22-145). Responding patients (CR+PR) had a longer median overall survival than non-responders (P=0.018). Six out of 18 (33%) responding patients are still in CR after a median follow-up of 425.5 days (range 208-758); 5 additional patients are alive with stable disease. Twenty-two patients died [while in aplasia (4), in CR (1), due to resistant disease (9) or due to progressive disease (8) after a median CR duration of 192 days (87-535)]. 29 patients had GEP analysis, and a molecular signature associated with the clinical response (CR vs. no CR) was identified. By supervised analysis, 212 genes differentially expressed based on the clinical response (complete remission (CR) vs no CR) were identified (Mann-Whitney, p2). The 212 genes differentially expressed were significantly associated with six relevant biological functions and pathways: β-catenin (βcat); TNFα signaling pathway via NFκB; ERB2; STK33/SKM (serine/threonine kinase 33 expression using the SKM cell line); inflammatory response; and epithelial-mesenchymal transition pathways. Conclusions: The tosedostat and low-dose cytarabine combo produced a CR rate superior to what expected (45.4% versus 25%), and thus met the primary endpoint of study. Further, potential biomarkers were identified by GEP. Specifically, the achievement of CR could be efficiently predicted by the gene expression patterns with an overall accuracy exceeding 90%. A validation analysis is currently being conducted on additional 14 patients in order to confirm the ability of GEP to identify potential responders to TST. The study was registered at European Union Drug Regulating Authorities Clinical Trials (EudraCT) n.2012-000334-19. Acknowledgments: CTI is gratefully acknowledged for providing Tosedostat for the patients. The study was supported in part by AIL Pesaro Onlus. Table 1. Characteristic N = 33 Median age, years (range) 75 (62-85) Median WBC count, × 109/L (range) 3.05 (0.26-24.53) Blasts, % (range) 60 (20-96) Cytogenetic risk group*, n Not evaluable 3 (9%) Intermediate Karyotype 17 (52%) Unfavourable Karyotype 13 (39%) AML, n De novo 16 (48%) Secondary, n 17 (52%) Disclosures Fanin: Novartis Farma: Speakers Bureau.

Description: Acute myeloid leukemia (AML) cells are poorly immunogenic and release soluble factors inhibiting T-cell function. AML-derived dendritic cells (AML-DCs) have better antigen presentation capacity than leukemic blasts but share with AML cells some immunosuppressive features. In this study, we show that AML-DCs generated from CD14− AML samples (which represent 80% of total AML patients) are defective in IL-12 production. We, then, transfected CD14−-derived AML-DCs with IL-12 gene through the novel non-viral method nucleofection. IL-12 gene-nucleofected AML-DCs produce significant amount of IL-12 while maintain leukemia-specific karyotype, DC-like phenotype and function. In presence of the supernatant from the human leukemic cell line K562, allogeneic T-cell proliferation and interferon (IFN)-γ production induced by mock-transduced AML-DCs are significantly reduced. This effect is mainly directed on T cells, since AML-DC phenotype and cytokine production are not affected by leukemic supernatant. However, when stimulated by IL-12-producing AML-DCs, T cells produce higher concentrations of IFN-γ, thus maintaining a Th1 cytokine profile. In conclusion, IL-12 gene can be expressed into AML-DCs defective in endogenous IL-12 production by using a novel non-viral method which does not modify their phenotypical, cytogenetic and functional features. IL-12 gene expression into AML-DC counteracts the inhibitory effect of leukemic microenvironment on T lymphocytes

Description: Background: In the ENESTnd trial, nilotinib (NIL) showed a superior efficacy to imatinib (IM) with higher response rates and less frequent progression to advanced phases (ABP). Based on these results, NIL has been approved for frontline treatment of chronic myeloid leukemia (CML). The treatment-free remission (TFR) is actually considered one of the most important treatment goals in CML and a sustained deep molecular response (DMR, MR4 or better) is a pre-requisite to achieve TFR. The 5-year update from the ENESTnd trial showed a superiority of NIL over IM in terms of both MR4 and MR4.5, but differences concerning the stability of DMR have not been reported. Moreover, a significant improvement in the long-term outcome has not been demonstrated yet. Despite the efficacy, cost and safety concerns may limit the NIL use as first line treatment in CML. Independent studies are extremely relevant to confirm or to extend the results of company-sponsored trials. Aims and Methods: To assess the efficacy of NIL frontline in terms of DMR, a phase 3b study was conducted by the GIMEMA CML WP (CML0811; NCT01535391). The primary endpoint was the rate of MR4 at 24 months. Key secondary objectives: evaluation of the kinetics of molecular response, assessment of the safety profile, analysis of the outcome. The starting NIL dose was 300 mg BID, with dose escalation to 400 mg BID in case of suboptimal response or failure according to ELN 2009 criteria, with the exception of progression to ABP and in absence of safety issues or BCR-ABL mutations insensitive to NIL. The molecular response was assessed in GIMEMA standardized molecular laboratories (LabNet network) and the results were expressed according to the International Scale. The MR4 was defined as either detectable disease ≤ 0.01% BCR-ABL or undetectable disease with ≥ 10.000 ABL copies; the MR4.5 was defined as either detectable disease ≤ 0.0032% BCR-ABL or undetectable disease with ≥ 32.000 ABL copies. Sustained MR4 or MR4.5: MR4 or MR4.5 for at least 1 year a, with at least 3 evaluable analysis. Adverse events were recorded continuously. A prospective evaluation of glucose metabolism and serum lipids was planned. All the analysis were performed according to the ITT principle. Results: 130 CML patients in early chronic phase have been enrolled in 32 italian hematologic centers; median age, 50 years (range 18-85); high risk patients, 22%, 6% and 8% according to Sokal, Euro and EUTOS scores, respectively; clonal chromosomal abnormalities in Ph+ cells at baseline, 5%; e13a2 BCR-ABL transcript, 34%. The median follow-up is actually 21 months (all patients had at least 18 months observation; a minimum observation of 24 months will be reached by October 2014). Data with at least 24 months follow-up will be presented on site. At the last contact, the patients still on treatment with NIL were 110/130, 85% (74% with 600 mg, 7% with 300 mg or less, 4% with 800 mg daily), while 20/130 patients, 15%, permanently interrupted the study drug for the following reasons: 3% progression to ABP, 2% failure or suboptimal response (dose escalation not feasible), 1% allogeneic stem cell transplantation, 5% toxicity, 4% other reasons (including consent withdrawal and pregnancy). The complete cytogenetic response rate and the major molecular response rate at 12 months were 76% and 53%, respectively. The rates of MR4 at 3, 6, 12 and 18 months were 2%, 12%, 27% and 29%, respectively. Fifty-four patients achieved a MR4 at least once; the patients with a sustained MR4 were 18/54 (33%, or 14% of the total). The rates of MR4.5 at 3, 6, 12 and 18 months were 0, 3%, 10% and 13%, respectively. Only 3 patients achieved a sustained MR4.5. A significant increase of glycosylated hemoglobin was not observed. The total cholesterol, and both LDL and HDL cholesterol fractions significantly increased during treatment. Triglyceride concentrations had not significant variations. Six patients (5%) had a cardiovascular event, including myocardial infarction and arterial thrombosis. All the patients are still alive. Conclusions: The molecular response rates seem to be superior to the historical data of IM. NIL 300 mg BID as frontline treatment of BCR-ABL+ CML, with dose optimization in case of non optimal response, may improve the proportion of patients able to discontinue TKI treatment. Due to the metabolic effects, a baseline selection is crucial to maximize the therapeutic benefit and to minimize the cardiovascular risks. Disclosures Castagnetti: Pfizer: Consultancy; Bristol Myers Squibb: Consultancy, Honoraria; Novartis Farma: Consultancy, Honoraria. Gugliotta:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rossi:NOVARTIS: Consultancy, Speakers Bureau; BRISTOL MYERS-SQUIBB: Consultancy, Speakers Bureau; ARIAD: Consultancy; ROCHE: Speakers Bureau. Turri:Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria. Tiribelli:Novartis: Consultancy, Honoraria; Bristol-Meyers and Squibb: Consultancy, Honoraria. Soverini:NOVARTIS: Consultancy; BRISTOL MYERS: Consultancy; ARIAD: Consultancy. Cavo:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Millenium Pharm: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Onyx: Honoraria. Martinelli:Pfizer: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau. Saglio:BMS: Consultancy, Fees for occasional speeches Other; Novartis: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; Pfizer: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other; ARIAD: Consultancy, Fees for occasional speeches, Fees for occasional speeches Other. Baccarani:ARIAD: Honoraria; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Rosti:ROCHE: Speakers Bureau; ARIAD: Consultancy; Bristol Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

Description: Background Myelofibrosis (MF) is featured by an inflammatory condition that can also drive the progression of disease. Ruxolitinib (RUX) is the-first-in-class Jak1/2 inhibitor approved for treatment for MF. Clinical benefits of RUX are presumably derived from reduction of inflammatory cytokines even if the exact mechanism remains unclear. Recent reports have identified the ratio between absolute neutrophils count (ANC) and absolute lymphocyte count (ALC), called NLR, as a simple parameter that mirrors the inflammatory status and the myeloid associated immune suppression. In various malignancies NLR has been indicated as predictor of progression free survival (PFS) and overall survival (OS). Our preliminary work in a single-center experience showed that patients with NLR〉6 before RUX start had a lower chance to obtain 〉 50% spleen reduction in the first 12 weeks or a complete resolution of splenomegaly at 24 weeks. Objective : We proposed to test NLR=6 as bio-marker in MF to apply into clinical practice as a possible predictor of response to RUX. Methods We used two separate cohorts to validate NLR (as a continuous variable and as a cut off 6) as predictor of response to RUX bases on our preliminary data from healthy volunteers (data not shown). Cohort (#1) including 111 MF patients from MD Anderson Cancer Center treated with RUX on phase 1/2 clinical trial from 2007 to 2010; and cohort (#2) including 367 patients treated at 18 Italian centers between years 2012 - 2018. Spleen responses to RUX treatment, PFS and OS were independently validated in cohorts #1 and 2. As cohort 1 included patients treated on clinical trial, spleen was assessed by MRI before and after 24 weeks of RUX therapy, and by physical examination at week 12. In cohort #2, spleen size was assessed by physical examination before, after 12 and 24 weeks of RUX continuous treatment in a real-life setting. NLR was calculated using data obtained from the complete blood count before RUX start and correlated with driver mutations, early spleen reduction, progression free survival (PFS), defined as time from RUX start to last follow-up or progressive disease (including progression to acute myeloid leukemia, ≥20% blasts in peripheral blood or bone marrow, AML) or death for any reason; and overall survival (OS). Results : Clinical and demographics characteristics of patients in each cohort are summarized in Table 1. In cohort #1 we found that NLR was lower in patients with lower bone marrow fibrosis (grade 0-1: 6.2±0.8 versus grade 2-3: 7.3±0.8, p=0.03). Similarly, in cohort #2, patients with grade 0-1 bone marrow fibrosis had lower NLR than those carrying grade 2-3 bone marrow fibrosis (7.7±0.7 versus 10.6±1.3, p=0.04). NLR was higher in patients carrying JAK2 (V617F) mutation (mean +/- SD, 6.4±0.6 vs 5.3±0.5, p=0.02 in cohort 1 and 9.1±0.6 vs 5.0±0.5, p=0.002 in cohort 2). While in cohort 1 NLR appeared lower in CALR (exon 9 indel) mutated patients, the difference was statistically significant in cohort 2 (5.4±0.8 vs 8.9±0.6, p=0.03). In both cohorts, there were no differences in NLR in either triple negative or MPL (exon 10) patients. In cohort 1, the mean percentage change from baseline in palpable spleen length was −47.7% at week 12 and −53.4% at week 24. NLR=6 was able to identify at baseline early response to RUX with 66.9% sensitivity and 72.3% specificity (HR 1.68, p=0.01). Patients with NLR〉6 before RUX start had a lower chance to obtain a complete resolution of splenomegaly at 24 weeks (p=0.001). These observations were confirmed in cohort 2 where NLR 〉 6 was able to identify at baseline early response to RUX with 50.3% sensitivity and 67.7% specificity (HR 1.56, p=0.01). The mean percentage change from baseline in palpable spleen length was −60.3% at week 12 and −66.7% at week 24. Patients with NLR〉6 before RUX start had a lower chance to obtain a complete resolution of splenomegaly at 24 weeks (p