Abstract: The aim of this study was to develop a predictive score for moderate-severe chronic graft-versus-host disease (cGVHD) on day +100 after allogeneic stem cell transplantation (HSCT). We studied 1292 patients allografted between 1990 and 2016, alive on day +100 after transplant, without cGVHD, and with full biochemistry laboratory values available. Patients were randomly assigned to a training and a validation cohort (ratio 1:1). In the training cohort, a multivariate analysis identified 4 independent predictors of moderate-severe cGVHD: gamma-glutamyl transferase ≥75 UI/l, creatinine ≥1 mg/dl, cholinesterase ≤4576 UI/l, and albumin ≤4 g/dl. A score of 1 was assigned to each variable, producing a low (0 to 1), intermediate (2 to 3), and high (4) score. The cumulative incidence of moderate-severe cGVHD was 12%, 20%, and 52% (P & lt; .0001) in the training cohort, and 13%, 24%, and 33% (P = .002) in the validation cohort, respectively. The 5-year cumulative incidence of transplant-related mortality (TRM) was 5%, 14%, 27% (P & lt; .0001) and 5%, 16%, 31% (P & lt; .0001), respectively. The 5-year survival was 64%, 57%, 54% (P = .009) and 70%, 59%, 42% (P = .0008) in the 2 cohorts, respectively. In conclusion, Day100 score predicts cGVHD, TRM, and survival and, if validated in a separate group of patients, could be considered for trials of preemptive therapy.

Abstract: Role of erythropoietin (EPO) in the treatment of late anemia in patients with Chronic Myeloid Leukemia (CML) is still undefined. Methods Fifty CML patients treated at 14 institutions with frontline imatinib for at least 12 months and in stable complete cytogenetic response who developed a late chronic anemia treated with EPO were retrospectively evaluated. Results Median time from imatinib start to EPO treatment was 42.2 months [interquartile range (IQR) 20.8‐91.9]. Median Hb value at EPO starting time was 9.9 g/dL (IQR 8.9‐10.3): Eleven patients (22.0%) were transfusion dependent. Alpha‐EPO (40 000 UI weekly) was employed in 37 patients, beta‐EPO (30 000 UI weekly) in 9 patients, zeta‐EPO (40 000 UI weekly) in 2 patients, and darbepoetin (150 mcg/weekly) in the remaining 2 patients. On the whole, 41 patients (82.0%) achieved an erythroid response, defined as a stable ( 〉 3 months) improvement 〉 1.5 g/dL of Hb level, and 9 patients (18.0%) indeed resulted resistant. Among responding patients, 10 relapsed after a median time from EPO start of 20.7 months (IQR 10.8‐63.7). No EPO‐related toxicity was observed. Conclusions Results of EPO treatment for late chronic anemia during long‐lasting imatinib therapy are encouraging, with a high rate of response.

Abstract: Introduction Some retrospective studies in tyrosine kinase inhibitor (TKI)-resistant Philadephia-positive (Ph+) leukemia patients (pts) have suggested that deep sequencing (DS) may provide a more accurate picture of BCR-ABL1 kinase domain (KD) mutation status as compared to conventional sequencing (CS). However, the frequency and clinical relevance of low burden mutations remains to be explored prospectively in large series of unselected pts. In addition, the implementation of routine BCR-ABL1 DS in multiple molecular diagnostic laboratories has never been attempted. These open issues led us to design a multi-center, multi-laboratory prospective study ('NEXT-IN-CML') aimed to assess the feasibility, performance and informativity of DS for BCR-ABL1 KD mutation screening. Aims The first phase of the study was aimed to establish a network of 5 reference labs sharing a standardized DS workflow, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and clinical reporting. The second phase of the study, involving 54 Italian Hematology Units, is aimed to assess the frequency and clinical significance of low burden mutations detectable by DS by prospective collection and analysis of samples from chronic myeloid leukemia (CML) pts who exhibit failure (F) or warning (W) responses and relapsed Ph+ acute lymphoblastic leukemia (ALL) pts. Methods A PCR and an amplicon DS protocol already set up and optimized for the Roche GS Junior in the framework of the IRON II international consortium was adopted. In the first phase, 5 batches of blinded cDNA samples were prepared and shipped to evaluate individual lab performances. The batches included archival samples with known BCR-ABL1 mutation status as assessed by CS and serial dilutions of BaF3 T315I+ cells in BaF3 unmutated cells, simulating mutation loads of 20% down to 1%. In the ongoing second phase prospectively, consecutively collected CML and Ph+ ALL samples are being analyzed in parallel by CS and DS. Clinical history and follow-up data are used for correlations. Results In the first phase of the study, 312/320 amplicons were successfully generated and sequenced. A median of 124,686 (range, 48,181-170,687) high quality reads were obtained across the 5 labs. Median number of forward and reverse reads was 1,757 (range 884-7,838), with no coverage dropouts for any amplicon or index. Comparison of observed vs expected mutations showed that 76/78 evaluable samples were accurately scored. In the remaining two, the analysis software failed to detect the 35bp insertion ('35INS') commonly detectable between exons 8 and 9. Quantitation of point mutation burden was highly reproducible across the entire range of frequencies, from 100% to 1%. The second phase of the study has started in Jan 2016. As of Jul 31st, a total of 106 consecutive pts (CML, n=96; Ph+ ALL, n=10) have been enrolled. The present analysis focuses on the first 75 CML pts (60 F and 15 W), for whom sequencing results are currently available (analysis of the entire population of patients enrolled up to Nov 2016 will be presented at the meeting). Clinically actionable mutations have been detected in 10/75 (14%) pts by CS and in 20/75 pts (27%) by DS. Notably, among the 10 pts positive for clinically actionable mutations by DS but not by CS, 3 had a low burden T315I (2 F [dasatinib, imatinib] and 1 W [dasatinib] ). In 5 additional pts negative for mutations by CS (3 F and 2 W), DS identified multiple low burden mutations with unknown IC50, suggesting that the cooperation of individually 'weak' mutants may be a new mechanism underlying reduced TKI efficacy. Longitudinal analysis and follow-up of pts are shaping the clinical significance of different types of low burden mutations and will be presented. Conclusions The 'NEXT-in-CML' study is demonstrating that DS of BCR-ABL1 can successfully be implemented in national lab networks and is an important step forward towards routine use of this technology. We have now adapted the protocol for both the Ion Torrent PGM and the Illumina Miseq platforms. For a minimum of 15 samples per sequencing run, DS costs are estimated to equal those of CS (cost per sample, reagents only: ≈100€ for PGM (314 chip) and Miseq (nano kit v2) vs ≈95€ for CS) with comparable turnaround times for delivery of results. Our study is also contributing useful data for the clinical interpretation of DS findings. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Ciceri:MolMed SpA: Consultancy. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Di Raimondo:Janssen-Cilag: Honoraria. Bassan:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Saglio:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy.

Abstract: While there is intense interest in the production of allogeneic CAR‐T cells from umbilical cord units, little is known about the reactivity and persistence of CAR‐T cells of umbilical origin. We report the case of a patient at our hematological center with multiple relapsing Ph+ B‐ALL, notably a Blinatunomab non‐responder, who underwent therapy with Brexucabtagene Autoleucel following relapse on Ponatinib post‐allogeneic hematopoietic stem cell transplantation. The patient achieved a rapid CAR‐T expansion and durable remission presenting in good clinical conditions 6 months post‐CAR‐T infusion, without manifestations of graft‐versus‐host disease. The case report provides insight into the reactivity and persistence of CAR‐T cells of umbilical origin, confirming the potential promise of allogeneic umbilical cord‐derived CAR‐T cells.

Abstract: The chimerism analysis after allogeneic haematopoietic stem cell transplantation (HSCT) is a useful tool to monitor the engraftment of donor cells and to early predict graft failure or disease relapse. Among several available methods to perform chimerism study, multiplex fluorescent short tandem repeat (STR) analysis represent a sensitive, accurate and reproducible technique. It provide a high rate of discrimination between donor and recipient cells since that fluorescent signal is proportional to the cells number, obtaining a quantitative assessment of chimerism. Aims Evaluation of a possible correlation between basal donor/recipient allelic discrepancies and post-transplant outcome in terms of graft versus host disease (GvHD) development, neutrophil engraftment, relapse, DFS and OS. Methods We enrolled 111 patients (pts) affected by onco-haematological diseases, consecutively submitted to HSCT at our division between February 2005 and December 2012. Pts were 67 males and 44 females with a median age of 51 years (range 14-70). Underlying diseases were: 58 AML, 10 MDS, 16 ALL, 8 CLL, 6 MM, 2 HL, 9 NHL, 1 IMF, 1 MPD. Myeloablative conditioning regimen was used in 26 pts while a reduced conditioning regimen was used for the other 85 pts. Graft was obtained from sibling donor and unrelated donor in 65 and 46 pts, respectively. GvHD prophylaxis was performed with cyclosporine (CSA) and methotrexate in 61 pts, CSA and mycophenolate mofetil in 43 pts and CSA in 7 pts. Donor and recipient allelic status was performed using a multiplex PCR amplification of ten STR loci: Amelogenin alleles X/Y, D3S1358, FGA, D8S1179, D18S51, D13S317, vWA, D21S11, D5S818, D7S820. We evaluated the donor/recipient (D/R) match or mismatch for each locus and the following variables: GvHD development and onset time, time to neutrophil engraftment, relapse, DFS and OS. Statistical analysis was performed by Kaplan Meier analysis using IBM SPSS Statistics 20 Core System. Results Pts with D/R mismatch for D8S1179 locus achieved neutrophil engraftment (ANC 〉 1* 109/L) at a median time of 18 days (CI 95% 17.202-18.798) compared with 21 days (CI 95% 17.328-24.672) of pts with D/R match for the same locus (p=0.007) (Figure 1A). Pts with D/R mismatch for D3S1358 locus developed acute GvHD at a median time of 20 days (CI 95% 15.781-24.219) compared with 29 days (CI 95% 15.421-42.579) of pts with D/R match for the same locus (p=0.031) (Figure 1B). Pts with D/R mismatch for D3S1358 locus showed an OS of 16 months (CI 95% 11.016-20.984) compared with 41 months (CI 95% 25.420-56.580) of pts with D/R match for the same locus (p=0.025) (Figure 1C). Pts with D/R mismatch for D8S1179 locus showed an OS of 16 months (CI 95% 8.528-23.472) compared with 56 months (CI 95% 11.254-78.057) of pts with D/R match for the same locus (p=0.012) (Figure 1D). No relationships were found with relapse, DFS neither for the other loci. Summary Our results highlight that pts who presented a D/R mismatch for D8S1179 locus showed an earlier neutrophil engraftment but a worse OS compared with the others. Moreover, for D3S1358 locus, D/R mismatch was associated with an earlier onset of acute GvHD after HSCT and with a shorter OS than the D/R match ones. Conclusion STRs are highly polymorphic di-, tri- and tetra-nucleotide repeat non-coding sequences, which are interspersed throughout the genome. Whether or not discrepancies between donor and recipient basal allelic status could be considered useful in predicting post-transplant outcome will require validation in a large sample of pts. Disclosures: No relevant conflicts of interest to declare.

Abstract: Median age of CML patients at diagnosis is reported to be around 66 years. Few data about the characteristics and outcome of patients younger than 40 years are available, and the clinical trials of dasatinib and nilotinib as first line treatment were not stratified by age. We retrospectively analyzed 251 young patients with CML in chronic phase from 12 different Italian Institutions, diagnosed between October 2001 and October 2016. Up to 2011 all patients were treated frontline with imatinib while from January 2011 onwards with imatinib or a second generation TKI (nilotinib or dasatinib), based on clinical judgment. At diagnosis median age was 32,6 years [interquartile range (IQR) 27,6- 36,9]; 143 (57%) were male. Splenomegaly was found in 129 out of 233 evaluable patients, in 29,2% of the cases spleen was palpable 〉 5 cm below the costal margin. The risk score was low in most of the cases (low risk Sokal 71%, low risk Eutos 91,3% vs high risk Sokal 9,8%, high risk Eutos 8,7%). Clinical features of the patients in treatment with different inhibitors are summarized in table 1. There were two main statistically significant differences in the group of patients treated with dasatinib: a higher proportion of high risk (30,8%) according to Eutos score and a lower median Hb level at diagnosis (8,5 gr/dL). These features probably reflected the trend of using dasatinib in patients with a more aggressive disease, as this drug was shown to be more potent since the first in vitro studies. Out of 251 patients 179 were treated with imatinib, 57 (22%) with nilotinib, 15 (5,9%) with dasatinib. Median follow up of the whole cohort was 76,5 months (IQR 41- 116); as expected, the follow up was longer in the imatinib group compared to the nilotinib and dasatinib groups (100 months vs 39,6 and 23,0 respectively). In the whole cohort, the cumulative incidence of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMolR) were 90,4% and 75,7% respectively; a deep molecular response (negative nested PCR, MR4.0, MR4,5) was achieved by 52.2% of patients, without differences among the 3 groups. Primary resistance occurred in 12,4% of patients, without differences among the 3 groups: secondary resistance occurred in 15.9% of patients, with a higher rate in the imatinib group (19,5%) as compared with nilotinib (7,0%) and dasatinib (6,7%) (p=0.047). Treatment discontinuation due to toxicity was observed in 6.0% of patients, without differences among the 3 groups. Blast transformation occurred in 7 out of 251 patients (2,8%), all in the imatinib group, after a median time from the diagnosis of 31 months (range 4-110). The 4-year cumulative Event-Free Survival (EFS) and Overall Survival (OS) were 72,4% (95%CI 66,7 - 78,5) and 98,1% (95%CI 96,4 - 99,8) respectively, without differences among the 3 groups. All deaths were related to blast transformation in imatinib group. In conclusion, as expected in a younger population, response to treatment and OS were excellent. However, the 4-year EFS was lower than expected, in spite of the presence of patients with predominantly low risk score. Furthermore, while the higher rate of secondary resistance in the imatinib group probably reflects the longer follow-up, it is worth of note that no statistically significant difference was observed between imatinib and 2nd generation TKI groups in terms of OS and EFS. Disclosures Breccia: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

Abstract: Background Dasatinib has been recently licensed for first line treatment of patients with chronic myeloid leukemia (CML). However, very few data are available as to toxicity and efficacy of dasatinib in unselected elderly CML patients. Aim To address this issue, we revised a “real-life” cohort of 43 CML patients in chronic phase aged 〉 65 years treated with frontline dasatinib in 19 Italian Centers from 6/2012 to 6/2014 focusing on toxicity and efficacy data. Methods The main clinical features of the patients at diagnosis were as follows: M/F 20/23 (46.5%/53.5%), median age 75.2 years [interquartile range (IQR) 70.3 – 79.8), median Hb 12.5 g/dl (IQR 11.0 – 13.7), median WBC 57.7 x 109/l (IQR 29.5 – 100.0), median PLTS 466 x 109/l (IQR 249 – 758). According to Sokal risk classification, 3 patients (6.9%) were low risk, 26 (60.4%) intermediate risk, 10 (23.2%) high risk while 4 (9.5%) were not classificable. 20/43 patients (46.5%) had ≥ 2 comorbidities requiring concomitant therapies: according to ECOG scale, performance status at baseline was 0 – 1 in 36 patients (83.7%) and 2 in 7 patients (16.3%). Results Median interval from diagnosis to dasatinib start was 23 days (IQR 14 – 32). Dasatinib starting dose was 140 mg/day in 1 patient (2.3%), 100 mg/day in 33 patients (76.7%) and 〈 100 mg/day in 9 patients (21.0%), respectively. After a median period of treatment of 9.7 months (IQR 4.3 – 17.5) all patients were evaluable for toxicity; on the whole, grade 3 – 4 hematological and extra-hematological toxicities were reported in 4 (9.3%) and 6 (13.9%) patients, respectively. Overall, 7 patients (16.2%) permanently discontinued dasatinib due to toxicity (2 patients in the first 3-month period of treatment and 5 beyond that period). Pleural effusions of all WHO grades occurred in 7 patients (16.2%): in 2 of them the pleural effusion occurred during the first 3-month period of treatment. As to treatment efficacy, 6 patients were considered too early to be evaluated ( 〈 3 months of treatment) and 37 were evaluable for cumulative response; on the whole, 33/37 patients (89.1%) achieved complete cytogenetic response (CCyR) and 23/37 (62.1%) also a major molecular response (MMolR). Response to treatment at different time-points is shown on Table.3rd month6th month12th monthNot evaluable: Too early Not performed11651311219190Evaluable323024Discontinuation2 (6.2%)4 (13.3%)6 (25%)Less than CCyR6 (18,7%)2 (6.7%)0CCyR only17 (53.1%)5 (16.6%)4 (16.6%)MMolR7 (21.9%)19 (63.3%)14 (58.4%) Conclusions Present data shows that dasatinib could have a major role in the treatment of unselected patients aged 〉 65 years; indeed, dasatinib seems very effective and has a favourable safety profile also in elderly subjects with comorbidities. Disclosures Latagliata: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Shire: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Breccia:novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy.

Abstract: Abstract 1229 In the “real world” of clinical practice, many very elderly CML patients have been treated with imatinib (IM), but there are few data on the results and the best initial dosage in such patients. To highlight peculiar aspects of toxicity and efficacy of IM in this subset which accounts for at least 10–15% of all CML cases, we retrospectively revised 156 CML patients in chronic phase treated with IM when aged 〉 75 years from 23 haematological Institutions in Italy; there were 85 males and 71 females, median age at IM start was 78.4 years (IR 76.1 – 81.4), Sokal Risk at diagnosis was low in 2 patients, intermediate in 90, high in 50 and not evaluable in 14. One or more concomitant diseases requiring specific treatments were present in 144/156 patients (92.3%), with 94 patients (60.2%) assuming 3 or more concomitant drugs. Thirty patients (19.2%) were in late chronic phase (≥ 12 months from diagnosis) and pretreated (25 with HU and 5 with IFN) before starting IM; on the whole, median time from diagnosis to IM was 1.2 months (IR 0.5 – 3.6). Starting dose of IM was 400 mg/day in 117 patients (75.0%) and 300 mg/day or less in 39 patients (25.0%); overall, 59 patients (37.8%) (52/117 at 400 mg starting dose and 7/39 at 3 300 mg starting dose) needed a dose reduction and 18 (11.5%) discontinued IM for toxicity (early toxicity in 13 and late toxicity in 5). Excluding the 13 patients who discontinued IM due to early toxicity, maximum tolerated daily dose during treatment was 400 mg in 63 patients, 300 mg in 51 patients and 〈 300 mg in 29 patients. According to CTC-AE, grade 3 – 4 hematological and extra-hematological toxicities were observed in 34 (21.7%) and 34 (21.7%) patients, respectively; 5 patients (3.2%) presented a pleural effusion during IM treatment. After a median treatment period of 29.4 months (IR 7.9 – 54.4), 8 patients (5.1%) are still too early ( 〈 6 months of treatment), 13 (8.3%) discontinued IM due to early toxicity, 3 (1.9%) were resistant and 1 (0.7%) died from unrelated cause early after IM initiation: the remaining 131 patients (84.6%) achieved a complete haematological response (CHR). Among these 131 patients in CHR, 11 refused any other karyotipic or molecular evaluation (1 lost CHR and shifted to hydroxyurea, 4 are still alive in CHR, 6 died in CHR from unrelated causes), 17 achieved CHR only and 103 (66.0% of all 156 patients) achieved a cytogenetic response (CyR), which was major in 11 patients and complete (CCyR) in 92 (58.9% of all 156 patients). In addition, among the 92 patients in CCyR, 62 (39.7% of all 156 patients) achieved a molecular response (major molecular response in 40 patients and complete molecular response with an undetectable BCR/ABL hybrid gene at qualitative nested PCR in 22 patients). After a median follow-up of 34.0 months (IR 12.9 – 60.0), 36 patients have died (5 from disease progression and 31 from unrelated causes), 4 patients were lost to follow-up and 116 are still alive: 2-year and 4-year overall survival were 90.2% (CI95% 84.8 – 95.6) and 76.8% (CI95% 68.6 – 85.0), respectively. In conclusion, results from this large unselected cohort of patients show that should be definitely considered unethical to avoid IM therapy to any elderly patient; no upper age limit should be given but also very elderly (and with concomitant severe diseases) patients should have this chance of cure. The role of a reduced starting dose of IM warrants further studies. Disclosures: No relevant conflicts of interest to declare.