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: Background and Rationale. Bosutinib (BOS), dasatinib (DAS) and nilotinib (NIL) are 2nd generation TKIs with similar second-line efficacy. The use of DAS and NIL may be burdened by pulmonary, infectious, cardiovascular and metabolic complications; these complications are more frequent and more clinically relevant in the elderly. BOS could represent an important therapeutic option in elderly patients intolerant to or failing a first-line TKI, but the dose of 500 mg OAD may be higher than necessary. Aims. All TKIs have been tested at a fixed initial dose, with dose adjustment in case of toxicity or treatment failure. On the contrary, the aim of our study was to evaluate in elderly CML patients if second-line BOS was effective and better tolerated at doses lower than 500 mg OAD, beginning with 200 mg OAD, then increasing the dose to 300 OAD or 400 mg OAD according to the molecular response, to find the minimum effective dose. Methods. A prospective phase 2 single-arm multicenter study has been designed by the GIMEMA CML Working Party (NCT02810990). Study design: all patients started with 200 mg OAD for 2 weeks ("run-in" period), then the dose was increased to 300 mg OAD; after 3 months, patients with BCR-ABLIS transcript ≤ 1% continued 300 mg OAD, while in patients with transcript & gt; 1% the dose is furtherly increased to 400 mg OAD. In responsive patients, BOS dose was maintained, 300 mg or 400 mg OAD. Key inclusion criteria: & gt; 60 yrs old, chronic phase CML, intolerance or failure of any first-line TKI (2013 ELN criteria), absence of T315I or V299L mutation. Sixty-three patients have been enrolled. The primary endpoint was the proportion of patients in MR3 at 1 year. Definitions: MR3, BCR-ABLIS & lt; 0.1%; MR4, BCR-ABLIS & lt; 0.01% with & gt; 10.000 copies; MR4.5, BCR-ABLIS & lt; 0.0032% with & gt; 32.000 copies. Results. Median age: 73 yrs (range 60-90). Age distribution: 60-69 yrs, 18 pts (29%); 70-79 yrs, 31 pts (49%); & gt; 80 yrs, 14 pts (22%). Sokal score at diagnosis: low 19%, intermediate 49%, high 32%. Reasons for switching to BOS: intolerance 63%, resistance 37%. First-line TKI: imatinib 83%, DAS 11%, NIL 6% (same TKI distribution in intolerant and resistant patients). Median follow-up: 9 mos (range 1-30). Overall, 10/63 patients had a dose-increase to 400 mg OAD, 49/63 to 300 mg OAD, while 4/63 continued on BOS 200 mg OAD without any dose increase. At baseline, 13 patients were already in MR3. The MR3 rates by 3 and 6 months were 43% and 56%, respectively. The cumulative rate of patients achieving or maintaining a MR3 by 12 months was 60% (65% in intolerant and 52% in resistant patients, p = 0.31). Interestingly, only 21% of patients & gt; 80 yrs old achieved or maintained a MR3 (p & lt; 0.001, compared to younger patients). Patients achieving MR4 or MR4.5 were 38% and 19%, respectively. Overall, 22%, 27% and 11% of patients had 1 log, 2 logs or & gt; 3 logs reduction from baseline BCR-ABLIS transcript level. Selected adverse events: cardiac ischemia, 2 patients; pericardial effusion, 2 patients; no pleural effusions. Events leading to permanent treatment discontinuation: 2 unrelated deaths, 7 adverse events (3 hypertransaminasemia, 1 nephrotoxicity, 1 diarrhea, 1 skin rash, 1 myalgia/fatigue), 3 unsatisfactory responses (without progressions). Fifty-one out of 63 patients are still on BOS at the last contact: 6 on 400 mg OAD, 34 on 300 mg OAD, 11 on 200 mg OAD. Conclusions. A gradual dose increase, based on prospective molecular monitoring, allowed the great majority of enrolled patients (approximately 70%) to remain on treatment with BOS 300 mg OAD or less, achieving a major molecular response (MR3) in 60% of the cases. These results trial showed that, in elderly patients intolerant to or failing a first-line TKI, BOS may be highly effective and better tolerated at a dose lower than 500 mg OAD, namely at 300 mg OAD. Disclosures Castagnetti: Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Bocchia:Novartis: Honoraria; Incyte: Honoraria; BMS: Honoraria. Trawinska:Novartis: Consultancy, Honoraria. Bonifacio:Novartis: Honoraria, Research Funding; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Elena:Novartis: Consultancy; Pfizer: Consultancy. Lucchesi:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Albano:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Lunghi:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Stagno:BMS: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Pregno:Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria. Iurlo:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Ferrero:Novartis: Honoraria. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: 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; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Jansen: Consultancy; Incyte: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Bosutinib is a second generation TKI already registered for the treatment of adult patients with chronic phase CML with resistance or intolerance to prior therapy. In the present study the initial dose is 200 mg instead of 500 mg OAD. A dose increase to 300 or 400 mg is scheduled according to molecular response and tolerabiliy

Abstract: Imatinib mesylate (Glivec, Novartis) is a tyrosine kinase specific inhibitor that kills BCR-ABL cells in vitro and in vivo. Cytogenetic abnormalities in Ph-negative cells emerging after treatment-induced suppression of the neoplastic clone have been described. A registry through the GWP in CML has been set and data on 23 patients collected. To acquire insights into the origin of the Ph-negative clone as well as the evolution of the coexisting Ph− and Ph+ cell populations, we have analyzed bone marrow cell segregation, cell culture and morphologic features. Patients characteristics and 28 months follow up are presented. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 23 patients after a median of 14.5 months after starting Imatinib. Median age was 51 years, median time from diagnosis 36 months. All patients started Imatinib while in chronic phase and none of the patients had ever presented accelerated or blastic phase. Five patients were treated with Imatinb at onset. Cytogenetics at diagnosis was characterized by the presence of Ph chromosome, except for one patient which presented with normal karyotype, but BCR-ABL B3A2 transcript. No additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 16 complete, 4 major and 3 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 13 patients, -Y in 2 patients, one −7, del(5q), del(7q), del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), and one patient presenting with both +8 and +21. The patient with dup(1q) maintained the abnormality while clearing the marrow from Ph positive cells (constitutional karyotype was normal). Retrospective analyses of stored pellet using FISH in patients presenting +8, −7, or −Y, did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 5 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. We performed cell culture on a subgroup of patients demonstrating normal growth in four patients and an abnormal growth in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone segregated into the CD34+ compartment suggesting the stem cells involvement. FISH on cultured cells did not demonstrate a growth advantage for Ph+ cells or for the new clone. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. While a longer follow up observation and laboratory analyses are required, we remark that after & gt;2 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS, nor it seems to be associated with CML clonal evolution and disease progression. Hypothesis regarding the biological significance of these abnormalities are formulated.

Abstract: Introduction: In chronic myeloid leukemia (CML), tyrosine Kinase Inhibitors (TKIs) treatment is a potentially life-time therapy for the majority of patients (pts), as few of them, only after achieving a deep and stable molecular response, may discontinue TKIs without recurrence of disease. Available data suggest that relapse after TKIs discontinuation is due to the persistence of leukemic stem cells (LSCs) intrinsically resistant to TKIs. Survival of CML LSCs may be the consequence of activation of several pathways BCR-ABL1 independent. qRT-PCR, the most sensitive assay to monitor disease status in CML pts, may be inappropriate to quantify residual quiescent CML LSCs that are transcriptionally silent. Therefore, the possibility to easily quantify LSCs during TKIs treatment is a great opportunity to better understand the behavior of residual LSCs and potentially to identify those pts candidates to safely discontinue TKIs. Recently, Valent et al described that CD34+/CD38-/Lin- CML LSCs specifically co-express dipeptidylpeptidase IV (CD26) and that CD26 is a potential biomarker for the quantification and isolation of CML LSCs, in bone marrow samples of CML patients. Furthermore, Culen et al. quantified CD26+ LSCs bone marrow compartment in 31 CML patients at diagnosis and their number appears to correlate with response to TKIs treatment. In the present study we wanted to explore the feasibility, rate and potential implication of detecting CD26+ LSCs in peripheral blood (PB) from CML pts during TKI treatment. Methods: CML pts during first line treatment with any approved TKIs, referring to several Italian Hematology Centers, entered this non interventional cross sectional study after signing a proper informed consent. During a routine follow up visit, in which pts were checked for molecular response by standard PB qRT-PCR BCR-ABL1 analysis, additional 3 mls of PB were collected in EDTA and sent within 24 hours to Siena Hematology Lab to detect CD34+/CD38-/CD26+ LSCs by multicolor flow cytometry. After red blood cells lysis, cells were incubated with anti CD45 (BD Biosciences), CD34 (581), CD38 (HIT2), CD26 (M-A261) (BD Pharmigen). After washing, acquisition and analysis were performed by FACSCanto II (BD Biosciences, NR Nannini) using DIVA 8 software (BD, Biosciences). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. In 5 pts a FISH analysis of PB sorted CML LSCs population was also performed. Results: to validate our assay we first performed a FISH analysis of both PB sorted CD34+/CD38-/CD26+ and CD34+/CD38-/CD26- in 5 CML patients at 3-6 months after starting treatment, confirming Ph+ cells only in the CD26+ fraction. Afterward, we checked for circulating CML LSCs a total of 202 CML pts in first line treatment with TKIs for a median of 39 months (range 1-175). Type of TKI, length of treatment, molecular response and quantification of LSCs are summarized in Table 1. PB CML LSCs were detectable in 146/202 (72.3%) pts with a median number of CD26+ of 0,0165 cells/µL (range 0,0018-0,66). Kendall rank correlation coefficient used to analyze the relation between the measurable variables showed no correlation between BCR-ABL/ABLIS ratio (median 0,004 range 0-61) and number of residual LSCs (r 0.118 p=0.097). In 56/202 (27.7%) pts CD26+ LSCs were undetectable, yet we found no correlation with the concomitant degree of molecular response. Conclusions: this study represents the first attempt to measure in a large cohort of CML patients residual circulating LSCs during TKIs treatment. In our hands PB LSCs flow-cytometry assay appeared feasible, specific and sensitive and thus suitable for routine monitoring. As expected, the majority of CML patients, even in deep molecular response, still harbor residual LSCs and the number of PB CD26+ did not correlate with the number of BCR-ABL1 copies. This evidence suggests that the molecular response refers to transcriptionally active CML progenitor cells and not to quiescent, TKIs resistant, CML LSCs. Prospective studies evaluating the behavior of PB CML LSCs during different TKIs treatment, as well as studies monitoring PB CD26+ in CML pts that discontinued TKIs treatment are ongoing. Our goal is to rule out the impact, if any, of a "stem cell response" in addition to the standard molecular response in the management of CML patients mainly to identify those pts candidates for a safe TKI discontinuation. Disclosures Bocchia: Janssen: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Aprile:Novartis: Honoraria. Castagnetti:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Abstract: Here, we report real-world evidence on the safety and efficacy of nilotinib as a first-line treatment in elderly patients with chronic phase CML, treated in 18 Italian centers. Sixty patients aged  〉  65 years (median age 72 years (65–84)) were reported: 13 patients were older than 75 years. Comorbidities were recorded at baseline in 56/60 patients. At 3 months of treatment, all patients obtained complete hematological response (CHR), 43 (71.6%) an early molecular response (EMR), while 47 (78%) reached a complete cytogenetic response (CCyR). At last follow-up, 63.4% of patients still had a deep molecular response (MR4 or better), 21.6% reached MR3 as best response and 11.6% persisted without MR. Most patients (85%) started the treatment at the standard dose (300 mg BID), maintained at 3 months in 80% of patients and at 6 months in 89% of them. At the last median follow-up of 46.3 months, 15 patients discontinued definitively the treatment (8 due to side effects, 4 died for unrelated CML causes, 1 for failure, 2 were lost to follow-up). One patient entered in treatment-free remission. As to safety, 6 patients (10%) experienced cardiovascular events after a median time of 20.9 months from the start. Our data showed that nilotinib could be, as first-line treatment, effective and relatively safe even in elderly CML patients. In this setting, more data in the long term are needed about possible dose reduction to improve the tolerability, while maintaining the optimal molecular response.

Abstract: We report the final analysis, with a 10-year follow-up, of the phase II study GIMEMA CML 0307 (NCT 00481052), which enrolled 73 adult patients (median age 51 years; range, 18-83) with newly diagnosed chronic-phase chronic myeloid leukemia to investigate the efficacy and the toxicity of front-line treatment with nilotinib. The initial dose was 400 mg twice daily; the dose was reduced to 300 mg twice daily as soon as this dose was approved and registered. The 10-year overall survival and progression- free survival were 94.5%. At the last contact, 36 (49.3%) patients were continuing nilotinib (22 patients at 300 mg twice daily, 14 at lower doses), 18 (24.7%) patients were in treatment-free remission, 14 (19.2%) were receiving other tyrosinekinase inhibitors and four (5.5%) patients have died. The rates of major and deep molecular responses by 10 years were 96% and 83%, respectively. The median times to major and deep molecular response were 6 and 18 months, respectively. After a median duration of nilotinib treatment of 88 months, 24 (32.9%) patients discontinued nilotinib while in stable deep molecular response. In these patients, the 2-year estimated treatment-free survival was 72.6%. The overall treatment-free remission rate, calculated on all enrolled patients, was 24.7% (18/73 patients). Seventeen patients (23.3%), at a median age of 69 years, had at least one arterial obstructive event. In conclusion, the use of nilotinib front-line in chronic phase chronic myeloid leukemia can induce a stable treatment-free remission in a relevant number of patients, although cardiovascular toxicity remains of concern.