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 Bosutinib is a 2nd generation tyrosine-kinase inhibitor (TKI) active in Chronic Myeloid Leukemia (CML) patients resistant or intolerant to frontline imatinib, dasatinib or nilotinib; the favourable toxicity profile makes bosutinib potentially useful in elderly patients, but at present there are no data in unselected cohorts of these subjects. Aim To highlight this issue, a real-life cohort of 91 patients followed in 21 Italian Centers and treated with bosutinib when aged 〉 65 years was retrospectively evaluated. Patients The main clinical features of the whole cohort at diagnosis and at baseline of bosutinib treatment are reported in the Table; all patients were in CP when bosutinib was started. Median interval from diagnosis to bosutinib treatment was 49.7 months [interquartile range (IQR) 14.2 - 117.5]. Results Starting dose of bosutinib was 500 mg/day in 20 patients (22.0%), 400 mg/day in 7 patients (7.7%), 300 mg/day in 28 patients (30.8%), 200 mg/day in 34 patients (37.3%) and 100 mg/day in 2 patients (2.2%), respectively. After a median period of treatment of 18.1 months (IQR 9.4 - 27.7) all patients were evaluable for toxicity; on the whole, all grade hematological and extra-hematological toxicities were reported in 12/91 (13.1%) and 45/91 (49.4%) patients, respectively. A grade 3 - 4 hematological toxicity occurred in 5/91 patients (5.4%); a grade 3 - 4 extra-hematological toxicity occurred in 16/91 patients (17.5%). Overall, 46 patients (50.5%) never discontinued bosutinib: a temporary discontinuation 〈 6 weeks was needed in 19 patients (20.9%) and a temporary discontinuation 〉 6 weeks in 2 patients (2.2%). A permanent bosutinib discontinuation was needed in the remaining 24 patients (26.4%): in particular, 11 patients (12.1%) permanently discontinued bosutinib due to toxicity (skin rash in 3 cases, gastro-intestinal toxicity in 3 cases, pleural effusion in 2 cases, transaminitis, QTc prolongation and myalgia in 1 case each), 6 patients (6.6%) due to resistance and 7 patients (7.7%) due to other reasons (unrelated death in 6 cases and patient decision in 1 case). As to response, 5 patients (5.5%) were considered too early for assessment ( 〈 3 months of treatment); among the 86 patients evaluable for response, 11 patients (12.7%) did not have any response (including 6 patients who discontinued bosutinib for early toxicity), 4 (4.6%) achieved hematological response only, and 71 (82.5%) achieved Cytogenetic Response (CyR) (Major CyR in 4, Complete CyR in 67). Among the 67 patients in Complete CyR, 58 (67.4% of all 86 evaluable patients) also achieved Molecular Response (MR) [Major MR (MR 3.0) in 19 (22.1%), Deep MR (MR 4.0/4.5) in 39 (45.3%)] . The 3-year Overall Survival and Event-Free Survival of the whole cohort of patients from bosutinib start were 83.0% (CI95% 71.6 - 94.4) (Figure 1) and 59.5% (CI95% 39.9 - 72.1), respectively. Conclusions Our real-life data show that bosutinib is effective, even if initial doses in many cases were lower than recommended, with a favourable safety profile also in elderly patients with important comorbidities resistant/intolerant to previous TKI treatments,: as a consequence, it could play a significant role in the current clinical practise for these frail patients. Disclosures Latagliata: Celgene: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Trawinska:Novartis: Consultancy, Honoraria. Annunziata:Pfizer: Consultancy; Incyte: Consultancy; Novartis: Consultancy. Elena:Novartis: Consultancy; Pfizer: Consultancy. Crugnola:Incyte: Honoraria; Novartis: Honoraria. Bonifacio:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. Sgherza:Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Novartis: Honoraria. Iurlo:Pfizer: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria. Breccia:Celgene: Honoraria; Incyte: Honoraria; Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria.

Abstract: Introduction. Haematological malignancies related to previous chemo and/or radio-therapy for neoplasia have been well investigated. This subgroup was defined as therapy-related myeloid neoplasms by ELN. In the field of chronic myeloid leukaemia (CML) the diagnosis of a secondary neoplasia after CML diagnosis has been studied but the presence of 'secondary CML' has not been completely defined. In clinical practice we collected patients with a CML diagnosis who had a previous history of malignancy. Aim and methods. We reviewed our databases to collect the cases of 'secondary CML' in 4 hematological Italian centres. Results. We found 51 patients who were diagnosed with previous malignancies out of 617 CML patients (8.3%). We conducted the analysis on 48 patients, removing the other three patients who were diagnosed before the era of tirosine-kinase inhibitors (TKIs). We then subdivided patients in two groups according to the treatment they received for the previous neoplasia: patients treated by surgery (group A: 27 patients) and patients treated by chemotherapy and/or radiotherapy (group B: 21 patients). Clinical characteristics at diagnosis of the patients of the two groups were summarized in Table 1. The majority of the patients showed bcr-abl b3a2 (59% in group A and 62% in group B), no one showed additional cytogenetic abnormalities. The median time between the diagnosis of the primary neoplasia and CML were not different: 78 months (range 1-276) in the group A and 75 months (range 1-371) in the group B. Upfront treatment was Imatinib in 55.6% of the patients in group A and 81% in group B; the remaining patients were treated with Dasatinib in 22.2% and 14% of the patients, respectively, and Nilotinib 22.2% and 5% of the patients, respectively. All patients achieved haematological response after 1 month, 24 out of 27 patients (89%) of the group A achieved complete cytogenetic response after a median time of 3 months (range 3-28) and 20 out of 21 patients (95%) of the group B achieved complete cytogenetic response after a median time of 3 months (range 3-12). The median time of major molecular response was 9 months in 20 out of 27 patients (74%) of group A and in 19 out of 21 patients (90%) of group B. Patients in group A showed a higher number of events who determined a change in the treatment: 9 patients (33.3%) in group A changed TKI due to resistance (3 patients: 2 primary resistance and 1 secondary resistance) or intolerance (6 patients) versus 4 patients (19%) of the group B because of resistance (3 patients: 2 primary resistance and 1 secondary resistance) or intolerance (1 patient). We recorded 3 deaths: 2 in the group A not related to CML and 1 in the group B due to CML. The median overall survival was 53 months (range 8-228) in the first group and 51 months (range 4-196) in the second group. Unexpectedly we found a 12.5% of patients with a previous diagnosis of lymphoma in patients developing CML and this reached statistical significance (p=0.004) when compared to patients in A group. We also detected 3 out of 48 CML patients (6%) with a previous history of 2 neoplasia and all of them were treated with chemo and/or radiotherapy. Conclusion. Our research found that "secondary CML" occurred in 8.3% of newly diagnosed CML patients. Patients were comparable in terms of bcr-abl transcript, risk assessment, clinical and laboratory presentation. Cytogenetic and molecular response to TKIs as well as survival was similar to CML standard population. A significant fraction of patients with "secondary CML" received prior chemo and /or radiotherapy for an haematological malignancies and poses new questions in terms of either genetic predisposition and/or exposure to anticancer treatment. Disclosures No relevant conflicts of interest to declare.

Abstract: In the current clinical practice, imatinib is widely used also in very elderly patients with chronic myeloid leukemia (CML) at different doses based on concomitant diseases and physician' judgment. However, data on long-term follow-up of these patients are still lacking. To address this issue, we revised in our retrospective database 233 CML patients aged ≥ 75 years and treated with imatinib frontline in 34 italian hematological centers from 2/2002 to 7/2014. Median age at diagnosis was 78.4 years [interquartile range (IQR) 76.3 - 81.3], there were 113 males (48.5%) and 120 females (51.5%), median WBC, Hb and PLT counts were 45.0 x 109/l (IQR 29.4 - 83.4), 12,4 g/dl (IQR 11.0 - 13.6) and 375 x 109/l (IQR 238 - 680), respectively. Sokal Risk at diagnosis was low in 1 patient (0.4%), intermediate in 149 (67.4%), high in 71 (32.2%) and not evaluable in 12. One or more concomitant diseases requiring specific treatments were present in 225/233 patients (96.5%). Median interval from diagnosis to imatinib start was 0.7 month (IQR 0.2 - 1.4): the initial imatinib dose was 400 mg/day in 161 patients (69.1%), 300 mg/day in 57 (24.5%) and 〈 300 mg/day in 15 (6.4%). According to WHO, a grade 3 - 4 hematological and extra-hematological toxicity was reported in 44 (18.8%) and 41 (17.6%) patients, respectively. As to cumulative response, 13 patients (5.6%) discontinued IM due to early toxicity, 4 (1.7%) were resistant and 2 (0.8%) died from unrelated cause early after IM initiation: the remaining 214 patients (91.9%) achieved a complete haematological response (CHR). Among these 214 patients in CHR, 13 refused any other karyotipic or molecular evaluation, 23 achieved CHR only and 178 (76.4% of all 233 patients) achieved a cytogenetic response (CyR), which was partial in 16 patients and complete (CCyR) in 162 (69.5% of all 233 patients). In addition, among the 162 patients in CCyR, 125 (53.6% of all 233 patients) achieved a molecular response (MolR) (ratio 〈 0.1). A blastic phase occurred in 11 patients (4.7%). After a median follow-up from imatinib start of 45.0 months (IQR 22.3 - 72.0), 70 patients have died (9 from disease progression and 61 from unrelated causes), 16 patients were lost to follow-up and 147 are still alive (115 of them still in treatment with imatinib): 5-year event-free survival (EFS) and overall survival (OS) were 51.4% (CI95% 43.9 - 58.9) and 68.5% (CI95% 61.2 - 75.8), respectively. At univariate analysis, only the initial dose of imatinib (400 vs ≤ 300, p=0.03) was a significant predictive factor for CCyR achievement while only PLT count ≤ 500 x 109/l (p=0.031) was a significant predictive factor for MolR achievement. At multivariate analysis for EFS, achievement of a MolR (OR 0.25, 95%CI 0.14 - 0.43, p 〈 0.001), achievement of a CCyR (OR 0.40, 95%CI 0.23 - 0.67, p=0.001) and spleen enlargement (OR 1.56, 95%CI 1.01 - 2.41, p=0.042) were independent prognostic factors; at multivariate analysis for OS, achievement of a MolR (OR 0.30, 95%CI 0.18 - 0.49, p 〈 0.001), age 〈 80 yrs (OR 0.53, 95%CI 0.33 - 0.86, p=0.011) and male gender (OR 1.80, 95%CI 1.11 - 2.91, p=0.016) were independent prognostic factors. In conclusion, the long term follow-up of very elderly CML patients who started imatinib is very good and justify any effort to treat these patients with standard doses, in order to achieve cytogenetic and molecular responses as in younger subjects. Disclosures Castagnetti: BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Gugliotta:BMS: Honoraria; Novartis: Honoraria. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy.

Abstract: 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), and the remaining patients with 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 〈 6 months CCyR 7-12 months 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.

Abstract: Abstract 2751 Comorbidities have been identified as significant determinants of response to therapy in elderly patients with acute myeloid leukemia, breast cancer, head and neck, and lung cancer. Charlson comorbidity index (CCI) is a list of comorbidities with a weight assigned from 1 to 6 derived from relative risk estimates of a proportional hazard regression model using clinical data. We applied CCI stratification on a large cohort of chronic myeloid leukemia (CML) very elderly patients ( 〉 75 years) treated with imatinib, in order to observe the impact of concomitant diseases on both compliance and outcome. One hundred and eighty-one patients were recruited by 21 Italian centers. There were 95 males and 86 females, median age 78.6 years (range 75–93.6). According to Sokal score, 106 patients were classified as intermediate risk and 55 as high risk (not determined in 20 patients). According to CCI stratification, 71 patients were score 0, 50 patients had a score 1, 37 patients had score 2 and 23 patients had score ≥ 3. Imatinib standard dose was reduced in 68 patients independently from the evaluation of baseline comorbidities but based only on physician judgement: 43.6% of patients with score 0 started with a reduced dose (200–300 mg/day) compared to more than 50% of patients with score ≥ 3. No significant differences were found in terms of further reduction of the dose (39% in patients with score 0 compared to 21% in patients with score ≥ 3) or in terms of discontinuation due to toxicity (58% in patients with score 0 vs 48% in patients with score ≥ 3). We did not find significant differences as regards the occurrence of hematologic side effects, probably due to the initial reduction of the dose: 39% of patients with score 0 experienced grade 3/4 hematologic toxicity (most commonly anemia) compared to 35% of patients with score ≥ 3. Independently from the initial dose, comorbidities again did not have an impact on development of grade 3/4 non-hematologic side effects (most common skin rash, muscle cramps and fluid retention): 62% of patients with score 0 compared to 35% of patients with score ≥ 3. Notwithstanding the reduced dose and the weight of comorbidities we did not find differences in terms of efficacy: 66% of patients with score 0 achieved a CCyR compared to 56.5% of patients with score ≥ 3. Comorbidities appeared to have an impact on EFS (34 months for patients with score 0 vs 23.5 months for patients with score ≥ 3) and influenced the median OS (40.8 months for patients with score 0 vs 10.6 months for patients with score ≥ 3). Our results suggested that treatment of very elderly CML patients might be influenced by personal physician perception: evaluation at baseline of comorbidities according to CCI should improve initial decision-making in this subset of patients. Disclosures: Russo Rossi: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Alimena:Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

Abstract: In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

Abstract: In Philadelphia-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) patients (pts), resistance to tyrosine kinase inhibitors (TKIs) is frequently associated with the selection of one or more mutations in the BCR-ABL1 kinase domain (KD). The swift emergence of mutant clones as early as during induction therapy supports the hypothesis that, at least in some cases, mutations may already be present at diagnosis. Next Generaton Sequencing (NGS) has been proposed as an alternative to Sanger sequencing (seq) for BCR-ABL1 KD mutation screening because of its greater sensitivity and accuracy, but no studies have so far evaluated its prospective use in Ph+ ALL. Between 2015 and 2018, we have used NGS in parallel to Sanger seq to analyze a consecutive series of 126 Ph+ ALL pts who were newly diagnosed (n=39) or who had relapsed/refractory disease (n=87) on TKI therapy. In 22 cases, both bone marrow and peripheral blood were analyzed and compared. NGS of ≈400bp amplicons generated by nested RT-PCR was performed on a Roche GS Junior (until April 2017) or on an Illumina MiSeq (from May 2017 on). Read alignment and variant calling (with a lower limit set to 3%) were done with the AmpSuite software (SmartSeq srl). When multiple mutations mapped within the same sequence reads, assessment of cis vs trans configuration was done correcting for the probability of PCR recombination. Three out of 39 (7.7%) de novo Ph+ ALL pts had low burden point mutations detectable by NGS: one had a V289A (variant frequency, 3.4%); one had a D276G (4.0%) and a F359V (3.5%); one had an E255K mutation (3.3%). The first pt was enrolled in the GIMEMA LAL1811 study of frontline ponatinib; the second and the third pts were enrolled in the GIMEMA D-ALBA study of frontline sequential treatment with dasatinib and blinatumomab. All pts achieved molecular remission, consistently with the mutations being sensitive to the TKIs received. The 35INS insertion/truncation mutant was detected in 27 (69%) pts, who all have so far achieved molecular remission. This is in line with the report by O'Hare et al (Blood 2011) suggesting that the 35INS variant is kinase-inactive and does not contribute to TKI resistance. For this reason, the 35INS was excluded from subsequent analyses. Relapsed/refractory pts positive for mutations by Sanger seq were 57 (65%); those positive for mutations by NGS were 69 (79%). Fifty-six out of 87 (49%) pts had 〉 1 mutation (up to 13) detected by NGS. NGS identified low burden mutations (i.e., mutations present in a proportion of transcripts between 3 and 20%) in 12 pts who were negative for mutations by Sanger seq. Most importantly, NGS provided a more accurate picture of BCR-ABL1 mutations status in 40 (46%) pts who turned out to have one or more low burden mutations in addition to the dominant mutation(s) detectable by Sanger seq. In all cases, each low burden mutation detected by NGS could be recognized as poorly sensitive either to the TKI the pt was receiving at the time of testing, or to the previous TKI. The clonal nature of NGS-based analysis further proved its utility i) in 4 pts where Sanger seq had shown 2 base substitutions in the same codon so that the actual amino-acid change(s) were impossible to infer (a ponatinib-resistant pt with a T315M mutation, 2 dasatinib-resistant pts with various combinations of F317I, F317C and/or F1317L, a dasatinib-resistant pt with 2 different nucleotide substitutions both leading to the V299L), and ii) in 48/56 pts who had ≥2 mutations whose clonal configuration could not be resolved. Twenty-eight out of these 48 pts were found to carry one or more (up to 3) compound mutants. Compound mutants were more common in pts who had failed ≥2 lines of therapy, whereas polyclonality was more common in pts who had failed first line therapy. The most frequent compound mutants were T315I+E255K and T315I+E255V. Interestingly, the latter was associated with poor or no response to ponatinib. Our results in a relatively large series of Ph+ ALL pts suggest that an NGS-based approach provides a more accurate characterization of the complexity of BCR-ABL1 KD mutation status, including compound mutants some of whom may be poorly sensitive even to ponatinib. Mutations may already be detected at the time of diagnosis. It remains to be assessed whether more sensitive techniques like digital PCR may identify a greater number of pts with pre-therapy mutations and whether the detection of pre-therapy mutations may be used to guide 1st-line treatment selection. Disclosures Soverini: Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Pagano:Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau; Pfizer: Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Martinelli:Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Consultancy; Janssen: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees.