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A variety of mechanisms underlie the lack or loss of response to TKIs in patients (pts) with CML, but acquisition of point mutations in the BCR-ABL1 KD is, at present, the only actionable one. Detection of a TKI-resistant mutation by Sanger Sequencing (seq) should trigger a change of therapy and, in some cases, guide TKI selection. Although research studies have shown that NGS may hold added value over Sanger seq for BCR-ABL1 KD mutation screening, routine NGS is technically demanding and expensive and is not yet widely available. Newer methods, like ddPCR, might represent an attractive alternative. Here we set out to 1) assess the actionability of results, hence the potential clinical benefit, of more sensitive NGS-based testing vs Sanger seq in a consecutive series of CML pts who had non-optimal response to TKI therapy according to the 2013 ELN recommendations, and 2) test a novel ddPCR-based multiplex assay for rapid screening for a panel of BCR-ABL1 KD mutations relevant for TKI selection. Between January 2015 and May 2019, samples from 712 CML pts followed at one of 66 GIMEMA CML Working Party hematology centers were referred to our laboratory for BCR-ABL1 KD mutation testing because of a Failure (n=251 pts) or Warning (n=461 pts) response to TKI therapy. In parallel to Sanger seq, NGS of amplicons generated by nested reverse transcription(RT)-PCR was performed on a Roche GS Junior instrument until April 2017, and on an Illumina MiSeq instrument from May 2017 on. Read alignment and variant calling was done using AmpSuite software (SmartSeq). Variants detected in Among Failures, pts positive for BCR-ABL1 KD mutations by Sanger seq were 88/251 (35%). NGS detected low level mutations in 38/251 (15%) additional pts who were negative for mutations by Sanger seq. Moreover, 31/251 (12%) Failure pts were found to have low level mutations additional to those detectable by Sanger seq. Among low level mutations detected by NGS, those actually relevant for TKI selection (2GTKI-resistant) were identified in 22/251 (9%) pts. Compound mutations were found in 10 pts (4%; all progressed to blastic phase). The average turnaround time (TAT) of routine NGS-based analysis was 15 working days (range, 11-32). Thus, in the setting of Failure, NGS-based mutation scanning of the whole KD is of real clinical benefit in a minority of pts while increasing the TAT. Among Warnings, pts positive for BCR-ABL1 KD mutations by Sanger seq were 65/461 (14%). NGS detected low level mutations in 97/461 (21%) additional pts who were negative for mutations by Sanger seq. All Warning pts positive for low level resistant mutations who were not switched to another TKI turned their response into Failure after 3 to 8 months. The multiplex digital PCR assay proved capable to accurately identify and separate the T315I/A, F317L/V/I/C, Y253H, E255K, F359V/I/C, E255V, V299L mutations in five spatially distinct clusters (pan-resistant, Dasatinib-resistant, Nilotinib-resistant, Nilotinib- and Bosutinib-resistant, Dasatinib- and Bosutinib-resistant, respectively) starting from as little as 30ng of total cDNA or 1ng of the 1.7kb 1st PCR product. Very good concordance was observed between ddPCR- and NGS-identified mutations irrespective of mutation frequency or cluster proximity, even for compound mutations. Extensive assay assessment will be presented. TAT of ddPCR was 1 day. In conclusion: - Mutation testing in the Failure setting aims to a timely and rational TKI switch. The incidence of low level mutations relevant to the selection of subsequent-line therapy and the TAT of routine NGS in our cohort suggest that multiplex ddPCR would be an easier and faster alternative. - Mutation testing in the Warning setting may identify pts who need a change in therapy rather than a 'watch and wait' approach. In our cohort, approx 1/5 of the Warning pts negative by Sanger seq had low level mutations resistant to the TKI they were receiving, that ultimately led to Failure. Earlier detection of emerging resistant mutations enabled by NGS (or by ddPCR in pts receiving 2GTKIs) should support proactive TKI switch. Disclosures Soverini: Incyte: Consultancy. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Bonifacio:BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. 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. Tiribelli:Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Stagno:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria. D'Adda:Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Crugnola:Incyte: Honoraria; Novartis: Honoraria. Corner:BioRad: Employment. Maar:BioRad: Employment. 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. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Pfizer: Consultancy; ARIAD: Consultancy; Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy. Cavo:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bms: 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; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

Next-Generation Sequencing (NGS)-based BCR-ABL1 kinase domain (KD) mutation screening has been shown to enable greater accuracy and sensitivity and straightforward identification of compound mutants (CM) as compared to Sanger sequencing (seq). However, the prevalence of CMs has never been assessed in prospective studies, and although in vitro data suggest that many of them may be challenging for all tyrosine kinase inhibitors (TKIs) including ponatinib, attempts to correlate such data with in vivo responses have never been made. To address these issues, we have reviewed the results of routine NGS-based BCR-ABL1 KD mutation screening performed over the past 3 years. Between 2015 and 2018, we have prospectively used NGS to analyze a consecutive series of 751 Ph+ leukemia patients (pts) on TKI therapy who were eligible for BCR-ABL1 KD mutation screening according to ELN/NCCN/ESMO recommendations. The study population included 664 chronic myeloid leukemia (CML) pts with failure or warning response (chronic phase [CP], n=593; accelerated or blastic phase [AP/BP], n=71) and 87 Ph+ acute lymphoblastic leukemia (ALL) pts with relapsed/refractory disease. 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) using custom protocols whose accuracy, sensitivity and reproducibility was checked by national and international (EUTOS) control rounds. Read alignment and variant calling was done using the AmpSuite software (SmartSeq srl), with a lower detection limit set to 3%. Cis or trans configuration of mutation pairs, indicating CMs or polyclonality, respectively, was determined correcting for the likelihood of PCR recombination. The 35INS insertion/truncation mutant was excluded from the analysis. NGS identified mutations in the BCR-ABL1 KD in a total of 313/664 (47%) CML pts (255/593 [43%] CP-CML and 58/71 [82%] AP/BP-CML) and 69/87 (79%) Ph+ ALL pts. Ninety-one percent of the mutations could be recognized as conferring resistance to at least one TKI on the basis of publicly available IC50 data or published reports. In 42/593 (7%) CP-CML, 6/71 (8.5%) AP/BP-CML and 12/87 (14%) Ph+ ALL pts, low burden mutations (i.e., mutations carried by a proportion of transcripts 15% - hence detectable by Sanger seq). Fifty-five (9.2%) CP-CML, 51 (72%) AP/BP-CML and 56 (49%) Ph+ ALL pts had ≥2 mutations (CP-CML: 1-5 mutations; AP/BP-CML: 1-6 mutations; Ph+ ALL: 1-13 mutations). Identification of CMs in pts with ≥2 mutations was fully possible (i.e., all the candidate pairs mapped within a distance of 400bp) in 71% of cases and partially possible (i.e., some, but not all the candidate pairs mapped within a distance of 400bp) in another 12% of cases. A total of 86 CMs (85 double and 1 triple) in 73 pts (21 [3.5%] CP-CML, 23 [32%] AP/BP-CML and 29 [37%] Ph+ ALL pts) could be catalogued (Figure 1A). All but two (T315I+D276G, M244V+E255K) were detected in pts who had received ≥2 TKIs and all included at least a 2nd-generation TKI-resistant mutation. The most frequent CMs were T315I+E255K, T315I+E255V, T315I+F359V, F317L+Y253H (Figure 1A). The triple CM, detected in a ponatinib-resistant pt, was F317I+Y253F+Q252H. Correlation of IC50 data with in vivo responses (the TKIs pts were clinically resistant to) confirmed only partially in vitro predictions (Figure 1B). In particular, although ponatinib was shown in vitro to be poorly effective against several CMs, only the T315I+E255V was consistently found to be associated with ponatinib failure. In conclusion, our results in a large unselected series of TKI-resistant pts analyzed by NGS show that:CMs are relatively infrequent in CP-CML, but may be a relevant issue in AP/BP-CML and Ph+ ALL;among pts with multiple mutations, those who have failed 1 line of therapy have most often polyclonality, whereas those who have failed ≥2 lines of therapy may have CMs or polyclonality;in vitro predictions of sensitivity and insensitivity based on IC50 data should be regarded with caution. In particular, the only compound mutant that we consistently found to be associated with ponatinib failure was the T315I+E255V. Supported by EUTOS 2016. Disclosures Soverini: Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy. Pagano:Pfizer: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Merck: Speakers Bureau; Janssen: Speakers Bureau. Gugliotta:Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Castagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Angelucci:Roche Italy: Other: Local (national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Celgene: Honoraria, Other: Chair DMC; Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC. Martinelli:Abbvie: Consultancy; Ariad/Incyte: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy. Cavo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: 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; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees.

According to the World Health Organization (WHO) 2008/2016 criteria for classification of myeloid neoplasms, a platelet (PLT) count ≥ 450X109/l, thus reduced from the previous WHO 2001 level ≥ 600 x 109/l, was considered the new PLT threshold for the diagnosis of Essential Thrombocythemia (ET). Aim of the study was to validate in a setting of current clinical practice this important diagnostic change and compare clinical and hematological features at diagnosis and during follow-up of patients with PLT ≥600 x 109/l versus patients with PLT < 600 x 109/l. We retrospectively analyzed data from 264 patients with ET according to WHO 2008/2016 criteria, enrolled in our center from 1/2008 to 12/2017. Patients were divided into Group A (G-A) (PLT ≥600 x 109/l at diagnosis) (199 patients - 75.4%) and Group B (G-B) (PLT ≥ 450 x 109/l < 600 x 109/l at diagnosis) (65 patients - 24.6%) and compared for clinical features at the onset, clinical course and follow-up. Main features and commonly recognized pro-thrombotic risk factors at diagnosis of the entire cohort as well as of G-A and G-B are reported in the Table 1. Among clinical features, only the median value of leukocytes was significantly higher in G- A [9.1 x 109/l, interquartile range (IQR) 7.8-10.3 vs 7.4 x 109/l, IQR 6.0-9.6; p = 0.001]. Among pro-thrombotic risk factors, only the median cholesterol value was significantly lower in the G-A [187 mg/dl (IQR 164-220) vs 204 mg/dl (RIQ 177-238); p = 0.048]. Cytostatic treatment was administered in 175 patients (71.1%) of entire cohort at different intervals from diagnosis, with a significantly higher rate in patients of G-A (76.9% versus 49.2%, p Our data indicate a substantial homogeneity among ET patients regardless of the PLT number at diagnosis, thus confirming the usefulness of 2008/2016 WHO diagnostic criteria. Furthermore, the counterintuitive lower CIT observed in G-A, due to a larger use of cytostatic treatments and/or to an acquired Von Willebrand phenomenon when PLT levels > 1.000 x 109/l, highlights how thrombotic risk is unrelated to PLT value and leads to consider the administration of adequate cytostatic therapy even in patients with relatively lower PLT count at diagnosis. Disclosures Breccia: Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. Foà:INCYTE: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; NOVARTIS: Speakers Bureau.

Deletion of the chromosome 20 long arm (del20q) has been reported in 3-7% of patients with Myelodysplastic Syndromes (MDS). In particular, isolated del20q seems to be associated with good prognosis, low risk of progression to AML and prolonged survival: however, very few reports addressed this subset of MDS patients up to now. To highlight this issue, we collected data from all patients with MDS and isolated del20q diagnosed and followed by Centers of the Gruppo Romano-Laziale Sindromi Mielodisplastiche (GROM-L). On the whole, 53 patients were analysed: the main features at diagnosis of these patients are reported in the Table. Platelet (PLT) count was < 100 x 109/l in 28 patients (52.8%), Hb level was < 10.0 g/dl in 23 patients (43.3%) and neutrophil count was < 1.0 x 109/l in 12 patients (22.6%). As to risk prognostication, according to IPSS score 51 patients (96.2%) had low/intermediate-1 risk and 2 (3.8%) had intermediate-2/high risk: according to r-IPSS, 8 patients (15.1%) had very low risk, 20 (37.7%) low risk, 19 (35.9%) intermediate risk and 6 (11.3%) high risk. During follow-up, 14 patients (26.4%) did not need any therapy and were only observed, 36 (67.9%) were treated with Erythropoietin (EPO), 1 (1.9%) with hypomethylating agents, 2 (3.8%) with other treatments (TNF-α inhibitor, interferon). Among the 36 patients treated with ESA after a median interval from diagnosis of 5.0 months [interquartile range (IR) 1.2 - 27.6], 24 (66.6%) received α-EPO, 11 (30.5%) β-EPO and 1 (2.9%) darbopoietin. Two patients were too early (< 3 months of treatment) for response evaluation: among the 34 evaluable patients, 21 (61.7%) achieved stable erythroid response according IWG criteria (Hb increase > 1.5 g/dl in 18 patients and reduction > 50% of basal transfusion requirement in 3 patients) while 13 (38.2%) were resistant to EPO. Nine patients (17%) progressed to Acute Myeloid Leukemia (AML) after a median time from diagnosis of 16.8 months (IR 4.1 - 51.7). At the last follow-up, 21 patients (39.6%) died (13 from MDS/AML related causes and 8 from unrelated causes), 12 (22.6%) were lost to follow-up and 20(37.8%) were alive. Median Overall Survival (OS) of the entire cohort was 61.6 months (95%CI 42.2 - 81.0): the 10-year cumulative OS was 38.6% (95%CI 18.6 - 58.6) (Figure 1). In conclusion, as MDS represent a heterogeneous group of pathologies, many efforts are ongoing to identify patients with similar biological, clinical and prognostic features (eg 5q- syndrome, MDS with ring sideroblasts). From the scarce data in the literature and from our results, it is reasonable that also patients with isolated del20q may represent a distinct clinical and biological entity, with specific clinical and prognostic features (low PLT count, low number of marrow blasts, low IPSS and r-IPSS risk score, good response to EPO, long OS). The mechanisms underlying del20q are still unclear and warrant future molecular analysis. Disclosures Breccia: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria.

Current treatment with Tyrosine-Kinase Inhibitors (TKI) has profoundly changed long-term prognosis of newly diagnosed Chronic Myeloid Leukemia (CML). However, the true disease/patient status at different time-points is still based on few data from controlled clinical studies (mainly based on "cumulative incidence" more than "effective situation" of any single patient at any single time from TKI start) and is still unclear in the real-life setting without selection criteria. In order to give an effective picture of their 5-year status, all consecutive CML patients newly diagnosed at our Institute until 6/2012 (i.e. with a minimum observation period of 5 years) and treated frontline with any TKI at any dosage in both controlled clinical trials or current clinical practice were retrospectively evaluated. On the whole, 259 patients, treated with imatinib (219) or 2nd generation TKI (2-TKI) (35 with nilotinib and 5 with dasatinib), were collected: the main features at diagnosis of the entire cohort and according to TKI are reported in the Table 1, without differences between the 2 groups. In the imatinib group, 13/219 patients (6%) received a reduced starting dose (< 400 mg/day), while all patients in the 2-TKI group started initial standard doses. At the 5-year evaluation, 227 pazienti (87.6%) were alive, 16 died (6.2%) and 16 (6.2%) were lost to follow-up. Among the 227 patients alive, 176 (67.9% of the entire cohort) were in Complete Cytogenetic Response (CCyR), 163 (62.9% of the entire cohort) were in Major Molecular Response (MMolR) and 121 (46.7% of the entire cohort) were in Deep Molecular Response (DMR). The remaining 51 patients alive at the 5th year (19.7% of the entire cohort) were in 2nd or subsequent line of treatment, due to intolerance in 13 cases (25.5%) or primary/secondary resistance in 38 (74.5%). Among the 16 deaths, 5 (1.9% of the entire cohort) were CML-related and 11 (4.3% of the entire cohort) were CML unrelated. Among the 16 patients lost to follow-up, 8 (3.1% of the entire cohort) were in response to 1st line treatment and 8 (3.1% of the entire cohort) were in 2nd line/resistant disease at the last visit. Six patients (2.3%) evolved in blastic phase and 8 (3.1%) developped a 2nd neoplasia. In the group treated frontline with 2-TKI there was a higher rate of patients alive in MMolR (p=0.038) but not in DMolR (p=0.137), with a lower rate of primary/secondary resistance (p=0.048) but a higher incidence of intolerance (p=0.002) compared to patients treated frontline with imatinib (Table 2). Five-year cumulative overall survival (OS), progression-free survival (PFS) and event-free survival (EFS) of the entire cohort were 93.2% (IC 95% 90.2-96.2), 78.2% (IC95% 73.1-83.3) e 70.2% (IC95% 64.5 - 75.9), respectively. According to initial treatment, 5-year PFS was significantly better in the 2-TKI group (p=0.003), but no difference was observed between the 2 groups as to 5-year EFS (p=0.274) and 5-year OS (p=0.077). In conclusion, results achievable at 5 years with frontline TKI treatment in the current clinical practice are excellent, with at least two thirds of patients alive in MMolR and about half of patients also in DMolR under frontline approach: deaths CML-related in the first 5 years of treatment are very rare and less than 1 out 5 patients needs to change frontline approach for intolerance/resistance. The role of frontline 2-TKI in this real-life setting deserves further examinations in larger unselected cohorts at longer time-points. Disclosures Breccia: BMS: Honoraria; Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Foà:ROCHE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; NOVARTIS: Speakers Bureau; INCYTE: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD.

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.

Background and Aims: The term Systemic Mastocytosis (SM) identifies a poorly understood group of rare and clinically heterogenous myeloproliferative neoplasms characterized by abnormal growth and activation of mast cells (MCs) and their precursors in the bone marrow and in various tissues and organs. Based on phenotype and extent of organ infiltration/dysfunction, a spectrum of disease variants can be recognized ranging from indolent SM (ISM) to aggressive SM (ASM) and mast cell leukemia (MCL). The fact that in all cases, including ISM who have a (near) normal life expectancy, neoplastic MCs display the same D816V KIT gene mutation points to additional mechanisms and molecular defects as responsible for ASM and MCL. So far, however, this issue has mainly been addressed with targeted resequencing studies of candidate gene panels. We thus decided to undertake an integrated molecular characterization study of ASM and MCL to identify novel, functionally relevant molecular lesions and/or clinically actionable signaling pathways. Methods: A discovery panel including 6 patients with ASM and 6 patients with MCL was studied using whole exome sequencing (WES) and copy number variation (CNV) analysis. WES (80x) was performed on a Hiseq 2500 (Illumina). CNV was done using Cytoscan HD Arrays (Affymetrix). Paired normal/MC DNA was analyzed in all but 2 archival MCL cases for whom germline DNA was not available. A validation panel of 30 ISM, 5 smoldering SM and 20 additional ASM was also included in this study. Results: In the discovery panel, WES identified a total of 1554 point mutations, small insertions and deletions. Seven hundred and eighty-five were non-silent mutations in 698 genes, with an average of 51 (range, 30-186) non-silent mutations per patient. Non-silent mutations included 354 missense mutations, 188 nonsense mutations, 145 frameshift insertions/deletions, 98 non-frameshift insertions/deletions. C to T transitions were by far the most frequent. Orthogonal validation estimated the accuracy of mutation calls at >95%. Interrogation of the COSMIC and OMIM databases revealed 42 known cancer genes. Among the missense mutations, 87 were predicted to have a high probability of being deleterious by Condel. MCL cases were found not to harbour a higher mutation load as compared to ASM cases. High resolution CN analysis showed that focal amplifications/deletions/loss-of-heterozygosity (LOH) were prevalent over arm-level alterations (found in 3 patients only). Genes were selected for further assessment when recurrently mutated in ≥2 patients or concurrently identified in WES and CNV analyses or previously associated with leukemogenesis or cancer pathogenesis. Among these, genes already reported to be affected by mutations in SM included TET2, NRAS, ASXL1, CBL, IDH1, SRSF2, SF3B1, RUNX1. We also identified genetic alterations in genes not previously implicated in SM pathogenesis including TP53BP1, RUNX3, NCOR2, CDC27, CCND3, EI24, MLL3, ARID1B, ARID3B, ARID4A, SETD1A, SETD1B, KDM1B, PRDM1, ATM, WRN. A long tail of infrequently mutated genes dominated, resulting in significant intertumoural heterogeneity. However, when genes were assigned to functional pathways to discern patterns of mutations across different patients, we found that PI3K/Akt and MAPK pathways, calcium pathway, chromatin modification, DNA methylation, and DNA damage repair were consistently affected (Figure 1). Further assessment of the mutation frequency of selected genes within each pathway and functional validation at the protein level are currently ongoing in the validation panel. Preliminary findings on a tumor suppressor selected among those identified by WES show transcript and/or protein downmodulation due to inactivating mutations, transcriptional silencing or enhanced degradation in 17/20 ASM. Detailed results will be presented at the meeting. Conclusions: WES and CNV analyses of ASM and MCL revealed a complex landscape, not unexpected when considering the clinical heterogeneity of these patients. Nonetheless, key pathways were found to be recurrently altered. Further investigation of selected candidate genes and pathways is warranted and will cast light on the cooperative genetic (and epigenetic?) events underlying the more aggressive forms of SM - paving the way to a better prognostic stratification and more effective treatment. <>This study was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; Ariad: Consultancy; AMGEN: Consultancy; MSD: Consultancy.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to mast cell leukemia (MCL). Somatic mutations in the Kit receptor tyrosine kinase (most frequently, D816V) can be detected in >90% of patients with SM and are thought to play a key pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL patient who came to our attention in 2012 – a 63 year-old woman diagnosed with MCL, aleukemic variant (50-60% atypical mast-cells in the bone marrow [BM] smear; CD117+/CD2+/CD13+-/CD33+/CD59+ immunophenotype; serum tryptase, 2500 µg/L; no C-findings). The patient had received imatinib for 6 months, with no clinical benefit. The disease, however, had had an overall chronic clinical course for 6 more months until severe anemia occurred. The patient rapidly progressed and died after 21 months from diagnosis. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified MCs isolated from BM at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA sequencing on an HiSeq1000 (Illumina, San Diego CA). Cytoscan HD arrays (Affymetrix, Santa Clara CA) were also used to scan for chromosomal gains and losses as well as for loss of heterozigosity (LOH). Among the mutated genes detected by WES, SETD2 stood out among others because two distinct putatively inactivating heterozygous mutations were identified, a frameshift insertion of a C in exon 20 (NM_014159:c.7595_7596insC: p.Gly2515ArgfsTer5) and a nonsense mutation in exon 15 (NM_014159:c.G6753T:p.Glu2234Ter). The two mutations were found to hit distinct alleles, pointing to a loss-of-function event. Western Blotting (WB), however, showed that only the 2234 a.a. Setd2 truncated isoform resulting from the nonsense mutation, losing the highly conserved WW and SRI functional domains, was detectable in the sample. The SETD2 gene encodes a histone methyltransferase nonredundantly responsible for trimethylation of lysine 36 of histone H3, a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. SETD2 gene mutations have been described in a variety of cancers and, more recently, have been found to be cooperating events in acute leukemia initiation and progression. In yeast, deletion of the SRI domain abolishes Set2-RNA polymerase II (PolII) interaction causing transcription elongation defects and abolishes K36 methylation. The truncated SETD2 isoform was actually found to lose the ability to bind RNAPolII, as shown by co-immunoprecipitation. Accordingly, RNA-sequencing showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes rather than from canonical promoters. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the recently reported role of SETD2-dependent H3K36Me3 in homologous recombination (HR) repair and genome stability, Cytoscan HD arrays showed that LOH and several gains and losses at many chromosomal loci, undetectable at diagnosis, had been acquired at the time of progression. Haploinsufficiency of PSIP1 (recruiting HR machinery at double strand breaks) at 9p24.3 might have represented a cooperating event. Downmodulation of the Setd2 protein (in the presence of LOH but in the apparent absence of sequence variations other than polymorphisms) and reduced H3K36Me3 levels were detected in two more MCL cases, in which putative cooperative lesions were also identified. Results of WES and high resolution karyotyping of additional SM cases will be presented. Our findings point to epigenetic regulation and/or DNA repair as two candidate pathways deserving further investigation in an attempt to identify novel actors or mechanisms contributing to the pathogenesis and progression of SM, or novel modulators of disease phenotype. They also extend the recent observation that the molecular landscape of SM is much more complex than the initial finding of KIT mutations allowed to imagine. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau.

Effective management of tyrosine kinase inhibitor (TKI) therapy for patients with CML requires frequent patient testing to monitor patient disease status. The gold standard for this testing is a PCR measurement of the BCR-ABL/ABL transcript ratio. Hence, standardized, accurate and reproducible molecular analyses are fundamental for clinicians in order to correctly address therapeutic decision and to achieve a better patient management. Based on these clinical needs, it is widely recognized that inter-laboratory reproducibility is a crucial issue that requires standardization and strict alignment of BCR-ABL values on the international scale (IS), as established by the International Randomized Study of Interferon and STI571 (IRIS) laboratories. In addition to this, another important aspect in terms of patient management is the availability of a rapid response, improving patient quality of life by reducing anxiety linked to delay in results delivery. Xpert® BCR-ABL Monitor, developed and manufactured by Cepheid (Sunnyvale, CA), is a cartridge-based automated assay for quantification of BCR-ABL1 mRNA, reporting results in International Scale (IS). Alignment to the IS, based upon the quality control standards derived from the WHO BCR-ABL Standards, is performed lot to lot automatically by the software of the Cepheid GeneXpert® DX Instrument. Xpert® BCR-ABL Monitor automates and integrates sample processing, nucleic acids extraction and amplification and target sequence detection in peripheral blood specimens collected either in EDTA or PAXgene tubes using real-time RT-PCR. The cartridge includes reagents to detect BCR-ABL fusion genes resulting from two major breakpoints, translocation e13a2 (b2a2) and e14a2 (b3a2) and the ABL transcript as an endogenous control. In the following study, Xpert® BCR-ABL Monitor was independently evaluated in the three Italian reference centers for BCR-ABL mRNA monitoring of the LabNet project. A total of 150 peripheral blood samples from CML patients, equally divided between centers, were analyzed both with the standard laboratory method and with Xpert® BCR-ABL Monitor Assay, in order to compare the results expressed in International Scale. BCR-ABL mRNA of the specimens covered a broad IS range, allowing to compare data also at low levels of disease (MR 4.5, 0.00316% BCR-ABL/ABL). Overall, linear regression demonstrated that there was a good correlation between Xpert® BCR-ABL Monitor and reference centers data (R2= 0.92). Correlation slightly increased when the data produced with Xpert® BCR-ABL Monitor were refined using a correction tool not automatically adopted by the current version of the assay software (R2= 0.93). Moreover, using assay comparison criteria proposed by Müller et al. (Leukemia 2009), Xpert® BCR-ABL Monitor was considered comparable to the standard laboratory methods of the reference centers, considering that all the three acceptance criteria were satisfied (58% of the samples between 0.5 and 2-fold variation, 78% of the samples between 0.33 and 3-fold variation, 91% of the samples between 0.2 and 5-fold variation). In conclusion, Xpert® BCR-ABL Monitor demonstrated to be a rapid, reliable and accurate test for monitoring of BCR-ABL mRNA transcript levels. Results were available within approximately 2 hours, potentially allowing clinicians to report results to patient the same day of the visit. From the technical point of view, cartridge-based automated system significantly reduced operator hands-on time from several hours to approximately 15 minutes. Concerning the assay comparative performance, Xpert® BCR-ABL showed to have a good inter-laboratory reproducibility, with no significant differences between the three reference centers standard methods. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Saglio: NOVARTIS: Consultancy. Pane:NOVARTIS: Consultancy, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

FOXM1 is a transcription factor of the Forkhead family which drives beta catenin nuclear import and transcriptional activation.Notably, it is a central component of a network which lets tumor cells bypass replicative stress and senescence and maintains the stem cell pluripotency. Accordingly, FOXM1 may have a role in the proliferative advantage and genomic instability of clonal hematopoiesis and leukemic stem cells (LSCs) of chronic myeloid leukemia (CML).Multiple events, including inactivation of protein phosphatase 2A (PP2A, which antagonizes cyclin A/Cdk-dependent activation of FOXM1), activation of Polo-like kinase 1 (PLK1, which directly promotes FOXM1 activating phosphorylation), suppression of Raf kinase inhibitor protein (RKIP, which down-regulates FOXM1 expression), depletion of APC/CDH1 E3 ligase (which degrades FOXM1 at mitotic exit) and down-regulation of micro RNA 370 (miR-370, which directly targets FOXM1 3’ UTR) may concur to BCR-ABL1-associated activation of FOXM1. Experiments carried in BCR-ABL1+ cell lines (K562 and 32D cell clones transduced with a temperature-sensitive mutant of BCR-ABL1 let establish that: 1-FOXM1 activation associated with BCR-ABL1 tyrosine kinase (TK) proceeds from post-transcriptional events encompassing the protein phosphorylation at serine-threonine residues and driving PLK1 activating phosphorylation; 2-FOXM1 phosphorylation is a key event for the interaction with beta catenin and PLK1; 3- the PLK1 inhibitor BI-2536, besides reducing the interaction of FOXM1 protein with beta catenin through events encompassing PLK1-induced phosphorylation of FOXM1, results in a significant reduction of FOXM1 transcript, suggesting a feedback mechanism involved in the regulation of FOXM1 expression in a BCR-ABL1+ cell context. Notably, a significant increment of FOXM1 transcript was found in mononuclear cells from bone marrow samples of 9 out of 15 CML patients at diagnosis compared to hematological healthy donors, which returned to control values in 4/4 patients when they reached a major molecular response to therapy with TK inhibitors. Computational analysis of FOXM1 sequences let distinguish the region encompassing a.a. 561-645 (ARM 10-12) as the one involved in FOXM1 interaction with a.a. 561-645 (ARM 10-12) of beta catenin. Site-directed mutagenesis of above mentioned FOXM1 and beta catenin regions (in progress) would let define FOXM1 role in the transformed phenotype associated with BCR-ABL1 and proceeding from FOXM1-mediated activation of beta catenin. The identification of FOXM1 as a component of BCR-ABL1-induced transformation may help in designing therapeutic strategies to specifically target beta catenin in clonal progenitors and LSCs, sparing the beta catenin signaling in other cell types, particularly, in normal hematopoietic stem cells. Acknowledgments: Umberto Veronesi Foundation, ELN, BolognaAIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project are acknowledged for financial support. MM is the recipient of a grant provided by the Umberto Veronesi Foundation. Disclosures Castagnetti: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Background and Aims- In Ph+ ALL pts treated with tyrosine kinase inhibitors (TKIs), the likelihood of acquiring TKI-insensitive mutations and the striking incidence of highly resistant T315I and compound mutants underscore the importance of BCR-ABL1 kinase domain (KD) sequence surveillance for timely and rational therapeutic reassessment. We used an amplicon DS strategy of the BCR-ABL1 KD to assess the following issues: i) whether DS allows earlier detection of emerging TKI-insensitive mutations in pts undergoing BCR-ABL1 KD mutation screening for minimal residual disease (MRD) persistence; ii) whether TKI-insensitive low burden mutations can be identified in relapsed pts with negative conventional sequencing results; iii) whether TKI-insensitive low burden mutations are necessary and sufficient to predict for treatment failure in all cases. Methods- This study was conducted in a total of 56 Ph+ ALL pts who received TKI-based therapies at our or collaborating institutions and were referred to our laboratory for MRD follow-up monitoring by RQ-PCR and for BCR-ABL1 KD mutation analysis in case of MRD positivity. These pts were divided into three different cohorts: i) 10 de novo and 24 advanced Ph+ ALL pts who relapsed and developed BCR-ABL1 KD mutations on TKI-based therapy administered 1st-line or for recurrent disease, respectively. To reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly-collected samples from the time of hematologic relapse backwards was performed by DS. Whenever samples were available, the analysis was done back to the time of diagnosis (n=10/10) or back to the time of first or former relapse (n=15/24), respectively. Two to 6 samples were analyzed for each pt, for a total of 109 samples. ii) 14 Ph+ ALL pts who were known to be negative for mutations at the time of hematologic relapse as assessed by conventional sequencing. Relapse samples were reanalyzed by DS. iii) 8 Ph+ ALL pts with long-term relapse-free survival despite persistent or intermittent MRD positivity at multiple timepoints. Up to 5 samples were analyzed for each pt, for a total of 28 samples. DS was performed on a Roche GS Junior. Lower mutation detection limit of DS was 1%. Results- In the 34 de novo or advanced Ph+ ALL pts who were known to have acquired TKI-insensitive mutations at the time of relapse on tyrosine kinase inhibitor (TKI) therapy, longitudinal retrospective reanalysis by DS allowed mutation backtracking in 13 (41%) cases. One patient was found to harbour a low burden Y253H at diagnosis. In 3 imatinib (IM)-resistant pts who switched to dasatinib (DAS), a low burden T315I mutation was already detectable at baseline. In the 14 pts with no mutations detectable by conventional sequencing at the time of relapse on IM or DAS, low burden TKI-insensitive mutations were detected by DS in 6 (43%) cases. In 2 cases who had relapsed on DAS, a T315I and an F317L mutation, respectively, were present just below the lower detection limit of conventional sequencing (15.9% and 12.4%, respectively); in the remaining 4 pts, DS identified multiple (2 to 3) low burden mutations, all of which known to confer a moderate to high degree of insensitivity to the ongoing TKI. In the 8 pts with persistently or transiently detectable BCR-ABL1 transcripts at multiple timepoints despite stable hematologic remission, DS detected low burden mutations in 9 samples from 4 pts. However, no mutation known to be truly insensitive to the ongoing TKI could be recognized. Conclusions MRD persistence in Ph+ ALL pts may hide emerging TKI-insensitive BCR-ABL1 KD mutations that DS may identify earlier than conventional sequencing - allowing a greater leeway before overt hematologic relapse occurs; polyclonal resistance sustained by multiple TKI-insensitive low burden mutations may explain relapse in a proportion of cases with unmutated BCR-ABL1 KD sequences as assessed by conventional sequencing; the type of mutation matters: detection of low burden mutations insensitive to the ongoing TKI was always found to predict/correlate with treatment failure. Detection of low burden mutations with low/unknown IC50 might explain low level MRD but does not predict for an impending relapse; MRD-triggered, BCR-ABL1 KD mutation screening by DS may be precious for earlier and more effective use of preemptive rescue therapies. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Abruzzese:BMS, Novartis, Pfizer, Ariad: Consultancy. Baccarani:ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:Onyx: Honoraria; BMS: Honoraria; Novartis: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Jansenn: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy; Ariad: Consultancy; MSD: Consultancy.

Imatinib (IM) targets the constitutively active BCR-ABL1 tyrosine kinase (TK) in chronic myeloid leukaemia (CML) and has become standard treatment based on good responses achieved in clinical trials. However, IM resistance can occur through several mechanisms including BCR-ABL1 kinase domain mutations, amplification, overexpression and clonal evolution. The discovery that Aurora kinases (AK) were abnormally expressed in malignancies including leukaemia prompted the development of agents that inhibit their activity. Aurora kinase A (AKA) is a central mitotic regulator necessary for mitotic entry, mitotic spindle assembly and accurate chromosome separation. The therapeutic potential of specifically targeting AKA activity as an anticancer strategy has not been rigorously investigated because all of the agents previously designed to target AKs have significant off-target effects on other family members and/or BCR-ABL1 kinase activity. However, the role of AKA in chemoresistance of CML is not well understood. In this study we investigated the mechanism of action of AKA in CML in vitro and ex vivo models (i.e. K562 cell lines and CD34+ cells derived from bone marrow aspirates of both CML at the clinical diagnosis and in mononuclear cell fraction of IM-resistant patients). Moreover, the role of two major players (i.e. FOXM1 and Polo-like kinase 1 (PLK1) a ser-thr kinase involved in G2/M progression) in the AKA signalling pathway has been investigated. First, we investigated AKA expression and activity by Western Blot and immunoprecipitation analyses. CD34+ cells from peripheral blood aphereses of hematologically healthy donors, pooled to avoid individual differences, were used as normal controls. Informed consent was obtained by all participants before their admittance to the study. Our data showed an over-expression and hyper-phosphorylation of AKA, FOXM1 and PLK1 in CD34+ cells of CML patients at clinical diagnosis compared to mononuclear cell fractions. Our results also showed a FOXM1 increment associated with IM resistance. An IM-resistant K562 cell line (LD50 0.25 mM vs 0.026 mM of parental cells) generated in our lab exhibited FOXM1 over-expression and hyper-phosphorylation contingent upon the upstream activation of AKA and PLK1. AKA, FOXM1 and PLK1 involvement in IM resistance was observed also in clonal progenitors from bone marrow samples of CML patients who developed IM resistance independent from BCR-ABL1 point mutations. Interestingly, the putative BCR-ABL1+/CD34+ LSC compartment, which is neither dependent on BCR-ABL1 TK for proliferation and survival nor killed by IM or second generation inhibitors, showed a hyper-phosphorylation of AKA and a consequent overexpression and hyper-activation of FOXM1 and PLK1. Taken together, these evidences may pave the way to the set up a new strategy to overcome drug resistance in CML. Moreover, our data identify a new signaling pathway involved both in drug resistance and in CD34+ cells survival, suggesting that AKA, FOXM1 and PLK1 are three very interesting druggable targets to eradicate the TKIs resistant CD34+ progenitors. Acknowledgments: Manuela Mancini was supported by a Fondazione Umberto Veronesi Fellowship. University of Bologna (RFO funds), Ministero della Pubblica Istruzione e della Ricerca (PRIN funds), Umberto Veronesi Foundation, AIRC, FP7 NCS-PLT project, Progetto Regione-Università 2010-12 (L. Bolondi) and BolognaAIL are acknowledged for financial support. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; AMGEN: Consultancy; MSD: Consultancy; Novartis: Consultancy, Speakers Bureau.

[Graphic][1] Background - Point mutations in the BCR-ABL kinase domain are associated with resistance to TKI therapy. The most recent (2013) European Leukemia Net (ELN) recommendations have re(de)fined the criteria for failure in pts receiving 1st-line and 2nd-line TKI therapy and introduced the concept of warning. Assessing in how many CML patients with failure and warning mutations can be identified, especially now that more sensitive NGS-based mutation screening methods are available, would advance our knowledge of the biology of TKI resistance as well as contribute useful data to revise the ELN recommendations as to when and how BCR-ABL mutation analysis should be performed. Aims - We aimed to determine the frequency of BCR-ABL mutations as assessed by NGS vs conventional Sanger sequencing (SS) in CML pts with failure and warning to 1st- or 2nd-line TKI therapy as per the latest, 2013 ELN definitions. Methods - Between May 2013 and June 2015, 298 consecutive CML pts on TKI therapy were referred to our laboratory for BCR-ABL mutation screening by SS. One hundred and fifty-eight cases had no clinical data available, or were not in CP, or were receiving ≥3rd-line TKI therapy, or had confirmed/suspected nonadherence, or had experienced dose reductions for toxicity - leaving 140 pts who could be included in this study. Pts who were negative for mutations as determined by SS (n=105/140) were retrospectively reanalyzed by NGS on a Roche GS Junior, using a protocol already set up and optimized in the framework of the IRON II (Interlaboratory RObustness of NGS) international consortium. Sequencing depth allowed to achieve a lower mutation detection limit of 1% in all samples. Results - Failures and warnings to 1st-line therapy (imatinib, n=57; nilotinib, n=22; dasatinib, n=13) were 63 and 29, respectively. BCR-ABL mutations were found in 15/63 (24%) failures and 3/29 (10%) warnings by SS (Table 1). NGS reanalysis of the 74 pts with no evidence of mutations by SS revealed low burden (median, 6.6%; range, 1.5-11.7%) mutations in 6 failures and 1 warning, so that, overall, 21/63 (33%) failures and 4/29 (14%) warnings turned out to have mutations (Table 1). Mutations were E462K, E279K, K262R, F359I, E255K, F317L, K378R, A399T, L364I, V280A. No compound mutation was detected. Failures and warnings to 2nd-line therapy (nilotinib, n=27; dasatinib, n=21) were 35 and 13, respectively. SS identified mutations in 13/35 (37%) failures and 2/13 (15%) warnings (Table 1). NGS reanalysis of the 33 pts with no evidence of mutations by SS revealed low burden (median, 5.4%; range, 1.9-10.0%) mutations in 5 failures and 2 warnings, so that, overall, 18/35 (51%) failures and 4/13 (31%) warnings turned out to have mutations (Table 1). Mutations were T315I, E255V, F317I, E258D, P480L, Y393C, W261L, L370P, V371A, L324Q, again with no compound mutations. | | All pts | Pts positive for mutations by SS | Additional pts positive for mutations by NGS | Total pts positive for mutations | | -------------------- | ------- | -------------------------------- | -------------------------------------------- | -------------------------------- | | 1ST -LINE FAILURES | | | No CyR @ 3 mo | 9 | 1 | | 1 | | BCR-ABL>10% @ 6 mo | 9 | | | | | mCyR @ 6 mo | 1 | 1 | | 1 | | BCR-ABL>1% @ 12 mo | 10 | | 2 | 2 | | No CCyR @ 12 mo | 2 | 1 | | 1 | | Loss of CCyR | 7 | 3 | 1 | 4 | | Loss of MMR | 20 | 6 | 3 | 9 | | Loss of CHR | 2 | 1 | | 1 | | Progression to BP | 3 | 2 | | 2 | | Total | 63 | 15 (24%) | 6 | 21 (33%) | | 1ST -LINE WARNINGS | | | BCR-ABL>10% @ 3 mo | 7 | 1 | | 1 | | BCR-ABL>1% @ 6 mo | 10 | 1 | 1 | 2 | | BCR-ABL>0.1% @ 12 mo | 12 | 1 | | 1 | | Total | 29 | 3 (10%) | 1 | 4 (14%) | | 2ND -LINE FAILURES | | | No CyR @ 3 mo | 3 | 1 | 1 | 2 | | BCR-ABL>10% @ 6 mo | 10 | 2 | 2 | 4 | | Loss of CCyR | 7 | 3 | | 3 | | Loss of MMR | 6 | 1 | 2 | 3 | | Loss of CHR | 4 | 3 | | 3 | | Progression to BP | 5 | 3 | | 3 | | Total | 35 | 13 (37%) | 5 | 18 (51%) | | 2ND -LINE WARNINGS | | | BCR-ABL>10% @ 3 mo | 6 | 2 | | 2 | | BCR-ABL>0.1% @ 12 mo | 7 | | 2 | 2 | | Total | 13 | 2 (15%) | 2 | 4 (31%) | Table. Conclusions 1) NGS allowed to identify BCR-ABL mutations in a greater proportion of cases as compared to SS. Low burden mutations included a T315I mutation in 2 pts on 2nd-line therapy classified as warnings: this would have turned them into failures. 2) Still, a substantial proportion of cases was found to not harbor any mutation, even when using a more sensitive NGS-based method. In particular, non-optimal achievement of the key molecular response milestones (10%, 1%, 0.1%) on 1st-line therapy was mostly not associated with BCR-ABL mutations, indicating that other mechanisms of molecular disease persistence have to be investigated in an attempt to optimize therapeutic outcomes. A national, multicenter study ('NEXT-IN-CML') aimed at the prospective assessment of NGS for routine BCR-ABL mutation screening of CML patients has just started. Supported by ELN, AIL, AIRC, FP7 NGS-PTL project, Progetto Regione-Universita 2010-12 (L. Bolondi) Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti: BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria. Bonifacio: Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Pfizer: Consultancy; Novartis Farma: Research Funding. Saglio: Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Rosti: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau. Baccarani: NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ARIAD Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Martinelli: Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; AMGEN: Consultancy; MSD: Consultancy; Ariad: Consultancy. [1]: /embed/inline-graphic-2.gif

Background and Aim although BCR-ABL kinase domain (KD) mutations can frequently be identified in patients who develop resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia-positive (Ph+) leukemias, other mechanisms may play a role. Small insertions and deletions within the BCR-ABL KD have occasionally been reported in chronic myeloid leukemia (CML) patients who failed TKI therapy and have been hypothesized to have a causative role in drug resistance. Some were in-frame insertions and deletions, others were predicted to result in truncated BCR-ABL proteins. However, the detection of these sequence variations is hampered by the fact that they are almost always confined to subclones co-exisiting with full length BCR-ABL. This has most likely resulted in an underestimation of their frequency and complexity, since i) they can be confused with background noise/reduced quality readings in direct sequencing chromatograms and ii) cloning would be needed to better resolve overlapping sequences in these samples. The recent development of Deep Sequencing (DS) technologies has opened the way to a more accurate characterization of molecular aberrations. DS enables greater sensitivity, quantitation of sequence variant abundance and clonal analysis of a given DNA region. We thus took advantage of DS to better characterize the spectrum of insertions and deletions in CML and Ph+ acute lymphoblastic leukemia (ALL) patients with response or resistance to TKI therapy. Methods a total of 110 samples of 41 CML and 16 Ph+ ALL patients who received one or multiple lines of TKI therapy were analyzed. DS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study. Runs were designed to achieve high sequencing depth; this allowed to reliably identify and characterize deletions or insertions with a lower detection limit of 0.1%. In order to reconstruct the dynamics of evolution of these sequence variations in relation to the TKI administered and to the level of response achieved, we evaluated their presence in serial follow-up samples collected during TKI therapy in 15 patients. Results DS revealed a 35-base pair (bp) insertion in 35/41 (85%) CML and 14/16 (87%) Ph+ ALL patients. This sequence variation, already reported in the literature as ‘35INS’, consists in the retention of 35 nucleotides (nt) from intron 8 at the exon 8 to exon 9 border. It leads to a truncated BCR-ABL variant having 10 a.a. encoded by intron 8 sequences but lacking 653 C-terminal a.a., including 22 a.a. of the KD, along with the entire C-terminal region. 35INS was detected with variable abundance (range 0.1%-96% of all BCR-ABL transcripts), but in only three samples abundance was higher than 15-20% - thus detectable also by conventional sequencing. Re-sequencing a set of samples in the same and independent runs confirmed the presence of the 35INS and demonstrated that this variant was not a PCR or sequencing artifact. Longitudinal analysis showed that the expression of 35INS fluctuated over time with no apparent correlation with response levels. In addition, DS detected one in-frame deletion in 20/41 (48%) CML patients and 7/16 (44%) Ph+ ALL patients, with an abundance ranging from 0.2% to 19%. This previously unreported variant consisted of a 72bp deletion (nt.1233-1304) at the junction of exon 6 to exon 7, that causes the loss of 24 residues (a.a. 359-383) of the KD. Conclusions Our results further underline that DS technologies allow more accurate sequence characterization in comparison to conventional methods. Minor clones harboring insertions or deletions (always involving intron/exon junctions - which implicates alternative or aberrant splicing mechanisms) were found to be very frequent both in CML and in Ph+ ALL patients but, apparently, did not correlate with response or resistance to TKI therapy. In line with our findings, a very recent functional study has demonstrated that the truncated BCR-ABL protein resulting from the 35INS is kinase-inactive and should not play any role in TKI-resistance - in contrast to what had initially been hypothesized. However, further analysis of a larger number of samples would be needed to better understand the biological and clinical meaning of these minor clones surviving TKI therapy. Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL’, IGA NT 11555/13899. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Martinelli:NOVARTIS : Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Background Ponatinib, a potent third generation pan BCR-ABL inhibitor, has recently shown relevant activity against native and mutant forms of BCR-ABL, including the TKI resistant T315I mutant. The aim of this compassionate protocol was to confirm and evaluate the efficacy and the safety of the compound in patients with advanced Ph+ ALL and CML. Design and Methods Ponatinib was obtained through a compassionate use named patient program, approved by ARIAD Pharmaceuticals and by the Bologna Ethical Committee. After informed consent was signed, 17 patients (M/F: 8/9) have been treated with Ponatinib (45 mg orally, once daily) between February 2012 and July 2013, including 14 Ph+ ALL (10 p190, 4 p210) and 3 blast phases of CML (2 Myeloid and 1 Lymphoid, p210). The median age of the patients was 64 years (range 23 -77). The median time from diagnosis was 754 days (range 46-2264). All the patients were resistant or intolerant to previous TKIs (median number of previous TKIs: 2; range 1-3). Standard chemotherapy was previously performed in 7/17 patients (41%). Four patients (23%) had previously received allogeneic stem cell transplantation. At the time of enrolment, median Hb, PLTs and WBC values were 10,9 g/dl (range 8.6-13.9), 139000/mmc (range 14000-325000) and 4300/mmc (range 1700-17000), respectively. In 6 out of 17 patients, additional cytogenetic alterations were revealed. Mutational analysis showed the presence of T315I mutation (9 pts), G250E (1 pt), T315I and Y253H (1 pt), T315I and Y253A (1 pt), V299L (1 pt). No mutations were detected in 4 patients. Results The median treatment duration was 139 days (range 14-540+). Causes of treatment stop were: progression disease (5 patients), savage allogenic stem cell transplantation (6 patients), drug intolerance (1 patient), consisting in grade III headache. With a median follow up of 284 days (range 8-540+), a maHR was obtained in 13/17 patients (76%). After one month of treatment, a reduction of BCR-ABL fusion transcript level was observed in 9/15 patients (60%). For two patients the follow up is too short to be evaluable. The level became undetectable in 4 patients (3 presenting with T315I mutation). After treatment, T315I mutation disappeared in 6 out of the 9 patients who showed this molecular alteration at the beginning of therapy. At the time of this report, 6/17 patients are still on study (35%). Five patients died due to progression disease. As expected, the drug was well tolerated. Non-hematologic adverse events were described in 7/17 patients (grade >III skin rash in 3 patients; grade>II serum lipase increase in 2 patients; grade>II myalgia in 1 patient; grade III headache). Conclusion In our experience, the activity of Ponatinib in advanced Ph+ leukemias, mainly in T315I mutated patients, was confirmed. No treatment-related deaths occurred. The understanding of molecular mechanisms responsible for resistance or lack of response to the drug will be necessary in order to identify patients early on who could take advantage of this treatment. Acknowledgments Work supported by European LeukemiaNet, AIRC, PRIN 2010-2011, University of Bologna and BolognAIL. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 692 Background and Aims: In chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (ALL), tyrosine kinase inhibitor (TKI) therapy may select for drug-resistant Bcr-Abl mutants. Mutation status of resistant patients is usually investigated by Sanger sequencing (SS) of the Bcr-Abl kinase domain (KD). Novel ultra-deep sequencing (UDS) technologies allow to conjugate higher sensitivity with the unprecedented possibility to perform instant cloning of thousands of DNA molecules. We thus decided to take advantage of an UDS-based approach in order to: 1. resolve qualitatively and quantitatively the complexity of mutated populations surviving TKIs; 2. investigate their clonal structure and evolution in relation to time and treatment. Methods: We retrospectively performed a longitudinal analysis of a total of 111 samples from 35 CML or Ph+ ALL patients who had received sequential treatment with multiple TKIs (two to four TKIs among imatinib, dasatinib, nilotinib, ponatinib) and had experienced sequential relapses accompanied by selection of TKI-resistant mutations. All samples had already been scored by SS; 74/111 (67%) were positive for one (n=33) or multiple (n=41) mutations. UDS of the Bcr-Abl KD was done using Roche 454 technology. UDS allowed to achieve a lower detection limit of at least 0.1% – as compared to 20% of SS. Results: Bcr-Abl KD mutation status was found to be more complex than SS had previously shown in 85/111 (77%) samples (representative examples are detailed in Table 1 ). In 33/74 (44%) samples known to harbour one or more mutations by SS, UDS revealed that up to four ‘minor' mutations with 1–20% abundance were present in addition to the ‘dominant' one(s). The type of mutations could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the current or to the previous TKI received. The higher degree of complexity was evident also when the clonal relationships of multiple mutations were reconstructed ( Table 1 ). This revealed that identical mutations may be acquired in parallel by independent populations (e.g., one wild-type and one already harboring a mutation), via the same or different nucleotide changes leading to the same amino acid substitution (convergent evolution). In addition, longitudinal quantitative follow-up of mutated populations revealed that: 1. complexity generally increases with increasing lines of TKI therapy; 2. with a few exceptions, double compound mutants have higher selective advantage over single mutants but also over triple; 3. however, whether a compound mutant will ultimately take over depends on TKI, treatment duration, competition with other coexisting populations - the same compound mutants behaved differently in different patients receiving the same TKI. Conclusions: 1. sequential changes in the selective pressure exerted by TKIs may result in a heterogeneous mosaic of subclones harbouring different mutations or mutation combinations; 2. the evolution of each subclone is shaped not only by its inherent sensitivity to the specific TKI administered (‘absolute' fitness) but also by the competition with all other coexisting subclones (‘relative' fitness); (nonlinear) acquisition of additional mutations dictates further dynamics of shrinkage/expansion over time; 3. information provided by SS may not always be sufficient to predict responsiveness to a TKI; 4. sensitivity of a single or a compound mutant to a TKI in vivo is dictated by more complex factors than the mere in vitro IC50 value. Table 1 . Sample TKI (line) Mutations by SS (%) Mutations by UDS (%) Mutated populations by UDS (%) CML-04 DAS (3rd, after IM and NIL) T315I (∼60) E255K (∼30) T315I (54.89) E255K (26.15) E255V (1.37) T315I (47.02) E255K (18.63) T315I+E255K (7.52) E255V (1.02) T315I+E255V (0.35) ALL-09 PON (3rd, after IM and DAS) E255K (∼70) T315I (∼50) E255K (76.25) T315I (52.19) Q252H (cag>cac) (14.20) Q252H (cag>cat) (7.49) G250E (0.95) E255K+T315I (51.60) E255K+Q252H (cag>cac) (13.94) E255K+Q252H (cag>cat) (7.34) T315I (5.75) E255K (2.52) E255K+ G250E (0.70) Q252H (cag>cac) (0.25) G250E (0.25) Q252H (cag>cat) (0.15) CML-21 DAS (2nd, after IM) Y253H (∼60) F317L (∼60) Y253H (54.90) F317L (54.40) Y253H+F317L (43.00) Y253H (11.90) F317L (11.40) Disclosures: Soverini: ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Castagnetti: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Luppi: CELGENE CORPORATION: Research Funding. Rosti: Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Baccarani: ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli: NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

High sequencing depths of NGS may cause false-positive variant calls of minor subclones (up to 10%). Errors inserted into the NGS pipeline during sample preparation and sequencing manifest by erroneous detections of mutations including point mutations. Thus, there is a need for algorithms for filtering data which occur by error from relevant biological information. We developed algorithms and a processing pipeline for error correction and detection of significant mutations after NGS of BCR-ABL kinase domain (KD). We validated our algorithms on the retrospective NGS analysis of 135 samples from 15 CML patients in chronic phase (median 8 samples per patient; range 5-19), who developed resistant mutations (confirmed by Sanger Sequencing, SS) during 2-4 lines of therapy. Amplicon libraries were prepared using reverse transcription and 2-step PCR. The second PCR was performed partly using fusion-primers designed within the IRON-II study research consortium (Roche Applied Science) which tested 4 overlapping amplicons and partly using alternative in-house set of fusion-primers that we have developed upfront and which utilized 3 overlapping amplicons covering the KD coding region. Key concept of our error control algorithm was to apply statistics used for bacterial mutation rate prediction, Lea-Coulson probability distribution (Lea and Coulson, J of Genet 1949), to distinguish sequencing pipeline errors from biologically relevant mutations. We postulated that spontaneous mutations in bacteria are similar phenomena as enzyme errors in vitro. In both processes there are new generations of bacteria or transcripts in which mutations or errors replicate exponentially. The error rate distributions based on analysis of c-ABL kinase domain of healthy donors (n=24) were fitted to Lea-Coulson distribution. From this analysis we derived, for each type of single nucleotide substitution, estimated thresholds based on which a particular mutation may be called significant by a self-developed statistical test. We cross-checked our results with results of standard Roche pipeline including GS Amplicon Variant Analyzer. Table 1 summarizes the estimated thresholds to be applied for transitions and transversions. Higher frequency of errors was found in case of using a 3-amplicon assay in comparison to a 4-amplicon assay. The PCR products in the 3-amplicon assay are 71 bp longer on average than in the 4-amplicon assay, thus the error frequency distribution may be dependent on the length of the sequence amplified. Using our algorithm we processed NGS data and reported significant mutations. Overall, no significant mutation that caused resistance during the treatment was detected at the time of diagnosis. During 1st line imatinib treatment 10 resistant mutations in 9 patients were detected as significant 2-5 months earlier than by SS. At the time of therapy switchover, in 3 patients the algorithm already detected minor populations of one of significant mutations F317L, T315I and M351T, while SS did not. These mutations manifested after the therapy switchover and caused treatment failure. After the therapy switch, baseline mutations were still significantly detectable by our algorithm in NGS data, but not by SS in 7 patients who achieved at the time of the analysis PCgR and MMR. In 5 patients, who subsequently failed therapy after switchover, resistant mutations were significantly detected by our algorithm in NGS data 2-9 months earlier than by SS. New minor mutations were revealed by NGS after the therapy switch in 8 patients.Table 1TRANSITIONS (%)TRANSVERSIONS (%)P valueA/GG/AT/CC/TA/C+C/A+T/A+A/T+T/G+G/T+C/G+G/C3-amplicon assay0.0112.24.5311.84.770.570.053.031.032.931.100.134-amplicon assay0.015.171.934.501.930.130.051.200.431.030.430.03 Since enzymes create errors during reverse transcription, 2-step PCR and sequencing process, the error correction is an essential part of the bioinformatics pipeline for relevant interpretation of BCR-ABL KD mutations detected with the highly sensitive NGS assay. Our validated algorithm and processing pipeline for significant mutation evaluations from NGS data is helpful for future clinical practice as it filters errors and allows reporting only significant mutations. This avoids false-positive results and misleading interpretations which may negatively influence treatment management of CML patients. Supported by IGANT11555 and NT13899 Disclosures: Machova Polakova: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:NOVARTIS, BMS(Consultancy and speaker bureau), PFIZER, ARIAD ( Consultancy): Consultancy, Speakers Bureau. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Klamova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.