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Our study aimed to investigate whether radiomics on MRI sequences can differentiate responder (R) and non-responder (NR) patients based on the tumour regression grade (TRG) assigned after surgical resection in locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiotherapy (nCRT). Eighty-five patients undergoing primary staging with MRI were retrospectively evaluated, and 40 patients were finally selected. The ROIs were manually outlined in the tumour site on T2w sequences in the oblique-axial plane. Based on the TRG, patients were grouped as having either a complete or a partial response (TRG = (0,1), n = 15). NR patients had a minimal or poor nCRT response (TRG = (2,3), n = 25). Eighty-four local first-order radiomic features (RFs) were extracted from tumour ROIs. Only single RFs were investigated. Each feature was selected using univariate analysis guided by a one-tailed Wilcoxon rank-sum. ROC curve analysis was performed, using AUC computation and the Youden index (YI) for sensitivity and specificity. The RF measuring the heterogeneity of local skewness of T2w values from tumour ROIs differentiated Rs and NRs with a p-value ≈ 10−5; AUC = 0.90 (95%CI, 0.73–0.96); and YI = 0.68, corresponding to 80% sensitivity and 88% specificity. In conclusion, higher heterogeneity in skewness maps of the baseline tumour correlated with a greater benefit from nCRT.

Background: COVID assessment can be performed using the recently developed individual risk score (prediction of severe respiratory failure in hospitalized patients with SARS-COV2 infection, PREDI-CO score) based on High Resolution Computed Tomography. In this study, we evaluated the possibility of automatizing this estimation using semi-supervised AI-based Radiomics, leveraging the possibility of performing non-supervised segmentation of ground-glass areas. Methods: We collected 92 from patients treated in the IRCCS Sant’Orsola-Malpighi Policlinic and public databases, each lung was segmented using a pre-trained AI method, ground-glass opacity was identified using a novel, non-supervised approach, radiomic measurements were collected and used to predict clinically relevant scores, with particular focus on mortality and the PREDI-CO score. We compared the prediction obtained through different machine learning approaches. Results: All the methods obtained a well-balanced accuracy (70%) on the PREDI-CO score but did not obtain satisfying results on other clinical characteristics due to unbalance between the classes. Conclusions: Semi-supervised segmentation, implemented using a combination of non-supervised segmentation and feature extraction, seems to be a viable approach for patient stratification and could be leveraged to train more complex models. This would be useful in a high-demand situation similar to the current pandemic to support gold-standard segmentation for AI training.

Background: Bile leak (BL) after hepato-pancreato-biliary (HPB) surgery is associated with significant morbidity and mortality. Aim of this study was to evaluate effectiveness and safety of percutaneous transhepatic approach (PTA) to drainage BL after HPB surgery. Methods: Between 2006 and 2018, consecutive patients who were referred to interventional radiology units of three tertiary referral hospitals were retrospectively identified. Technical success and clinical success were analyzed and evaluated according to surgery type, BL-site and grade, catheter size and biochemical variables. Complications of PTA were reported. Results: One-hundred-eighty-five patients underwent PTA for BL. Technical success was 100%. Clinical success was 78% with a median (range) resolution time of 21 (5–221) days. Increased clinical success was associated with patients who underwent hepaticresection (86%,p = 0,168) or cholecystectomy (86%,p = 0,112) while low success rate was associated to liver-transplantation (56%,p < 0,001). BL-site,grade, catheter size and AST/ALT levels were not associated with clinical success. ALT/AST high levels were correlated to short time resolution (17 vs 25 days, p = 0,037 and 16 vs 25 day, p = 0,011, respectively) Complications of PTA were documented in 21 (11%) patients. Conclusion: This study based on a large cohort of patients demonstrated that PTA is a valid and safe approach in BL treatment after HPB surgery.

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.

Purpose: Patients with hepatocellular carcinoma (HCC) of intermediate stage (BCLC-B according to the Barcelona Clinic Liver Cancer classification) are a heterogeneous group with different degrees of liver function impairment and tumour burden. The recommended treatment is transarterial chemoembolization (TACE). However, patients in this group may be judged as poor candidates for TACE because the risk-benefit ratio is low. Such patients may receive transarterial radioembolization (TARE) only by entering a clinical trial. Experts have proposed that the stage could be further divided into four substages based on available evidence of treatment benefit. We report here, for the first time, the outcome in patients with BCLC-B2 substage HCC treated with TARE. Methods: A retrospective analysis of the survival of 126 patients with BCLC-B2 substage HCC treated with TARE in three European hospitals was performed. Results: Overall median survival in patients with BCLC-B2 substage was not significantly different in relation to tumour characteristics; 19.35 months (95% CI 8.27–30.42 months) in patients with a single large (>7 cm) HCC, and 18.43 months (95% CI 15.08–21.77 months) in patients with multinodular HCC (p = 0.27). However, there was a higher proportion of long-term survivors at 36 months among those with a single large tumour (29%) than among those with multiple tumours (16.8%). Conclusion: Given the poor efficacy of TACE in treating patients with BCLC-B2 substage HCC, TARE treatment could be a better choice, especially in those with a large tumour.

In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL, n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%–18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.

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.

Background The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. Case presentation We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. Conclusions This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an ‘act’ to ‘atc’ codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3511-2) contains supplementary material, which is available to authorized users.

FOXM1 transcription factor is a central component of tumor initiation, growth, and progression due to its multiple effects on cell cycle, DNA repair, angiogenesis and invasion, chromatin, protein anabolism, and cell adhesion. Moreover, FOXM1 interacts with β-catenin promoting its nuclear import and transcriptional activation. Here, we show that FOXM1 is involved in the advantage of chronic myeloid leukemia hematopoiesis over the normal counterpart. FOXM1 hyper-activation associated with BCR-ABL1 results from phosphorylation by the fusion protein kinase-dependent activation of Polo-like kinase 1. FOXM1 phosphorylation lets its binding with β-catenin and β-catenin transcriptional activation, a key event for persistence of the leukemic stem cell compartment under tyrosine kinase inhibitor therapy. Polo-like kinase 1 inhibitor BI6727, already advanced for clinical use, breaks β-catenin interaction with FOXM1, hence hampering FOXM1 phosphorylation, β-catenin binding, nuclear import, and downstream signaling. In conclusion, our results support Polo-like kinase 1/FOXM1 axis as a complementary target to eradicate leukemic early progenitor/stem cell compartment in chronic myeloid leukemia. J. Cell. Biochem. 118: 3968-3975, 2017. © 2017 Wiley Periodicals, Inc.

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic ‘autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found in 11/20 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBank X06182.1: c.1550_1561delGTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBank X06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 2/20 ISM. This mutation is known to retain sensitivity to imatinib mesylate. Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alternative to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a starting point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Cavo: Celgene: Consultancy, Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Background According to the 2013 European LeukemiaNet recommendations, BCR-ABL kinase domain (KD) mutation screening by conventional Sanger sequencing (SS) is recommended in all patients (pts) with progression, failure or warning, since detection of second-generation (2G) TKI-resistant mutations (T315I, F317L/V/I/C, V299L, Y253F/H, E255K/V, F359V/I/C) helps in the selection of the most suitable alternative therapy. However, SS has limited sensitivity. A recent study (Parker et al, J Clin Oncol 2011) has shown that low level mutations (i.e., undetectable by SS) may be identified by mass spectrometry and this may aid in more appropriate selection of the second-line treatment strategy for imatinib-resistant pts. Aims We sought to determine i) whether an UDS-based approach for BCR-ABL KD mutation screening might allow more sensitive detection of 2GTKI-resistant mutations at the time of switchover to second or third-line therapy and ii) whether low level mutations identified by UDS undergo clonal expansion, thus predicting for subsequent failure, if the 2GTKI to which they are insensitive to happens to be chosen. Methods To this purpose, we used UDS to scan the BCR-ABL KD in a cohort of 75 chronic myeloid leukemia (n=50) or Philadelphia chromosome-positive acute lymphoblastic leukemia (n=25) pts who were treated with a 2GTKI (dasatinib or nilotinib) after having failed first-line (n=62) or second-line (n=13) TKI therapy. A total of 235 samples collected at the time of switchover and at subsequent timepoints during second-or third-line treatment were analyzed. All the samples had already been evaluated by SS. UDS 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. However, raw data analysis with Amplicon Variant Analyzer software (Roche Applied Science) was performed filtering out variants Results Forty-three imatinib-resistant pts failed subsequent second- (n=37) or third-line (n=6) treatment with 2GTKIs (dasatinib, n=28; nilotinib, n=15). By routine SS analysis, 32 BCR-ABL KD mutations had been identified in 29/43 (67%) pts at the time of switchover - informing subsequent-line treatment selection in 12 pts who were found to harbor 2GTKI-resistant mutations. At the time of treatment failure (after a median of 9 months; range, 2-30), SS had detected newly acquired 2GTKI-resistant mutations in all 43 pts. We thus wondered how many of these mutations could have been detected at the time of switchover using a more sensitive approach. By UDS re-analysis, 73 mutations were identified in 35/43 (81%) pts at switchover – UDS detected all the 32 mutations previously identified by SS plus 41 low level mutations (i.e., with an abundance between 1 and 15%). Twenty-four of the 41 low level mutations were resistant to the 2GTKI that happened to be selected (T315I=10; E255K=2; E255V=1; Y253H/F=4; F359V=1; F317L=4; V299L=1) and all invariably expanded becoming detectable by SS at relapse, alone or in combination with pre-existing mutations. Thus, mutations that would have influenced therapeutic decision after first or second TKI failure could have been detected in 17 more pts by UDS (p Thirty pts who achieved a stable response to second- (n=26) or third-line (n=4) treatment with 2GTKIs after having failed first- or second-line treatment, respectively, were also analyzed for comparison. By UDS, a total of 13 mutations were detected – including 6 mutations (6 pts) that had already been identified by SS plus 7 low level mutations (7 pts). No low level mutation resistant to the 2GTKI the pts actually received was detected. Conclusions In comparison to SS, UDS could identify 2GTKI-resistant mutations at the time of switchover in a significantly greater proportion of pts. Low level mutations (down to 1% abundance) detected by UDS were consistently found to expand when the 2GTKI they are insensitive to happened to be chosen. UDS-based screening of the BCR-ABL KD at switchover might thus have enabled more effective therapeutic tailoring. Further evaluation of whether this technology may be implemented in a diagnostic setting is highly warranted. Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL’, IGA NT 11555 and 13899. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Castagnetti:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Baccarani:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees, Speaker fees Other; Teva: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speaker fees Other. Martinelli:Novartis: Consultancy, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Speaker fees, Speaker fees Other; Ariad: Consultancy, Speaker fees, Speaker fees Other.

Chronic myeloid leukemia (CML) has been the first human malignancy to be associated, more than 50 years ago, with a consistent chromosomal abnormality--the t(9;22)(q34;q11) chromosomal translocation. The resulting BCR-ABL1 fusion gene, encoding a tyrosine kinase with deregulated activity, has a central role in the pathogenesis of CML. Ancestral or additional genetic events necessary for CML to develop have long been hypothesized but never really demonstrated. CML can successfully be treated with tyrosine kinase inhibitors (TKIs). Mutations in the BCR-ABL1 kinase domain might arise, however, that confer resistance to 1 or more of the currently available TKIs. Hence, the critical role of BCR-ABL1 mutation screening for optimal therapeutic management, with the current gold standard technique, conventional sequencing, likely to be replaced soon by ultra-deep sequencing. Mutations in genes other than BCR-ABL1 include ASXL1, TET2, RUNX1, DNMT3A, EZH2, and TP53 in chronic phase patients and RUNX1, ASXL1, IKZF1, WT1, TET2, NPM1, IDH1, IDH2, NRAS, KRAS, CBL, TP53, CDKN2A, RB1, and GATA-2 mutations in advanced phase patients. The latter also display additional cytogenetic abnormalities, including submicroscopic regions of gain or loss that only single nucleotide polymorphism arrays or array comparative genomic hybridization can detect. Whether whole genome/exome sequencing studies will uncover novel mutations relevant for pathogenesis, progression, and risk-adapted therapy is still unclear.

Abstract 2601 Background: Tyrosine Kinase Inhibitors (TKI) have been shown to be very effective for the treatment of Acute Lymphoblastic Leukemia (ALL), with a Complete Hematologic Remission (CHR) rate close to 100%, and a high rate of Complete Cytogenetic and Molecular responses (CCgR and CMR). However, when they are used alone, as single agents, most patients relapse, so that they are currently used in combination with chemotherapy and as a preparation to allogeneic stem cell transplantation (SCT). Since Ph+ ALL is more frequent in the elderly, many patients cannot tolerate intensive chemotherapy and are not eligible for SCT. We have explored if the administration of two TKIs, Nilotinib (NIL) and Imatinib (IM) can improve the results without increasing the toxicity. Aims: To evaluate the response and the outcome of Ph+ ALL patients treated with the sequential administration of NIL and IM, to investigate the type and number of BCR-ABL kinase domain mutations developing during and after the study. Methods: We have designed a study (ClinicalTrials.gov. NCT01025505) in which patients more than 60 years old or unfit for intensive chemotherapy and SCT where treated with two TKIs, NIL 400 mg twice daily, and IM 300 mg twice daily, alternating for 6 weeks for a minimum of 24 weeks (study core) and indefinitely in case of response. The 6-weeks rotation schedule was respected, irrespectively of temporary discontinuations. The primary end-point was the rate of Disease Free Survival (DFS) at 24 weeks (4 courses of treatment); the secondary end points included the evaluation of CHR, CCgR and CMR rates. Mutation analysis was performed by nested RT-PCR amplification of the ABL kinase domain of the BCR-ABL transcript (codons 206 through 421). Amplified products were screened by denaturing-high performance liquid chromatography (D-HPLC). Samples scored positive for the presence of sequence variations were then subjected to direct automatic sequencing to characterize the mutation. Results: 39 patients have been enrolled in 15 Italian hematologic Centers (median age 66 years, range 28–84). Among these, 8 patients were unfit for standard chemotherapy or SCT (median age 50 years, range 28–59). 27 patients were p190, 5 were p210 and 7 were p190/p210. After 6 weeks of treatment, 36 patients were evaluable for response: 34 were in CHR (94%) and 2 in PHR (6%). 23 patients have already completed the study core (24 weeks), 87% were in CHR and 17 are currently continuing therapy in the protocol extension phase. Thus, the OS at 1 year is 79%, and 64% at 2 years. Overall, 1 patient was primarily resistant and 13 patients have relapsed, with a median time to relapse of 7.6 months (range 0.8–16.1 months), for a DFS of 51.3% at 12 months (Figure 1). Mutations detected were T315I in 2 cases, Y253H in 3 cases, T315I and Y253H in 1 case, E255K in 1 case, T315I and E255K in 1 case, E255V and Y253H in 1 case. Two patients were WT. A detailed kinetics of Molecular responses is shown in Table 1. Data on mutational analysis are reported in Table 2. Further details about Cytogenetic and Molecular responses, and about Adverse Events will be provided on site. Conclusions: In this small cohort of Ph+ ALL elderly/unfit patients, the rates of relapse and progression were not likely to be different from the rates observed with Imatinib alone (Vignetti et al, Blood 2007, May 1;109(9):3676-8) and Dasatinib alone (Foà, Blood 2011, Dec 15;118(25):6521-8). It's important to notice that the mutations that occurred at the time of relapse were sensitive to other TKIs (Dasatinib and Ponatinib). Acknowledgments: COFIN, Bologna University, BolognAIL, PRIN, Fondazione del Monte di Bologna e Ravenna, INPDAP. Disclosures: Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria. Luppi:CELGENE CORPORATION: Research Funding. Vallisa:CELGENE CORPORATION: Research Funding. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau.

Abstract 1749 Introduction. Mastocytosis is a myeloid neoplasm characterized by abnormal accumulation and frequent activation of mast cells (MCs) in various organs, mostly bone marrow, skin, liver and gastrointestinal tract. In most adult patients, the systemic form of mastocytosis (SM) is diagnosed, which includes an indolent, an aggressive and a leukemic subvariant. The c-kit mutation D816V is detectable in most adult patients with SM. Treatment of SM usually focuses on symptom relief by histamine receptor antagonists and other supportive therapy. However, in aggressive and leukemic variants, cytoreductive and targeted drugs must be applied. Methods. From 2008, 11 patients (male/female=3/8) affected by Mastocytosis, have been referred to our Institution. The median age was 53 years. All the patients underwent a bone marrow biopsy, with flow citometry and molecular biology analysis, in order to identify the presence of D816V mutation of c-Kit gene. Serum tryptase level was tested, resulting elevated in all cases. According to 2008 WHO diagnostic criteria, 9 patients presented with SM, whereas in the other cases a skin isolated involvement was detected. Systemic symptoms were characterized by nausea, diarrhoea, asthenia, weight loss, pruritus and serotine fever, identifying an aggressive form of the disease in 5/11 patients, due to skeletal involvement in three cases, ascitis and liver function impairment in another one and bone marrow disfunction in the fifth one. Therefore, since a first line therapy with supportive care and histamine receptor antagonists wasn't followed by a significant benefit, a personalized use of PKC412 was asked and obtained for all these five patients. Results. From March 2011 three out of the five patients with aggressive SM have received a prolonged period of treatment with PKC412, which was administered orally, at the dosage of 100 mg twice daily, without rest periods. The drug was well tolerated. No serious adverse events were observed. All the patients obtained a quick and prolonged improvement of clinical symptoms, in terms of weight gain, bowel function and skeletal pain. At the bone marrow evaluation, the persistence of the D816V c-kit mutation was observed, despite a significant decrease of mast cell marrow involvement. In one case we observed, after a first good response, a disease progression, characterized by the sudden reoccurrence of the same symptoms detected at diagnosis, confirmed by a relevant expansion of a pathologic mast cell population in the bone marrow. After a rest period, the drug was readministered, and a second remission was obtained. Conclusions. PKC412 is safe and effective in patients with SM, being able to significantly improve not only the gastrointestinal and systemic symptoms, but also the haematological profile. The persistence of the D816V c-kit mutation, despite a morphologic remission, suggests that many other oncogenic factors may be responsible for the pathogenesis of the disease. Acknowledgments. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, Fondazione del Monte di Bologna e Ravenna, University of Bologna. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy, Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Abstract 284 Background and Aims: Selection of drug-resistant mutations in the Bcr-Abl kinase domain (KD) is a critical problem undermining the long-term efficacy of tyrosine kinase inhibitor (TKI)-based therapies in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients. Bcr-Abl KD mutation screening is routinely performed by Sanger sequencing (SS). Before the advent of ultra-deep sequencing (UDS) technologies, no method was available that could conjugate the possibility to scan the KD for the so many mutations known to be associated with TKI resistance with a sensitivity higher than that of SS. UDS technologies also allow high throughputness and accurate quantitation of mutated clones and their application in a diagnostic setting is not far to come. We used an UDS strategy for Bcr-Abl KD mutation screening in order to study the dynamics of expansion of mutated clones in Ph+ ALL patients receiving TKI-based therapies and to test the ability of UDS to highlight emerging clones harboring critical mutations. Methods: 72 samples from 25 Ph+ ALL patients who had developed resistance to one or multiple lines of TKI (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) therapy were selected for this retrospective analysis. All the patients had previously been analyzed by Sanger sequencing (SS) and were known to have developed one or more TKI-resistant Bcr-Abl KD mutations on treatment. In order to reconstruct the dynamics of mutation emergence, longitudinal re-analysis of monthly collected samples was perfomed with UDS on a Roche GS Junior. UDS allowed to achieve a lower detection limit of at least 0.1% (by generating a minimum of 5,000 sequence reads/patient), as compared to 20% of SS. Results: 39 samples were known to harbor one (n=27 samples) or more (n=12 samples) TKI-resistant mutations with >20% abundance, as assessed by SS. UDS could successfully detect all the 54 mutations previously identified by SS. In addition, UDS detected one or multiple lower-level (20% abundance. The type of lower-level mutations detected by UDS could easily be accounted for by TKI exposure history, since the majority were known to be poorly sensitive either to the TKI being administered or to the previous TKI received. Overall, 44 samples turned out to carry multiple (two to five) mutations at any level, distributed in the same and/or in different subpopulations with a complex clonal architecture that UDS allowed to reconstruct. Of note, in 14/25 (56%) patients with molecularly detectable disease but not yet evidence of cytogenetic or hematologic relapse, UDS could identify emerging TKI-resistant mutations 1 to 2 months before they became detectable by SS. These outgrowing mutations were detected at 1–19% abundance in 12 patients and at 0.1–1% abundance in 2 patients. In the remaining 11 patients, dynamics of outgrow of the TKI-resistant mutations (five T315I, two Y253H, two E255K, one E255V and one F317L) was so rapid that not even strict monthly monitoring could allow to pick them up before they became dominant. Conclusions: Now that multiple options are available, Bcr-Abl KD mutation monitoring has become a precious tool for rational decision-making in order to maximize the efficacy of TKI-based regimens as induction or salvage therapy for Ph+ ALL patients. UDS proved as reliable as SS for the detection of mutations with >20% abundance and to have comparable costs. As a key advantage, UDS added precious quantitative and qualitative information on the full repertoire of mutated populations, that SS failed to appreciate in more than half of the samples analyzed. TKI-resistant mutations leading to patient relapse were not necessarily preexisting at low levels at diagnosis or at the time of switchover to another TKI, underlining the importance of regular monitoring of patients. Although TKI-resistant populations may arise and take over very rapidly, in approximately half of the patients monthly monitoring with UDS would have allowed to identify them earlier than SS and well in advance of clinical relapse, thus allowing a more timely therapeutic intervention. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Luppi:CELGENE CORPORATION: 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.

The course of chronic myeloid leukemia (CML) and the response to treatment with tyrosine kinase inhibitors (TKIs) are best monitored and assessed using two molecular tests: the first is real-time quantitative reverse transcription-polymerase chain reaction (RQ-PCR), which measures the size of residual disease that is expressed as BCR-ABL1% (the ratio between BCR-ABL1 and a control gene) and the other is mutational analysis by Sanger sequencing, which checks for the presence of BCR-ABL1 kinase domain point mutations. Both tests are technically demanding and require a high level of specialization and standardization. RQ-PCR, when performed on a regular basis, allows for the defining of molecular response (MR) levels as log reduction from a standardized baseline: major molecular response (MMR or MR3) that is the best predictor of survival; and the deeper molecular response (MR4, MR4.5, and MR5) that is necessary to enroll a patient in a trial aiming at treatment-free remission (TFR). Mutational analysis, to be performed in case of failure or warning by Sanger sequencing, allows for screening of the BCR-ABL1 kinase domain for mutations conferring resistance to TKIs. Since different mutations have different degrees of sensitivity to each of the currently available TKI, the knowledge of BCR-ABL1 kinase domain–mutation status is necessary for subsequent treatment choice. Optimal patient management requires that MR and mutational information be rationally interpreted at both the technical and at the biologic level, and put into context—therapeutic decisions also take into account other factors, such as age, comorbidities, side effects, compliance, and treatment-related complications.

BACKGROUND Patients with Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) frequently relapse on imatinib with acquisition of BCR-ABL kinase domain (KD) mutations. To analyze the changes that second-generation tyrosine kinase inhibitors (TKIs) have brought in mutation frequency and type, a database review was undertaken of the results of all the BCR-ABL KD mutation analyses performed in the authors' laboratory from January 2004 to January 2013. METHODS Interrogation of the database retrieved 450 mutation analyses in 272 patients with Ph+ ALL. Prescreening of samples was performed with denaturing high-performance liquid chromatography (D-HPLC), followed by direct sequencing of D-HPLC–positive cases. RESULTS BCR-ABL KD mutations were detected in 70% of imatinib-resistant patients, with T315I, E255K, and Y253H mutations accounting for 75% of cases. Seventy-eight percent of the patients reported to be resistant to second-generation TKIs after imatinib failure were positive for mutations, and 58% of them had multiple mutations. Analysis of patients relapsing on dasatinib revealed a newly acquired T315I mutation in almost two-thirds of the cases. Direct sequencing detected no mutations at diagnosis, even in patients who relapsed after a few months. CONCLUSIONS Second-generation TKIs ensure a more rapid debulking of the leukemic clone and have much fewer insensitive mutations, but long-term disease control remains a problem, and the T315I mutation is revealed to be an even more frequent enemy. BCR-ABL KD mutation screening of patients with Ph+ ALL who are receiving imatinib or second-generation TKIs would be a precious ally for timely treatment optimization. In contrast, the clinical usefulness of conventional direct sequencing at diagnosis seems to be very low. Cancer 2014;120:1002–1009. © 2013 American Cancer Society.

Abstract 1396 Introduction. Mutations in the kinase domain (KD) of BCR-ABL are a known mechanism of resistance to the tyrosine kinase inhibitor (TKI) treatment of patients with chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). The gold standard for the routine examination of mutations in patients with therapy failure is the Sanger sequencing of amplified cDNA region of BCR-ABL KD with the sensitivity of ∼15–20%. More than 100 BCR-ABL KD mutations were described in association with development of resistance to imatinib. It was suggested that early ultra-sensitive detection of KD BCR-ABL mutations, at the time of diagnosis or shortly after imatinib start and before switch to 2nd generation TKI, may predict their subsequent expansion and failure of the treatment if the patient is treated with TKI against which is the type of mutation resistant. Ultradeep amplicon pyrosequencing represents a promising tool to study BCR-ABL mutation development and their expansion under TKI treatment. In our study, the detection of mutations reached the sensitivity of 0.03-0.01%. At this level we may expect detection of artifacts introduced during amplicon library preparation and pyrosequencing. Aims. The aim is to define the threshold for relevant detection of published BCR-ABL mutations (n= 124). Methods. We evaluated 155 CML or Ph+ALL patient samples. As the non-mutated control, the cDNA of KD c-ABL of healthy individuals (n= 9) and cDNA of wild type BCR-ABL from Ph+ cell lines (n=3) were used. To cover cDNA region of kinase domain (950 bp), multiple amplicons with the length up to 400 bp should be prepared to apply 454 technology. The experiments consist of: 1) comparison of two amplicon designs with 3 and 4 fragments covering KD to follow the amplicon-related artifacts; 2) the reproducibility test of 5 samples using 3 and 4 PCR fragments for amplicon library preparation; 3) tests with 2 different reverse transcriptases and Taq polymerases to detect artifactual nucleotide substitutions. Results. Using 454 technology we detected high number of ultralow-level single nucleotide substitutions (∼ 200) with the frequency < 1 in 100 in all samples analyzed including non-mutated controls using 3 and also 4 amplicon design. Surprisingly, we found that almost all low-level variants were T#x2610;#x0025;C or A#x2610;#x0025;G substitutions (Fig. 1), including those that may result in clinically relevant mutations (V288A, M244V, V289A, F317L, F359L, M351T, E355G, E453G). Evaluation of pyrograms suggested that they were unlikely to be errors of pyrosequencing, because they were not necessarily associated with hompolymer regions. The frequency of T#x2610;#x0025;C or A#x2610;#x0025;G low-level variants was lower when using proof-reading Taq polymerase. The pattern of these nucleotide substitutions is similar to that reported in IGH hypermutation analysis in CLL (Campbell et al. PNAS 2008) showing that T#x2610;#x0025;C or A#x2610;#x0025;G substitutions are errors caused by Taq polymerases. This is supported by the studies on PCR fidelity suggesting that Taq polymerase caused this pattern of base substitution even when using a proof-reading enzyme. Thus, the T#x2610;#x0025;C or A#x2610;#x0025;G substitution in BCR-ABL KD should not be evaluated below the level of 1%. The other but less frequent transitions caused by Taq polymerase are G#x2610;#x0025;A or C#x2610;#x0025;T substitutions that were detected in our study with the frequency < 1 in 1000, thus, the variants detected below 0.1% should not be examined as the true-positive result. Conclusion. Mutation data from ultradeep amplicon sequencing of BCR-ABL KD below 1% must be evaluated cautiously to avoid misleading interpretations. Attention must be paid to the fact that T#x2610;#x0025;C or A#x2610;#x0025;G substitution may arise spontaneously in vivo, but there is no way to differentiate in vivo spontaneous mutations from Taq polymerase errors in vitro. Disclosures: Soverini: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. 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. Machova Polakova:Novartis: Honoraria, Research Funding; BMS: Honoraria.

Abstract 112 BCR-ABL kinase domain (KD) mutation analysis may be an useful tool for physicians and is being performed in a growing number of laboratories. Recommendations aimed to rationalize the use of mutation testing in chronic myeloid leukemia (CML) have recently (Blood 2011) been compiled by a panel of experts appointed by European LeukemiaNet (ELN) – including specific recommendations as to when mutation analysis should be performed. They came from the expert opinion of the panel members whenever published data were insufficient or contradictory. In order to provide further data to validate or refine these recommendations, we have analyzed the GIMEMA CML WP database recording the results of mutation analyses performed in CML pts (n=1301) receiving imatinib and/or 2nd generation TKIs between January 2004 and July 2011. At dagnosis, mutation analysis was recognized to be useful in the few pts who present in accelerated phase or blast crisis (BC), while it was not recommended in chronic phase (CP) pts. Interrogating our database, we could retrieve 58 mutation analyses in newly diagnosed pts in CP and 12 in newly diagnosed pts in BC. Imatinib-resistant mutations were detected in 0 and 2 pts, respectively. In pts receiving 1st-line imatinib, mutation analysis was recommended both in case of failure and in case of suboptimal response. We have analyzed 399 chronic phase (CP) CML pts receiving first-line imatinib because they were found to meet one of the criteria for failure or suboptimal response. Overall, 45/166 (27.1%) failures were found to be positive for one or more BCR-ABL KD mutations. In particular, mutations were detected in 3/16 (18.8%) pts with less than CHR at 3 months, 1/9 (11.1%) pts with no CyR at 6 months, 4/24 (16.7%) pts with less than PCyR at 12 months, 6/36 (16.7%) pts with less than CCyR at 18 months, 15/49 (30.6%) pts who lost CCyR and 16/32 (50%) pts who lost CHR. More interestingly, only 11/233 (4.7%) suboptimal responders we analyzed were positive for mutations. Among ‘cytogenetic' suboptimal responders, mutations were detected in 1/15 (6.7%) pts with no CyR at 3 months, 1/20 (5.0%) pts with less than PCyR at 6 months, 5/51 (8.2%) pts with less than CCyR at 12 months. Among ‘molecular' suboptimal responders, mutations were detected in 0/52 pts with less than MMR (but having achieved CCyR) at 18 months and in 4/95 (4.2%) pts who lost MMR (but not CCyR). Which rise in Bcr-Abl transcript level should trigger a mutation analysis was the most difficult issue to provide recommendations upon, given the lack of convincing and reproducible data in the literature. It was finally agreed to recommend mutation analysis only in case of MMR loss. In 159 of the CP pts we have analyzed, mutation analysis was specifically requested because of a transcript increase at a single RQ-PCR assessment: 29 pts had less than 1-log increase and 41 pts had a 1-log increase or more – but with no loss of MMR. None of these pts was found to have mutations. Another 36 pts had less than 1-log increase and 53 had a 1-log increase or more, leading to loss of MMR. Mutations were detected in 1 (2.8%) and 3 (5.7%) pts, respectively. In pts receiving dasatinib or nilotinib as 2nd-line agents, mutation analysis was recommended at baseline and then in case of failure according to the provisional definitions proposed by Baccarani et al (J Clin Oncol 2009). Nineteen among the pts we analyzed met these criteria; overall, mutations were detected in 11 (57.8%), including 5/7 pts with no CyR at 3 months, 6/9 pts with minimal CyR at 6 months, 1/4 pts with less than PCyR at 12 months. In addition, newly acquired mutations were detected in 93/131 (71%) pts who lost a previously achieved HR or CyR. We also tested 19 pts who met the provisional definitions for suboptimal response to dasatinib or nilotinib 2nd-line. Mutations were detected in 4/19 pts (21%), including 2/5 pts with minor CyR at 3 months, 1/7 pts with PCyR at 6 months, 1/7 pts with less than MMR at 12 months. Our data indicate that: a) pts harbouring mutations can more frequently be found among cytogenetic suboptimal responders than among molecular suboptimal responders; b) any Bcr-Abl transcript increase that is not associated with MMR loss shouldn't indeed trigger a mutation analysis; c) although definitions of response to dasatinib or nilotinib 2nd-line are still provisional and might soon be refined, not only failures but also suboptimal responses are frequently associated with mutations. Supported by AIL, AIRC, PRIN and FIRB. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Martinelli:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

BACKGROUND: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. METHODS: a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. RESULTS: DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. CONCLUSIONS: DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.