Kurzfassung: This is an Italian multicentric cohort study evaluating efficacy and safety of venetoclax + hypomethylating agents (HMA) in a real‐life setting of newly diagnosed, relapsed, and refractory patients with acute myeloid leukemia (AML). The data show that the combination of venetoclax and HMA offers an expectation of remission and long‐term survival not only to newly diagnosed but also to relapsed or refractory elderly patients with AML.

Kurzfassung: The SARS-CoV-2 (COVID-19) outbreak is upending current life and generating much anxiety and uncertainty. The effects of home confinement, social isolation, cancelled schools, closed businesses, and negative economic impacts have had serious consequences. Hematologic patients (HP) are a subset of highly vulnerable population with increased risk of developing severe COVID-19 symptoms due to their immunocompromised status. These risks have been augmented during the COVID outbreak because of deviations from current standards of care, e.g., reduced visits, treatment supply and access to routine exams. This study investigated the impact of the current pandemic on HP assessing demographics, medical information, mental health and caregiver practical management. In collaboration with AIL (Italian Association against Leukemia) and CNR (National Research Council), a survey was generated and distributed to Italian HP. The general population (GP) were used as controls. The assessment used the DASS-21 questionnaire, a self-reported, 21 item screening instrument that provides independent measures of depression, stress and anxiety with recommended severity thresholds subscales. The survey was self-administered between April and August, 2020. The questionnaires' reliability was verified based on an analysis of its internal consistency using Cronbach's alpha. As of 30 June 2020, 1113 HP and 1125 GP completed the survey from 20 Italian Regions. The two population groups were homogeneous by age, gender and distribution and included regions at both high (CHP) and low (CLP) prevalence of infections at the time of the survey. HP and GP median age was 50 years (range: HP 11- 93; GP 13-85). 61% HP and 68% GP were female; the rest were male. The year of diagnosis of hematological disease ranged from 1965 to 2020; 21.9% had chronic myeloid leukemia, followed by Hodgkin (15.7%) and non-Hodgkin (15.9%) lymphomas, chronic myeloproliferative neoplasms (15.9%), multiple myeloma (8.9%), chronic lymphocytic leukemia (4.9%), acute myeloid (5.6%) and lymphoblastic (3.25%) leukemias, other (7%). 1071 HP and 1125 GP responded about their occupation as follows: employed full/part time (38.7% HP , 47.7% GP), retired (19.2% HP,10.8% GP), freelancer (9.2% HP, 12.2% GP), unemployed (6.2% HP, 3.4% GP) , students (4.6% HP, 3.2% GP), company executive (4.1% HP, 4.8% GP), manager (2% HP, 4.7 % GP), cooperative member (0.7% HP , 0.7% GP), housewife (8.5% HP 4.8% GP), other (6.8% HP,7.7% GP). During the pandemic 63.7% HP didn't work and 36.3% did work compared to the GP group (33.8% didn't; 66.2 % did). Where specified, the reasons for not working were: layoffs (10.9% HP, 15.9% GP), lack of work (8.1%HP, 15.6 % GP), vacation/ parental leave (4.4% HP, 3.5% GP), reduced business activity for economic reasons (2.2% HP, 3.3% GP), occasional work (2.1% HP, 5.7% GP), seasonal employment, (0.6% HP,1.1 % GP). 625/1073 HP (58.2%) were in active treatment. Of these, 40.1% were in Day Hospital, 56.4% were outpatient; and 3.5% inpatients. The remaining HP were off therapy (448/1073; 41.8%). 1105 HP and 1127 GP responded to the DASS questionnaire. Extremely severe depression was found in 12.9% HP vs 7% GP; 18.1% HP expressed severe anxiety vs 9.6% GP and extremely severe stress was present in 7.1% HP and 5.3% GP. The Cronbach's alpha coefficient for the internal homogeneity of the questionnaire was 0.95, confirming the cohorts correctness (0.8 or greater, indicates a very good level of reliability). Providing care to HP during the pandemic has been challenging for both patients and doctors. Restrictions on visits and lab/instrumental exams, reduced equipment supply and a paucity of personal protection equipment (PPEs) for health care providers and patients have penalized normal routine care. As a result, 38.2% HP postponed or did not attend scheduled therapy and & gt;50% had difficulty obtaining PPEs through normal sources; 57% in CHP and 36% in CLP regions had to buy them themselves. To our knowledge this is the first report of the impact of the pandemic on psychological distress, work consequences and illness management in HP. Most of HP in this study are outpatients in active treatment. Interesting data emerged from job losses, which is more common among HP. The DASS-21 instrument revealed higher anxiety and depression levels in HP. Detailed results including longitudinal analysis and high versus low geographic prevalence of COVID-19 infections will be presented. Figure 1 Disclosures Abruzzese: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria.

Kurzfassung: Introduction. Chronic myeloid leukemia (CML) patients (pts) treated with imatinib first line achieve complete cytogenetic response (CCyR) in 〉 70% of cases and major molecular response (MMR) in 18-58%. These pts have a life expectancy similar to the general population. However even undetectable BCR-ABL may not equate to eradication of the disease because of the sensitivity of Q-RT-PCR. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, has been recently developed (Goh HG et al., 2011). dPCR corresponds to a 100 fold increase in sensitivity as compared to Q-RT-PCR. Therefore, dPCR by assessing the presence of minimal residual disease with higher sensitivity, could potentially identify pts in whom CML has been eradicated. Aims. The Imatinib Suspension And Validation (ISAV) study is aimed at validating the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods. This study involves 15 sites, 10 in Italy and 1 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. CML pts (Chronic or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible for this study. Patients had to be in CMR for at least 18 months (mts), with a minimum of 3 Q-RT-PCR performed at their own sites. After signing the informed consent, blood samples are obtained for dPCR and the pts discontinue imatinib therapy. Standard Q-RT-PCR is performed monthly (mts 1-6) and then bimonthly for 36 mts to assess the maintenance of the molecular remission. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resume imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is evaluated through the EORTC – C30 Quality of Life questionnaire. Results. The enrolment in ISAV began in November 2011 and ended in July 2013. The study enrolled 112 pts: Italy 69.6%, Germany 21.4%, Canada 5.3%, Spain 2.6% and Israel 0.9%. Among the 112 pts, 59.3% were male and 37.0% were aged 65 or older; median duration of imatinib treatment was 103.1 mts with median duration of CMR of 25.8 mts before imatinib discontinuation. To date, the median follow-up (FUP) time is 16.6 mts [95% CI: 14.9-18.2]. Forty-seven pts (43.5%, 95% CI: 34.0-53.4) of the 108 eligible pts relapsed and resumed imatinib; 38/47 (80.9%) of them relapsed in the first 9 mts and the last relapse occurred 19.6 mts after imatinib discontinuation. A loss of CCyR occurred in 11 pts (23.4%): 10/11 CCyR losses were recovered; 1 patient withdrew the consent shortly after obtaining a partial cytogenetic response. No case of CML progression was observed. After the resumption of imatinib the median time to either MMR or CMR was 1.9 [95% CI: 1.2-2.4] mts. Of the 61 not-relapsed pts, 43 (39.8% of the total) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity was 3.6 mts [95% CI: 3.0-4.8] and the range of duration of Q-RT-PCR positivity (below 0.1%) was between 5.7 and 29.2 mts. No significant correlation between relapse and previous duration of imatinib treatment, use of interferon, time to CCyR or duration of CMR was identified. An inverse relationship between pts age and risk of relapse is evident: 90% of pts 〈 45 years relapsed vs 37.5% in the class ≥ 45 - 〈 65 years and 27.5% of pts ≥ 65 years, p(χ2) 〈 0.0001. dPCR results showed that 23.4% of pts were positive and 76.6% negative, with a dPCR Negative Predictive Value (NPV) of 63.4% (Tab.1) and a significant NPV ratio (dPCR/Q-RT-PCR) of 1.131 [95% CI: 1.032-1.239]. Age and dPCR results predicted the risk of relapse: pts with less than 45 years and with a positive dPCR had the highest risk of relapse (100%) as opposed to pts ≥ 45 years and with negative dPCR (30.6%; Fig.1). Conclusions. After 32 mts from the beginning of the study, with a median FUP of 16.6 mts, 43.5% of pts relapsed; the majority of relapses developed in the first 9 months after imatinib discontinuation. Age 〈 45 years and dPCR positivity are significantly associated with relapses. Funded by Regione Lombardia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR‐ABL1 by Q‐RT‐PCR does not equate to eradication of the disease. Digital‐PCR (dPCR), able to detect 1 BCR‐ABL1 positive cell out of 10 7 , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q‐RT‐PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q‐RT‐PCR for 36 months. Patients losing molecular remission (two consecutive positive Q‐RT‐PCR with at least 1 BCR‐ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow‐up of 21.6 months. Fifty‐two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not‐relapsed patients, 40 (37.0% of total) regained Q‐RT‐PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013–1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients 〈 45 years relapsed versus 42% in the class ≥45 to 〈 65 years and 33% of patients ≥65 years [ P (χ 2 )  〈  0.0001]. Relapse rates ranged between 100% ( 〈 45 years, dPCR+) and 36% ( 〉 45 years, dPCR‐). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse. Am. J. Hematol. 90:910–914, 2015. © 2015 Wiley Periodicals, Inc.

Kurzfassung: Patients with hematological malignancies are at an increased risk of SARS‐CoV‐2 disease (COVID‐19) and adverse outcome. However, a low mortality rate has been reported in patients with chronic myeloid leukemia (CML). Preclinical evidence suggests that tyrosine kinase inhibitors (TKIs) may have a protective role against severe COVID‐19. Methods We conducted a cross‐sectional study of 564 consecutive patients with CML who were tested for anti‐SARS‐CoV‐2 IgG/IgM antibodies at their first outpatient visit between May and early November 2020 in five hematologic centers representative of three Italian regions. Results The estimated serological prevalence of SARS‐CoV‐2 infection in patients with CML after the first pandemic wave was similar to that in the general population (about 2%), both at national and regional levels. CML patients with positive anti‐SARS‐CoV‐2 serology were more frequently male ( p  = 0.027) and active workers ( p  = 0.012), while there was no significant association with TKI treatment type. Only 3 out of 11 IgG‐positive patients had previously received a molecular diagnosis of COVID‐19, while the remainders were asymptomatic or with mild symptoms. Conclusions Our data confirm that the course of SARS‐CoV‐2 infection in patients with CML is generally mild and reassure about the safety of continuing TKIs during the COVID‐19 pandemic. Furthermore, we suggest that patients with CML succeed to mount an antibody response after exposure to SARS‐CoV‐2, similar to the general population.

Kurzfassung: The SETD2 protein is a histone methyltransferase that specifically catalyzes the trimethylation of Lysine 36 on histone H3 (H3K36me3). SETD2/H3K36me3 are implicated in transcript elongation and splicing, DNA repair, chromosome segregation. SETD2 gene deletions and/or mutations (mostly frameshift or nonsense) have been reported in solid tumors (clear cell renal cell carcinoma, bladder cancer, lung cancer, melanoma, endometrial cancer) and in acute leukemias. Using a Western Blotting (WB) approach to screen for SETD2 protein expression and for H3K36me3 levels in a relatively large cohort of 80 advanced-phase chronic myeloid leukemia (CML) patients (pts), we could detect reduced or null SETD2 and H3K36me3 in 86% of pts as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis who achieved optimal responses to TKI, but neither mutations/deletions nor transcriptional down-regulation were the underlying causes. Inhibition of proteasome-mediated degradation in primary cells from pts with undetectable SETD2 restored H3K36me3 and led to accumulation of hyper-ubiquitinated SETD2, suggesting that a functional protein is produced but rapidly degraded. Moreover, proteasome inhibition was found to induce apoptosis and to reduce clonogenic growth. In K562 cells (SETD2/H3K36me3low), co-immunoprecipitation (co-IP) performed before and after proteasome inhibition showed accumulation of the hyper-ubiquitinated form of SETD2 bound to MDM2. MDM2 inhibition by SP-141 resulted in cytostatic effects and restored SETD2 expression and activity. Superimposable results were achieved by siRNA-mediated silencing of MDM2, suggesting that MDM2 is implicated in SETD2 reduced stability. Co-IP also showed that SETD2 interacts with Aurora Kinase A a Ser-Thr kinase frequently overexpressed in CML. We found that Aurora Kinase A phosphorylates SETD2, and both pharmacological inhibition by Danusertib and siRNA-mediated silencing rescued SETD2 expression and activity. Next, to investigate whether SETD2/H3K36me3 loss may contribute to genetic instability, LAMA 84 (SETD2/H3K36Me3high) and K562 (SETD2/H3K36me3low) cells were studied by WB and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci in steady state conditions and after sub-lethal DNA damage by UV exposure. The same studies were performed after SETD2 silencing for 3 months. Cells with low or silenced SETD2 had significantly higher levels of γH2AX and were unable to induce homologous recombination (HR) repair after DNA damage. Clonogenic assays performed in LAMA 84 cells before and after SETD2 silencing, in K562 (SETD2/H3K36me3low) and in imatinib-resistant (IM-R) K562 cells which have lost SETD2 expression and activity, suggested that reduction of clonogenic growth after proteasomal or MDM2 inhibition is strictly dependent on SETD2 expression and functional status (Figure 1A). First and second generation proteasome inhibitors (bortezomib, carfilzomib and ixazomib) inhibited the clonogenic potential of the mononuclear cell fraction from both CP (n=2) and blast crisis (BC) (n=4) CML pts at subnanomolar concentrations, with the extent of anti-tumor activity clearly anti-correlated with SETD2 expression and H3K36me3 levels: pts with lower SETD2 expression showed lower LD50 when compared with pts with higher SETD2 expression and H3K36me3 levels (Figure 1B). Similarly, clonogenic assays performed by administrating increasing doses of SP-141 (from 0.25 to 1.25 µM) suggested that MDM2 specific inhibition had more significant effects in BC-CML pts showing low SETD2 levels and activity as compared to BC-CML pts showing intermediate SETD2 levels and activity and to CP CML pts. In conclusion, phosphorylation by Aurora Kinase A and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in advanced-phase CML. Loss of SETD2/H3K36me3 is associated with increased DNA damage and impaired HR repair. Restoring physiological H3K36me3 levels may help improve the outcome of this critical subset of pts. Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). Figure 1. Figure 1. Disclosures Castagnetti: Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Incyte: Honoraria. Abruzzese:Pfizer: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Martinelli:Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo:Adaptive Biotechnologies: 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; Bristol-Myers Squibb: 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; AbbVie: 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Bristol Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy.

Kurzfassung: Imatinib induces complete cytogenetic response (CCyR) in up to 80% of chronic myeloid leukemia (CML) patients (pts) and major molecular response (MMR) in 33-60% of them. These patients enjoy life expectancy similar to general population. However even undetectable BCR-ABL may not equate to eradication of the disease because the sensitivity of the standard diagnostic method, the Q-RT-PCR, is limited. A new diagnostic method, the digital-PCR (dPCR), able to detect 1 BCR-ABL+ cell out of 107 cells, corresponding to a 100 times increased sensitivity as compared to conventional Q-RT-PCR, was developed (Goh HG et al., Leuk Lymphoma 52(5): 896-904. 2011). Therefore dPCR, assessing with more sensitivity the presence of minimal residual disease, could potentially identify pts in whom CML is eradicated. The Imatinib Suspension And Validation (ISAV) study is aimed at assessing the capability of dPCR to predict relapses after imatinib discontinuation in CML pts with negative Q-RT-PCR results. Methods This study involves 15 sites, 10 in Italy and 5 in each of the following countries: Germany, Spain, The Netherlands, Canada and Israel. In this study CML patients (Chronic Phase or Accelerated Phase) under imatinib therapy since more than 2 years and in complete molecular remission (CMR) were eligible. Patients had to be in CMR for at least 18 months, with a minimum of 3 Q-RT-PCR performed in their own centers. After signing the informed consent, pts were tested for dPCR and discontinued imatinib therapy. They are being monitored by standard Q-RT-PCR for 36 months to assess the maintenance of the molecular remission. At the end of this period, a peripheral blood sample for dPCR analysis will be obtained from those pts who will still have undetectable BCR-ABL transcripts by Q-RT-PCR, to verify CML eradication. The loss of molecular remission is defined as two consecutive positive Q-RT-PCR tests with at least one BCR-ABL/ABL value above 0.1%. Patients losing molecular remission resumed imatinib treatment at the same dosage used before interruption. Patients’ quality of life during imatinib discontinuation/resumption is being evaluated trough the EORTC – C30 Quality of Life questionnaire. Results The enrollment in the ISAV study began in November 2011 and ended in June 2013. The study enrolled 112 pts: Italy 69.6%, Berlin 21.4%, Montreal 5.3%, Zaragoza 2.6% and Tel Hashomer 0.9%. Sixty-one percent of the pts were male and 38% were aged 65 or older; median duration of imatinib treatment is 102 months with median duration of CMR of 32 months before imatinib discontinuation. To date, the median follow-up (FUP) time is 4.6 months [95% CI: 4.1-5.8] and 92 pts out of 112 (82%) had at least one Q-RT-PCR performed after imatinib discontinuation. The following analysis is restricted to 48 pts with a minimum of 6 months of FUP. Of these 48 pts, 20 remained Q-RT-PCR negative (42%, 95% CI:29-56%, median duration of negativity after imatinib discontinuation: 10.3 months). Nineteen pts (40%, 95% CI:27-53%) relapsed and resumed imatinib. All relapses occurred in the first 10 months and all but 3 of them in the first 6 months. A loss of CCyR happened in 5 pts out of 19 (26%): 1 pt regained CCyR after 3 months of re-treatment and is now in CMR, 1 pt died shortly after the diagnosis of relapse because of lung adenocarcinoma and 3 pts are now being monitored after imatinib resumption. No case of progression of CML was observed. After the resumption of imatinib the median time to either MMR or CMR, whichever came first, was 2.1 [95% CI: 0.9-5.8] months. Finally, nine pts (18%, 95% CI:10-31%) regained Q-RT-PCR positivity but never lost MMR. The median time to Q-RT-PCR positivity in this group of pts was 2.92 months (range 1-5 months), and the range of duration of Q-RT-PCR positivity (below 0.1%) is between 2 and 14 months. No significant correlation between relapse and previous duration of imatinib treatment, time to CCyR or duration of CMR was present. Patients previously treated with interferon showed a trend toward lower risk of relapse which is not significant so far. Finally, 19% of pts complained of musculoskeletal/articular pain after imatinib discontinuation. Conclusions After 21 months from the beginning of the study with a median follow-up of 4.6 months, 40% of pts relapsed; the majority of relapses happened in the first 6 months after imatinib discontinuation. The correlation of dPCR results with clinical outcomes will be presented at the meeting. Disclosures: le Coutre: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Novartis: Research Funding. Gozzini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy.

Kurzfassung: 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.