Abstract: 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.

Abstract: 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. Table.All ptsPts positive for mutations by SSAdditional pts positive for mutations by NGSTotal pts positive for mutations1ST -LINE FAILURESNo CyR @ 3 mo9101BCR-ABL 〉 10% @ 6 mo9000mCyR @ 6 mo1101BCR-ABL 〉 1% @ 12 mo10022No CCyR @ 12 mo2101Loss of CCyR7314Loss of MMR20639Loss of CHR2101Progression to BP3202Total6315 (24%)621 (33%)1ST -LINE WARNINGSBCR-ABL 〉 10% @ 3 mo7101BCR-ABL 〉 1% @ 6 mo10112BCR-ABL 〉 0.1% @ 12 mo12101Total293 (10%)14 (14%)2ND -LINE FAILURESNo CyR @ 3 mo3112BCR-ABL 〉 10% @ 6 mo10224Loss of CCyR7303Loss of MMR6123Loss of CHR4303Progression to BP5303Total3513 (37%)518 (51%)2ND -LINE WARNINGSBCR-ABL 〉 10% @ 3 mo6202BCR-ABL 〉 0.1% @ 12 mo7022Total132 (15%)24 (31%) 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-Università 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.

Abstract: 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 〈 1% to reduce the likelihood of amplification artifacts and sequencing errors. 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 〈 0.001). 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.

Abstract: Imatinib (Glivec, Novartis) is a tyrosine kinase specific inhibitor used for the treatment of CML. The occurrence of cytogenetic abnormalities in Ph-negative cells emerging after suppression of the Ph-positive clone has been described; however the origin as well as the biological and clinical significance are unknown. We collected data on 32 patients through the GWP in CML registry. Bone marrow cell segregation, cell culture and morphologic features in a subgroup of these patients were studied to acquire insights into the origin of the Ph-negative clone. Patient characteristics and clinical follow up (up to 49 months) are presented together with hypothesis regarding their biological significance. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 32 patients after a median of 16.2 months after starting Imatinib. Median age was 51 years, F:M=18:14, median time from CML diagnosis 35 months. All patients but one have started Imatinib while in chronic phase and were in chronic phase at detection of the abnormal Ph-negative clone. Eight patients were treated with Imatinb at onset. At diagnosis no additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 21 complete, 5 major and 6 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 14 patients, −Y in 5 patients,, three del(20q), two del(5q) and del(7q), one −7, del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), with one patient presenting with both +8 and +21, and one three clones with +8, double +8, and double +8 and −X. Retrospective analyses of stored pellet using FISH did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 7 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. Two patients that lost response to Glivec were treated with a second generation tyrosine kinase inhibitor Dasatinib (Sprycel, Bristol-Myers Squibb) and, at reduction of Ph+ clone, the del (7q) reappeared in one patient, while the +8 clone of the second patient was not further evidenced. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone was present into the CD34+ compartment suggesting the stem cells involvement. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. We performed cell culture on a subgroup of 6 patients demonstrating normal growth in five patients and an abnormal growth pattern in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. While a longer follow up observation and laboratory analyses are required, we remark that after 〉 4 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS/AML, nor it seems to be associated with CML clonal evolution and disease progression.

Abstract: One of the hallmarks of chronic myeloid leukemia (CML) is genomic instability, that fosters the acquisition of tyrosine kinase inhibitor (TKI)-resistant BCR-ABL1 mutations and/or of additional chromosomal aberrations leading to progression to blast crisis (BC). Inactivating mutations in the SETD2 tumor suppressor occur in solid tumors and acute leukemias. SETD2 trimethylates histone H3 Lysine 36 (H3K36Me3) playing a key role in maintaining DNA integrity. We have recently demonstrated that, in CML, SETD2 loss of function may occur at the post-translational level. Reduced or null SETD2 and H3K36Me3 was detected in 83/96 (86%) patients (pts) with BC CML as compared to a pool of healthy donors and to chronic phase (CP) pts at diagnosis. Proteasome inhibition in primary cells from pts with undetectable SETD2 restored H3K36Me3 and led to accumulation of hyper-ubiquitinated SETD2. In K562 cells (SETD2/H3K36Me3low), we observed that after proteasome inhibition hyper-ubiquitinated SETD2 co-immunoprecipitates with MDM2. MDM2 inhibition rescued SETD2 expression and activity, suggesting that MDM2 is implicated in SETD2 reduced stability. Co-IP also showed that SETD2 interacts with Aurora Kinase A (AKA) a S/T kinase frequently overexpressed in CML. We found that AKA phosphorylates SETD2, and its inhibition rescued SETD2 expression and activity. To investigate whether SETD2/H3K36Me3 loss may be a druggable lesion, we performed clonogenic assays in LAMA84 (SETD2/H3K36Me3high) cells before and after SETD2 silencing, in imatinib-sensitive K562 (SETD2/H3K36Me3low) cells and in IM-resistant K562 cells, that are characterized by complete SETD2 loss. The extent of reduction of clonogenic growth after proteasomal, AKA or MDM2 inhibition was found to be inversely correlated to SETD2 residual expression. These observations were confirmed in cells from both CP (n=2) and BC (n=4) CML pts showing different levels of SETD2 expression and activity. Further experiments were performed in the aforementioned cell lines to verify if reduced clonogenic potential was due to cytostatic or cytotoxic effects. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Proteasomal inhibition by bortezomib, carfilzomib and ixazomib and AKA de-phosphorylation by Danusertib caused a time-dependent increase of annexin-V-positive cells by activating the mitochondrial apoptotic pathway as reflected by an increase in Bax expression and induction of the cleavage of caspase-3,-9 and PARP. Moreover, all drug treatments as single agent, at nanomolar doses (Bortezomib: 10 nM, Carfilzomib: 5 nM, Ixazomib: 10 nM and Danusertib: 500 nM) induced a significant increase of the DNA double-strand break marker γH2AX, suggesting that in a SETD2 knock-down context, proteasomal and AKA inhibition propagates genomic instability by forcing the cells through successive replication cycles, ultimately resulting in apoptosis from mitotic catastrophe. Reduced SETD2/H3K36Me3 levels, in association with MDM2 and AKA hyper-activation, were also detected when the CD34+ cell fraction of 10 CML-CP pts, was compared to the total mononuclear cell fraction or to the CD34+ compartment obtained from a pool of healthy donors. We thus hypothesized that leukemia progenitor cells, showing higher MDM2 and AKA activity and consequent SETD2 loss, accumulate genetic aberrations despite inhibition of BCR-ABL1 kinase. Studies are ongoing to verify if MDM2 or AKA inhibition may restore SETD2 expression and function and induce cell death. Finally, it has already been shown that alterations of epigenetic regulators such as the KDM4 family members control tumor cell proliferation in a variety of cancers including acute myeloid leukemia. Recent findings have identified KDM4 demethylases as putative therapeutic targets in a SETD2 mutated context and illustrated the efficacy of KDM4 inhibitors in AML therapy. Starting from these evidences, we will test the same approach in BC CML models. In conclusion, phosphorylation by AKA and ubiquitination by MDM2 contribute to SETD2 non-genomic loss of function in BC CML and in CD34+ leukemic progenitors. Restoring physiological H3K36Me3 may help to improve the outcome of this critical subset of pts. Acknowledgments: Study supported by AIRC (project code 16996), AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma), Italian Ministry of Health, project GR-2016-02364880. Disclosures Gugliotta: Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, 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. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Abruzzese:Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; BMS: Consultancy. Pregno:Incyte: Consultancy, Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Albano:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Bonifacio:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. 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. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Roche: Consultancy; Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau. Soverini:Incyte: Consultancy.

Abstract: Introduction Some retrospective studies in tyrosine kinase inhibitor (TKI)-resistant Philadephia-positive (Ph+) leukemia patients (pts) have suggested that deep sequencing (DS) may provide a more accurate picture of BCR-ABL1 kinase domain (KD) mutation status as compared to conventional sequencing (CS). However, the frequency and clinical relevance of low burden mutations remains to be explored prospectively in large series of unselected pts. In addition, the implementation of routine BCR-ABL1 DS in multiple molecular diagnostic laboratories has never been attempted. These open issues led us to design a multi-center, multi-laboratory prospective study ('NEXT-IN-CML') aimed to assess the feasibility, performance and informativity of DS for BCR-ABL1 KD mutation screening. Aims The first phase of the study was aimed to establish a network of 5 reference labs sharing a standardized DS workflow, a joint database for clinical and mutational data storage and a common pipeline of data analysis, interpretation and clinical reporting. The second phase of the study, involving 54 Italian Hematology Units, is aimed to assess the frequency and clinical significance of low burden mutations detectable by DS by prospective collection and analysis of samples from chronic myeloid leukemia (CML) pts who exhibit failure (F) or warning (W) responses and relapsed Ph+ acute lymphoblastic leukemia (ALL) pts. Methods A PCR and an amplicon DS protocol already set up and optimized for the Roche GS Junior in the framework of the IRON II international consortium was adopted. In the first phase, 5 batches of blinded cDNA samples were prepared and shipped to evaluate individual lab performances. The batches included archival samples with known BCR-ABL1 mutation status as assessed by CS and serial dilutions of BaF3 T315I+ cells in BaF3 unmutated cells, simulating mutation loads of 20% down to 1%. In the ongoing second phase prospectively, consecutively collected CML and Ph+ ALL samples are being analyzed in parallel by CS and DS. Clinical history and follow-up data are used for correlations. Results In the first phase of the study, 312/320 amplicons were successfully generated and sequenced. A median of 124,686 (range, 48,181-170,687) high quality reads were obtained across the 5 labs. Median number of forward and reverse reads was 1,757 (range 884-7,838), with no coverage dropouts for any amplicon or index. Comparison of observed vs expected mutations showed that 76/78 evaluable samples were accurately scored. In the remaining two, the analysis software failed to detect the 35bp insertion ('35INS') commonly detectable between exons 8 and 9. Quantitation of point mutation burden was highly reproducible across the entire range of frequencies, from 100% to 1%. The second phase of the study has started in Jan 2016. As of Jul 31st, a total of 106 consecutive pts (CML, n=96; Ph+ ALL, n=10) have been enrolled. The present analysis focuses on the first 75 CML pts (60 F and 15 W), for whom sequencing results are currently available (analysis of the entire population of patients enrolled up to Nov 2016 will be presented at the meeting). Clinically actionable mutations have been detected in 10/75 (14%) pts by CS and in 20/75 pts (27%) by DS. Notably, among the 10 pts positive for clinically actionable mutations by DS but not by CS, 3 had a low burden T315I (2 F [dasatinib, imatinib] and 1 W [dasatinib] ). In 5 additional pts negative for mutations by CS (3 F and 2 W), DS identified multiple low burden mutations with unknown IC50, suggesting that the cooperation of individually 'weak' mutants may be a new mechanism underlying reduced TKI efficacy. Longitudinal analysis and follow-up of pts are shaping the clinical significance of different types of low burden mutations and will be presented. Conclusions The 'NEXT-in-CML' study is demonstrating that DS of BCR-ABL1 can successfully be implemented in national lab networks and is an important step forward towards routine use of this technology. We have now adapted the protocol for both the Ion Torrent PGM and the Illumina Miseq platforms. For a minimum of 15 samples per sequencing run, DS costs are estimated to equal those of CS (cost per sample, reagents only: ≈100€ for PGM (314 chip) and Miseq (nano kit v2) vs ≈95€ for CS) with comparable turnaround times for delivery of results. Our study is also contributing useful data for the clinical interpretation of DS findings. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Castagnetti:ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Ciceri:MolMed SpA: Consultancy. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Di Raimondo:Janssen-Cilag: Honoraria. Bassan:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Baccarani:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Saglio:Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Ariad: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Genentech: Consultancy.

Abstract: 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.