Abstract: 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.

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: 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.

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.

Abstract: The overwhelming success of tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) patients has opened a discussion among medical practitioners and the lay public on the real possibility of pregnancy and conception in females and males with CML. In the past 10 years this subject has acquired growing interest in the scientific community and specific knowledge has been obtained “from bench to bedside”. Embryological, pharmacological, and pathophysiological studies have merged with worldwide patient databases to provide a roadmap to a successful pregnancy and birth in CML patients. Male conception does not seem to be affected by TKI therapy, since this class of drugs is neither genotoxic nor mutagenic, however, caution should be used specially with newer drugs for which little or no data are available. In contrast, female patients should avoid TKI therapy specifically during the embryonic stage of organogenesis (5–12 weeks) because TKIs can be teratogenic. In the last 15 years, 41 pregnancies have been followed in our center. A total of 11 male conceptions and 30 female pregnancies are described. TKI treatment was generally terminated as soon as the pregnancy was discovered (3–5 weeks), to avoid exposure during embryonic period and to reduce the risk of needing treatment in the first trimester. Eleven pregnancies were treated with interferon, imatinib or nilotinib during gestation. Nilotinib plasma levels in cord blood and maternal blood at delivery were studied in 2 patients and reduced or absent placental crossing of nilotinib was observed. All of the patients were managed by a multidisciplinary team of physicians with obligatory hematological and obgyn consultations. This work provides an update on the state of the art and detailed description of pregnancy management and outcomes in CML patients.

Abstract: Abstract 3375 Introduction. Telomere are effective sensors of cell integrity and their accelerated shortening is a marker of genetic and/or proliferative stress in several tissues including the hematopoietic compartment. Severe telomere attrition has been indeed observed in aplastic anemia and post-transplant setting. Little is currently known on the genetic integrity of Ph-negative hematopoietic cells (HCs) repopulating the bone marrow (BM) after successful chronic myeloid leukemia (CML) treatment. We thus decided to verify whether severe telomere shortening might occur in this setting and to assess whether its presence might correlate to genetic and functional impairment of Ph-negative hematopoiesis. Patients and methods. We investigated 81 CML patients with persistent (≥12 months) complete cytogenetic remission (CCyR). Median age was 62 years (23-88), M/F ratio was 1.5. Median time from diagnosis and CCyR were 4 years (1-18) and 3 years (1-12) respectively. 15 patients had acquired cytogenetic abnormalities (CA) (del7: 4 patients, +8: 5 patients, del5q: 2 patients, del or +Y: 2 patients, other CA: 2 patients). Telomere length (TL) analysis was performed by Southern Blotting on polymorphonucleates (PMN) and on monocyte-depleted PBMC (MD-PBMC) to monitor both the myeloid and lymphoid compartments. As control group we analyzed 76 age-matched healthy donors. Prospective follow-up monitoring of TL was performed on 56 CML patients with a median time of 22 months from the first determination (range 12–20). Results. PMN (but not MD-PBMC) from CML patients showed a major erosion of their telomeric DNA (median loss 1294 bp p 〈 0.001). Correlations were sought by using a multivariate general linear model on the whole population (CML patients and controls) and then exclusively on the CML population. In the whole population a previous history of CML was a predictor of TL attrition together with age (both p 〈 0.001). In the CML-only population we found no association between TL and sex, Sokal score, or treatment schedule. Most notably we found a correlation between TL attrition and presence of acquired CA (p=0.02, figure 1A), particularly in case of del7 and +8. Somehow more surprisingly we found an increased TL shortening among patients lacking complete molecular remission (CMolR) (p=0.001). The physiological correlation between age and TL persisted also among CML patients (p=0.003). Moreover we found an association between the presence of short telomeres and G≥2 hematological toxicity of any kind (p=0,005), anemia (p=0,007) and a trend with the presence of neutropenia (p=0,080). The association persisted also when G1-4 toxicities were considered (hematological toxicity of any kind p=0.030, anemia p=0.010). We than made a prospective assessment of the telomere dynamics over time performing a second TL determination after at least 12 months on 56 patients. The overall population showed further significant ongoing telomere shortening that was superior to the expected yearly loss for healthy subjects (median annual telomeric loss of 261 bp). We then performed a patient by patient analysis of TL dynamics over time. None of the patients had evidence of telomere recovery. Moreover even considering the maximal recorded interassay variability of 300 bp and the maximal physiological annual telomeric loss (50 bp), a non-physiological telomeric loss was observed in 17 patients (30% of CML population, median loss of 534 bp, range 1290-357 bp, figure 1C). Conclusions. i) Ph-negative HCs display severe telomeric loss, compared to healthy controls; ii) telomere erosion is more pronounced in patients with CA and without CMolR; iii) a strong association between shorter telomeres and hematological toxicity (particularly anemia) was observed; iv) telomere loss is persistent over time in the whole population. Moreover one third of them has a clear evidence of ongoing telomere erosion during the remission phase. Our results indicate that Ph-negative hematopoiesis emerging after successful CML treatment suffers from severe and ongoing telomeric stress whose biological and clinical consequences need to be carefully investigated. Disclosures: No relevant conflicts of interest to declare.