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The chromosomal translocation t(4;11)(q21;q23), a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL), encodes mixed-lineage leukemia (MLL)-AF4 oncogenic chimera that triggers aberrant transcription of genes involved in lymphocyte differentiation, including HOXA9 and MEIS1. The scaffold protein 14-3-3θ, which promotes the binding of MLL-AF4 to the HOXA9 promoter, is a target of MiR-27a, a tumor suppressor in different human leukemia cell types. We herein study the role of MiR-27a in the pathogenesis of t(4;11) ALL. Reverse transcription quantitative PCR (qPCR) reveals that MiR-27a and 14-3-3θ expression is inversely correlated in t(4;11) ALL cell lines; interestingly, MiR-27a relative expression is significantly lower in patients affected by t(4;11) ALL than in patients affected by the less severe t(12;21) leukemia. In t(4;11) leukemia cells, ectopic expression of MiR-27a decreases protein level of 14-3-3θ and of the key transcription factor RUNX1. We show for the first time that MiR-27a also targets AF4 and MLL-AF4; in agreement, MiR-27a overexpression strongly reduces AF4 and MLL-AF4 protein levels in RS4;11 cells. Consequent to AF4 and MLL-AF4 downregulation, MiR-27a overexpression negatively affects transcription of HOXA9 and MEIS1 in different t(4;11) leukemia cell lines. In agreement, we show through chromatin immunoprecipitation experiments that MiR-27a overexpression impairs the binding of MLL-AF4 to the HOXA9 promoter. Lastly, we found that MiR-27a overexpression decreases viability, proliferation, and clonogenicity of t(4;11) cells, whereas it enhances their apoptotic rate. Overall, our study identifies the first microRNAthat strikes in one hit four crucial drivers of blast transformation in t(4;11) leukemia. Therefore, MiR-27a emerges as a new promising therapeutic target for this aggressive and poorly curable form of leukemia.

Simple Summary The introduction to clinical practice of a treatment-free remission approach in chronic myeloid leukemia patients with a stable deep molecular response highlighted how crucial it is to monitor the molecular levels of BCR–ABL1 as accurately and precisely as possible. In this context, the droplet digital PCR (ddPCR) presents an alternative methodology for such quantification. To hypothesize the introduction of this technology in routine practice, we performed a multicentric study that compares ddPCR with the standard methodology currently used. Our results demonstrate that the use of ddPCR in clinical practice is feasible and could be beneficial. Abstract BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTM BCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.

In chronic myeloid leukemia (CML) patients, tyrosine kinase inhibitors (TKIs) may select for drug-resistant BCR-ABL1 kinase domain (KD) mutants. Although Sanger sequencing (SS) is considered the gold standard for BCR-ABL1 KD mutation screening, next-generation sequencing (NGS) has recently been assessed in retrospective studies. We conducted a prospective, multicenter study (NEXT-in-CML) to assess the frequency and clinical relevance of low-level mutations and the feasibility, cost, and turnaround times of NGS-based BCR-ABL1 mutation screening in a routine setting. A series of 236 consecutive CML patients with failure (n = 124) or warning (n = 112) response to TKI therapy were analyzed in parallel by SS and NGS in 1 of 4 reference laboratories. Fifty-one patients (22 failure, 29 warning) who were negative for mutations by SS had low-level mutations detectable by NGS. Moreover, 29 (27 failure, 2 warning) of 60 patients who were positive for mutations by SS showed additional low-level mutations. Thus, mutations undetectable by SS were identified in 80 out of 236 patients (34%), of whom 42 (18% of the total) had low-level mutations somehow relevant for clinical decision making. Prospective monitoring of mutation kinetics demonstrated that TKI-resistant low-level mutations are invariably selected if the patients are not switched to another TKI or if they are switched to a inappropriate TKI or TKI dose. The NEXT-in-CML study provides for the first time robust demonstration of the clinical relevance of low-level mutations, supporting the incorporation of NGS-based BCR-ABL1 KD mutation screening results in the clinical decision algorithms.

More than 15 years ago, imatinib entered into the clinical practice as a “magic bullet”; from that point on, the prognosis of patients affected by chronic myeloid leukemia (CML) became comparable to that of aged-matched healthy subjects. The aims of treatment with tyrosine kinase inhibitors (TKIs) are for complete hematological response after 3 months of treatment, complete cytogenetic response after 6 months, and a reduction of the molecular disease of at least 3 logs after 12 months. Patients who do not reach their goal can switch to another TKI. Thus, the molecular monitoring of response is the main consideration of management of CML patients. Moreover, cases in deep and persistent molecular response can tempt the physician to interrupt treatment, and this “dream” is possible due to the quantitative PCR. After great international effort, today the BCR-ABL1 expression obtained in each laboratory is standardized and expressed as “international scale.” This aim has been reached after the establishment of the EUTOS program (in Europe) and the LabNet network (in Italy), the platforms where biologists meet clinicians. In the field of quantitative PCR, the digital PCR is now a new and promising, sensitive and accurate tool. Some authors reported that digital PCR is able to better classify patients in precise “molecular classes,” which could lead to a better identification of those cases that will benefit from the interruption of therapy. In addition, digital PCR can be used to identify a point mutation in the ABL1 domain, mutations that are often responsible for the TKI resistance. In the field of resistance, a prominent role is played by the NGS that enables identification of any mutation in ABL1 domain, even at sub-clonal levels. This manuscript reviews how the molecular tools can lead the management of CML patients, focusing on the more recent technical advances.

Molecular detection of the BCR-ABL1 fusion transcripts is necessary for the genetic confirmation of a chronic myeloid leukemia diagnosis and for the risk classification of acute lymphoblastic leukemia. BCR-ABL1 mRNAs are usually identified using a conventional RT-PCR technique according to the BIOMED-1 method. In this study, we evaluated 122 BCR-ABL1-positive samples with the Q-LAMP assay to establish if this technology may represent a valid alternative to the qualitative BIOMED-1 PCR technique usually employed for the detection and the discrimination of the common BCR-ABL1 transcripts (p190 and p210 isoforms). We found a 100% concordance rate between the two methods. Specifically, the p190- and p210-positive samples were amplified by Q-LAMP with a median threshold time (Tt) of 26.70 min (range: 24.45&ndash, 31.80 min) and 20.26 min (range: 15.25-34.57 min), respectively. A median time of 19.63 was observed in samples displaying both (e13a2/e14a2) p210 isoforms. Moreover, the Q-LAMP assay allowed recognition of the BCR-ABL1 e13a2 and e14a2 isoforms (median Tts 18.48 for e13a2 vs. 26.08 min for e14a2, p &lt, 0.001). Finally, 20 samples harboring rare BCR-ABL1 isoforms (e1a3, e13a3, e14a3, and e19a2) were correctly identified by the Q-LAMP assay. We conclude that the Q-LAMP assay may represent a faster and valid alternative to the qualitative BIOMED-1 RT-PCR for the diagnosis at BCR-ABL1-positive leukemias, especially when samples are analyzed in centers with restricted resources and/or limited technical expertise.

Background: Monitoring of BCR-ABL1 molecular levels is essential for the management of Chronic Myeloid Leukemia (CML) patients treated with Tyrosine Kinases Inhibitors (TKIs). Real Time Quantitative PCR (RT-qPCR) is currently the standard method for assessing molecular remission (MR) in patients with CML. Recently, droplet digital PCR (ddPCR) has emerged to provide a more accurate detection of minimal residual disease (MRD). In order to hypothesize the use of this new technology in the clinical practice, in the era of TKI discontinuation, we designed a multi-centric study to evaluate the potential value of ddPCR in diagnostic routine. Aims of the study were:1) the evaluation of the agreement between the measures obtained by ddPCR and RT-qPCR and 2) the assessment of the repeatability of the two methods. Methods: Total RNA was extracted from 37 CML patients using Maxwell 16 LEV simplyRNA Blood kit (Promega), following the manufacturer's instructions. Samples were divided in 5 groups based on molecular response (MR) as follow: group 1, MR Statistical analysis: Bland-Altman analysis was performed. For the measurement of the agreement we reported the bias, which is the mean of the difference between the methods, the 95% limits of agreement and the coefficient of variation. Residual variance and coefficients of repeatability (i.e. the upper limits of a prediction interval for the absolute difference between two measurements by the same method on the same item) were computed to achieve the second endpoint. An analysis of sensitivity on the labs was also carried out. All analyses were stratified by the level of disease. Results: A total of 370 measures were included in the analysis, 185 for ddPCR and 185 for RT-qPCR divided as follow: 50 for group 1 and for group 2, 90 for group 3, 4 and 5. In Table 1 we reported the median and interquartile range (IQR) for all levels of disease. Results of the Bland-Altman analysis are shown in Table 2. The coefficients of variation, which expresses the standard deviation as a percentage of the mean, were 2.35, 2.31, 1.10, 1.34, 39.12 in group 1, 2, 3, 4 and 5 respectively. The repeatability coefficients of ddPCR were smaller than qRT-PCR across all the levels of disease, showing a slightly better precision of ddPCR (Table 2). Conclusions: Higher coefficients of variation in group 1 and 2 were probably due to a greater heterogeneity of the patients. In fact, BCR-ABL1/ABL1 levels by RT-qPCR ranged between 1.43 and 6.94 and between 0.10 and 0.25 in group 1 and in group 2 respectively (Table 1). Sensitivity analysis showed that the high coefficient of variation in group 5 can be explained almost all by the variability observed in Lab B. Coefficient of repeatability of ddPCR was always smaller than RT-qPCR for all level of disease showing a slightly better precision. Our results showed that ddPCR has a good agreement with RT-qPCR and it is more precise to quantify BCR-ABL1 transcript levels, particularly for MR 4 and MR 4.5. Thus, ddPCR may be valuable in diagnostic routine. Disclosures Fava: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Martino:Bio-Rad: Employment. Saglio:Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy. Martinelli:Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Cilloni:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

a b s t r a c t The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co- cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

Molecular tests are the best way to monitor CML course in patients under treatment with tyrosine kinase inhibitors (TKI). International guidelines indicate the absolute copy number of the control gene ABL1 as reference for the definition of the sensitivity of the analytical method. A general implementation of the International Guidelines (EUTOS) and the moving forward of current technologies towards one-step reactions, that allow direct testing from the patient RNA, require continuous verification of the method performances. Here, as Italian laboratory network for the standardization of CML diagnosis (LabNet), we performed a comparative study across the three reference laboratories in order to evaluate the inclusion of "BCR-ABL P210 ELITe MGB® Kit" (ELITechGroup S.p.A.) one-step assay among the technologies indicated in the Laboratory Recommendations and Indications (R.I.L.) of the Italian Network for CML monitoring. "BCR-ABL P210 ELITe MGB® Kit" is a new assay that allows to perform in a unique reaction the retro-transcription and the amplification of the extracted RNA sample. In this study 30 RNA extracted from whole blood samples of CML patients at different stages of the disease and centrally distributed to the other reference labs have been analyzed. All laboratories tested 300 ng per reaction of each RNA according the one-step approach and the same RNA according each own routine method. Moreover, in the same experiments, the European Reference Material certified plasmid ERM-AD623 has been evaluated. Our results show an increased analytical sensitivity in detection of both genes (BCR/ABL1 and ABL1): the limit of detection of the one-step reaction is as low as 0.001% IS BCR/ABL1. By testing the ERM-AD623 at 1 copy/reaction the rate of PCR positivities is 63%, and the average estimated quantity is 2.5 (SD = ± 1.5) copies/reaction. The linear measurement range of BCR/ABL1 and of the control gene ABL1 evaluated using the ERM-AD623 reference material are linear and equivalent in the range of 102-107 copies/reaction. Quantifications obtained with this kit are aligned to the European Reference Material. Using 7500 Fast Dx Real-Time PCR Instrument or 7900 Real-Time PCR System (Applied Biosystem, Thermo Fisher Scientific), we confirm that the calibrator of the "BCR-ABL P210 ELITe MGB® Kit" is aligned to the ERM-AD623 DNA international standard and we demonstrated the inter-laboratory low variability and good linearity of the method by processing the secondary reference material aligned to WHO primary reference material. By analysis of 30 RNA of CML patients we observed high results reproducibility among laboratories (figure 1). In addition, at comparison with the individual routine methods (ipsogen BCR-ABL1 Mbcr IS-MMR DX, P210 PHILADELPHIA Q-PCR Alert kit. and an home-made assay) we report up to 97.4% correlation of BCR-ABL P210 ELITe MGB® kit results. In conclusion, our data demonstrate that "BCR-ABL P210 ELITe MGB® Kit" is a rapid, reproducible assay, aligned and calibrated towards the current goal standards BCR/ABL1 assays. It allows direct testing from RNA samples while maintaining the desired sensitivity. By requiring reduced hands on time of the operators and by allowing direct testing of RNA, "BCR-ABL P210 ELITe MGB® kit" will provide a significant improvement in the standardization of the molecular approach to CML monitoring. Figure 1 BCR-ABL P210 ELITe MGB® Kit reproducibility with clinical samples. Data of the individual laboratory were plotted against the mean assigned value. The regression fit of all data is R-Sq=96.8%. Figure 1. BCR-ABL P210 ELITe MGB® Kit reproducibility with clinical samples. Data of the individual laboratory were plotted against the mean assigned value. The regression fit of all data is R-Sq=96.8%. Disclosures Castagnetti: Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria. Martinelli:ARIAD: Consultancy; Genentech: Consultancy; Roche: Consultancy; Amgen: Consultancy; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; Novartis: Speakers Bureau; MSD: Consultancy; Roche: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy, Speakers Bureau. Saglio:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Honoraria.

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.

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+ hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+ hematologic malignancies.

// Giorgia Montano 1,2 , Karina Vidovic 2 , Chiara Palladino 1 , Elena Cesaro 1 , Gaetano Sodaro 1 , Concetta Quintarelli 3 , Biagio De Angelis 3 , Santa Errichiello 3 , Fabrizio Pane 3 , Paola Izzo 1 , Michela Grosso 1 , Urban Gullberg 2 and Paola Costanzo 1 1 Department of Molecular Medicine and Medical Biotechnology University of Naples Federico II, Naples, Italy 2 Department of Haematology and Transfusion Medicine, BioMedical Center, Lund University, Lund, Sweden 3 Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy Correspondence to: Paola Costanzo, email: // Keywords : ZNF224, chronic myeloid leukemia, BCR-ABL, WT1, tyrosine kinase inhibitors Received : March 23, 2015 Accepted : July 08, 2015 Published : July 22, 2015 Abstract The Kruppel-like protein ZNF224 is a co-factor of the Wilms’ tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML.

Introduction Tyrosine Kinase Inhibitors (TKI) have completely changed the scenario of CML and dramatically improved the outcomes. Thus, early identification of patients expecting poor outcome is crucial to offer alternative TKI regimens or in some selected cases stem cell transplantation before disease progression may occur. The Evaluating Nilotinib Efficacy and Safety in Trial as First-Line Treatment (ENEST1st) is a phase 3b is an open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL positive CP-CML. Aim of the ENEST1st sub-study N10 was to investigate BM microenvironment markers that regulate leukemic stem cells in the bone marrow (BM) niche of Nilotinib-treated patients. Methods The study enrolled patients in 21 Italian ENEST1st participating centers. Response was based on ELN recommendations (Baccarani M, et al. Blood 2013 122:872-884). In an interim analysis, molecular and cytogenetic response by 24 months was assessed. Mononuclear cells were collected from BM and PB samples at the screening visit (V0) and after 3 months of treatment (V4). RT-qPCR for the expression of 10 genes (ARF, KIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the stemness and hematopoietic stem cells survival signaling regulation was conducted. RT-qPCR data were normalized by the expression of GUS mRNA (normalized copy number, NCN). Plasma samples were collected at different time points from both BM or PB samples. Concentrations of 20 different analytes, including IL-1a, IL-3, M-CSF, SCF, SDF1-a, TRAIL, HGF, PDGF-bb, IL1b, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, G-CSF, GM-CSF, MIP-1a, TNF-a, and VEGF, were simultaneously evaluated using commercially available multiplex bead-based sandwich immunoassay kits. Results 33 out of 37 patients enrolled were available for an interim molecular analysis at 24 months: an optimal response was achieved in 25 patients, a warning response in 5 patients and a failure response in 3 patients. We observed a significant correlation between the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) or cytokine signaling (SET) and patient outcome. Indeed, NANOg and SET mRNA were significantly down-regulated in PB samples at diagnosis of patients with optimal response compared to patients with warning/failure response (NANOg mRNA: 0.3±0.25 NCN vs 0.6±0.7 NCN, respectively; p=0.05; SET mRNA: 0.2±0.3 NCN vs 2.3±4.2 NCN, respectively; p=0.03). We also investigated the plasma level of several factors involved in the hematopoietic stem cells (HSCs). Some of these markers showed a significant correlation with patient's outcome when evaluated at diagnosis in either PB or BM samples. Indeed, high level of IL12 (in the BM samples), or HGF, mCSF and SCF (in the PB samples) were associated to a worst prognosis markers, since significantly correlating with no MMR@12months (IL12, p=0.03), intermediate/high Socal score (mCSF, p=0.03; SCF, p=0.03), no reduction of MMR below to 1 at 3 month (SCF, p=0.04) or warning/failure response to Nilotinib treatment (HGF, p=0.03; SCF, p=0.04). Indeed, we find a lower levels of PDGFb, SDF1, TNFa, TRAIL (in the BM samples), and HGF, SDF1, TRAIL (in the PB samples) in those patients with intermediate/high Hasford or Sokal score (PDGFb, p=0.0007; SDF1, p=0.02), warning/failure response to Nilotinib treatment (HGF, p=0.03) or lacking of MMR4.0 (SDF1, p=0.01; TNFa, p=0.02; TRAIL, p=0.05). Conclusion/Summary Taken together, our results suggest that the expression analysis of genes involved in cell pluripotency (NANOg) and/or cell signaling (SET) at baseline, may indicate early achievement of deep molecular response in shown CML-CP patients treated with nilotinib. In addition, in patients with optimal response to Nilotinib, high concentration of SDF-1, TRAIL (inversely correlated with BCR-ABL, and associated to an higher susceptibility to apoptosis in the leukemic blasts) were observed as well as BM TNF (cell-extrinsic and potent endogenous suppressor of HSC activity). A lower concentration of several factors associated to hematopoietic progenitor cell growth and survival (including HGF, SCF and IL12) were observed compared to patients failing to achieve response to Nilotinib. These data strongly suggest that stromal microenvironment supports the viability of BCR-ABL cells in BM niches through direct feeding, or environment releasing of survival factors. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy. Saglio:Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis Pharmaceutical Corporation: Consultancy, Honoraria. Galimberti:Novartis: Employment. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract 2795 In the era of molecular target therapy, whereas Imatinib has shown an overall survival rate of 85% and an estimated event-free survival of 55% in a 8 year result update of IRIS trial, several in vitro data have confirmed that Ph+ stem cells housed within BM niches are resistant to TKI treatments. Second generation TKI Nilotinib has been designed with improved target specificity over Imatinib. Its efficacy in the treatment of patients with CML who are resistant to or intolerant to Imatinib led to the registration of a clinical trial CAMN107EIC01, a phase IIIb, multicentre, open-label study applying Nilotinib in the treatment of newly diagnosed CML. The aim of the current study (CAMN107EIC01 sub-study N10) is to define BM microenvironment markers that nurture and determine leukemic stem cell fate in the BM niches of Nilotinib treated patients. We enrolled 37 patients involving 21 Italian centers, from whom written informed consent has been obtained for sub-study N10. Patients have been monitored by Real Time RT-PCR (RT-qPCR) for the expression of the fusion mRNA BCR-ABL, as specified by the core protocol. Major Molecular Response (MMR) is defined as detectable disease ≤0.1% BCR-ABL according to the international scale (IS). Plasma and mononuclear cells have been collected from BM and PB samples of the enrolled patients at the screening visit (V0) and after 3 months of treatment (V4). We purified total RNA from BM and PB mononuclear cells of the enrolled patients to screen by RT-qPCR the expression of 10 genes (ARF, cKIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the regulation of the stemness and survival signaling of hematopoietic stem cells. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN). Moreover, we evaluated by multiplex ELISA assay BioPlex, the BM plasma concentration level of 20 cytokines (IL1a, IL1b, IL3, IL6, IL7, IL8, IL10, IL12, IL15, G-CSF, M-CSF, SCF, SDF1, TRAIL, HGF, PDGFbb, GM-CSF, MIP-1a, TNFa and VEGF), known to be key factors in the interaction of Ph+ stem cells to BM microenvironment. The interim analysis of MMR until the 12th month is available in 26 out of 37 enrolled patients. We observed that MMR is achieved during the first 12 months of treatments in 17 out of 26 (65%) patients. Molecular analysis showed that the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) and cytokine signaling (SET) were significantly down-regulated at V0 in PB mononuclear cells of patients achieving MMR in comparison to cells from non-responding patients (4vs21 NANOg NCN, p=0.05; 8vs32 SET NCN, p=0.05). These data were also confirmed on BM patient's sample. Moreover, we investigated the concentration level of the above-mentioned soluble factors in BM plasma samples of all 37 enrolled patients at both V0 and V4. We observed that the BM plasma level of several cytokines produced by leukemic cells significantly decreased during the first 3 months of Nilotinib treatment: IL3 (54vs3ng/ml; p=0.02), M-CSF (56vs12 ng/ml; p=0.005), SCF (170vs104 ng/ml; p=0.007), HGF (7565vs705 ng/ml; p Disclosures: Paratore: Novartsi: Employment. Galimberti:Novartis: Employment. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Introduction. Interferon alpha (IFN-α) is an attractive agent for the treatment of Essential Thrombocythemia (ET) due to its ability to induce clonal complete remission, sometimes lasting beyond treatment discontinuation, and to its recognized non-leukemogenicity. However, despite decades of clinical experience with IFN-α in patients with MPNs, optimal dose schedules, treatment duration and the ultimate molecular basis of the heterogeneous response still remain undefined. Hence, the early identification of IFN-sensitive patients may help limit IFN-α exposure to those who really benefit from treatment. Aim. Here we report the results of a trial involving 61 ET patients treated with IFN-α, aimed to identify the baseline molecular and clinical parameters able to predict response to treatment. Methods. IFN treatment schedule implied an initial induction phase with 3MU/five times a week; in patients who reached a platelet count 600x109/L or platelet reduction was Careful medical history, main laboratory data and spleen volume, assessed by ultrasonography scan were recorded in all patients at presentation and during follow-up. Complete hematological response (CHR) is defined as the normalization of both platelet and WBC counts ( mRNA levels of JAK1, JAK2, STAT1, STAT3, SOCS1, SOCS3 and TYK2 were assayed in pre-treatment bone marrow specimens by Real-Time PCR using the SYBR Green method. Results. After a median follow-up of 41.2 months, 72% of patients achieved CHR and were considered Good-Rs for subsequent analysis, whereas the remaining 17 were considered Bad-Rs. Among the Good-Rs, 24 (54%) are still on therapy with standard IFN-α doses (i.e. 3 MU 3 or 2 times a week), whereas 10 (23%) are maintained in CHR by the administration of very low doses of IFN-α-2b (3 MU every 7 or 15 days), and 3 (7%) have maintained CHR after therapy discontinuation (up to a median time of 31 months). The initial univariate analysis indicated that the mRNA levels of JAK1, STAT3, SOCS3 were significantly lower in Good-R than in Bad-R patients. Interestingly, among the different genes involved in the IFN-α receptor pathway, the expression levels of JAK1, together with spleen volume and platelet count, were selected by the stepwise multivariate analysis as the variables that independently correlate with IFN-α response. We used the relative HRs and the optimal cut-offs for response calculated for each variable by the ROC analysis to develop a prognostic score able to predict IFN-α response. This score has an overall 87% diagnostic efficiency in discriminating IFN-α response and unambiguously identifies the response to IFN-α in most patients, avoiding treatment in those with no probability of gaining benefit from this therapy. In addition, this score is able to identify unambiguously the response to IFN in a sizeable proportion of patients: an IFN-R score of 3 or 4 (31 patients, corresponding to 70.4% of the Good-R) indicates a 100% odd to obtain CHR, while a score of 0 indicates no chance of achieving a response. Conclusion. In conclusion, this study shows for the first time that the use of three simples parameters predicts the response to IFN in ET patients. Disclosures No relevant conflicts of interest to declare.

Introduction Interferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders. IFN binding its receptor results in tyrosine cross-phosphorylation and auto-phosphorylation of the JAKs proteins (Tyk2 and Jak1). These phosthyrosines recruit and activate STAT family member such as STAT1 and STAT3. These proteins induce the transcription of SOCSs, whose role is to extinguish cytokine signaling by inhibition of JAK kinase-activity directly through the KIR-domain, and indirectly promoting the proteasomal degradation of Jak2, by SOCS-box-motif. In summary, IFN induces the expression of SOCSs, which inhibit TPO mediated signaling through Jak2 double inhibition. This allows IFN-α and TPO pathway to cross-talks by means of the JAK-STAT-SOCS cascade. Aims To identify molecular markers that identify those patients who respond to IFN, we analyzed bone marrow cells transcript levels of specific genes involved in the IFN receptor pathway, whose signal cross-talks with the TPO dependent JAK-STAT pathway. In particular we investigated the mRNA expression of JAK1, TYK2, STAT1, STAT3, SOCS1 and SOCS3. Methods We analyzed 60 ET patients treated with 3 million units of IFN-α-2b 5 times a week as induction (3 months), and 3 times a week as maintenance. Responses were classified as follow: Good-Responders(R) (n=44), those who achieved complete response according to European Leukemia Net criteria, and Bad-Responders(NR) (n=17) who didn’t reach the criteria. The mRNA expression of genes of interest was measured in bone marrow samples from ET patients by RTq-PCR and tested for their predictive value using receiver operating characteristics (ROC) curves. Data were normalized as following: [mRNA normalized copy number (NCN)=mRNA target gene/mRNA GUSB]. An IFN score was calculated as an average in log2 of mRNA levels of genes differently expressed between Good-R and Bad-R. Results Main clinical characteristics were similar between the two groups of response. JAK2 V617F mutation was detected in 56,8% of Good-R and 58,8% of Bad-R (p=0,81) and no difference was found in JAK2V617F allele burden (p=0,17) and mRNA expression (p=0,2). Patients showed a median spleen volume of 500 ml in Good-R and 250 ml in Bad-R group (p=0.01). Bad-R compared with Good-R showed higher mRNA expression of JAK1 (13.4 vs 4.7; p Average expression in log2 of these three genes was calculated and used as IFN score. The ROC curve AUC analysis for IFN-score revealed an AUC of 0.9 (95% CI:0.8-1.0). The score value with highest combined sensitivity (94.1%, 95% CI: 71.3-99.8) and specificity (88.6%, 95% CI: 75.4-96.2) was 4.74, with a likelihood ratio of 8.28. In our cohort, all Bad-R patients but one, showed IFN-score higher than the established cut off, and this further support the accuracy of our IFN-responsive score for ET patients. Conclusions We identified a set of three genes whose expression could be compounded into IFN score that showed a significant correlation with response in ET patients. The IFN score could represent a predictive biomarker for responsiveness to IFN and may likely become a substantial aid to the physician. Disclosures: No relevant conflicts of interest to declare.