ALBERT

Description: Abstract 2291 Background: Nilotinib is a potent and the most selective inhibitor of BCR-ABL. In the phase 3 ENESTnd trial, nilotinib demonstrated superior efficacy vs imatinib with higher and faster molecular responses and a significantly lower rate of progression on treatment to accelerated or blast phases of CML. Nilotinib has been previously shown to prolong the QT interval. The cardiac safety profile of nilotinib was previously described in pts with imatinib-resistant and -intolerant CML-CP enrolled in phase 2 clinical trials. Here, we report cardiac safety data on nilotinib 300 and 400 mg twice daily (bid), and imatinib, in pts with newly diagnosed CML-CP from the ENESTnd trial. Methods: A total of 836 pts were included in the safety analysis of ENESTnd (279, 277, and 280 pts in the nilotinib 300 mg bid, nilotinib 400 mg bid, and imatinib arms, respectively) with a median follow-up of 18 months. Pts were excluded from study participation if they had known uncontrolled or medically significant cardiac disease, left ventricular ejection fraction (LVEF) 〈 45%, or QTcF interval 〉 450 msec. Prospective cardiac monitoring was conducted throughout the study for QT prolongation (via electrocardiogram) and LVEF (via echocardiogram) at regular intervals. Results: QTcF increases of 〉 30 msec from baseline occurred in 26% of pts in each nilotinib arm and in 18% of pts in the imatinib arm. QTcF increases of 〉 60 msec from baseline were uncommon, occurring in 〈 1% of pts in all three arms (Table). The highest mean changes from baseline in QTcF interval were 10.4, 12.4, and 7.9 msec in nilotinib 300 mg bid, nilotinib 400 mg bid, and imatinib arms, respectively, which occurred between months 3–6 of therapy in all arms. Furthermore, there was a modest linear correlation between nilotinib serum concentration and QTcF change from baseline that was similar to previously reported results. No pt in any treatment arm demonstrated an absolute QTcF interval 〉 500 msec. Analysis was conducted to identify any potential case of clinically symptomatic QT prolongation. Only events of syncope were identified (1 pt in each arm with drug-related events) by this analysis and none of these could be attributed to QT prolongation; there were no episodes of torsades de pointes in any treatment arm. Additionally, there was no decrease from baseline in mean LVEF observed anytime on treatment in any arm (Table). No patient in any treatment arm had a LVEF of 〈 45% on treatment or an absolute reduction from baseline in LVEF of 〉 15%. Importantly, no pt discontinued therapy due to QT prolongation or LVEF change. An analysis of grouped adverse event terms was performed to identify cases consistent with ischemic heart disease (IHD) or left ventricular dysfunction. A total of 11 pts in all treatment arms experienced IHD events after median treatment duration of 18 months: 3 (1%) on nilotinib 300 mg bid, 6 (2%) on nilotinib 400 mg bid, and 2 (〈 1%) on imatinib. Of these 11 pts, 10 had preexisting cardiac disease or risk factors and only 1 pt discontinued treatment due to an IHD event. Nine pts reported angina pectoris. Two pts experienced myocardial infarction (MI) and 1 of these pts died during coronary artery bypass surgery (perisurgical MI). A total of 7 pts in all treatment arms with events consistent with left ventricular dysfunction were identified: 1 (〈 1%) on nilotinib 300 mg bid, 4 (1%) on nilotinib 400 mg bid, and 2 (〈 1%) on imatinib. Of these 7 pts, 4 pts experienced reduction in LVEF (3 were asymptomatic grade 1/2) and 3 pts experienced cardiac failure/congestive failure; 5 of these 7 pts had prior history of cardiovascular disease and/or preexisting cardiovascular risk factors. None of these 7 pts discontinued treatment due to events with left ventricular dysfunction. There were no sudden deaths reported on study. Conclusions: Overall, QT prolongation, changes in LVEF, and clinical cardiac events were uncommon in all arms and seldom led to discontinuation. There was no cumulative effect of nilotinib exposure on cardiac safety with a median of 18 months follow-up. Nilotinib at both doses had a favorable cardiac safety profile that was similar to imatinib in pts with newly diagnosed CML-CP. Disclosures: Larson: Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Hochhaus: Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Saglio: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria. Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau. Lopez: Novartis: Consultancy; Bristol Myers Squibb: Consultancy. Goldberg: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Gallagher: Novartis Pharma AG: Employment, Equity Ownership. Hoenekopp: Novartis: Employment. Ortmann: Novartis: Employment. Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria. Kantarjian: Novartis: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding.

Description: Earlier evaluation of therapy effect in patients with CML would assist in optimal use of available tyrosine kinase inhibitors (TKI). Single cell analysis by mass cytometry has enabled the quantification of up to 46 antibody epitopes, making it ideally suited for exhaustive immunophenotyping of the haematological hierarchy, and evaluation of associated dynamic signal transduction events, in a clinical setting. By integrating time resolved single cell signalling data with clinical parameters, we searched for prognostic and efficacy-response mass cytometry biomarkers within a month of TKI therapy. We report data from experiments used to validate the custom panels of antibodies, highlighting the power of mass cytometry in the analysis of primary patient material obtained on clinical studies. Peripheral Blood (PB) samples were collected before, 3 hours, 7 days and 28 days, after start of nilotinib (300 mg BID) treatment in a subset of patients (n=55) enrolled in the ENEST1st trial. PB cells were stained with two panels of antibodies, allowing a comprehensive immunophenotyping of numerous cellular subsets, and also the evaluation of intracellular phosphorylation status of several epitopes. Moreover, using a straightforward barcoding scheme, the time-resolved samples from each individual patient were pooled after barcoding and stained with the antibody panels to minimize sample variation. In a pilot study, 7 and 10 cell subsets were identified in PB samples from 4 untreated healthy donors and 2 complete sets of 4 patients enrolled in this sub study, respectively. Furthermore, a robust signal was measured for pCrkL, pStat5, pStat3, pCreb, pAbl Y412 and pAbl Y245. The two sets of samples from study patients showed substantial changes in activation status over the course of therapy. Some changes, such as pStat3 alterations are only detectable in neutrophils and monocytes, while the activity of others i.e. pCreb was found to be ubiquitous. CD34+ cells indicated decreased phosphorylation of CrkL, Stat5, and Abl Y412/245. To increase the immunophenotyping resolution of the myeloid lineage, 3 additional cell surface markers were incorporated into the cell surface panel. In 1 healthy donor, and in diagnostic samples from three patients enrolled in this sub study, this allowed the identification of 13 cell subsets: CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-) (Figure 1 A,B). With respect to the relative number of cells identified for each cell type, the three diagnosis samples differed from the single healthy control. In the patients, we observed an expansion of the granulocytic compartment, as well as the emergence of CD34+ progenitor and stem cells in the peripheral blood. In conclusion, the here presented developed assay is able to resolve most of the cell subpopulations found in the hematopoietic tree, and also robustly measure the activity of central signalling substrates known to be involved in CML pathogenesis. With the addition of new phospho-specific antibodies, the methodology may facilitate the detailed characterization of CML in an immunological context, and may shed new light on both the disease and therapeutic mechanism. Analysis of variation in signal responses and immune profile are now in progress in the subset of patients (n=55) in the ENEST1st trial. Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Disclosures Thaler: AOP Orphan: Research Funding. Lang:Celgene: Consultancy. Hjorth-Hansen:Bristol-Myers Squibb: Research Funding; Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding. Hellmann:Novartis: Consultancy, Other: funding of travel, accomodations or expenses, Research Funding, Speakers Bureau; BMS: Consultancy, Other: funding of travel, accomodations or expenses, Speakers Bureau. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Janssen:ARIAD: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Ossenkoppele:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Pfizer: Honoraria, Research Funding; the Finnish Cancer Societies: Research Funding; Academy of Finland: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Gjertsen:Bergen University Hospital: Research Funding.

Description: Abstract 1702 The use of imatinib (ima) and other tyrosine kinase inhibitors (TKI) has dramatically improved the clinical outcome for patients (pts) with chronic myeloid leukemia in chronic phase (CMLcp). Although most pts respond well, there is a subpopulation that fails to reach internationally defined clinical treatment goals, as assessed by techniques such as cytogenetics (CG) and quantitative polymerase chain reaction (PCR). Pts with a poor initial treatment response appear to have an increased risk of subsequent disease progression to accelerated phase or blast crisis, which still constitute a serious medical challenge. It was recently shown that landmark analysis assessing PCR levels after 3 months (mo) of ima treatment was linked to major cytogenetic response (mCyR) at 12 mo and risk of long-term progression (Hanfstein ASH2010 abstract 360). Similar data based on fluorescence in-situ hybridization (FISH) landmark assessment of larger CMLcp pt groups are still lacking. We have followed a cohort of 45 newly diagnosed CMLcp pts, all initiated on ima (400mg qd), by sequentially assessing their treatment responses by CG, PCR and interphase ES-FISH and relating early BCR-ABL expression levels to subsequent clinical response. A complete cytogenetic response (CCyR) was observed in 56% of evaluable pts after 6 mo, 80% after 12 mo and 94% after 24 mo of treatment. Corresponding figures for major molecular response (MMR) were 9%, 47% and 71%, respectively. Early levels of BCR-ABL as assessed by FISH could predict subsequent response. Thus, of patients with FISH-pos ≤10% at 3 mo 95% achieved CCyR at 12 mo, while this response was noted in only 67% of pts with FISH-pos 〉10% at 3 mo (p=0.042; Fisher's exact test, n=37). Similarly, all evaluable pts with FISH-pos ≤10% at 3 mo were alive and event-free (EFS) at 36 mo, as compared to 67% EFS at 36 mo among pts with FISH-pos 〉10% at 3 mo (p=0.008, n=37). Performing similar landmark analyses with FISH ≤ or 〉10% at 6 and 12 mo also yielded significant differences regarding EFS at 36 mo (p=0.046 and p=0.003, respectively). Employing corresponding landmark analyses with PCR at 3 mo and 6 mo, no significant association to EFS at 36 mo could be detected. In conclusion, our landmark analysis data indicate that ES-FISH can be used effectively already after 3 mo of ima treatment in order to identify a patient cohort with inferior long-term survival and with a higher risk for disease progression. FISH can unlike CG, be efficiently performed on peripheral blood cells which facilitates its clinical use. In comparison to PCR earlier data have shown that FISH, although less sensitive, may be more reliable and reproducible at higher BCR-ABL expression levels, typically seen in the early treatment phase. This may explain the superior outcome of early landmark analysis using FISH, as compared to PCR, in our study. Expanded trials on larger CMLcp pt cohorts are warranted to further clarify the prognostic value, and possible impact on early treatment alterations, of early FISH analyses during TKI treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML. Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1〉0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times. Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22). Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation. The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group. Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. Disclosures Själander: Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: Background: Ponatinib is an approved potent oral tyrosine kinase inhibitor active against native and mutated forms of BCR-ABL, including T315I. The phase 2 PACE study demonstrated that ponatinib is highly active in heavily pretreated Philadelphia chromosome‒positive leukemia patients. Ponatinib efficacy and safety were evaluated in newly diagnosed CP-CML patients in the EPIC trial. Methods: EPIC was a multicenter, international, phase 3, randomized, 2-arm, open-label trial of ponatinib (45 mg once daily) compared with imatinib (400 mg once daily) in newly diagnosed CP-CML; patients were stratified by Sokal risk score (low [1.2]). On 18 October 2013,EPIC was terminated due to the observation of arterial thrombotic events in the ponatinib development program. Consequently, none of the prospectively defined endpoints could be analyzed. Data as of 1 April 2014 are presented for endpoints that could be analyzed: BCR-ABLIS

Description: Background: Tyrosine kinase inhibitors (TKIs) have significantly improved the treatment of CML. Even though TKI treatment is generally not considered curative, recent studies have shown that nearly half of CML patients who have achieve good and durable responses are able to stop the TKI treatment. However, patients who have successfully discontinued TKI treatment still have residual disease. We hypothesized that the immune system plays a role in treatment free remission (TFR), and our preliminary results in the EURO-SKI trial showed that patients who relapse early after imatinib discontinuation have decreased numbers and frequencies of NK cells. In EURO-SKI trial relapse was defined as the loss of major molecular response (MMR). We now aimed to analyze in more detail the phenotype and function of the NK cells in order to understand their role in TFR. Methods: Lymphocyte subclass analysis (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1 month after the imatinib discontinuation in patients participating in the EURO-SKI stopping trial in the Nordic countries (n=105, results are presented from patients who have reached 6 months follow-up). More detailed immune phenotype and functional assays (NK-cell degranulation and secretion of Th1 type of cytokines IFN-γ/TNF-α) were analyzed from a proportion of patients (n=31). Results: Imatinib treated patients remaining in remission for 6 months (non-relapsing, n=48, median age 60,5 years) displayed an increased amount of NK cells at the time of drug discontinuation (18.6% vs. 11.0%, p=0.02, NK-cell count 0.25 x109 cells/L vs. 0.184 x109 cells/L m, p=0.059) compared to patients who relapsed early (before 5 months, n=29, median age 60,5 years). Furthermore, the NK cell frequency in non-relapsing patients was even higher than in healthy controls (11.5%, n=48, p=0.001). T and B cell counts and frequencies showed no differences between the groups. Detailed analysis of the NK cell compartment displayed a more mature phenotype for the NK cells in non-relapsing patients. Larger frequencies of NK cells from early relapsing patients was CD56bright compared to non-relapsing patients (4.8% vs. 2.7% of CD56 NK cells, p=0.04). Furthermore, patients who had higher frequencies of CD56bright NK cells than median had decreased TFR at 6 months (42%) compared to patients with lower frequency (70%, p=0.01). In addition, there was a trend towards more CD57pos (78% (n=21) vs. 66% (n=10), p=0.09) CD56dim NK cells in non-relapsing patients. To further study the mature NK cells in non-relapsing patients, recently identified markers (FceRgneg, PLZFneg, SYKneg, EAT-2neg) for adaptive NK cells were analyzed. Interestingly, there was a trend that non-relapsing patients had higher frequencies of adaptive-like NK cells. For example, non-relapsing patients had more CD56dim NK cells that had down regulated EAT-2 (2.8% (n=6) vs. 1.3% (n=5) of lymphocytes, p=0.03) and more CD56dim NK cells expressing NKG2D (11.2% vs. 2.6% of lymphocytes, p=0.02) and NKp46 (13.6% vs. 3.9% of lymphocytes, p=0.05). Moreover, after imatinib discontinuation the expression of transcription factor Eomes increased in the CD56dim NK cells of the early relapsing group (baseline MFI 2045 vs. 1 month 3480, p=0.06), while in non-relapsing group it seemed to even decrease (baseline MFI 2273 vs. 1 month 1980, p=0.13) pointing towards an adaptive phenotype. No significant differences between the groups were observed when degranulation against K562 cell line was studied. However, CD16neg NK cells from non-relapsing patients responded to K562 stimulation by secreting more TNFα/IFNγ compared to the early relapsing patients (21% vs. 13% of CD56pos CD16neg NK cells, p=0.01). Furthermore, patients whose CD16neg NK cells had higher than median TNFα/IFNγ secretion when stimulated with K562 cells showed an increased TFR at 6 months (78%) compared to patients who had lower TNFα/IFNγ secretion than median (37%, p=0.005). Conclusions: CML patients who successfully discontinued imatinib therapy displayed a higher number and frequency of peripheral blood mature, adaptive-like NK cells capable of secreting cytokines TNFα/IFNγ relative to relapsing patients. How such NK cells may contribute to maintenance of treatment free remission is still unknown. Nonetheless, our results warrant further clinical studies with NK-cell modulating agents. Disclosures Muller: Novartis: Honoraria, Other: Consulting or Advisory Role, Research Funding; ARIAD Pharmaceuticals Inc.: Honoraria, Other: Consulting & Advisory Role, Research Funding; BMS: Honoraria, Other: Consulting or Advisory Role, Research Funding. Hjorth-Hansen:Novartis: Honoraria; Ariad: Honoraria; Bristol-Myers Squibb: Research Funding; Pfizer: Honoraria, Research Funding. Saussele:Pfizer: Honoraria, Other: Travel grant; BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; ARIAD: Honoraria. Mahon:ARIAD: Consultancy; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Mustjoki:the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Academy of Finland: Research Funding; Sigrid Juselius Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Description: Rationale: Dasatinib (DAS) and interferon have different modes of action and may have synergistic activity in CML, due to both antineoplastic and immunostimulatory mechanisms. Addition of pegylated interferon (PegIFN) to imatinib therapy in CP-CML has in previous clinical trials (French SPIRIT and NordCML002) resulted in deeper molecular responses. Thus, an optimal combination of DAS and PegIFN may increase the proportion of patients who reach deep molecular response with potential for treatment-free remission (TFR). Design: Newly diagnosed CP-CML patients were treated with DAS (Sprycel, BMS) 100 mg OD as single drug for three months. Thereafter weekly subcutaneous injections of Peg-IFN α2b (PegIntron, MSD) were added to DAS; from end of month 3 (M3) to M6, 15µg/week, thereafter 25µg/week until M15. Primary end points were safety and the rate of MMR at M12. The doses of PegIFN were lower than in the SPIRIT and NordCML002 studies to increase adherence. Population: Forty patients were included at 14 university centers. One patient was lost to follow-up after M6. All patients were included in analysis up to M12. Mean and median age was 48 years (range 19-71). The proportions of high risk patients were 25% (Sokal), 15% (Hasford), and 15% (EUTOS). Safety and dosing: Treatment was well tolerated with expected DAS and PegIFN related side effects. Six patients had seven serious adverse events (AEs), all hospitalizations. 1 episode each of bradycardia/atrial fibrillation (possibly PegIFN-related), headache (DAS), fever (PegIFN), anaphylaxis-like reaction (PegIFN), fever/malaise/headache (PegIFN), pneumonia and a knee effusion (both unrelated). One pleural effusion occurred (grade 2, 3%). Grade 3-4 neutropenia and thrombocytopenia occurred in 6 and 9 patients respectively. Prolonged hematological toxicity (〉2 months) occurred in 8 patients, causing dosing problems in 5. One patient suffered grade 3 depression. Grade 3 flu-like symptoms occurred in 2 patients. One patient had lipase elevation grade 3 and one patient developed hypothyroidism attributed to PegIFN. Grade 2 dermal AEs like rash and acne occurred in about 20%, attributable to both drugs. 94% (DAS) and 76% (PegIFN) of assigned dose was given. Dose reductions occurred in 19 patients for DAS and 20 patients for PegIFN. Two patients discontinued DAS and switched to nilotinib, 1 for headache at M3 and 1 at M12 for lack of efficacy/hematological toxicity. Two patients could not start PegIFN for hematological toxicity (one lost to follow-up after M6). PegIFN was discontinued because of bradycardia/atrial fibrillation (1 patient), anaphylaxis (1 patient), flu-like syndrome (2 patients) and long-term hematological toxicity (2 patients). At 12 months 31/38 pats (82%) were still on PegIFN, a higher proportion than in the French Spirit or NordCML002 studies. Efficacy: We have used the DAS arm of the Dasision study (Kantarjian NEJM 2010) as a historical control. Early response at M3 was very similar between studies. In the present and the Dasision cohorts respectively, 18% vs 16% missed the 10% BCR-ABLIS landmark, 66% vs 56% achieved a CCyR and 8% vs 8% achieved MMR. At M6, three months after introduction of PegIFN, a steep increase in MMR rate was observed compared with Dasision. This was also reflected in deep responses, MR4.0 (see tables) and MR4.5 at M12, 18% vs 5%. The primary efficacy endpoint was MMR at M12, 82% vs 46%. Table 1.MMRDAS+PegIFN (%)DAS (Dasision)(%)Difference (%)M3880M6532726M9663927M12824636Table 2.MR4.0DAS+PegIFN (%)DAS (Dasision) (%)Difference (%)M3303M620614M938830M12481236 Progressions and treatment failure defined by ELN 2013: Failures: No progression was noted. At M3, 2 patients still had 〉95% Ph+ metaphases (MF). At M6, four patients (11%) had 〉 35% Ph+MF or 〉10% BCR-ABL levels. At M12, one patient failed CCgR and two more patients failed

Description: Abstract 2520 INTRODUCTION. Age, cytogenetics, FLT3 and NPM1 mutations are the most significant prognostic factors (PFs) for adult AML treated with standard regimens, but the predictive significance of FLT3 and NPM1 with contemporary treatments is unknown. We examined the clinical significance of NPM1 and FLT3 mutations in adult de novo AML pts enrolled on SWOG study S0106. METHODS. S0106 was a randomized phase III clinical trial for pts of age 18–60 with de novo non-M3 AML, evaluating the effects of adding Gemtuzumab Ozogamicin (GO) to standard induction therapy (Cytosine Arabinoside and Daunomycin, AD), and of post-consolidation GO vs. no additional therapy (ASH, 2009, Abstract 790). Samples from 198 of the 600 eligible pts were evaluated. Analyses for nucleotide insertions in exon 12 of the NPM1 gene and internal tandem duplications (ITD) within exons 14–15 of FLT3 were performed using fragment analyses in diagnostic bone marrow (BM, N=190) and peripheral blood (PB, N=8) samples. Mutant/wild-type (WT) allelic ratios (AR) were computed for all mutations. Effects of mutations and other PFs on complete response (CR), resistant disease (RD), overall survival (OS) and relapse-free survival (RFS) were analyzed by logistic and Cox regression. P-values are 2-sided. RESULTS. Patient characteristics and outcomes are shown in Table 1. In univariate analyses, NPM1-Mut pts had significantly higher CR (81% vs. 58%, P=.0018) and lower RD (13% vs. 28%, P=.028) rates, better OS (64% vs. 47%, P=.045) and RFS (54% vs. 41%, P=.50). FLT3-ITD was not associated with CR or RD, but was associated with poorer OS (hazard ratio [HR] 2.28, P=.0011) and RFS (HR 2.74, P=.0009). FLT3-ITD length (range 18–366, median 46), FLT3 AR (range 0.18–8.2, median 0.98), and NPM1 AR (range 0.2–1.0, median 0.8) were not associated with CR, RD, or OS, but RFS tended to be lower with higher ITD length (P=.076). In multivariate analyses with other PFs, neither NPM1 nor FLT3 was associated with CR or RD rates, however the combined effects of FLT3 and NPM1 identified 3 mutation risk groups for OS (P=.0044, Fig 1A) and RFS (P=.0003, Fig 1B), since NPM1 did not significantly affect outcomes within the FLT3-ITD pts. These risk groups are FLT3-WT/NPM1-Mut (Good Risk: 3-yr OS 82%, RFS 69%), FLT3-WT/NPM1-WT (Intermediate Risk: OS 49%, RFS 43%), and FLT3-ITD (Poor Risk: OS 29%, RFS 14%). The impact of adding GO to induction therapy was examined within each risk group. In each risk group, CR rates were higher in the AD+GO arm, though not significantly so. Likewise, the RD rates were lower in the AD+GO arm, but this difference was significant only in the largest group: Intermediate Risk, FLT3-WT/NPM1-WT, 17% vs. 34% (P=.026). Treatment arm did not significantly affect OS and RFS in any mutation risk group. CONCLUSION. This study confirmed prognostic effects of FLT3 and NPM1 mutations in de novo AML pts treated with AD or AD+GO. Analyses of the joint impact of NPM1 and FLT3 mutations do not rule out the possibility that they act independently. With the small numbers of pts in the “good” and “poor” risk groups, there was no clear evidence that mutation status predicts clinical benefit from adding GO to therapy. We are evaluating additional samples and will update these results as data matures. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4269 Background Imatinib (IMA) is recommended as first-line therapy for patients diagnosed with CML in chronic phase (CMLcp). Earlier studies, based particularly on the IRIS trial, have indicated that a favorable long-term clinical outcome to therapy is clearly associated with a reduction of the Ph-pos cell population and of BCR-ABL transcripts, as measured with cytogenetics and qPCR, respectively. We have investigated the response to IMA in a CMLcp cohort treated at our institution and assessed the prognostic value of early and repeated determinations BCR-ABL expression, using both ES-fluorescence in situ hybridization (FISH) and qPCR. Methods 45 pts with newly diagnosed CMLcp (24M/21F; median age 54 yrs, range 19-87; 5 with 9q del, 5 with variant translocations; Sokal scores 16 LR, 20 IR, 9 HR) were started on IMA (400mg qd) and hematologic, cytogenetic and molecular responses were followed at regular 3-mo intervals. Median IMA treatment time at follow-up was 29 mo (range 9-94). Regular cytogenetic karyotyping (CG) and interphase ES-FISH were performed on bone marrow cells (the latter method by scoring BCR-ABL in at least 500 cells on smear preparations, detection limit 20.2%) while qRT-PCR was performed on peripheral blood leukocyte samples with ABL as control gene. Major molecular response (MMR) was defined as the ratio BCR-ABL/control gene 20.1%. Results At diagnosis the pts displayed a median of 100% (range 67-100) Ph-pos metaphases by CG, while ES-FISH revealed BCR-ABL gene expression in a median of 86,2% (range 45,9-97,3) of cells. The response to IMA-treatment during the first 12 mo, displayed in Table I, included CCyR in 80%, MMR in 48% and reduction of FISH-detected BCR-ABL to 20). A total of 6 pts showed ‘event‘ (4 AP, 1 BC, 1 loss of CHR; 2 died). Table III provides landmark data related to FISH and PCR levels at 3 time points. Patients with a high remaining BCR-ABL expression were more likely to develop ‘events‘, as compared to those with low expression. Conclusion Our single centre data from an unselected CMLcp pat cohort on first-line IMA shows good clinical and molecular responses, well in line with results from the IRIS study. We propose that repeated, longitudinal interphase ES-FISH analyses may provide additional and important prognostic information regarding later targeted clinical endpoints. The method gives reliable information also early during the treatment, at higher disease penetration when PCR may be less reproducible, and can also be performed on peripheral blood samples. Disclosures: No relevant conflicts of interest to declare.