European LeukemiaNet Recommendations Research Articles

Plasma Imatinib Trough Level Is a Predictor For 3-Month Early Molecular Response In New CP CML Patients

BackgroundRecent studies have demonstrated that measurements of BCR-ABL1 transcript levels at 3 and 6 months were able to identify high-risk patients treated with IM. However, the value of early molecular response has not been fully defined. New European Leukemia Net (ELN) recommendations concluded that a single measurement of BCR-ABL1 transcripts level after 3 months of treatment is not sufficient to define failure necessitating a change of treatment. The aim of this study was to identify predictive factors for an achievement of 3-month EMR. For the purpose, we explored contributing factors including trough plasma concentrations of IM, precise IM dose schedule. Additionally, in the same population, prognostic implications of 3- month EMR were analyzed. MethodsBetween December 2009 and June 2012, 102 patients with newly diagnosed CP CML were enrolled. They started IM (400 mg/day) therapy without prior treatment except hydroxyurea or anagrelide and molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). All qRT-PCR tests were performed in a single laboratory (Cancer Research Institute, The Catholic University of Korea, Seoul, Korea). Measurements of IM plasma concentrations were performed on day 29. ResultsA total of 102 newly diagnosed CP CML patients (including 61 men and 41 women) were analyzed. With a median age of 41 years (range, 18-75 years), the distribution of low, intermediate and high Sokal risk scores were 42%, 42% and 16%, respectively. All patients, except one patient who showed less than complete hematologic response, were evaluable for molecular analyses at 3 months. Day 29 trough IM level data were available from 99 patients. The median trough concentrations of IM were 1,252 (range, 439-3,491) and cut-off IM levels for Q1 and Q4 quartiles were 958 and 1767 ng/mL on day 29, respectively.Univariate analyses revealed that age of ≥ 40 years (P = 0.046), high Sokal risk (P = 0.066), high Euro risk (P = 0.004), increased platelet count (P = 0.028), increased blast percentage (0.023), and large spleen size (P = 0.046) were potential predictive factors for no achievement of 3-month EMR. In addition, plasma IM trough level of ≤ Q1 quartile on day 29 was associated with no achievement of 3-month EMR. After adjusting for factors affecting achievement of 3-month EMR on univariate analyses, multivariate analyses showed that large spleen size (RR of 0.79, P = 0.030) was predictor for no achievement of 3-month EMR and patients in ≤ Q1 of plasma IM trough level on day 29 had a lower 3-month EMR, compared with those in Q2-4 (RR of 15.61, P = 0.005).To evaluate the prognostic value of 3-month EMR in our cohort, we analyzed CCyR at 6 months, MMR at 12 months, and the 3-year CI of CCyR, MMR, and UMRD. In addition, the 3-year EFS, FFS, and PFS were also assessed. In patients with BCR-ABL1 ≤10% at 3 months, significantly higher rates of CCyR at 6 months (79% vs 13%, P < 0.001) and MMR at 12 months (54% vs 10%, P = 0.014) were observed, compared with that of patients with BCR-ABL1 >10%. They also had significantly better 3-year CI of CCyR (100% vs 63.0%, P<0.001) and MMR (100% vs 30.4%, P = 0.001), EFS (62.2% vs 26.3%, P = 0.002), and FFS (89.6% vs 73.7%, P = 0.044). However, there were no significant differences in 3-year PFS. ConclusionsThis study analyzed various factors, such as baseline biological characteristics, adherence to IM, IM dose intensity, pharmacokinetics. It provides predictors for 3-month EMR and re-confirmed the prognostic significance of 3-month EMR. The considering of IM plasma concentrations for 3-month EMR should be needed in the clinical decision of changing therapy at this time point. Further clinical investigations in a larger patient population with longer follow-up are needed. Disclosures:No relevant conflicts of interest to declare.

Imatinib-Treated CML Patients With Discordant Response Between Cytogenetic and Molecular Tests At 3 and 6 Month Timepoints Have a Reduced Probability Of Subsequent Optimal Response: A Study From The “Gruppo Triveneto LMC”

IntroductionThe 2013 version of the European LeukemiaNet (ELN) recommendations for the management of chronic myeloid leukemia (CML) patients defines as optimal response the achievement of at least a partial cytogenetic response (PCyR) and/or BCR-ABL <=10% at 3 months, and of a complete cytogenetic response (CCyR) and/or BCR-ABL<1% at 6 months. Obtaining less than PCyR (i.e. Ph+ 36-95%) and/or BCR-ABL >10% at 3 months, and less than CCyR and/or BCR-ABL 1-10% at 6 months are regarded as warning. Patiens with discordant response between cytogenetic and molecular tests (e.g. PCyR and BCR-ABL >10% at 3 months) may be alternatively considered at the same timepoint as optimal or warning. Aims and MethodsTo evaluate the outcome of CML patients with discordant results between cytogenetic and molecular tests, we retrospectively analyzed our cohort of early chronic phase CML patients for which both cytogenetic and molecular responses were evaluable at 3 and/or 6 months. All patients received front-line imatinib 400 mg daily. PCyR and CCyR were defined as 1-35% and 0% Ph+ metaphases, respectively; major molecular response (MMR) was defined as BCR-ABL <=0.1%IS. Failure-free survival (FFS) was measured from the start of imatinib to the date of any of the following events: progression to accelerated or blastic phase (ABP), death for any cause at any time, imatinib dose increase (>=600 mg/day) or switch to nilotinib/dasatinib for primary or secondary hematologic or cytogenetic resistance. Cumulative responses and survival probabilities were estimated by the Kaplan-Meier method and compared by log rank test; differences among variables were evaluated by the Fisher's exact test. ResultsA total of 201 patients were analyzed. Median age at diagnosis was 55 (range 20-84) years. The distribution according to the Sokal score was: 86 (42.8%), 79 (39.3%) and 36 (17.9%) patients for low, intermediate and high risk, respectively. We observed that patients with concordant optimal (n=110) and discordant (n=19) results at the 3 month timepoint had significantly different chances of subsequent 6-month CCyR (88% vs 40%, p<.0001) and 12-month MMR (68% vs 12%, p<.0001), while there were no significant differences between patients with discordant or concordant warning (n=21) results (6-month CCyR 40% vs 14% and 12-month MMR 12% vs 0%, respectively). Also, patients with discordant results, compared to concordant optimal patients, had a significantly longer median time to CCyR (10.5 vs 3.5 months, p<.0001) and to MMR (49.6 vs 9.1 months, p<.0001), while there were no differences between discordant and concordant warning patients. Similarly, considering the 6-month timepoint, patients with discordant (n=28) results had a significantly inferior probability of subsequent 12-month MMR compared to concordant optimal (n=104) patients (16% vs 82%, p<.0001) but not different from concordant warning (n=38) cases (7%). Long-term FFS was significantly different between concordant optimal, discordant and concordant warning patients both considering the 3-month (82.8% vs 52.7% vs 9.6%, respectively) and the 6-month (90.4% vs 67.9% vs 23.7%, respectively) timepoints (figure). ConclusionsOur results suggest that only CML patients with concordant cytogenetic and molecular optimal response at the earlier timepoints have an excellent probability of obtaining subsequent MMR and a favourable long-term FFS, while patients with at least one warning result should be carefully monitored, since their risk of treatment failure is higher. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High BCR-ABL/GUSIS Levels At Diagnosis Are Associated With Unfavorable Responses To Imatinib

The approval of three tyrosine kinase inhibitors (TKIs) for the first line treatment of Chronic Myeloid Leukemia (CML) has generated an urgent need for molecular parameters predictive of unfavorable therapeutic outcomes. Recent evidence suggests that failure to achieve early molecular responses (i.e. BCR-ABL/ABLIS levels <10% after 3 months or <1% after 6 months of TKI treatment) results in inferior rates of both overall and progression-free survival.With the current study, we wanted to establish if high BCR-ABL transcripts at diagnosis would be associated with unfavorable responses to Imatinib Mesylate (IM). Thus, we correlated quantitative determinations of BCR-ABL levels at diagnosis with the outcome of 230 newly diagnosed CML patients that were assigned to receive IM 400 mg/die. BCR-ABL transcripts were measured from peripheral blood samples drawn before exposure to any form of treatment. Real-Time Quantitative PCR (RQ-PCR) determinations were subsequently performed in triplicates using glucuronidase-beta (GUS) as the reference gene, since previous evidence has demonstrated that ABL is not a reliable control gene in samples collected at diagnosis. Values were then reported on the International Scale employing a conversion factor obtained from the laboratory of the University of Heidelberg in Mannheim, Germany.Median follow-up of the study population was 50 months. Estimated 5-year cumulative incidences of complete hematologic response, complete cytogenetic response (CCyR) and major molecular response were 97.9%, 89.5% and 64.7%. Five-year probabilities of overall survival (OS), transformation-free survival (TFS: survival without disease transformation to the accelerated phase or blast crisis) and failure-free survival (FFS: survival without IM failure as defined by the 2009 European Leukemia Net recommendations) were 93.8%, 97.8% and 76%. Elevated BCR-ABL/GUSIS correlated with inferior probabilities of optimal response (p<0.001), and lower rates of CCyR after 12 months of IM (p<0.001). Moreover, high BCR-ABL/GUSIS transcripts were associated with lower probabilities of FFS (p<0.001) and TFS (p=0.01). When we employed the 2009 European Leukemia Net criteria to subdivide our patient cohort in optimal responders, suboptimal responders and individuals failing IM, we found that increasingly elevated BCR-ABL/GUSIS transcripts accurately distinguished the three patient groups (optimal vs suboptimal p<0.001; optimal vs resistant p<0.001; suboptimal vs resistant p<0.001). Furthermore, using receiver operating characteristic curves we found that progressively higher BCR-ABL/GUSIS levels at diagnosis defined quantitative transcript thresholds (15.96% for Optimal Response, 16.01% for EFS, 16.09% for FFS, 20.36% for TFS and 22.04% for OS) that separated low risk from high risk patients. Finally, we wanted to determine the concordance rates between BCR-ABL/GUSIS levels at diagnosis and early molecular responses (eMRs) at 3 and 6 months. We therefore employed the 15.96% BCR-ABL/GUSIS threshold to identify subjects with high (<15.96%) or low (>15.96%) probabilities of obtaining an Optimal Response and found that 69% of patients displaying <15.96% BCR-ABL/GUSIS achieved <10% BCR-ABL/ABLIS levels after 3 months of IM (p<0.001). Likewise, 78% of subjects presenting <15.96% BCR-ABL/GUSIS at diagnosis attained <1% BCR-ABL/ABLIS after 6 months of therapy (p<0.001).We conclude that high BCR-ABL transcripts at diagnosis measured by RQ-PCR employing GUS as a reference gene allow the identification of CML patients unlikely to benefit from IM that should receive alternative forms of treatment. Disclosures:Muller:Novartis, BMS, ARIAD: Consultancy; Novartis, BMS: Research Funding; Novartis, BMS, ARIAD: Honoraria.

Congestive Heart Failure (CHF) During Induction In ACUTE Promyelocytic Leukemia (APL) Patients

BackgroundAPL is a highly curable malignancy with cure rates of greater than 90% in most co-operative group trials. Population-based studies show that the survival is only about 65-70% with up to 30% early deaths. The most common reasons of early deaths are bleeding, differentiation syndrome (DS) and infection. Differentiation syndrome is very peculiar to this disease and potentially fatal unless recognized early. The wide variety of clinical presentations associated with DS might lead to delay in diagnosis in some patients which may lead to poorer outcomes. European Leukemia Net recommendations suggest that congestive heart failure (CHF) is one of the presenting features of DS and most of the reports on cardiac abnormalities focus on pericardial effusion. Cardiac stunning is only briefly reported in the literature. Cardiac stunning might be a result of cytokine storm attributable to tumor lysis in addition to being part of the DS. Here we report the incidence of CHF in patients undergoing induction for APL. MethodsWe performed a retrospective chart review on patients diagnosed with APL who received induction between December 1, 2004 and July 31, 2013 at Georgia Regents University and also patients who were referred to us from surrounding treatment centers with whom we co-manage these patients. Baseline and follow up ejection fractions (EF) were recorded by echocardiogram or nuclear medicine scan. We evaluated patients who had a drop in EF during the induction period. Results41 consecutive patients with APL with normal ejection fraction at diagnosis were evaluated. 1 patient refused treatment and was excluded. 38/40 patients received idarubicin and ATRA remission induction and 2 patients received Arsenic and ATRA. There were seven deaths during induction phase of treatment In the surviving patients, 10 patients had a repeat ECHO during the first 30 days of induction phase for suspected cardiomyopathy. 5 patients (15.1% of surviving patients) demonstrated a decrease in EF and all five were in the anthracycline group. The age range of patients with drop in EF was 30-75 years. Absolute drop in EF was between 10- 35%. Only one patient had mild elevation in troponins while others had no elevation. 3 out of the 5 patients had significant DS. Of the surviving patients, 4 out of 5 patients recovered their EF completely with one patient recovering partially to 45-50% (from 20-25%). ConclusionsAnthracyclines, along with ATRA, are still the mainstay of treatment for this curable malignancy. Although the incidence of cardiac abnormalities is described with repeated courses of anthracyclines, a single dose of anthracyclines can also play a role in cardiac injury. The highly inflammatory state present during the early treatment of APL might also play a role in cardiac injury resulting in higher number of patients with decreased EFs. DS clinical presentation most commonly involves dyspnea and edema, which are also symptoms of heart failure. Prompt cardiac evaluation should be undertaken to rule out congestive heart failure as an early start to therapy will lead to improved outcomes. Disclosures:Awan:Lymphoma Research Foundation: Research Funding; Spectrum Pharmaceuticals Inc.: Speakers Bureau. Jillella:Lymphoma Leukemia society: Research Funding. Kota:Teva: Speakers Bureau; Ariad: Advisory board, Advisory board Other.

High Dose Imatinib Is Effective In Children and Adolescents With Chronic Myelogenous Leukemia In Chronic Phase. The Italian Experience

Imatinib (IM) is an established first-line treatment for children with chronic myeloid leukemia (CML). However, the most effective dosage and duration of IM treatment are not well defined. This study was designed to evaluate the response to high-dose IM and long-term outcome in pediatric CML patients, previously untreated or resistant to IFN. Patients aged <18 years with a diagnosis of CML in chronic phase (CP) were treated with IM at a dosage of 340 mg/m2/day. Cytogenetic analysis was performed on bone marrow (BM) cells before and during IM therapy, at planned intervals; quantitative RT-PCR was assessed on peripheral blood (PB) monthly and on BM every 3 months, according to the European LeukemiaNet recommendations for minimal residual disease quantification. Major molecular response (MMR) is defined as <0.1% BCR-ABLIS, while complete MR (CMR) is considered as <0.01% BCR-ABLIS.From March 2001 to February 2013, 45 CML patients in CP (18 females, 27 males; median age: 119/12 years) were recorded from 9 Italian pediatric centers. Eight patients had previously received IFN. IM was started in all patients, including 16 with an HLA-matched sibling. The dosage was modulated according to hematologic toxicity and/or appearance of WHO >2 side- effects, mostly during the first 6 months of treatment (median administered dose: 309 mg/m2/day). Hematologic toxicity (medullary hypoplasia[n=1], neutropenia [n=11] and/or thrombocytopenia [n=6], anemia [n=1]) was observed in14/44 evaluable patients (32%); 13 patients (29.5%) experienced isolated or combined side effects: arthralgia/myalgia (n=10), nausea (n=1), vomiting (n=1), diarrhea (n=1), hepatitis (n=1), edema (n=1). After 3 months of IM treatment, 7/25 of tested patients (28%) obtained complete cytogenetic response (CCyR). Overall, 34/36 evaluable patients (94%) obtained a CCyR at a median time of 6 months.A molecular response (<0.1% BCR-ABLIS) was achieved in 21/26 tested patients (81%) on PB and in 30/33 evaluable patients (91%) on BM. Seventeen of 26 patients (65%), including 2 with a HLA-matched sibling, obtained a CMR on PB cells and 19/33 (55%) on BM cells at a median time of 15 and 19 months, respectively. With the aim of reducing the risk of longitudinal growth impairment or to improve treatment compliance, 9 patients with sustained CMR and 2 adolescents with MMR lasting >12 months received IM at the same daily dosage for 3 weeks a month (intermittent therapy). IM given without interruption was resumed in 3 of these 9 patients because of an increased BCR-ABL transcript. The growth rate showed a delay in height, which recovered over time, in 6 children who received IM prior to puberty.Overall, IM was stopped in 22/44 evaluable patients (50%) because of various reasons: stem cell transplant (SCT) in 8 patients (3 in CP, 1 in CCyR, 3 in MMR, 1 in CMR); hematologic (n=2) or extra-hematologic toxicity (n=2) (WHO grading >3) during the first 6 months in 4; recurrent disease in 6 (3 increased BCR-ABL transcript, 2 cytogenetic relapse,1 blast crisis [BC]); no response in 1; CMR ( <0.0032% BCR-ABL IS) lasting >88 months in 2 and pancreatitis in 1 patient in CMR for 75 months.Twelve patients underwent a SCT after a median time of 8 months: 8 from an identical siblings (5 responders to IM [1 CMR, 3 MMR, 1 CCyR]), 3 MUD (2 in CCyR and 1 in MMR) and 1 cord blood in CCyR after chemotherapy for CB. Three patients, transplanted from an identical sibling, had disease recurrence after 24 (molecular relapse), 36 (cytogenetic relapse) and 83 (BC) months, respectively.At the last follow-up, all patients are alive (CMR= 25; MMR=14; CCyR=3; minor CyR=2; too early=1) at a median of 52 months (range: 3-146). Of 42 patients evaluable for treatment, 23 are receiving IM at a dosage of 340 mg/m2 (4 intermittent IM); 11 are in CMR without any treatment (8 after SCT; 3 at 32, 33 and 50 months after IM discontinuation); 4 are in treatment with dasatinib, 3 with nilotinib and 1 with IFN.In our experience, higher dose IM is an effective treatment for childhood CP CML, associated with sustained responses. Moreover, IM can be safely discontinued in pediatric CML patients with a deep CMR lasting more than 7 years. For patients candidates to a SCT, IM provides a safe bridging option to transplant, at no increased risk. Since patients may lose their response, a close and regular monitoring should be performed, mostly for those who stop IM, as SCT and new TK inhibitors may be successfully employed in patients failing IM. Disclosures:Saglio:Celgene: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Can Children and Adolescents With Chronic Myelogenous Leukemia Be Cured Without Stem Cell Transplant? A Single Center Experience

For many years stem cell transplant (SCT) has been considered the only curative treatment for adults and children with chronic myeloid leukemia (CML). The availability of therapies, potentially capable of eradicating the Ph+ clone, has changed the treatment approach to CML patients. In this study, we report the long-term outcome of CML patients aged <20 years treated with interferon alpha (IFN) and/or imatinib (IM), and of those submitted to SCT.From 1990 to 2011, 35 consecutive CML patients in chronic phase (CP) (20 males, 15 females; median age1210/12 years) were treated according to the local guidelines. Before 2002, CML patients with a HLA-matched sibling underwent an allogeneic SCT, while the remaining patients were systematically treated with IFN. Since 2002, IM at a dose of 340 mg/m2/day was used in all newly diagnosed patients in CP, including those with a HLA-matched sibling. Cytogenetic analysis and qualitative RT-PCR were performed on bone marrow (BM) cells before and during IM therapy, at planned intervals, while FISH was carried out at complete cytogenetic response (CCyR). Since 2000, quantitative RT-PCR was assessed on peripheral blood (PB) monthly and on BM every 3 months, according to the European LeukemiaNet recommendations for minimal residual disease quantification. Major molecular response (MMR) is defined as<0.1% BCR-ABLIS, while complete MR (CMR) is considered as <0.01% BCR-ABLIS. The terms of MR4, MR4.5 and MR5 are used to indicate levels of disease <0.01% BCR-ABLIS,<0.0032% BCR-ABLIS,<0.001% BCR-ABLIS, respectively.From 1990 to 2001, 23/27 patients received IFN at a median dose of 6 ML IU/day (range: 2.5–9) and 4/27 patients with a HLA-matched sibling underwent a SCT. Twelve of 23 patients treated with IFN (52%) obtained a cytogenetic response (6 complete) and 5 of them (22%) achieved a molecular response (4 CMR and 1 MMR) after a median of 76 months (range: 43-122). IFN was stopped in 20/23 patients (87%) because of: no response (n=7), recurrent disease (cytogenetic relapse [n=1], blast crisis [BC] [n= 5], accelerated phase [AP] [n=1]), SCT (n=1 in CP), intolerance (n=3, with molecular response), autoimmune disease (n=1), lack of compliance (n=1). The remaining 3 patients continued IFN, that was discontinued in 2 of them in CMR5lasting >99 and 120 months, respectively.Since 2002, IM was started in 8 newly diagnosed patients in CP, including 3 with a HLA-matched sibling, and in 7 patients previously treated with IFN (1 in MR4.5, 1 in MMR, 5 in CP). Twelve of 13 evaluable patients (92%) obtained a CCyR after a median of 6 months (range 3-12). Of 12 patients evaluable for molecular response, 8 achieved a CMR after a median of 13 months and 3 a MMR; 1 patient had a MR4 on PB and a MMR on BM. Two patients stopped IM because of extra-hematologic toxicity after 1 and 2 months, respectively; 2 patients had a disease recurrence (cytogenetic relapse [n=1] and BC [n=1]). IM was discontinued in 4/8 patients (including 1 with a HLA identical sibling) in sustained CMR (MR4.5-MR5) lasting >75, 88, 89 and 123 months, respectively.Overall, 15 patients underwent a SCT after a median of 17 months: 5 from an identical sibling (4 in 1st and 1 in 2nd CP), 6 from a MUD (3 in CP, 2 in AP and 1 in CCyR), 2 from UCB (1 in aplasia and 1 in 2ndCCyR ), 1from a haploidentical-related donor and 1 autologous. Two patients (1 MUD and 1 UCB) died of SCT-related toxicity and 4 patients had disease recurrence (BC [n=3] and AP [n=1]).At the last follow-up, 5/15 transplanted patients have died (3 of disease progression) after a median of 29 months (range 19-216), 2 are lost to follow-up and 8 patients are alive in CMR (4 in MR4.5-5) at a median of 205 months (range: 48-236). Of 20 patients treated with IFN and/or IM, 5 have died of disease progression after a median of 41 months (range 27-72), 1 is lost to follow-up, and 14 patients are alive at a median of 161 months (range: 22-246): 2 are in treatment with IFN, 6 with IM and 6 are in CMR (7 in MR4.5-5) without any treatment at 31, 31, 37, 38, 50 and 60 months from IFN (n=2) + IM (n=2) or IM (n=2) discontinuation.In our experience, IM + IFN has proven to be an effective therapy in CML children and adolescents in CP, offering a valid alternative to SCT. These drugs provide sustained responses and can be safely discontinued in CML patients with a deep sustained CMR (MR4.5-MR5). Larger cohorts of pediatric patients are necessary to confirm these promising results which raise the possibility that children with CML might be cured without SCT. Disclosures:Saglio:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

Ultra Deep Sequencing (UDS) Allows More Sensitive Detection Of Tyrosine Kinase Inhibitor (TKI)-Resistant BCR-ABL Mutations That Would Influence Therapeutic Decision At The Time Of Switchover To Second- Or Third-Line Therapy

Ultra Deep Sequencing (UDS) Allows More Sensitive Detection Of Tyrosine Kinase Inhibitor (TKI)-Resistant BCR-ABL Mutations That Would Influence Therapeutic Decision At The Time Of Switchover To Second- Or Third-Line Therapy

Early Responses At 3 Months and 12 Months After Starting Imatinib As Predictive Factors For The Achievement Of Deep MR In Japanese CML Patients

Background and PurposeImatinib therapy shows excellent efficacy for chronic myeloid leukemia (CML) patients. The five-year overall survival of chronic-phase CML patients treated with imatinib can be expected to reach over 90%. Now, the goal of therapy has become the achievement of therapy-free remission (TFR) for most patients. However, predictive markers for achieving deep molecular response (MR) or TFR are yet to be elucidated. The recently published European LeukemiaNet recommendations 2013, which are mostly based on Caucasian studies, show the importance of an early response at 3 months or 12 months after starting imatinib treatment to assess optimal response. Using the registry of our study group, we assessed whether early cytogenetic or molecular responses at 3 months and 12 months after starting imatinib are associated with the achievement of deep MR or long-term outcome in Japanese CML patients. Patients and MethodWe reviewed 135 CML patients in the registry of our study group. Imatinib was started between December 2001 and June 2008. We retrospectively analyzed 92 CML patients (35 patients with prior therapy before imatinib) who could be assessed for partial cytogenetic response (PCyR: Ph<35% or bcr/abl transcript <10%) at 3 months after starting imatinib treatment, and 81 patients (25 patients with prior therapy before imatinib) who could be assessed for major molecular response (MMR: bcr/abl transcript <10% or 300 copies/μg) at 12 months in our multicenter observation study group. The clinical data was reviewed in August 2010. We excluded patients who had been switched from imatinib to a second tyrosine kinase inhibitor (TKI) before August 2010. We compared overall survival and the cumulative achievement of deep MR (negative bcr/abl transcript by PCR or the TMA-HPA method) between patients with and without PCyR at 3 months, also between patients with and without MMR at 12 months. The probability of overall survival and the cumulative incidence of deep MR were calculated by the Kaplan-Meier method and compared by the log-rank statistic. ResultsSeventy-six out of 92 patients (82.6%) achieved PCyR at 3 months. Forty out of 81 patients (49.3%) achieved MMR at 12 months. The patients with PCyR at 3 months showed significantly better overall survival (p=0.004) and higher cumulative achievement of deep MR (p=0.009) than the patients without PCyR. Overall survival between the patients with and without MMR at 12 months did not show a significant difference (p=0.06). However, the patients with MMR at 12 months showed significantly higher cumulative achievement of deep MR (p<0.001) than the patients without MMR. ConclusionEarly cytogenetic and molecular responses at 3 months and 12 months after starting imatinib are also predictive factors for good prognosis and the achievement of deep MR in a registry of Japanese patients. Our data confirm the criteria of optimal response in European LeukemiaNet recommendations 2013 was appropriated for Japanese CML patients in practical setting. Disclosures:No relevant conflicts of interest to declare.

Immunophenotyping of selected hematologic disorders – focus on lymphoproliferative disorders with more than one malignant cell population

Currently, clinical laboratories face increasing demand for flow cytometry testing combined with limited funding. Therefore, many laboratories search for panels that would provide sufficient immunophenotyping information and meet economical requirements. At the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada, we apply two 10-color tubes of surface markers for diagnosis of lymphoproliferative disorders (LPDs). These tubes contain most of the mandatory B- and T-cell markers according to European Leukemia Net (www.leukemia-net.org) recommendations. The B-cell-oriented panel includes the following antibodies: Kappa-FITC/lambda-PE/CD19-ECD/CD38-PC5.5/CD20-PC7/CD34-APC/CD23 APC-AF700/CD10 APC-AF750/CD5-PB/CD45-KO. A different combination is applied to detect cytoplasmic Ig light chain expression and aberrant immunophenotype of plasma cells. The T-cell panel allows enumeration of various T- and NK-cell subsets: CD57-FITC/CD11c-PE/CD8-ECD/CD3-PC5.5/CD2-PC7/CD56-APC/CD7-APC-AF700/CD4-APC-AF750/CD5-PB/CD45-KO. The reported overall incidence of B-cell chronic LPDs presenting with more than one aberrant population is approximately 5%. Multicolor analysis facilitates the detection of multiple aberrant populations in the same sample because expression of multiple antigens can be studied simultaneously in each defined population. Examples of LPDs with multiple aberrant populations are presented.

Prognostic Impact of the Number of Methylated Genes in Myelodysplastic Syndromes and Acute Myeloid Leukemias Treated with 5-Azacytidine

AbstractAbstract 1717 Background:Aberrant DNA methylation of tumor suppressor genes is a common event in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). Therefore hypomethylating agents like azacytidine (AZA) and decitabine seem to be a good therapeutic approach for the treatment of these diseases. Clinical experience and recent published data have demonstrated that AZA is effective for the treatment of MDS and AML patients. However, the prognostic impact of the aberrant hypermethylation on response and outcome to AZA treatment remains to be determined. For this reason the influence of the methylation status of a selected set of tumor suppressor genes on the overall survival and clinical response in MDS and AML patients, prior to treatment with AZA was studied. Patients and Methods:A total of 78 patients with MDS or AML who had been treated with AZA were evaluated. Among these, 25 were excluded because they had received less than 4 cycles, AZA was used after allogeneic stem cell transplantation, response was not assessable, or there was not enough quality DNA available. So finally, the study was focused in 53 patients: 36 MDS and 17 AML. Most of the AML included in the study had low blast count (20–30%). Responses were assessed according to the IWG MDS criteria in accordance to Fenaux et.al, and IWG AML criteria following European LeukemiaNet recommendations. DNA methylation status was analyzed using the Methylation Specific Multiplex Ligation Probe Amplification (MS-MLPA), with a panel of 24 different tumor suppressor genes related to cell cycle control, apoptosis regulation, DNA repair, cell adhesion and cell growth. Results:In the study cohort 47% of patients had cytogenetic alterations prior to AZA treatment, 4 with isolated -5/del(5q), 7 with isolated −7/del(7q), 4 with trisomy 8, 4 with not isolated -5/del(5q), 1 with trisomy 14, and 5 with complex cytogenetics. Methylation analysis showed that most patients (74%) had at least one methylated gene, but only 10% of patients displayed more than 3 methylated genes. The most frequently methylated genes were IGSF4 (28.3%), CDKN2B (24.5%), ESR1 (22.6%), CDH13 (17%) and CDKN1B (11.3%).The presence of a high number (≥2) of methylated genes (p=0.02), an adverse cytogenetics (p=0.03) or anemia (p=0.04) were independent prognostic factors associated with shorter overall survival. Moreover, the analysis of those patients displaying “no methylation”, patients with 1 methylated gene, patients with 2 methylated genes and those with more than 3 methylated genes, showed that as the number of methylated genes increases, the survival was shorter. The patients displaying the highest level of methylation (more than 3 genes), had a very short survival (median OS of 9.3 months, p<0.001).Forty-nine per cent of all patients responded to azacytidine. The statistical analysis revealed that the number of methylated genes did not correlate in general with the clinical response to AZA, although four of the five patients with more than 3 genes methylated did not respond. By contrast, cytogenetics was the only factor that independently influenced the response to AZA. Thus 64.7% of patients with favorable cytogenetics responded to AZA, while only 28.6% of those with adverse cytogenetics responded (p=0.03). Interestingly, our study included 3 MDS patients with isolated chromosome 7 alterations, and all of them responded. Conclusion:In summary, these results suggest that the analysis of the methylation status before AZA treatment by a feasible methodology such as MS-MLPA could be used in a clinical setting in order to identify a group of patients with poor survival, in which other alternative therapeutic approaches might be considered. [Display omitted] [Display omitted] Disclosures:Ramos:Celgene, Novartis, Amgen, Pfizer, and Merck Sharp & Dohme: Honoraria. Hernández:Celgene: Research Funding.

EUTOS Score Identifies Cases with Poor Outcome in Patients with Early Chronic Phase Chronic Myeloid Leukemia, Though Not Predictive for Optimal Response to Imatinib

AbstractAbstract 3778 Introduction.The EUTOS score has recently been developed by the European Leukemia-Net (ELN) to predict the achievement of an 18-month complete cytogenetic response (CCyR) and progression-free survival in imatinib-treated early chronic phase (ECP) chronic myeloid leukemia (CML) patients. The score uses the percentage of basophils and spleen size to divide patients in 2 groups of low- and high-risk. Since its publication in 2011, however, there have been conflicting reports about the efficacy of EUTOS score. Moreover, scanty data are available on the power of this scoring system to foresee optimal response to imatinib, as defined by ELN recommendations. Aims and Methods.To test the power of EUTOS score in predicting achievement of optimal response to imatinib, as defined by ELN, time to imatinib failure (TTF) and progression-free survival (PFS), we evaluated 265 ECP CML patients treated with front-line standard dose imatinib (400 mg daily) at 5 major hematology centres in the north-eastern area of Italy. Partial cytogenetic response (PCyR) and CCyR were defined as 1–35% and 0% Ph+ metaphases, respectively; major molecular response (MMR) was defined as BCR-ABL <0.1%IS. TTF was measured from the start of imatinib to the date of any of the following events: progression to accelerated or blastic phase, death for any cause at any time, imatinib dose increase (≥ 600 mg/day) for primary or secondary hematologic or cytogenetic resistance. PFS was measured from the start of imatinib to the date of progression to accelerated or blastic phase or death for any cause at any time. Survival probabilities were estimated by the Kaplan-Meier method and compared by log rank test; differences among variables were evaluated by the Fisher’s exact test or by Student’s t-distribution. Results.A total of 265 consecutive patients with ECP CML were included in this study. The median age was 55 years (range 19–84), with 149 males and 116 females. The median follow-up was 61 months (range 6–136). The median time from diagnosis to imatinib therapy was 0.7 months (range 0 – 7.6). The distribution according to the EUTOS score was: 248 patients (93.6%) in the low risk group and 17 patients (6.4%) in the high risk group. The “optimal response” endpoints to imatinib (i.e. PCyR at 6th months, CCyR at 12th months and MMR at 18th months) were higher in low-risk patients, but did not achieve statistical significance. Specifically, the values were as following: PCyR 86% vs 67% (p=0.055), CCyR 80% vs 63% (p=0.117) and MMR 61% vs 36% (p=0.126). Cumulative incidence of CCyR was comparable in the two groups (88%% in low-risk and 80% in high risk), but time to CCyR was shorter in low-risk patients (6 months) compared to the one in high-risk patients (9 months) (p=0.048) [figure 1]. More importantly, EUTOS score was able to predict long term response to therapy. Indeed, 59% of patients in the high-risk group experienced imatinib failure, compared to 30% in the low-risk group (p=0.027). Moreover also TTF was significant shorter in the high-risk group [figure 2]. Fifty-three patients in the low-risk group (21%) were switched to 2nd-generation TKIs (29 dasatinib, 22 nilotinib, 1 bosutinib, 1 ponatinib), compared to six (35%) in the high-risk group (4 dasatinib, 2 nilotinib). Also PFS rate was significantly worse in patients with high EUTOS score, with 11/248 events (4%) in the low-risk group and 4/17 (23%) in the high-risk cases (p=0.01) [figure 3]. Conclusions.In our study group, the EUTOS score was predictive for long-term outcome of imatinib therapy, both in terms of treatment failure and of progression-free survival. Taking into consideration the ELN definitions of optimal response, there was a trend toward better cytogenetic and molecular response in low-risk patients; the lack of statistical significance could be due to the relatively small number of high-risk cases. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Retrospective Real-Life Analysis of Chronic Myeloid Leukemia Patients in Suboptimal Response to Imatinib

AbstractAbstract 4446Life expectancy of CML patients has greatly improved in tyrosine-kynase inhibitor (TKI) era, but still some questions remain about the management of suboptimal responders (SR) to imatinib standard dose. This group of patients appears to be heterogeneous, with significant differences in terms of event-free survival between cytogenetic SR and molecular SR (Alvarado et al, Cancer 2009)European Leukemia Net (ELN) recommendations did not clarify those differences and there isn't a clear agreement on SR: maintaining imatinib at standard or higher dose or switching to another TKI are all considered acceptable options (Baccarani et al, JCO 2009).We retrospectively analyzed 63 CML patients, diagnosed in chronic phase between 1988 and 2011, SR to imatinib 400 mg/d, treated according to the 3 different ELN options. Fifty-two patients received imatinib front line and 11 had been previously treated with an interferon based therapy. Sokal score, evaluable in 44 patients, was high in 7, intermediate in 24 and low in 12, respectively. Twenty-five patients were cytogenetic SR and 38 molecular SR. The median follow-up from diagnosis was 76 months (range 10–292).At suboptimal response detection 47 patients (74%) continued imatinib 400 mg/d (30 of them afterword switched to high dose imatinib or new TKI), 12 patients (19%) increased imatinib dose to 600 mg/d (7) or 800 mg/d (5) (8 of them later changed TKI) and only 4 patients switched immediately to new TKI.Twenty-three percent of the 47 patients who continued imatinib 400 mg/d obtained a stable complete cytogenetic response (CCyR) and major molecolar response (MMR) while 27% underwent to a failure. Globally thirty-tree SR patients increased imatinib dose, 36% at suboptimal response detection and 64% after a median of further 12 months of standard dose treatment (range 3–50): 48% of them obtained a durable CCyR and MMR. Twenty-six evaluable patients switched to new TKI (9 to nilotinib and 17 to dasatinib), 13 after high dose imatinib: 62% of the patients achieved a stable CCgR and MMR. Both high dose imatinib and new TKI were significantly more effective in achieving CCyR and MMR than maintaining imatinib 400 mg/d (p<0,05).Considering separately the subgroup of cytogenetic SR patients, a stable CcyR has been reached by 35% of patients continuing imatinib standard dose, by 50% of the patients who increased imatinib dose and by the totality of the patients treated with new TKI (option significantly superior to the other two, p<0,05).Among molecular SR, 26% of the patients obtained a stable MMR with imatinib 400 mg/d, 52% with imatinib 600 or 800 mg/d and 63% switching to new TKI. The difference was statistically significant between new TKI and imatinib 400 standard dose only (p<0,05).Cytogenetic SR maintained at imatinib 400 mg/d had a higher risk of event (defined as loss of hematologic or cytogenetic response or death) compared to molecular SR (40% vs 5%, p<0,01), although at the last follow-up, after a change in therapeutical strategy, no difference in response rate was detected between cytogenetic and molecular SR (68% vs 71%) in stable CCyR and MMR.Two patients progressed to accelerated phase (clonal evolution) but then obtained an optimal response after switching to new TKI; 2 patients died of unrelated disease. Among the 61 living patients, 71% was in MMR, 26% in CCyR only and 3% didn't reach CCyR (for these patients the efficacy of the therapeutic change is not evaluable yet).In our casistics cytogenetic SR had a higher risk of negative events than molecular SR, as reported in literature, although they obtained similar responses after changing therapeutic strategy. No clear advantage in maintaining imatinib 400 mg/d after suboptimal response was observed, since it led to a few optimal responses and was associated with a significant risk of treatment failure. Imatinib dose increment might represent a more reasonable option, at least for molecular SR.Considering the global casistic no significant difference in response rates were found between new TKI and high dose imatinib even if dasatinib and nilotinib showed a trend towards a superior efficacy in patients mostly unresponsive to the last option, suggesting that an earlier switch to new TKI might further increase the proportion of optimal responders. For cytogenetic SR the switch to new TKI brought better results than those obtained with high dose imatinib: therefore it seems to be the best choice in this subgroup of patients. Disclosures:No relevant conflicts of interest to declare.

Prognostic Impact of the Levels of Expression of Cell Surface Proteins Commonly Expressed by Blasts and Hematopoietic Precursor Cells in De Novo AML Patients: A Report From the Spanish Cetlam Study Group

AbstractAbstract 1452▪▪This icon denotes a clinically relevant abstract Introduction:Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods:Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results:Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion:In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures:No relevant conflicts of interest to declare.

Comparison of Sokal, Hasford and EUTOS Scores in Terms of Long-Term Treatment Outcome According to the Risks in Each Prognostic Model: A Single Center Data Analyzed in 255 Early Chronic Phase Chronic Myeloid Leukemia Patients Treated with Frontline Imatinib Mesylate.

AbstractAbstract 2794 BackgroundThe main difference of recently developed new prognostic classification, EUTOS score, from the conventionally used Sokal and Hasford scores is the simplicity of adopting only 2 clinical parameters, basophil counts and size of spleen, to discriminate high vs. low risks in chronic myeloid leukemia (CML). Concerns may rise from possible inter-observer gap when measuring the size of spleen. While there is currently controversial debate on its prognostic power, we present our single center results of comparing EUTOS score with other two scores. Patients and MethodsAmong 380 adult patients, consecutively registered as early chronic phase (CP) CML at Seoul St. Mary’s Hospital (Jan. 2004 – Apr. 2010), who received imatinib (IM) as frontline therapy, 255 were evaluable for analysis: subjects were strictly limited to those with documented clinical parameters measured at very early time of diagnosis by a single doctor, before any cytoreduction therapies. The median age was 42 years (range, 19–77) with a slight male predominance (57%). Cytogenetic and molecular responses were calculated at specific time points and cumulative rates of response were compared according to risk scores. Event-free survival (EFS) was analyzed by time from start of IM to the time of events, defined by death from any cause, primary or secondary resistance to IM, progression to advanced phases, intolerance to IM and switch to second line tyrosine kinase inhibitor (TKI) or allografting. Response assessment followed current European Leukemia Net recommendation. ResultsMedian time from diagnosis to IM start was 14 days (range, 1–183). After a median follow-up duration of 56.6 months (range, 13.0–102.2) in survivors, there were 10 deaths and 3 patients with disease progression to accelerated phase of blastic crisis. Sixty-four patients had switched IM to second TKI due to either IM resistance or intolerance.Overall comparisons are described in Table 1. Of 42 EUTOS high risk patients, only 25 high risk patients by Sokal and 15 by Hasford were included. Almost 90% of either low or intermediate (INT) risk patients by all risk models achieved complete cytogenetic response (CCyR) at 12 months after IM, whereas high risk groups had lower rates, especially in EUTOS high risk (57%, p = <0.0001). Regarding major molecular response (MMR) at 18 months, significant differences were observed according to risk stratifications by Sokal and EUTOS, but not in Hasford risks, in which the same proportion of low and INT risks achieved MMR (48%, P = 0.308). This seemingly less prominent prognostic difference of low vs. INT groups of Sokal and Hasford scores were further demonstrated by insignificant difference between these groups in cumulative incidence (CI) of CCyR and MMR. All 3 high risk groups showed significantly lower incidence of both CCyR and MMR compared to low risk groups. INT risk groups by Sokal and Hasford models failed to show statistical significance in all three outcomes: CI of CCyR and MMR, and 5-year EFS. Of note, high risk stratified by EUTOS score demonstrated the least favorable outcomes with strongest p-values (Table 1). ConclusionBy simply comparing the p-values, our data may implicate that high risk group discriminated by the EUTOS score is predicted with more unfavorable outcomes in achieving CCyR, MMR and survival after IM frontline therapy. Furthermore, the lack of statistically significant difference between outcomes of low and INT risk groups by conventional risk models may challenge the need for future classification of INT from low risk in this era of TKI.Table 1Comparison of Sokal, Hasford and EUTOS scoresSokalHasfordEUTOSn (%)Low 118 (46)INT 92 (36)High 45 (18)Low 135 (53)INT 100 (39)High 20(8)Low 213(84)High 42(16)CCyR rate at 12 months (%, n=245)9084698883638957P0.0080.020<0.0001MMR rate at 18 months (%, n=250)5249274848305124P0.0120.3080.002Median months to CCyR5.95.96.15.95.96.35.97.3HR (95% CI) for CI of CCyR10.8 (0.6–1.1)0.6 (0.4–0.9)10.9 (0.7–1.2)0.5 (0.3–0.8)10.4 (0.3–0.7)P0.1470.0040.7820.0180.0002Median months to MMR14.514.017.215.213.322.514.018.8HR (95% CI) for CI of MMR10.9 (0.7–1.3)0.5 (0.3–0.9)10.9 (0.6–1.2)0.5 (0.2–1.0)10.4 (0.2–0.7)P0.8980.0200.5430.0610.0015–year EFS (%¡¾SD)76.7 ± 4.169.0 ± 5.150.8 ± 7.975.5¡¾3.967.1 ± 4.840.7 ±73.9¡¾3.247.0 ± 8.2HR (95% CI)11.4 (0.8–2.5)2.5 (1.4–4.4)11.6 (0.9–2.6)2.8 (1.4–5.6)12.4 (1.50–4.04)P0.1610.0020.0600.003<0.0001 Disclosures:No relevant conflicts of interest to declare.

High BCR-ABL Levels At Diagnosis Are Associated with Unfavorable Responses to Imatinib Mesylate.

AbstractAbstract 2790The approval of second-generation tyrosine kinase inhibitors (TKIs) for the first line treatment of Chronic Myeloid Leukemia (CML) has generated a need for early molecular parameters associated with inadequate responses to Imatinib Mesylate (IM).Recent evidence suggests that CML patients presenting BCR-ABL/ABLIS levels >10% after 3 months of IM or >1% after 6 months of treatment have inferior outcomes in terms of both overall survival (OS) and progression-free survival. We wanted to establish if high BCR-ABL transcripts at diagnosis would also be associated with unfavorable responses to IM. To this end, we correlated quantitative determinations of BCR-ABL measured at diagnosis with the outcome of 230 newly diagnosed CML patients receiving IM 400 mg/die. BCR-ABL transcripts were measured from peripheral blood samples drawn at diagnosis using Real-Time Quantitative PCR (RQ-PCR). All molecular determinations were performed twice (in triplicates) on the same sample using either ABL or glucuronidase-beta (GUS) as reference genes.Median follow-up of the study population was 42 months. Estimated 5-year cumulative incidences of complete hematologic response, complete cytogenetic response (CCyR) and major molecular response were 97.9%, 89.5% and 64.7%. Five-year probabilities of OS, transformation-free survival (TFS: survival without disease transformation to the accelerated phase or blast crisis) and failure-free survival (FFS: survival without IM failure as defined by the 2009 European Leukemia Net recommendations) were 93.8%, 97.8% and 76%. Correlations between high BCR-ABL transcripts at diagnosis and unsatisfactory IM responses were much stronger using GUS in place of ABL as a reference gene. Indeed, while elevated BCR-ABL/GUSIS (p<0.0001) and elevated BCR-ABL/ABLIS (p<0.0001) both correlated with inferior probabilities of optimal response, high BCR-ABL/GUSIS (p<0.0001) but not high BCR-ABL/ABLIS (p=0.18) was associated with lower rates of CCyR after 12 months of IM. Neither BCR-ABL/GUSIS nor BCR-ABL/ABLIS at diagnosis were predictive of OS. However, high BCR-ABL/GUSIS was more accurately associated with lower probabilities of FFS (p<0.0001) and TFS (p=0.01) as compared to BCR-ABL/ABLIS (p=0.02 for FFS; p=0.36 for TFS). When we employed the 2009 European Leukemia Net criteria to subdivide our patient cohort in optimal responders, suboptimal responders and individuals failing IM, we found that elevated BCR-ABL/GUSIS (p<0.0001) was superior to elevated BCR-ABL/ABLIS (p<0.004) in distinguishing patient outcome. Furthermore, while BCR-ABL/ABLIS at diagnosis only discriminated optimal from resistant subjects (p=0.005), BCR-ABL/GUSIS transcripts were significantly different between the three patient groups (optimal vs suboptimal p=0.0002; optimal vs resistant p<0.0001; suboptimal vs resistant p<0.0001). Using receiver operating characteristic curves and the achievement of an optimal response as a specific endpoint, we found that 16.46% BCR-ABL/GUSISat diagnosis defined a threshold distinguishing low risk from high risk patients.High BCR-ABL transcripts at diagnosis measured by RQ-PCR employing GUS as a reference gene allow the identification of CML patients unlikely to benefit from IM that should receive alternative forms of treatment. Disclosures:Turri:Novartis: Consultancy, Novartis Other; Bristol Myers Squibb: Bristol Myers Squibb, Bristol Myers Squibb Other, Consultancy. Morabito:Celgene: Honoraria.

Prognostic Impact of Mutant to Wild-Type Ratio and Insertion Site in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication

AbstractAbstract ▪785▪This icon denotes a clinically relevant abstract Background:FLT3 internal tandem duplications (FLT3-ITD) occur in about 25% of acute myeloid leukemia (AML), are associated with cooperating gene mutations (NPM1, DNMT3A), and confer an adverse prognosis. Several studies have indicated that the unfavorable impact of FLT3-ITD is influenced by a number of factors, such as the mutant to wild-type ratio (allelic ratio), insertion site of FLT3-ITD in the beta1 sheet of the tyrosine kinase domain 1, and the molecular background of cooperating mutations. Aims:To evaluate the relative impact of FLT3-ITD allelic ratio and insertion site, as well as cooperating genetic lesions on prognosis and treatment decision making in a large cohort of homogeneously treated younger adult patients. Methods:The basis of the study were 2377 younger adults (median age, 48 years; range, 16–62 years) with newly diagnosed AML enrolled on three prospective treatment trials of the German-Austrian AML Study Group (AMLSG) between 1993 and 2008. Patients with acute promyelocytic leukemia (n=99), core-binding factor AML (n=279) and AML with adverse-risk cytogenetics (n=436) according to the European LeukemiaNet recommendations were excluded. Based on material availability, the presence of FLT3-ITD could be analyzed in 1414 patients; NPM1 and DNMT3A mutational status was available in 97% and 84% of the patients, respectively. In FLT3-ITD positive AML (n=394), the allelic ratio, determined by Genescan-based fragment-length analysis, was available in 86% and the insertion site in 72%. Allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission was performed in 41% and 29% of FLT3-ITD positive and negative patients, respectively. Results:We first evaluated the prognostic impact of the different FLT3-ITD characteristics within the subgroup of FLT3-ITD positive patients. The allelic ratio was categorized into quartiles ranging from low to high. For the endpoints event-free (EFS), relapse-free (RFS) and overall survival (OS), only the fourth quartile with the highest allelic ratio showed a prognostic impact for all endpoints, whereas no difference was identified between the other three quartiles. For further analyses, the allelic ratio was dichotomized comparing the fourth quartile versus the other three quartiles. FLT3-ITD insertion site in the beta1 sheet was significantly associated with an unfavorable outcome for all endpoints. Additionally, FLT3-ITD size was directly correlated with the insertion site: the more C-terminal the ITD inserted in the FLT3 gene the longer the FLT3-ITD size. There was no prognostic impact of FLT3-ITD size neither as continuous nor as quartile-categorized variable. Multiple FLT3-ITDs, present in 13% of AMLs, were associated with an unfavorable prognosis. The presence of either NPM1 and/or DNMT3A mutations in FLT3-ITD positive patients did not alter the original FLT3 prognosis. In multivariable models for the endpoint OS of the total cohort of intermediate-risk AML, an independent prognostic impact beyond the variable FLT3-ITD was shown for the allelic ratio (fourth quartile) [HR, 1.4; p=0.037] and in trend for insertion site in the beta1 sheet [HR, 1.33; p=0.06]. Survival of patients exhibiting a high allelic ratio (n=43) or insertion site in the beta1 sheet (n=60) was comparable, with a median of 10 and 13 months and 4-year survival of 19% and 24%, respectively. Of note, outcome of patients with both high allelic ratio and insertion site in the beta1 sheet (n=21) was very poor with a median OS of 10 months and 4-year OS of 5%, respectively. In patients with FLT3-ITD positive AML without these unfavorable factors (n=144), median and 4-year OS were 15 months and 42%, respectively. Of note, a clear benefit of allogeneic HSCT in first CR was only seen in FLT3-ITD positive patients without these two unfavorable factors, with a 4-year OS of 63%. In comparison, the 4-year OS of the same subgroup of patients achieving a CR after induction therapy without proceeding to allogeneic HSCT during first CR was 35%. In contrast, outcome in patients with high allelic ratio and/or insertion site in the beta1 sheet remained poor despite allogeneic HSCT in first CR. Conclusion:High FLT3-ITD allelic ratio and ITD insertion site in the beta1 sheet presented as prognostic indicators for poor outcome in patients with the presence of a FLT3-ITD. Only patients without these unfavorable FLT3-ITD features significantly benefitted from allogeneic HSCT. Disclosures:Schlenk:Roche: Research Funding; Pfizer: Research Funding; Amgen: Research Funding.

Chronic Myelogenous Leukemia Patients with Highly Nilotinib-Resistant BCR-ABL Mutations Demonstrate Similar Efficacy to Dasatinib, but Have a Higher Likelihood of Developing New Mutations in the Event of Clinical Resistance

AbstractAbstract 3755 Objective:Assess the outcome of imatinib- and/or nilotinib-failure chronic myelogenous leukemia (CML) patients with pre-existing, highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C), during dasatinib therapy. Methods:Sixty-one CML patients who failed imatinib (n=44) or imatinib and nilotinib (n=17), including 20 in chronic phase (CP), 16 in accelerated phase (AP) and 25 in blast phase (BP), were switched to dasatinib, except for patients with highly dasatinib-resistant mutations (T315I/A, F317L/V/I/C and V299L). Patients were grouped into 3 cohorts: no mutation (n=22), no-specific mutation (any mutation except highly nilotinib-resistant mutations; n=15), or specific mutation (Y253H, E255K/V or F359V/C; n=24), according to baseline BCR-ABL mutation status. Cumulative incidence frequencies of hematological response (HR), complete hematological response (CHR), major cytogenetic response (MCR), complete cytogenetic response (CCR) and major molecular response (MMR) were compared. Probabilities of clinical resistance to dasatinib (according to European LeukemiaNet recommendations), progression-free survival (PFS) and overall survival (OS), and dynamics of BCR-ABL mutations during dasatinib therapy were also assessed. Results:Prior nilotinib was associated with a higher proportion of specific mutation versus no mutation (50.0% vs 13.6%, P=0.001) and non-specific mutation (50.0% vs 13.3%, P=0.02). Patients harboring specific mutation also had a higher frequency of advanced phase CML before dasatinib treatment than patients with no mutation (83.3% vs 50.0%, P=0.008), and a similar frequency to those with non-specific mutation (83.3% vs 66.7%, P=0.266). Response, PFS and OS rates, and follow-up duration were similar among the 3 cohorts by baseline mutation status, as shown in Table. Probability of clinical resistance to dasatinib in patients with specific mutation was not significantly different to those with no mutation (54.2% vs 77.3%, P=0.100) or non-specific mutation (54.2% vs 33.3%, P=0.204) despite that there was a significant difference between the cohort with no mutation and non-specific mutation (77.3% vs 33.3%, P=0.008). Among 32 patients who developed clinical resistance to dasatinib and were assessed for BCR-ABL mutation status, newly detectable mutations were identified in 14 patients and most frequently occurred in those already harboring specific mutation versus those with no mutation (76.9% vs 26.7%, P=0.008) and non-specific mutation (76.9% vs 0%, P=0.015) at baseline. T315I (9/14, 64.3%) was the most common newly acquired BCR-ABL mutation. Harboring specific mutation compared with other mutation states at baseline (76.9% vs 16.7%, P=0.001), and developing hematological resistance rather than cytogenetic resistance during dasatinib therapy (65.0% vs 8.1%, P=0.002), were identified as risk factors associated with the development of new mutations. Conclusions:Imatinib- and/or nilotinib-failure patients with highly nilotinib-resistant BCR-ABL mutations demonstrate similar efficacy to dasatinib as those with no or any other mutation. However, patients with highly nilotinib-resistant mutations had a higher likelihood of developing new mutations at the time of clinical resistance. More extensive studies are needed to assess the significance of various BCR-ABL mutations prior to and during tyrosine kinase inhibitor therapy for CML.Table:Response, survival and clinical resistance to dasatinib by baseline BCR-ABL mutation statusMutation statusP valueNo mutation n=22Non-specific mutation n=15Specific mutation n=24HR, n (%)16 (72.7)14 (93.3)21 (87.5)0.202CHR, n (%)16 (72.7)12 (80.0)19 (79.2)0.832MCR, n (%)a9 (69.2)8 (80.0)12 (70.6)0.826CCR, n (%)a8 (61.5)8 (80.0)10 (58.8)0.511MMR, n (%)a3 (23.1)5 (50.0)4 (23.5)0.2813-year PFS, %84.673.765.20.4693-year OS, %75.680.854.00.798Clinical resistance to dasatinib, n (%)17 (77.3)5 (33.3)13 (54.2)0.027Follow-up (months), median (range)31 (2..C48)24 (2..C48)7 (1..C48)0.277a40 patients were evaluated including 13 with no mutation,10 with non-specific mutation and 17 with specific mutation Disclosures:No relevant conflicts of interest to declare.

Additional chromosomal abnormalities in Philadelphia-positive clone: adverse prognostic influence on frontline imatinib therapy: a GIMEMA Working Party on CML analysis

AbstractAdditional chromosomal abnormalities (ACAs) in Philadelphia-positive cells have been reported in ∼ 5% of patients with newly diagnosed chronic myeloid leukemia (CML) in chronic phase (CP). Few studies addressing the prognostic significance of baseline ACAs in patients treated with imatinib have been published previously. The European LeukemiaNet recommendations suggest that the presence of ACAs at diagnosis is a “warning” for patients in early CP, but there is not much information about their outcome after therapy with tyrosine kinase inhibitors. To investigate the role of ACAs in early CP CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective trials of the Gruppo Italiano Malattie Ematologiche dell'Adulto Working Party on CML: 378 patients were evaluable and ACAs occurred in 21 patients (5.6%). The overall cytogenetic and molecular response rates were significantly lower and the time to response was significantly longer in patients with ACAs. The long-term outcome of patients with ACAs was inferior, but the differences were not significant. The prognostic significance of each specific cytogenetic abnormality was not assessable. Therefore, we confirm that ACAs constitute an adverse prognostic factor in CML patients treated with imatinib as frontline therapy. This study was registered with clinicaltrials.gov as NCT00514488 and NCT00510926.

Comprehensive therapeutic outcomes of frontline imatinib mesylate in newly diagnosed chronic phase chronic myeloid leukemia patients in Korea: feasibility assessment of current ELN recommendation

Optimal responses during imatinib therapy are commonly defined following the European LeukemiaNet (ELN) recommendations. Achievements of these optimal responses have not, however, been comprehensively tested as response-related prognostic factors using single center data sets. We evaluated the parameters using long-term (median 63months) outcomes from 363 chronic phase chronic myeloid leukemia patients treated with imatinib as frontline therapy at our center. Intention-to-treat analysis showed comparable rates of complete cytogenetic response (86%), major molecular response (MMR, 54%), and complete molecular response (MR(4.5), 8%). Estimated overall survival, progression-free survival, and event-free survival at 7years were 94, 88 and 84%, respectively. Achievement of recommended optimal response at 6months (major cytogenetic response) and 12months (complete cytogenetic response) yielded significantly better overall, progression-free, and event-free survival. However, achievement of recommended optimal response at 18months (MMR) provided marginal benefit only in event-free survival. Most ELN criteria were predictive of long-term outcomes, with the exception of the clinical significance of achieving MMR at 18months. Treatment adherence in the early treatment period was one of the important independent predictors of favorable long-term outcome. Durable cytogenetic and molecular responses were maintained in a majority of patients treated with optimal dose intensity.

Quantitative monitoring of WT1 expression in peripheral blood before and after allogeneic stem cell transplantation for acute myeloid leukemia – a useful tool for early detection of minimal residual disease

Overexpressed Wilms tumor gene 1 (WT1) has been found in a majority of patients with acute myeloid leukemia (AML). The aim of this study was to confirm the applicability of WT1 expression measurement as a marker of minimal residual disease (MRD). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood (PB) according to European Leukemia Net (ELN) recommendations. The WT1 expression was related to the expression of a reference gene Abelson (ABL) and the results were calculated as a number of WT1 copies related to 104 copies of ABL gene. The upper normal limit of WT1 expression was set at 50 copies of WT1 to 104 copies of ABL. Morphological, flow cytometry and chimerism examinations were evaluated according to standard protocols.A total of 51 AML patients with overexpressed WT1 gene were analyzed. The median follow-up after transplantation was 14 (2-72) months. WT1 expression levels exceeding the upper normal limit were considered as a sign of impending hematological relapse, in accord with morphological, flow cytometry and chimerism data, as well as with the expression of the specific fusion genes. Moreover, in 7 patients the rise of WT1 expression preceded all other standard methods. Patients with high WT1 expression before allogeneic hematopoietic stem cell transplantation (allo-HSCT) had significantly worse outcome than patients with low WT1 level. Examination of WT1 expression in PB of patients with AML is a useful tool for MRD monitoring. Moreover, the WT1 gene expression before stem cell transplantation seems to be of prognostic significance.