ALBERT

Description: Abstract 2765 In CML, achievement of major molecular response (MMR) is a significant prognostic factor as it has been shown to be associated with longer duration of complete cytogenetic response (CCyR) and long-term progression-free survival. In IRIS study, patients who achieved both CCyR and MMR showed higher progression-free survival rates, compared to those who had CCyR without MMR. Higher doses of imatinib are expected to yield higher CCyR and MMR rates, compared to standard dose of imatinib, and second-generation tyrosine kinase inhibitor, nilotinib also produces high CCyR and MMR rates in patients with CP CML who are resistant to imatinib. In this prospective study, the efficacy of nilotinib and high-dose imatinib was investigated in suboptimal molecular responders who received standard-dose imatinib as first-line therapy. Early CP CML patients who have achieved CCyR but no MMR after at least 18 months and up to 24 months (≥ 18 to ≤ 24 months) on first-line imatinib therapy at a daily dose of 400 mg were enrolled in this clinical trial, and informed consents were obtained from all patients prior to participation. In nilotinib arm, patients received oral dose of 400 mg BID (800 mg/day), and patients received 800 mg/day administrated as 400 mg BID in imatinib dose-escalation arm. To assess the drug efficacy, cytogenetics and RQ-PCR analysis were performed at regular intervals, and baseline mutational analysis was conducted for every patient with subsequent mutational analyses performed in patients demonstrating either lack of response or disease progression. Primary endpoint is to evaluate the cumulative MMR rates by 12 months, and secondary endpoints are to evaluate the cumulative CMR rates and time to and duration of MMR and CMR during further 24 month follow-up. Progression-free survival and safety profiles will also be assessed as secondary endpoints. Patients showing lack of response (lack of complete hematologic response (CHR) at 6 months, increasing WBC, no major cytogenetic response (MCyR) at 24 months), loss of response (loss of CHR or MCyR) or severe intolerance to treatment were allowed to crossover to the alternative treatment arm. With a data cut-off date of 18 Jul 2011, a total of 30 patients were randomized into nilotinib arm (n =13) or imatinib arm (n = 17), and 6 patients have crossed-over to nilotinib arm due to lack of response. With a median follow-up of 11 months (range, 0.2–28 mos), all patients have maintained CCyR without progression to advanced disease, and progressive decrease in BCR-ABL transcript levels was observed in all patients. Cumulative MMR rates at 20 months were significantly higher in nilotinib arm compared to imatinib dose-escalation arm (59.00% vs. 27.40%, P = 0.047), and patients treated with nilotinib also showed faster molecular response rates, with 5 patients achieving MMR within 3 months of nilotinib therapy. At the last follow-up, 7/13 (53.85%) and 2/11 (18.18%) patients achieved MMR in nilotinib arm and in high-dose imatinib arm, respectively, with 1 patient in nilotinib arm achieving 4-log reduction of BCR-ABL transcripts. Although toxicity was observed more frequently in imatinib dose-escalation arm, all patients currently maintain the initial dose (except 1 patient who interrupted imatinib therapy due to neurosurgical operation), and based on the toxicity data, no additional or serious adverse events were developed except for pre-existing toxicities before randomization. These preliminary results demonstrate that early intervention using nilotinib or dose escalation of imatinib could be recommended in suboptimal molecular responders, with nilotinib being more preferable. Through further clinical investigation on a large patient population and longer period of observation, efficacy and safety of early intervention of suboptimal molecular response using nilotinib or dose escalation of imatinib will be assessed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: Woodman: Novartis: Employment, Equity Ownership. Szczudlo:Novartis: Employment, Equity Ownership. Kim:Novartis: Employment.

Description: Most CML patients are sensitive to imatinib mesylate (IM), however, a small fraction develop resistance, mostly through the emergence onset of BCR-ABL mutation. Dasatinib and nilotinib, novel tyrosine kinase inhibitors (NTKIs), are active against most of IM resistant BCR-ABL kinase domain mutants except T315I. Although T315I mutation has been highly resistant to IM and both NTKIs, precise clinical characteristics and outcome have not been known yet. A total of 81 patients with various phases of CML who were intolerant or were resistant to IM (M:F-52:29, median age: 43 years, range; 12–74 years) were enrolled in this study between May 2005 and Sep 2006. Eighty one patients had received dasatinib and/or nilotinib in phase II or extended access program. At the time of IM failure and every 3 months during NTKIs treatment, mutations were screened by both ASO-PCR and direct sequencing. Clinical characteristics and outcome probabilities were statistically analyzed. T315I was detected in 20 of 31 patients (65%) harboring kinase domain mutations; 9 patients had mutation before NTKIs treatment and 11 patients were developing mutation after NTKIs treatment. Median age was 33 years (range, 19–74 years). Transcripts were Major BCR for all patients and 8 patients had received prior interferon therapy. 6 patients had additional chromosomal abnormalities (ACA) at diagnosis. At the time of T315I emergence, 8 patients were CP, 6 patients were AP, and 6 in BC (4 in myeloid and 2 in lymphoid). Median accumulate dose per day was 398.8 mg/day (range, 205.3–600.0 mg/day). 13 patients were received dasatinib, 5 received nilotinib and 2 received both dasatinib and nilotinib. The best response to NTKIs was complete cytogenetic response (CCyR) in 6 and complete hematologic response (CHR) in 8. Median overall survival (OS) from NTKIs start was 7.0 months for advanced phase and 12.3 months for chronic phase. The 3 year survival rate was 21.5%, with median value was 8.4 months in all patients. With NTKIs therapies [median follow-up, 9.7 months (range, 0.7–27.3 months)], 8 patients are alive (40%); 6 patients are alive with active disease and 2 patients are alive with ongoing response. 10 patients died of disease progression and 2 patients died of pneumonia. Low grade disease phase at discovery of T315I mutation (log-rank P=0.0237) demonstrated significantly favorable outcome but after NTKIs treatment, there was no difference in OS between disease phases (log-rank P=0.271). And there was no difference in OS between T315I mutation and other mutations (P=0.147). However the ACA at diagnosis (P=0.029) and achievement of best CCyR during NTKIs treatment (P=0.085) was different in OS. And there was significant difference in survival between patients with and without ACA and achievement of best CCyR during NTKIs treatment (log-rank P=0.0235), suggesting they have prognostic influences on survival as T315I mutation. In summary, our findings confirm that ACA at diagnosis and achievement of best CCyR during NTKIs treatment are statistically significant prognostic information with respect to survival probability at patients with T315I mutation. However T315I mutation was highly resistant to NTKIs as well as IM, and are associated with poor outcome. As diverse outcomes in patients with T315I mutation have been demonstrated, different strategies such as MK-0457 and/or novel T315I mutation inhibitor should be applied.

Description: Abstract 3443 BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD. We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available. Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable. Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Table 1. Patients Tx at mutation detection (mg) Compound type Compound % 1 Nilotinib400 G250E+T315I 6.7 G250E+D444G 33.3 T315I+D444G 6.7 2 Nilotinib400 M244V+T315I 95.0 3 Dasatinib100 Y253H+T315I 95.0 4 Dasatinib140 T315I+E459K 55.6 5 Dasatinib200 T315I+M351T 66.7 6 Dasatinib100 NCM Dasatinib80 NCM Dasatinib100 M244V+F359V 16.7 7 Bosutinib500 NCM 8 Dasatinib140 T315I+F359C 35.3 9 Imatinib400 E255K+T315I 5.6 10 Dasatinib80 E255V+T315I 90.0 11 Imatinib800 E255K+T315I 10.5 12 Nilotinib800 E255K+T315I 12.5 13 Dasatinib100 F311I+T315I 35.0 F311I+F317Lb 10.0 Imatinib400 F311I+T315I 10.0 F311I+F317La 15.0 F311I+F317Lb 55.0 14 Nilotinib800 Y253H+F359I 5.6 15 Bosutinib500 V299L+E459K 95.0 Nilotinib400 + Dasatinib100 V299L+F359I 5.0 V299L+E459K 55.0 V299L+F317La+E459K 15.0 V299L+F359I+E459K 15.0 V299L+F317La+F359I+E459K 5.0 16 Imatinib600 NCM 17 Imatinib400 NCM NCM: no compound mutation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1240 Use of reliable predictable factors in newly diagnosed chronic phase (CP) CML patients could ensure the most appropriate selection of therapy in individual patients, and the Sokal score which was developed in the pre-interferon era still retains prognostic values in patients treated with imatinib. In addition to the Sokal score, patients with a high BCR-ABL transcript level at diagnosis might respond differently to therapy. However, to our knowledge, there has been no distinct study to investigate the association between BCR-ABL transcript level at diagnosis and the response to therapy. It has been generally agreed that baseline BCR-ABL transcript levels are not relevant to predict the response to therapy, and the use of international scale (IS) is also based on the concept in which 100%IS is defined as the standardized baseline and 0.1%IS corresponds to major molecular response (MMR) in all CML patients. In this study, cytogenetic and molecular responses to the therapy was investigated in patients with high BCR-ABL transcript levels at diagnosis and patients with low BCR-ABL transcript levels at diagnosis to assess if baseline BCR-ABL transcript levels could be the reliable predictable factor in CP CML patients. Between May 2001 and Aug 2008, total 756 CML patients were treated with imatinib in St. Mary's Hospital of the Catholic University of Korea, and among them, 113 CP patients have been treated with imatinib at a dose of 400 mg/day for more than 6 months without interferon or stem cell transplantation prior to imatinib therapy. BM or PB samples were obtained at regular intervals from diagnosis for hematologic response (HR), cytogenetic response (CyR) and molecular response (MR) monitoring. For additional statistical analysis, 102 patients with Sokal scores available at the time of diagnosis were divided into 3 groups: low, intermediate and high Sokal groups. The median baseline BCR-ABL transcript level for 113 patients was 103.79%(IS), and the median BCR-ABL transcript levels were compared between 57 patients in a group of high baseline BCR-ABL levels and 56 patients in a group with low BCR-ABL levels by 3, 6, 12 and 18 months of imatinib therapy (Table 1). No difference was observed when the median values at month 3 and month 6 were considered, but from month 12 to month 18 after initiation of imatinib, the median BCR-ABL levels in patients with high baseline BCR-ABL levels was at least twice as high as median BCR-ABL levels in patients with low baseline BCR-ABL levels. However, differences cannot be considered to be significant due to the small number of patients under analysis. Regarding the CCyR rate by 3, 6, 12 and 18 months of imatinib therapy, no significant difference was observed between 2 groups of high and low baseline BCR-ABL levels, and there was also no statistically significant difference in CCyR and MMR rates depending on the baseline BCR-ABL levels even after grouping the patients according to Sokal scores.Table 1:Median BCR-ABL levels over time under imatinib treatment depending on the baseline BCR-ABL transcript levels.BaselineBCR-ABLtranscriptlevels (IS%)Month 3Month 6Month 12Month 18〉 103.79n25534625median3.900.850.330.20range0.74 - 38.870 - 77.840 - 26.660 - 3.00〈 103.79n36513122median4.110.950.150.07range0 - 183.300.001 - 41.980 - 8.040 - 2.51 In this study, no evidence of the correlation of the baseline BCR-ABL levels and the response to therapy was observed, and BCR-ABL level at diagnosis does not seem to give additional prognostic information to predict the response to therapy. Thus, the value of baseline BCR-ABL levels is still questionable to be used to identify patients who require higher dose of imatinib or more potent tyrosine kinase inhibitors, and further study with larger cohort and longer follow-up would be necessary to confirm whether baseline BCR-ABL levels are relevant to predict the response to therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3441 Resistance to imatinib can occur in chronic myeloid leukemia (CML). Mutations in BCR-ABL kinase domain have been known as the clinically most relevant mechanisms of imatinib resistance. Several studies have shown that 40~80% of the imatinib resistant patients have BCR-ABL kinase domain mutations. However, there has not been much information about mutation status in CML patients with suboptimal response to imatinib. With samples from Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) study which investigates efficacy of imatinib 400 mg daily and 800 mg daily with Philadelphia positive CML CP patients, and relationship between various responses and emergence of mutation was investigated by both standard and highly sensitive mutation assays. As a clinical correlative study (CCS) program with samples from TOPS study, we analyzed BCR-ABL mutation from 51 patients with optimal response, suboptimal response or treatment failure to imatinib in order to investigate their mutation status at different time points including diagnosis, 6 months, 12 months and 18 months. In addition, 53 non-clinical trial patients from Seoul St. Mary's hospital were involved in this analysis. All patients were in CP. Suboptimal responders and treatment failures were selected based on European Leukemia Net (ELN) 2009 guideline. CML patients' samples from the TOPS study and Seoul St. Mary's hospital were collected at particular time points after initiation of imatinib treatment and stored as cryopreserved cells or isolated RNAs. Mutations in BCR-ABL kinase domain were analyzed using direct sequencing and allele-specific oligonucleotide (ASO)-PCR (for Y253H, Y253F, G250E, E255K, E255V, T315I, F359V, and M351T). We performed mutation analysis with total 164 samples collected from 104 patients at different time points including diagnosis, 6 months, 12 months and 18 months after initiation of imatinib treatment. Serially collected samples at all time points were not available for all the patients; samples from 31 patients were available at diagnosis, 41 patients at 6 months, 58 patients at 12 months and 34 patients at 18 months (Table 1). We found ten BCR-ABL kinase domain point mutations including G250E, Q252H, Y253H, T315I, F317L, E355G, F359V, F359I and D444Y in 13 patients. In addition, we also found other mutations including 35 base pair insertion between exon 8 and exon 9 of ABL, and deletion of exon 7 of ABL in other 10 patients. No mutation was found from the patients' samples collected at diagnosis. At 6 months, mutation was found 5% (1 of 21), 18% (2 of 11) and 22% (2 of 9) patients in optimal response, suboptimal response and treatment failure group, respectively. ASO-PCR revealed that one patient in optimal response group had T315I. The same mutation status of the patient maintained at 12 months and the patients showed treatment failure at 12 months. At 12 months, mutation portion was 0% (0 of 15), 13% (2 of 15) and 25% (7 of 28) in optimal response, suboptimal response and treatment failure group, respectively. At 18 months, 36% (5 of 14) of suboptimal molecular responders who achieved CCyR, but no MMR showed mutation, and 62% (8 of 13) of failure group who showed less than CCyR had mutations. No particular difference in mutation frequency was found between 400mg group and 800mg group. Patients with suboptimal response or treatment failure showed much higher chance of BCR-ABL point mutation, 35 base pair insertion or exon 7 deletion in comparison with optimal responders, suggesting that mutation screening is important for patients with suboptimal response as well as treatment failure on the basis of ELN guideline. Treatment failure who achieved less than CCyR at 18 months and suboptimal responders who achieved CCyR, but no MMR at 18 months were highly recommended for mutation screening based on these data. Highly sensitive ASO-PCR provided early detection of point mutation in BCR-ABL kinase domain. However, clinical relevance of low level mutant clone, 35 base pair insertion and exon 7 deletion require long-term follow up for better understanding. Table 1. Response Diagnosis 6 months 12 months 18 months Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Optimal response 0 31 1 (5%) 21 0 (0%) 15 0 (0%) 7 Suboptimal response 2 (18%) 11 2 (13%) 15 5 (36%) 14 Failure 2 (22%) 9 7 (25%) 28 8 (62%) 13 Total Patients 31 41 58 34 Disclosures: Pane: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hochhaus:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 2766 Superior efficacy of Bcr-Abl tyrosine kinase inhibitors in chronic myeloid leukemia (CML) therapy has increased the demand for more sensitive monitoring system in checking clinical response from CML patients. Molecular response measuring Bcr-Abl transcript level by quantitative real-time PCR has been introduced and then widely used as important routine monitoring methodology (Müller et al. Leukemia 2009; 23: 1957–19633; Nick Best Pract Res Clin Haematol 2009; 22:355–65). European LeukemiaNet 2009 guide line recommended achievement of major molecular response (MMR) as optimal response at 18 months after initiation of imatinib (Baccarani et al. J Clin Oncol 2009; 27: 6041–6051). However, several studies have reported different clinical implications of MMR at specific time points to long-term survival outcomes. IRIS study proved MMR at 12 and 18 months can predict significantly better progression free survival (Hughes et al. N Engl J Med 2003; 349: 1423–1432; Druker et al. N Engl J Med 2006; 355: 2408–17). However, other studies reported that such differences may not be clinically relevant and only achievement of CCyR was significant to anticipate survival (de Lavallade et al. J Clin Oncol 2008; 26: 3358–3363, Kantarjian et al. Cancer 2008; 112: 837–84511). To find the level of molecular response (MR) at 18 months which can provide survival benefits, we selected newly diagnosed CP CML patients who registered at Seoul St. Mary's Hospital between January 2001 and June 2011. Selection conditions included initiation of imatinib therapy (400mg/d) within 6 months of diagnosis without prior treatment for leukemia (except for hydroxyurea or anagrelide), treatment with imatinib for more than 18 months and available molecular response record at 18 months. Total 237 patients met the above conditions. Their median age was 40 years (range; 18–74 years) and median duration of imatinib treatment was 44 months (range; 18–119 months).We investigate effect of different levels of MR (0.1%, 0.2%, 0.4% and 0.8% in Bcr-Abl transcript level) at 18 months on survivals including overall survival (OS), progression free survival (PFS) and event free survival (EFS) with these patients. Achievement of MMR did not provide survival benefits in OS, PFS and EFS. However other level of MR showed significant survival benefits in PFS and EFS between patients who achieved the level and patients who did not achieve the level. In achievement of 0.2% in Bcr-Abl transcript level, patients with MR level of 0.2 or less % showed better survival benefit than patients with MR level of more than 0.2% in PFS (98.0% vs.91.1%; P=0.017) and in EFS (94.2% vs. 85.5%; P=0.009) at 84 months. Bcr-Abl transcript level of 0.4% also showed survival benefits at 84 months between the patients with 0.4% or less and patients with more than 0.4% in PFS (97.7 vs. 88.9, P=0.007) and EFS (94.6 vs. 80.8, P=0.005). Similar results were observed in Bcr-Abl transcript level of 0.8%. However, achievement of all the levels of MR investigated in this study failed to provide clear benefit in OS. It may be due to extremely low portion of death (2.5%; 6 of 237 patients). The 0.2% of Bcr-Abl transcript at 18 months was the lowest value which showed clear correlation with survival benefits. MMR is defined as a Bcr-Abl transcript level of 0.1% or lower on international scale (IS), which is equivalent to a reduction in the Bcr-Abl transcript level by at least 3 log from the baseline level. The baseline level was determined as a median ratio of Bcr-Abl to Abl obtained from 116 untreated CP-CML patients in our institution. Survivals according to different level of molecular response (MR)Level of MR at 18 months0.1%0.2%0.4%0.8%Group=, 0.1=,〈 0.2〉 0.2=,0.4=,0.8Patients117120147901766119443OSDeath24242433Cumulative % at 7 yrs97.494.898.091.197.788.996.987.9P value0.5550.2280.0520.108PFSProgression27273645Cumulative % at 7 yrs97.493.398.091.197.788.996.987.9P value0.1210.0170.0070.005EFSEvent51151161079Cumulative % at 7 yrs92.689.194.285.594.680.893.976.7P value0.1360.0090.00050.0001 Disclosures: No relevant conflicts of interest to declare.

Description: Diagnosis of chronic myeloid leukemia (CML) is based on detection of the BCR-ABL gene or Philadelphia chromosome, and the BCR-ABL tyrosine kinase inhibitor imatinib has been the standard therapy for CML patients. Although imatinib therapy is effective in CML, it is still unclear whether imatinib can be safely discontinued without relapse. This study was designed to investigate the outcome of 26 CML patients after discontinuation of imatinib and to determine whether intermittent imatinib therapy can be employed in CML patients. Between May 2001 and Jun 2007, 555 patients have been treated with imatinib in St Mary’s Hospital of the Catholic University of Korea, and 26 patients discontinued imatinib when they achieved either complete cytogenetic response (CCyR) or complete molecular response (CMR). These 26 patients were diagnosed as Philadelphia positive (Ph+) CML between November 1995 and May 2002, and 22 patients were in chronic phase (CP) and 4 patients were in accelerated phase (AP) at diagnosis. The median age was 35 years (22–56), and 12 patients (46%) were female and 14 (54%) were male. Among 26 patients, 7 received interferon prior to imatinib therapy and 7 underwent SCT. Five patients received both interferon and SCT before imatinib therapy, and the remaining 7 patients received the imatinib as a front line therapy. Imatinib was started at oral dose of 400mg and 600mg daily for patients in CP and AP, respectively, and when they achieved CCyR or CMR, imatinib was discontinued after informed consent of the patient. In case of cytogenetic or molecular relapse, patients in all phases were retreated with imatinib at 400mg daily. Bone marrow (BM) or peripheral blood (PB) samples were obtained at regular intervals from diagnosis for hematologic response (HR), cytogenetic response (CyR) and molecular response (MR) monitorings. Eleven patients discontinued imatinib when they achieved CCyR, and 15 patients discontinued imatinib after achieving CMR. After the median duration of 7 month (4–48) observation without imatinib therapy, hematologic, cytogenetic and molecular relapses occurred in 4, 7 and 10 patients, respectively, and imatinib at oral dose of 400mg daily was reintroduced to all patients except 2 who continued to remain in CMR after imatinib discontinuation. Except 1 patient who expired and 2 patients who are in persistent molecular remission, all of 23 patients are maintaining the best response achieved after imatinib resumption with a median duration of 38 months (16–58). In conclusion, although imatinib cannot be discontinued completely, intermittent therapy can be considered for the treatment of CML patients. Intermittent imatinib treatment should not be restricted to CP patients who achieve CMR, and AP patients or patients with CCyR also can be considered for intermittent imatinib treatment. We will continue the follow-up of the patients enrolled in this study, and long-term study of intermittent imatinib treatment with expanded pool of patients will enable us to determine the accurate consequences of discontinuation of imatinib and intermittent imatinib treatment.

Description: Abstract 889 Background. BCR-ABL mutations are a major mechanism of acquired imatinib (IM) resistance in CML. Consequently, mutation analysis is recommended at milestones indicative of IM failure or suboptimal response (Baccarani JCO 2009: ELN recommendations). These criteria include lack of specified treatment responses up to 18 months of therapy, loss of response at any time, or progression to accelerated phase (AP) or blast crisis (BC). Mutation analysis was performed for patients (pts) enrolled in the TOPS trial (400 mg vs 800 mg of IM) for failure to achieve a major cytogenetic response (MCyR) by 6 months, failure to achieve a major molecular response (MMR) at 12 months, clinical evidence of resistance (including loss of any response and progression to AP/BC) and a significant rise in BCR-ABL. We determined whether the ELN recommendations for mutation screening were appropriate to optimally identify pts who acquired mutations and to limit the number of samples requiring mutation screening. Methods. 462 pts were on study for at least 3 months and were included in the analysis (median 39 months, range 3.7–54). Of the 462 pts, 280 had mutation analysis performed at least once by HPLC (high performance liquid chromatography) and/or direct sequencing. If mutation analysis was performed at a particular timepoint and no mutation was detected, then for the purposes of this analysis it was assumed the patient did not have a mutation at prior timepoints. When a mutation was detected, prior samples were tested to determine when the mutation first emerged. Results. Twenty-six mutations were detected in 20 pts (median 13 months, range 6–36). Multiple mutations were detected in 5/20 pts (25%). The mutations were confirmed at several timepoints in 17/20 pts, whereas mutations in the remaining 3 pts became detectable at the last sample collected before discontinuation. Sixteen of the 20 pts with mutations discontinued treatment. The frequency of mutations for the treatment arms was similar; 400 mg 6/155 (3.9%), 800 mg 14/307 (4.6%). However, the median month of mutation detection was earlier for pts treated on the 800 mg arm compared to the 400 mg arm, 10 versus 27 months, respectively. Pts treated on the 800 mg arm also had a higher frequency of the T315I mutation compared to the 400 mg arm; 6/14 pts (43%) on 800 mg compared to 1/6 pts (17%) on 400mg. In the total patient cohort, mutations were more commonly detected in those with high Sokal score (10/111 pts, 9%) compared to intermediate (6/160 pts, 3.8%) or low (4/191 pts, 2.1%). Furthermore, highly IM-resistant mutations (T315I, Y253H/F, E255V/K, L248V, G250E, F486S; Baccarani JCO 2009) were common in pts with a high or intermediate Sokal score, 9/10 (90%) and 4/6 pts (67%) respectively, compared to 0/4 with low Sokal score. Early indicators of IM failure, as recommended by the ELN, are no complete hematologic response (CHR) by 3 months and 〉95% Philadelphia chromosome (Ph) at 6 months. No mutations were detected in the total cohort of pts who met these failure criteria and who were tested for mutations (Table). This is consistent with previous studies indicating a low frequency of mutations in pts with primary IM resistance. However, lack of some milestone responses and loss of response were associated with mutations (Table, some pts lost more than 1 type of response). Failure to achieve an MMR by 18 months is an ELN criterion to perform mutation analysis. However, only 5% of pts in this category had a mutation and the majority of these pts also failed to achieve a CCyR. Lack of an MMR by 18 months when CCyR is achieved does not appear to be an indication for mutation analysis. Conclusion. The data suggest that the ELN recommendations for performing mutation analysis that would most frequently detect mutations in IM-treated pts are failure to achieve MCyR/CCyR at 12 months, loss of any response and AP/BC. IM doses of 800 mg were associated with the earlier acquisition of mutations, including those that are highly resistant. Disclosures: Branford: Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ortmann:Novartis: Employment. Duniec:Novartis: Employment. Jin:Novartis: Employment. Woodman:Novartis: Employment. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kim:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Radich:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Hughes:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria.

Description: Abstract 2763 Approximately 50% of CP CML patients achieve complete molecular response (CMR) at 6–7 years of first-line imatinib therapy. Although imatinib therapy is effective in CML patients and a substantial portion of patients achieve CMR with prolonged imatinib therapy, up to 10^7 leukemic cells can still be present in the absence of detectable BCR-ABL in RQ-PCR assay due to the sensitivity limit of current RQ-PCR technology. The recent data from STIM (Stop Imatinib) trial showed that the probability of persistent CMR at 12 month follow-up after imatinib discontinuation was 41%, and the conclusion was that imatinib can be safely discontinued, at least in some patients with persistent CMR. However, it is still not clearly defined whether discontinuation of imatinib therapy can be safely employed in patients with sustained CMR. In our prospective study, we examined if imatinib therapy can be safely discontinued in CML patients with sustained CMR4.5 according to strict PCR sensitivity criteria, and CMR4.5 was defined as undetectable BCR-ABL using RQ-PCR assay with at least 4.5-log sensitivity. CML patients who were treated with imatinib for more than 3 years and whose BCR-ABL was undetectable in RQ-PCR for at least 2 years were enrolled in this study. Our primary objectives were to evaluate the probability of persistent CMR4.5 at 12 month follow-up after discontinuation, and to measure the duration of persistent CMR4.5 after discontinuation. The secondary objective was to evaluate the probability of major molecular response (MMR) loss and the time taken to lose MMR at 12 month follow-up after discontinuation. In patients with loss of MMR, the probability of re-achieving MMR/CMR4.5 and the time taken to re-achieve MMR/CMR4.5 after imatinib resumption were also evaluated. After discontinuation, molecular response was monitored using RQ-PCR assay every month up to 6 month follow-up, every 2 months up to 12 month follow-up, and every 3 months thereafter. Digital PCR methodology with higher sensitivity compared to RQ-PCR assay was also applied before discontinuation and every year after discontinuation for more accurate estimation of BCR-ABL transcript levels. In case of relapse, defined as loss of MMR on 2 consecutive assessments, imatinib therapy was re-introduced and molecular response after resumption was observed using both RQ-PCR and digital PCR assays. As of data cut-off date of 15 Jul 2011, 20 patients (13 females, 7 males) who were diagnosed in Seoul St. Mary's Hospital between 20 Mar 1996 and 25 Apr 2005 were enrolled in this study with a median follow-up of 7 months (range, 2–9), and informed consents were obtained from all patients prior to participation. With a median age of 44 years (range, 25–67), the percentages of patients with low, intermediate and high Sokal risk scores were 30%, 30% and 15%, respectively with unknown Sokal risk scores in 25%. Ten patients (50%) received SCT and/or interferon therapy prior to imatinib therapy, while 10 patients (50%) received first-line imatinib therapy. The median time on imatinib therapy and the median duration of sustained CMR4.5 were 91 months (range, 40–112) and 60 months (range, 23–104), respectively, prior to discontinuation. Since discontinuation of imatinib therapy, all of 20 patients remained off therapy at the last follow-up with persistent CMR4.5 in 18 patients (90%) and loss of CMR in 2 patients (10%). Although loss of CMR was observed in 2 patients, both patients have not resumed imatinib therapy as MMR was maintained at the last follow-up. Our preliminary data show lower relapse rate after discontinuation compared to previous discontinuation studies. Strict PCR sensitivity criteria should be employed to assess the accurate measurement of BCR-ABL transcript levels prior to discontinuation, and then it might be possible to safely stop imatinib therapy in CML patients with stable CMR4.5. Through further clinical investigation on a large patient population and longer period of observation, more concrete conclusion can be made regarding the outcome of imatinib discontinuation. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1726 With prolonged imatinib therapy, BCR-ABL transcript levels measured by RQ-PCR assay show a progressive reduction, and some patients achieve complete molecular remission (CMR), which is defined as sustained undetectable BCR-ABL using RQ-PCR assay with a sensitivity of at least 4.5-log below the standardized baseline. However, due to the sensitivity limit of the current RQ-PCR technology, PCR negativity should not be considered as cure as more than 106 leukemic cells can still remain in the absence of detectable BCR-ABL transcripts by RQ-PCR. Although the numbers of patients with undetectable BCR-ABL are increasing with prolonged imatinib therapy and also with advent of more potent novel tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib and bosutinib, currently there is no methodology to further classify the patients in CMR. In this study, digital PCR (dPCR) assay was applied for measurement of BCR-ABL transcript levels in patients with CMR to assess if more sensitive detection methodology can be implemented for molecular monitoring in CML. Between May 2001 and Aug 2008, total 757 CML patients were treated with imatinib in St. Mary's Hospital of the Catholic University of Korea, and 192 chronic phase (CP) CML patients have been under imatinib therapy for more than 2 years. Among them, 36 patients achieved PCR negativity at least once during imatinib treatment, and serial PB samples collected from the patients who have maintained CMR for at least 3 years in RQ-PCR were screened by dPCR. In dPCR assay, each sample was partitioned into hundreds to tens of thousands of reaction chambers, and this sample partitioning enables detecting extremely low copy numbers that would normally be undetectable by conventional RQ-PCR platforms. Using the BioMark Real-Time PCR System (Fluidigm) and 12.765 Digital Array (Fluidigm) in dPCR assay, only 1 liquid-transfer step is required to automatically partition each of 12 samples into 765 reaction chambers of approximately 4.6 ul (6 nl × 765), and pre-amplification step was performed prior to dPCR assay to improve the sensitivity. Regarding the detection limit, whereas down to 10-5 of cell line dilutions and down to 10-4 of patient sample dilutions were detectable using conventional RQ-PCR, dPCR showed 2–3 log improvement in the detection sensitivity limit by detecting down to 10-7 of cell line dilutions and patient sample dilutions. In all the patient samples collected at the first time point of PCR negativity, positive BCR-ABL signals were detected in dPCR assay, and gradual decrease in the number of positive signals were observed in the serial samples collected on yearly basis after achieving CMR. PB samples collected from 5 healthy individuals were also screened to confirm the significance of positive results in dPCR, and no amplification was detected in all of 5 samples. This study shows that the patients in CMR who is currently categorized based on the data derived from conventional RQ-PCR assay could be classified further using more sensitive methodology such as dPCR assay. In the previous studies on imatinib discontinuation, the selection criteria of candidates was solely based on the duration of PCR negativity prior to discontinuation, and conventional RQ-PCR was employed to measure PCR negativity. The fact that the absolute number of residual leukemia clones could not be measured under the detection limits of conventional RQ-PCR might have resulted in relapse in more than half of the patients, and it reflects that firm conclusions cannot be drawn about if a patient could safely discontinue the therapy solely based on conventional RQ-PCR. It might be necessary to have more sensitive assays which will allow further classification of patients who could be candidates for imatinib discontinuation without relapse. This study shows the potential of highly sensitive PCR approach for molecular monitoring, and dPCR examined here can be extended to expand our understanding of molecular profiles in CML patients and to correlate to clinical significance. Detection and quantitation of low copy numbers may help to more precisely follow-up the course of disease and thereby to more accurately tailor personalized therapeutic choices. Disclosures: No relevant conflicts of interest to declare.