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The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+ Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance.

The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance.

In chronic myeloid leukemia (CML) patients, a breakpoint cluster region–Abelson (BCR-ABL1) value >10% at 3 months of therapy is statistically associated with poorer outcome, yet many of these patients still achieve satisfactory outcomes. We investigated 528 first-line imatinib-treated patients to determine whether patients with the poorest outcome can be better discriminated at 3 months. All outcomes were significantly superior for the 410 patients with BCR-ABL1≤10% at 3 months (P< .001). However, the poorest outcomes among the 95 evaluable patients with BCR-ABL1>10% at 3 months were identified by the rate of BCR-ABL1decline from baseline, assessed by estimating the number of days over which BCR-ABL1halved. Patients with BCR-ABL1halving time <76 days (n = 74) had significantly superior outcomes compared with patients whose BCR-ABL1values did not halve by 76 days (n = 21; 4-year overall survival, 95% vs 58%, P= .0002; progression-free survival, 92% vs 63%, P= .008; failure-free survival, 59% vs 6%, P< .0001; and major molecular response, 54% vs 5%, P= .008). By multivariate analysis, the halving time was an independent predictor of outcome in this poor risk group. Our study highlighted that the rate of BCR-ABL1decline may be a critical prognostic discriminator of the patients with very poor outcome among those >10% at 3 months. The International Randomized IFN vs STI571 (IRIS) trial was registered at http://www.clinicaltrials.govas #NCT00006343. The Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial was registered at http://www.clinicaltrials.govas #NCT00124748. The Therapeutic Intensification in DE-novo Leukaemia (TIDEL) I trial was registered at http://www.ANZCTR.org.auas #ACTRN12607000614493. The TIDEL II trial was registered at http://www.ANZCTR.org.auas #ACTRN12607000325404.

In chronic myeloid leukemia (CML) patients, a breakpoint cluster region–Abelson (BCR-ABL1) value >10% at 3 months of therapy is statistically associated with poorer outcome, yet many of these patients still achieve satisfactory outcomes. We investigated 528 first-line imatinib-treated patients to determine whether patients with the poorest outcome can be better discriminated at 3 months. All outcomes were significantly superior for the 410 patients with BCR-ABL1 ≤10% at 3 months (P < .001). However, the poorest outcomes among the 95 evaluable patients with BCR-ABL1 >10% at 3 months were identified by the rate of BCR-ABL1 decline from baseline, assessed by estimating the number of days over which BCR-ABL1 halved. Patients with BCR-ABL1 halving time <76 days (n = 74) had significantly superior outcomes compared with patients whose BCR-ABL1 values did not halve by 76 days (n = 21; 4-year overall survival, 95% vs 58%, P = .0002; progression-free survival, 92% vs 63%, P = .008; failure-free survival, 59% vs 6%, P < .0001; and major molecular response, 54% vs 5%, P = .008). By multivariate analysis, the halving time was an independent predictor of outcome in this poor risk group. Our study highlighted that the rate of BCR-ABL1 decline may be a critical prognostic discriminator of the patients with very poor outcome among those >10% at 3 months. The International Randomized IFN vs STI571 (IRIS) trial was registered at http://www.clinicaltrials.gov as #NCT00006343. The Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial was registered at http://www.clinicaltrials.gov as #NCT00124748. The Therapeutic Intensification in DE-novo Leukaemia (TIDEL) I trial was registered at http://www.ANZCTR.org.au as #ACTRN12607000614493. The TIDEL II trial was registered at http://www.ANZCTR.org.au as #ACTRN12607000325404.

Recent studies have demonstrated that some patients with chronic myeloid leukemia (CML) can maintain remission after discontinuation of imatinib. A prerequisite is stable, undetectable BCR-ABL1. It is not known how many patients achieve this response or the factors associated with its achievement. We examined 423 de novo imatinib-treated patients to determine the cumulative incidence of achieving the discontinuation criteria as defined in the CML8 study (≥2 years of undetectable BCR-ABL1[Stable MR4.5]), and predictive factors. After 8 years of imatinib, the cumulative incidence of Stable MR4.5was 36.5%. Therefore, 9% to 15% of first-line imatinib-treated patients would maintain remission after discontinuation. The BCR-ABL1level at 3 months and factors at diagnosis were examined for association with Stable MR4.5: Sokal risk, age, sex, and assigned imatinib dose. The only independent predictors were female sex (54.4% vs 27.2%; P= .018) and the 3-month BCR-ABL1(P< .001). The highest cumulative incidence of Stable MR4.5after 8 years was 78.2% for patients with BCR-ABL1≤ 0.10%ISat 3 months (n = 38). Time to major molecular response (MMR) influenced the time to reach Stable MR4.5(P< .001), suggesting slower dynamics of response with a delayed MMR. The findings justify the focus on rapid reduction of BCR-ABL1as a strategy to maximize potential suitability for imatinib discontinuation studies. The Iris trial was registered at http://www.clinicaltrials.govas NCT00006343. The Tops trial was registered at http://www.clinicaltrials.govas NCT00124748. The TIDEL I trial was registered at www.ANZCTR.org.auas ACTRN12607000614493. The TIDEL II trial was registered at www.ANZCTR.org.auas ACTRN12607000325404.

Recent studies have demonstrated that some patients with chronic myeloid leukemia (CML) can maintain remission after discontinuation of imatinib. A prerequisite is stable, undetectable BCR-ABL1. It is not known how many patients achieve this response or the factors associated with its achievement. We examined 423 de novo imatinib-treated patients to determine the cumulative incidence of achieving the discontinuation criteria as defined in the CML8 study (≥2 years of undetectable BCR-ABL1 [Stable MR4.5]), and predictive factors. After 8 years of imatinib, the cumulative incidence of Stable MR4.5 was 36.5%. Therefore, 9% to 15% of first-line imatinib-treated patients would maintain remission after discontinuation. The BCR-ABL1 level at 3 months and factors at diagnosis were examined for association with Stable MR4.5: Sokal risk, age, sex, and assigned imatinib dose. The only independent predictors were female sex (54.4% vs 27.2%; P = .018) and the 3-month BCR-ABL1 (P < .001). The highest cumulative incidence of Stable MR4.5 after 8 years was 78.2% for patients with BCR-ABL1 ≤ 0.10%IS at 3 months (n = 38). Time to major molecular response (MMR) influenced the time to reach Stable MR4.5 (P < .001), suggesting slower dynamics of response with a delayed MMR. The findings justify the focus on rapid reduction of BCR-ABL1 as a strategy to maximize potential suitability for imatinib discontinuation studies. The Iris trial was registered at http://www.clinicaltrials.gov as NCT00006343. The Tops trial was registered at http://www.clinicaltrials.gov as NCT00124748. The TIDEL I trial was registered at www.ANZCTR.org.au as ACTRN12607000614493. The TIDEL II trial was registered at www.ANZCTR.org.au as ACTRN12607000325404.

BCR-ABL1 mutations (muts) frequently cause resistance to tyrosine kinase inhibitors (TKIs) in CML. T315I, the most common mut, confers resistance to 1st and 2nd generation TKIs. In vitro studies suggest that all common individual muts are sensitive to the pan-BCR-ABL1 TKI ponatinib, and durable responses have been observed in patients (pts) with T315I. We have shown that low level muts below the detection limit of Sanger sequencing (SS) are important clinically, and that pts with >1 mut are a poor risk subgroup with lower CCyR and MMR rates and higher risk of treatment failure on 2nd-line therapy with nilotinib or dasatinib (Parker Blood 2012).To determine whether the presence of low level BCR-ABL1 muts detected by mass spectrometry (mass spec) adversely impacts response to ponatinib.We assessed 376 of the 392 pts treated with ponatinib in the phase II PACE trial who consented for sensitive mass spec mut analysis (detects 32 muts, limit ∼0.2%; 240 CP, 77 AP, 42 BP, 17 Ph+ ALL). RNA from samples collected at trial Day 1 were received from the commercial lab that performed SS. We performed mass spec analysis on duplicate cDNA samples blinded to the SS results. Muts were considered present if detected in both replicates. Cumulative incidence of MCyR, CCyR and MMR were calculated and tested with Gray's K-sample test. Progression free survival (PFS) was assessed by Kaplan-Meier with the log-rank test. 146 pts had disease progression. Mut analysis was performed by SS at discontinuation for 98 pts at the commercial lab. Pts remaining on study had a minimum follow up of 18 months (mo).Of the 376 pts, 373 were evaluable at Day 1 by SS, and 359 by mass spec. Muts (242) were detected in 196 pts (53%) by SS. Among these muts, 95% were included in the mass spec assay and were potentially detectable; mass spec detected all but 6. At Day 1, >1 mut was detected in more pts by mass spec (64, up to 8 per pt) than SS (42, up to 3 per patient), 17% v 11%. T315I was the most common mut detected. Using mass spec, we detected an additional 12 pts with T315I compared with SS (107 v 95). Of these pts, 73 had T315I only and 34 had T315I plus at least 1 additional mut by mass spec. By SS, 75 pts had T315I only and 20 had T315I plus another mut.At Day 1, mass spec detected 77 low level muts in 53 pts that were not detected by SS. Mut analysis was performed at discontinuation for 10/53 pts. In contrast to our previous studies where certain low level mutants known to confer resistance rapidly expanded with 2nd generation TKI treatment after imatinib resistance, only 2 of the low level mutants expanded during ponatinib therapy and were detected by SS at discontinuation (2 T315I). This supports the pan-BCR-ABL1 inhibition predicted from pre-clinical studies.To investigate relationships between mass spec mut status and response and PFS, pts were grouped according to presence of 1) T315I only, 2) T315I plus additional mut, 3) >1 mut (not T315I), 4) 1 mut (not T315I), 5) no mut. The cumulative incidence of MCyR, CCyR and MMR and probability of PFS for AP, BP and Ph+ ALL pts was not significantly different according to mut status. However, among CP pts, significant differences were seen for response and PFS (Figure).Among the CP pts without T315I, pts with no mut had significantly inferior cytogenetic and molecular responses than pts with >1 mut, but PFS was not significantly different.For CP pts with T315I, the presence of additional mut was associated with significantly inferior cytogenetic responses and PFS. At 18mo, the cumulative incidence of MCyR, CCyR and MMR for pts with T315I only was 76%, 74% and 64%, respectively, whereas it was 50%, 44% and 33%, respectively, for pts with T315I plus additional mut. PFS at 18 mo was 88% for pts with T315I only, compared with 59% for pts with T315I plus additional mut. Notably, 56% (10/18) of CP pts with T315I plus additional mut as determined by mass spec were not classed as such by SS.Mass spec identified a subgroup of CP pts with the T315I mut who had relatively inferior responses and outcome. These pts had T315I plus additional mut at Day 1 by mass spec. These pts also had inferior PFS as compared to pts without muts. Multiple muts in pts with T315I may be a marker of longer disease duration or poor disease control, but suggests that BCR-ABL1 mut status determined by mass spec has prognostic value. Ponatinib may overcome the poor responses to 2nd generation TKIs observed for pts with >1 mut without T315I and for pts with a single T315I mut.Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria. Lustgarten:ARIAD: employees of and own stock/stock options in ARIAD Pharmaceuticals, Inc Other, Employment. Hodgson:ARIAD: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership. Hughes:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Branford:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

BCR-ABL1mutations (muts) frequently cause resistance to tyrosine kinase inhibitors (TKIs) in CML. T315I, the most common mut, confers resistance to 1stand 2ndgeneration TKIs. In vitro studies suggest that all common individual muts are sensitive to the pan-BCR-ABL1 TKI ponatinib, and durable responses have been observed in patients (pts) with T315I. We have shown that low level muts below the detection limit of Sanger sequencing (SS) are important clinically, and that pts with >1 mut are a poor risk subgroup with lower CCyR and MMR rates and higher risk of treatment failure on 2nd-line therapy with nilotinib or dasatinib (Parker Blood2012).

BCR-ABL1 kinase domain mutations are the most common known cause of resistance to tyrosine kinase inhibitors (TKIs) in CML. Some imatinib resistant mutations also confer resistance to second generation TKIs nilotinib and/or dasatinib. Therefore, it is recommended that mutation analysis be performed before changing therapy. However, BCR-ABL1 mutant clones are often de-selected upon TKI cessation or change of therapy, and may become undetectable (Hanfstein et al, Haematologica 2011). It is not known whether treatment discontinuation or long term alternative TKI therapy leads to eradication of these mutant clones. If mutant clones persist at sub-clonal levels they have the potential to be re-selected and expand clonally given favorable conditions, such as change to a TKI for which they confer resistance. We examined longitudinal data of patients with imatinib resistant mutations that became undetectable by direct sequencing to determine whether these “long dormant” mutations could reappear, and the circumstances related to reappearance.All chronic phase patients who had been monitored at our institution since starting imatinib, and had mutations detectable by sequencing during imatinib therapy were analyzed; 49 patients, median follow up since starting imatinib was 4.3 years (range 0.6–11.6 years). Sensitive mutation analysis using mass spectrometry (detection limit 0.2% mutant) was performed at selected times when the mutations became undetectable by direct sequencing (detection limit 10–20%).Of the 49 patients with mutations detected by sequencing during imatinib therapy, mutations became undetectable by sequencing in 21 patients (29 mutations), at a median of 2 months after changing therapy (range 1–20 months). This was associated with increased imatinib dose (3 mutations), stopping imatinib (2), hematopoietic cell transplant (6), chemotherapy (1), switching to nilotinib (3), or switching to dasatinib (14). All mutations that became undetectable by sequencing when the patient switched to nilotinib or dasatinib were those known to be sensitive to the inhibitor received (e.g. F359V in a patient treated with dasatinib).In 16 of the 21 patients whose mutations became undetectable by direct sequencing, the mutations have not been detected again with 0.1 to 6.9 years of follow up since the mutations were last detected (median 1.1 years). Of these 16 patients, 15 maintained a stable complete cytogenetic response and 1 lost a major cytogenetic response. In the other 5 patients, the same mutations as those originally detected (identical nucleotide exchange) became detectable by sequencing between 1.7 and 5.4 years after last detection (median 4.4 years), Figure. The original mutations in 4 of these patients confer resistance to nilotinib as well as imatinib (Y253H and F359V), and their reappearance was associated with initiation of nilotinib therapy, Figure. Three of these 4 patients died of their disease, and 1 lost a major cytogenetic response. Sensitive mutation analysis could detect the mutation in 1 of these patients during the time of “dormancy” and before nilotinib therapy. The 5th patient received an autologous hematopoietic cell transplant upon detection of F359V, and the mutation became undetectable by sequencing. The patient subsequently received dasatinib for 3 years and the mutation remained undetectable. Dasatinib therapy was stopped due to intolerance and F359V rapidly reappeared while the patient was off TKI therapy, having been undetectable for 4.8 years. Using sensitive mutation analysis, F359V could be detected at low levels after the transplant, suggesting that the mutant clone had not been eradicated.The data suggest that some BCR-ABL1 mutations may persist at sub-clonal levels for many years after changing therapy. This could lead to clonal expansion under the selective pressure of a TKI for which the mutation confers insensitivity. Alternatively, the reappearance of the mutation could be a new occurrence of the same mutation. The study highlights the importance of knowing the mutation history of individual patients to enable informed therapy choices.Yeung: Novartis Pharmaceuticals: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Hughes:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Branford:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid: Consultancy.

Abstract 1692