Patients with chronic myeloid leukemia who are eligible for treatment-free remission (TFR) may still relapse after tyrosine kinase inhibitor (TKI) cessation. There is a need for accurate predictors of outcome to enable patients with a favorable profile to proceed while avoiding futile attempts. Sensitive detection of residual disease in total leukocytes at treatment cessation is associated with relapse but is not highly discriminatory, likely because it is a composite measure of residual leukemia derived from different cell line-ages, whereas only some lineages are relevant for relapse. We prospectively measured BCR::ABL1 DNA as a predictive yes/no binary test in 5 cellular fractions from 48 patients meeting conventional criteria for TKI discontinuation. The median BCR::ABL1 DNA level was higher in granulocytes and T cells, but not in other lineages, in patients who relapsed. Among the 40 patients undergoing their first TFR attempt, we defined 3 groups with differing relapse risk: granulocyte-positive group (100%), granulocyte-negative/T-cell- positive group (67%), and granulocyte-negative /T-cell-negative group (25%). These data show the critical importance of lineage-specific assessment of residual disease in the selection of patients who can attempt to achieve TFR with a high expectation of success and, concurrently, defer patients who have a high prob-ability of relapse.

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML); however, evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGA) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGA at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival, and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS), and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGA included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the patient's genetic profile and other baseline factors. AGA were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements) were detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the EUTOS long-term survival clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGA had poorer response rates. These data provide evidence for the incorporation of genomically-based risk assessment for CML.

With treatment-free remission (TFR) rapidly becoming the ultimate goal of therapy in chronic myeloid leukemia (CML), there is a need to develop strategies to maximize sustained TFR by improving our understanding of its key determinants. Chronic-phase CML patients attempting TFR were evaluated to identify the impact of multiple variables on the probability of sustained TFR. Early molecular response dynamics were included as a predictive variable, assessed by calculating the patient-specific halving time of BCR-ABL1 after commencing tyrosine kinase inhibitor (TKI) therapy. Overall, 115 patients attempted TFR and had ≥12 months of follow-up. The probability of sustained TFR, defined as remaining in major molecular response off TKI therapy for 12 months, was 55%. The time taken for the BCR-ABL1 value to halve was the strongest independent predictor of sustained TFR: 80% in patients with a halving time of <9.35 days (first quartile) compared with only 4% if the halving time was >21.85 days (last quartile) (P < .001). The e14a2 BCR-ABL1 transcript type and duration of TKI exposure before attempting TFR were also independent predictors of sustained TFR. However, the BCR-ABL1 value measured at 3 months of TKI was not an independent predictor of sustained TFR. A more rapid initial BCR-ABL1 decline after commencing TKI also correlated with an increased likelihood of achieving TFR eligibility. The association between sustained TFR and the time taken for BCR-ABL1 to halve after commencing TKI was validated using an independent dataset. These data support the critical importance of the initial kinetics of BCR-ABL1 decline for long-term outcomes.

Atypical BCR-ABL1 transcripts occur in around 3% of patients with chronic phase chronic myeloid leukemia (CML) [1]. Due to the rarity of each individual transcript type there are limited data on th...

Naranie Shanmuganathan, Jodi A. Braley, Agnes S. M. Yong, Devendra K. Hiwase, David T. Yeung, David M. Ross, Timothy P. Hughes and Susan Branford

Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10 −4.9 vs 10 −5.7 ; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P =.007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1. The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P =.07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

Accurate quantification of minimal residual disease (MRD) during treatment of chronic myeloid leukemia (CML) guides clinical decisions. The conventional MRD method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first three months of treatment, BCR-ABL1 mRNA values declined more rapidly than DNA. By six months, the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in MRD being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first three months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond three months, falling levels of BCR-ABL1 mRNA are proportional to the depletion of leukemic cells.

Following the achievement of deep molecular response on tyrosine kinase inhibitors (TKIs), approximately half of patients with chronic myeloid leukemia (CML) can discontinue TKI and remain in treatment-free remission (TFR). The ALLG CML8 study enrolled 40 imatinib-treated patients with undetectable BCR-ABL1 mRNA (approximately MR 4.5 ). Molecular relapse was defined as detectable BCR-ABL1 on two consecutive tests or any single value >0.1%. With a median follow-up of 8.6 years (range 5.7–11.2 years), 18 patients remain in continuous TFR (45.0%; 95% confidence interval 31.9−63.4%). The latest relapse detected was 27 months after stopping imatinib. No patient progressed to advanced phase. Twenty-two patients met criteria for imatinib re-treatment and all regained undetectable molecular response. Nine patients in long-term TFR were monitored by highly sensitive individualized BCR-ABL1 DNA PCR in a sufficient number of samples to enable more precise quantification of residual leukemia. BCR-ABL1 DNA decreased from a median of MR 5.0 in the first year of TFR to MR 6.1 in the sixth year of TFR. Our results support the long-term safety and remarkable stability of response after imatinib discontinuation in appropriately selected CML patients. Serial high sensitivity testing provides a new and unexpected finding of gradually reducing CML cells in patients in long-term TFR.

Abstract Background: Discontinuation of tyrosine kinase inhibitor (TKI) treatment for chronic myeloid leukaemia (CML) patients in stable deep molecular response leads to treatment-free remission (TFR) in approximately 50% of cases. In most studies, monthly PCRs was performed for 12 months followed by 2-3 monthly testing thereafter. Around 80% of molecular relapses occur within the first 6 months after TKI cessation. The current recommendation for TKI recommencement is a single BCR-ABL1 value ≥0.1% IS (International scale), indicating loss of major molecular response (MMR). Not all institutions can offer monthly PCR monitoring due to financial constraints, particularly relevant in developing countries. For some patients, remaining on TKI is a cheaper alternative. Aim: To assess the safety of less frequent BCR-ABL1 monitoring for detection of loss of MMR for CML patients attempting TFR. Methods: We monitored 85 patients who ceased TKI with the aim of achieving TFR. Patients had a minimum of 24 months of sustained MR4 (n=3) or MR4.5 (n=82) prior to TKI cessation. At the time of TKI cessation, 64 patients were on imatinib (75%), 17 on nilotinib (20%) and 4 on dasatinib (5%). Forty of the patients were enrolled in the TWISTER study where the criteria for TKI recommencement was loss of MMR or 2 consecutively rising BCR-ABL1 positive values. The remaining patients were on a registry study and the trigger for TKI recommencement was loss of MMR. Results: TKI recommencement occurred in 49 of 85 patients. Median time to TKI recommencement was 4 months (range 2-28 months) at a median BCR-ABL1 value of 0.27% on the International Scale (IS), range 0.002-24% IS. Thirty-six of the 49 patients (73%) lost MMR prior to TKI recommencement; the median time to loss of MMR was 3 months (range 1 to 10 months). One patient lost MMR within the first month. Figure A demonstrates the time to loss of MMR in the 36 patients with PCR values ≥ 0.1%. Eighteen of the 36 patients (50%) lost MMR by the 3 month BCR-ABL1 assessment and 35 of 36 patients (97%) lost MMR by 6 months. The latest loss of MMR was at 10 months. Fourteen patients recommenced TKI at a BCR-ABL1 value of >1% and 1 recommenced at a value >10%. Clinician delay in TKI recommencement of 1 month resulted in a BCR-ABL1 rise from 0.84% to 24% with associated loss of complete hematological response. We propose monthly BCR-ABL1 testing between 2 and 6 months post TKI cessation followed by 2 monthly testing. Detection of a BCR-ABL1 value of ≥0.1% would trigger TKI recommencement. In the presence of a rising BCR-ABL1, which remains ≤0.1%, monthly monitoring should ensue in order to avoid hematological relapse. If this strategy were employed in this cohort of patients, only 1 patient would have had the trigger for TKI recommencement delayed by 1 month (estimated BCR-ABL1 at recommencement ~2.5%). This patient had loss of MMR in the first month post TKI cessation. If this molecular monitoring strategy was applied to patients in our cohort who had not lost MMR at TKI recommencement, we estimate that 1 other patient would have had TKI recommencement delayed by 1 month based on the average BCR-ABL1 doubling time of 1 log per month. A proportion of patients maintain low levels of BCR-ABL1 after TKI cessation and do not lose MMR. There were 2 such patients in our cohort. Conclusion: The critical time for molecular monitoring to trigger TKI recommencement is the first 6 months. A monthly monitoring strategy beginning 2 months after cessation would capture the majority of patients at loss of MMR. The data suggest that after 6 months, 2-monthly monitoring could follow. Monthly BCR-ABL1 testing can be re-introduced in the event of a positive result in those that ceased TKI with undetectable BCR-ABL1 or if there is a BCR-ABL1 result higher than the cessation value. This approach would reduce BCR-ABL1 testing by approximately 33% in the majority of cases while minimizing hematological relapse. Therefore this strategy would reduce the cost and inconvenience of molecular monitoring for a trial of TKI cessation, making the option of TFR available to some patients for whom it is otherwise not feasible. Disclosures Branford: Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Yeung:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Bristol-Myers Squibb: 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; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group.

The Therapeutic Intensification in De Novo Leukaemia (TIDEL)-II study enrolled 210 patients with chronic phase chronic myeloid leukemia (CML) in two equal, sequential cohorts. All started treatment with imatinib 600 mg/day. Imatinib plasma trough level was performed at day 22 and if <1000 ng/mL, imatinib 800 mg/day was given. Patients were then assessed against molecular targets: BCR-ABL1 ≤10%, ≤1%, and ≤0.1% at 3, 6, and 12 months, respectively. Cohort 1 patients failing any target escalated to imatinib 800 mg/day, and subsequently switched to nilotinib 400 mg twice daily for failing the same target 3 months later. Cohort 2 patients failing any target switched to nilotinib directly, as did patients with intolerance or loss of response in either cohort. At 2 years, 55% of patients remained on imatinib, and 30% on nilotinib. Only 12% were >10% BCR-ABL1 at 3 months. Confirmed major molecular response was achieved in 64% at 12 months and 73% at 24 months. MR4.5 (BCR-ABL1 ≤0.0032%) at 24 months was 34%. Overall survival was 96% and transformation-free survival was 95% at 3 years. This trial supports the feasibility and efficacy of an imatinib-based approach with selective, early switching to nilotinib. This trial was registered at www.anzctr.org.au as #12607000325404.

Abstract Background: Clinical scoring systems, such as Sokal risk, continue to have prognostic relevance for patients (pts) treated with tyrosine kinase inhibitors (TKI) and may have utility in combination with emerging biomarkers. The BCR-ABL value at 3 and 6 months (mo) of TKI are the strongest predictors of response. However, recent data demonstrate that the rate of BCR-ABL decline from the pre imatinib level adds significant predictive information (Hanfstein, Leukemia 2014; Branford, Blood 2014). Among poor risk pts with >10% at 3 mo in our cohort of first line imatinib, those with a slow rate of decline from their pre imatinib value, assessed by calculating the number of days over which BCR-ABL halved (halving time), predicted significantly poorer outcomes. Notwithstanding the importance of the 3 and 6 mo values, a prognostic biomarker obtained at an earlier timepoint may allow opportunities for therapy optimization. We therefore examined the prognostic significance of the rate of BCR-ABL decline at 1 mo in the context of other predictors of response. Aim: To determine whether baseline factors (age, gender, Sokal risk and imatinib starting dose: 400, 600 or 800 mg) and the BCR-ABL halving time at 1 mo of imatinib have predictive significance. Method: 528 first line imatinib treated pts were evaluated (median 45 mo of imatinib). Molecular assessment was performed pre imatinib and at 1 mo for 470/528. 453 of these 470 pts had a Sokal score available and were included in the analysis of outcome. Results: The median BCR-ABL halving time at 1 mo of imatinib was 17 days, quartiles 11, 29. An initial rapid BCR-ABL decline, indicated by halving times in the lowest quartile of ≤11 days (n=115), was associated with significantly superior rates of MMR by 12 mo, MR4.5 and failure-free survival (FFS) by 4 years compared with longer halving times of >11 days (n=338), Table. MMR by 12 mo was assessed since it represents an optimal response and is associated with subsequent deep molecular response. By univariate and multivariate regression analysis only the 1 mo halving time and Sokal risk significantly predicted MMR, MR4.5 and FFS. These factors were independent and there was no difference between the median 1 mo halving times among the Sokal risk groups, P = .36. The high Sokal risk pts had significantly poorer outcomes. To improve response prediction, these pts were divided into 2 groups according to their 1 mo halving time; ≤11 days (n = 28) and >11 days (n=90). A 1 mo halving time of ≤11 days was associated with significantly improved outcomes for these pts, Table and Figure. The responses equated to those of pts with low Sokal risk: high risk ≤11 days vs low risk: MMR by 12 mo 57% vs 59%, P = .95; MR4.5 by 4 years 36% vs 40%, P = .82; FFS by 4 years 79% vs 84%, P = .39. The high Sokal risk pts with the rapid initial BCR-ABL decline also had a lower probability of BCR-ABL >10% at 3 mo (early molecular response [EMR] failure), which is considered a warning or treatment failure; ≤11 days vs >11 days: 14% vs 33%, Table. Table 1 Outcome* by Sokal risk and BCR-ABL halving time at 1 mo of imatinib Factor No. of pts EMR % 3 mo MMR % 12 mo MR4.5 % 48 mo FFS % 48 mo Sokal Low 195 90 59 40 84 Intermediate 140 79 50 35 71 High 118 71 43 26 59 P value <.0001 .008 .006 <.0001 1 mo BCR-ABL halving time ≤11 days 115 92 63 41 84 >11 days 338 78 49 30 70 P value <.0001 .0005 .01 .002 High Sokal risk ≤11 days halving time 28 86 57 36 79 >11 days halving time 90 67 39 16 53 P value .004 .03 .02 .03 *EMR, MMR, MR4.5 cumulative incidence; FFS Kaplan Meier In contrast to the improved response observed with a rapid initial decline for high Sokal risk pts, a slow decline for pts with low or intermediate Sokal risk (halving times in the upper quartile of >29 days) was associated with a significantly lower cumulative incidence of MMR by 12 mo: low Sokal risk ≤29 days (n = 151) vs >29 days (n = 44) 65% vs 39%, P = .002; intermediate Sokal risk ≤29 days (n = 103) vs >29 days (n=37) 57% vs 31%, P = .004, Figure. Conclusion: Imatinib treated high Sokal risk pts have a higher rate of treatment failure and poorer molecular response. However, our data suggest their prognosis can be refined by taking into account the kinetics of BCR-ABL decline after only 1 mo of treatment. A rapid initial decline defined a subgroup of high Sokal risk pts with outcomes equivalent to those of low Sokal risk pts. Frequent molecular monitoring in the critical first months of treatment could enhance outcome prediction and limit the indication for a change of treatment. Figure 1 Figure 1. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding. Yeung:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ross:Novartis: Honoraria, Research Funding; BMS: Honoraria. Seymour:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria, Research Funding.

Background: Discontinuation of tyrosine kinase inhibitor (TKI) treatment for patients (pts) with CML is desirable for both clinical and economical reasons. Two studies, STIM and TWISTER, demonstrated that treatment free remission (TFR) is achievable in a proportion of pts. A qualifying period of 3 years of imatinib and sustained undetectable BCR-ABL with a sensitivity of MR5 or MR4.5 for at least 2 years was required before discontinuation. Due to the very slow clearance of BCR-ABL after an initial rapid reduction in the first year of imatinib, the number of pts meeting discontinuation criteria potentially requires many years of imatinib. New discontinuation studies may require at least 3 years of TKI treatment but are exploring less stringent molecular criteria for discontinuation. The first requirement is the achievement of MR4.5 (≤0.0032%) or even MR4 (≤0.01%), and these responses must be maintained. More potent TKIs when administered as first line or after switch from imatinib are associated with higher rates of deep molecular response, which potentially leads to a greater number of pts who qualify for a discontinuation trial. It is unknown what proportion of imatinib treated pts who have not achieved a deep molecular response at 3 years will subsequently do so, and in which subgroup of patients a switch to a more potent TKI is warranted if TFR is the goal.

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.