Detectable BCR-ABL Research Articles

Hematological profile and its correlation with reverse transcription–polymerase chain reaction-based serial quantitative detection of BCR-ABL in chronic myeloid leukemia patients at a referral cancer center

Abstract BACKGROUND: There are variations in the hematological profile among the Indian population affected with chronic myeloid leukemia (CML). The range of white blood cell count (WBC) was 0.46 × 109/mm3 to 1.86 × 109/mm3. Similarly, range for hemoglobin was 9–11 g/dl. WBC count can be as low as 0.18 × 109/mm3 even though CML is a myeloproliferative disorder. OBJECTIVE: The objective of this study was to correlate BCR-ABL expression with the hematological and myeloid proliferative parameters. METHODS: The present study was a single-center, hospital-based follow-up study among 66 cases of CML. The sample of patients was analyzed for BCR-ABL gene expression; hematological parameters and myeloid proliferative parameters such as band forms, myelocytes, metamyelocytes, and blast were also measured. Some patients reported twice, some thrice, and the total count after all follow-up for all 66 cases was 179. RESULTS: The mean age was 43.15 years. Men were more than women with male-to-female ratio of 1.6:1. The mean hemoglobin was lesser at 10.8 g/dl. Median total count was normal at 5610 (interquartile range = 2750) per μl. Mean neutrophils, lymphocytes, monocytes, basophils, and platelets were normal. The eosinophil count was raised. The BCR-ABL gene expression was positively and significantly correlated with total count. It was negatively and significantly correlated with lymphocytes and monocytes. It was not correlated with other hematological parameters. All the myeloid proliferative parameters, namely, band forms, myelocytes, metamyelocytes, and blast, were positively and significantly correlated with BCR-ABL gene expression. CONCLUSION: Total count and all myeloid proliferative parameters were positively and significantly correlated with BCR-ABL gene expression. Lymphocytes and monocytes were negatively correlated. These parameters can be used to track the disease progression in patients with CML.

Ponatinib and blinatumomab for Philadelphia chromosome-positive acute lymphoblastic leukaemia: a US, single-centre, single-arm, phase 2 trial

Ponatinib and blinatumomab are effective therapies in patients with Philadelphia chromosome-positive (Ph-positive) acute lymphoblastic leukaemia, and their combination might be a promising treatment option. In this study, we aimed to evaluate this chemotherapy-free strategy. We did a single-centre, single-arm, phase 2 study at the University of Texas MD Anderson Cancer Center, Houston, TX, USA, in patients aged 18 years or older with newly diagnosed or relapsed or refractory Ph-positive acute lymphoblastic leukaemia or chronic myeloid leukaemia in lymphoid blast phase. Patients with an ECOG performance status of 2 or less who had a total bilirubin concentration two-times the upper limit of normal (ULN) or less (≤2·4 mg/dL), alanine aminotransferase and aspartate aminotransferase concentration no more than three-times the ULN, and serum lipase and amylase concentrations no more than three-times the ULN were eligible for inclusion. Ponatinib 30 mg orally and continuous intravenous blinatumomab 28 μg over 24 h (for 28 days each cycle) were given in combination for up to five 42-day cycles, followed by ponatinib monotherapy. Patients received 12 doses of intrathecal chemotherapy as CNS prophylaxis. The primary endpoints were complete molecular response (defined as absence of a detectable BCR-ABL1 transcript by PCR at a sensitivity of 0·01%) in patients with newly diagnosed disease and overall response in patients with relapsed or refractory disease or chronic myeloid leukaemia in lymphoid blast phase. All assessments were done according to the intention-to-treat principle. The trial completed its original target accrual and was amended on March 23, 2022, to enrol an additional 30 patients, thus increasing the sample size to 90 patients. The trial is registered with ClinicalTrials.gov, NCT03263572, and it is ongoing. Between Feb 6, 2018, to May 6, 2022, 60 (83%) of 72 patients assessed were enrolled and received ponatinib and blinatumomab (40 [67%] patients had newly diagnosed Ph-positive acute lymphoblastic leukaemia, 14 [23%] had relapsed or refractory Ph-positive acute lymphoblastic leukaemia, and six [10%] had chronic myeloid leukaemia in lymphoid blast phase). 32 (53%) patients were men and 28 (47%) were women; 51 (85%) patients were White or Hispanic; and the median age of participants was 51 years (IQR 36-68). The median duration of follow-up for the entire cohort was 16 months (IQR 11-24). Of patients with newly diagnosed Ph-positive acute lymphoblastic leukaemia, 33 (87%) of 38 evaluable patients had a complete molecular response. 12 (92%) of 13 evaluable patients with relapsed or refractory Ph-positive acute lymphoblastic leukaemia had an overall response. 11 (79%) had a complete molecular response. Five (83%) of six patients with chronic myeloid leukaemia in lymphoid blast phase had an overall response. Two (33%) had a complete molecular response. The most common grade 3-4 adverse events that occurred in more than 5% of patients were infection (22 [37%] patients), increased amylase or lipase concentration (five [8%] patients), increased alanine aminotransferase or aspartate aminotransferase concentration (four [7%] patients), pain (four [7%] patients), and hypertension (four [7%] patients). One (2%) patient discontinued blinatumomab due to tremor. Three (5%) patients discontinued ponatinib secondary to cerebrovascular ischaemia, portal vein thrombosis, and coronary artery stenosis in one patient each. No treatment-related deaths were observed. The chemotherapy-free combination of ponatinib and blinatumomab resulted in high rates of complete molecular response in patients with newly diagnosed and relapsed or refractory Ph-positive acute lymphoblastic leukaemia. Patients with newly diagnosed Ph-positive acute lymphoblastic leukaemia could be spared the toxicities associated with chemotherapy and the need for allogeneic haematopoietic stem-cell transplantation in first response. Takeda Oncology and Amgen.

Highly Sensitive Droplet Digital PCR to Identify CML Patients with a High Probability of Achieving Treatment-Free Remission

Introduction: Treatment Free Remission (TFR) is the ultimate goal of therapy for most CML patients. Despite adopting consensus eligibility criteria of a sustained deep molecular response and more than 4 years of TKI therapy, the relapse rate after TKI cessation is still around 50%. More sensitive detection of residual leukaemia has the potential to improve our capacity to predict TFR outcomes for individual patients.Aim: To correlate droplet digital PCR (ddPCR) assay results with TFR outcome, especially in the setting of undetectable levels measured by qRT-PCR.Method: ddPCR was performed on blood samples from 51 TFR-eligible CML patients at the time of TKI cessation. 5 µg RNA per sample was used in 8 wells/sample using the BioRad QXDx BCR-ABL %IS kit on QX200 ddPCR system which yielded BCR-ABL1% (IS) directly. All these patients achieved MR4.5 that was sustained for ≥ 2 years.Results: 100% of patient were in MR4.5 via qRT-PCR at the time of stopping. 61% of the 51 patients evaluated relapsed within 12 months. Median duration of TKI therapy for the whole group was 5.8 years (range 2.2- 14 years). 20 patients achieved TFR success with a median follow up of 24 months (TFR group; sustained BCR-ABL1 <0.1% (IS) after TKI discontinuation for ≥12 months), while 31 patients relapsed (Relapse group; BCR-ABL1 >0.1% (IS) after stopping; median time of relapse 3 months, range 1-10 months).A ROC curve analysis correlated TFR outcome with ddPCR results, with BCR-ABL1 level ≥0.003% via ddPCR at the time of stopping identified as an optimal cut-off. Kaplan-Meier analysis showed that 89% of the patients with ddPCR ≥0.003 relapsed after TKI cessation, whereas the ddPCR <0.003 demonstrated a significantly reduced relapse rate to 54% (p=0.01, Figure 1A). In addition, the TFR group (median BCR-ABL1 0.00065%) demonstrated approximately two-fold lower levels of BCR-ABL1transcript level compared to the relapse group (median 0.0012%). Interestingly, 7/31 (23%) of the relapsed group had undetectable BCR-ABL1 transcript even with the current highly sensitive method, while this undetectable level was only observed in 35% of the TFR group.We next assessed other known predictors of TFR success relative to ddPCR results in a Cox proportional hazard model. We have previously demonstrated that the BCR-ABL1 halving time after commencing therapy is highly predictive of TFR. At a univariate level, transcript type (e13a2 versus e14a2, p=0.01), BCR-ABL1 halving time (p>0.0001), and mRNA quantitation by ddPCR ≥ 0.003% (p=0.02) were all significantly associated with clinical outcome. Other variables including gender, age, ELTS score, Sokal score, MR4.5 duration and TKI duration were not associated with clinical outcome in this cohort (Figure 1B). In the multivariate analyses (Figure 1C), ddPCR remained an independent predictor after adjusting for ELTS, TKI duration and MR4.5 duration. Interestingly, ddPCR was not an independent predictor after adjusting for BCR-ABL1 transcript type or halving time.Conclusion: QXDx ddPCR assay is a promising tool for molecular residual disease monitoring in CML, especially when the BCR-ABL1 is undetectable by conventional method. The CML patients with levels of detectable BCR-ABL1 ≥0.003% measured by ddPCR have a significantly higher probability of relapse compared to patients with lower levels of the transcript. The ≥0.003% BCR-ABL1 level cut-off value could be a potential tool to aid decision-making when attempting TKI discontinuation in CML. However, even though a measurable level of BCR-ABL1 above 0.003% via ddPCR identified patients at high risk of relapse after a TFR attempt, it does not rule out the possibility of TFR; and a negative ddPCR result does not exclude the risk of molecular relapse. ddPCR may be most useful where other TFR predictive factors including BCR-ABL1 transcript type and halving time are not available.In-kind support was received from Bio-Rad for this study. [Display omitted] DisclosuresBranford: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; Incyte: Honoraria; BMS: Honoraria, Research Funding. Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

Interim Results of a Phase II Study of Dasatinib and Prednisone As an Oral, Chemo-Free Induction and Consolidation Regimen for Adults with Newly Diagnosed Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

BackgroundTyrosine kinase inhibitors (TKIs) combined with intensive chemotherapy as a first-line treatment regimen significantly improved the prognosis of Philadelphia (Ph) chromosomal positive acute lymphoblastic leukemia (Ph+ALL). However, it has been reported that 74% of patients fail to complete 8 cycles of chemotherapy, and the early mortality rate during induction was as high as 13%. Our previous retrospective study determined an oral, chemo-free regimen (dasatinib plus prednisone) as induction and consolidation yields 100% of complete remission (CR) rates in Ph+ ALL with minimal induction death (Li XY, et al. Br J Haematol. 2020;189:e231-e234). To further verify the effectiveness of this completely oral and chemo-free regimen, we conducted a phase II, one-arm and multicenter trial (ChiCTR200038053).MethodsAdults ≥16 years of age with new diagnosed Ph+ALL were eligible. Induction (Course I) used dasatinib 100 mg daily on days 1-28 and prednisone 1mg/kg/daily on days 1-21, 0.5 mg/kg/daily on days 22-28. Patients not in CR/CRi received Course I as second induction again. Two courses of consolidation (Course II and III) used dasatinib 100 mg daily on days 1-28 and prednisone 1mg/kg/daily on days 1-7. CNS prophylaxis used intrathecal cytarabine and dexamethasone. Patients 16-65 years old underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) if they had a HLA-matched donor; otherwise they received 6 cycles of hyper-CVAD chemotherapy alternating regimen-B (methotrexate, cytarabine) and regimen-A (cyclophosphamide, doxorubicin, vincristine, dexamethasone). The dose of chemotherapy was reduced by a third in patients 60-70 years old and by half in patients over 70 years. The primary objectives were to determine induced complete remission rate and major molecular response of the dasatinib/prednisone oral, chemo-free regimen.ResultsTo date, 32 patients from 10 centers enrolled. Median age was 54 years (range: 15-84). The dominant BCR-ABL1 isoform was p190 in 72%, p210 in 18%, rare types in 6.3% and not reported in 3.1%. Patients received an average of 2.5 courses of dasatinib/prednisone regimen (Course I-III). Most patients were tolerable the dose of dasatinib, and two patients were reduced to 50mg daily due to severe pleural effusion. Allo-HSCT was performed in 10 patients, 50% after remission and 50% after marrow relapse.As shown in Figure 1, the CR rate was 97% with one induction death due to sepsis. The major molecular response (MMR was defined as: 3-log reduction in BCR-ABL1 transcript) was 50% and the complete molecular response (CMR was defined as: the absence of a detectable BCR-ABL1 transcript with 0.01% sensitivity/4.5-log) was 40% either at 3 months after starting induction or before HSCT, respectively. From Course I to Course II, the CMR increased from 18.5% to 48%, but accompanied by an increase in marrow relapse rates, which were 16% and 18.75% at course II and course III, respectively. Marrow relapse occurred in 9 patients, all occurred within 6 months after starting induction. T315I mutation in ABL1 KD was detected in 5 of 7 marrow relapses (72%). With a median follow up of 232 days (range: 77-392 days), 1-year OS and 1-year event-free survival (events were defined as marrow relapse and death) was 97% and 54%, respectively (Figure 2). The cumulative molecular relapse rate (defined as BCR-ABL1:ABL1 ratio rises after reaching the CMR level) was 35% at 3 months, 48% at 6 months, and 54% at 1 year (Figure 2).ConclusionsThe chemotherapy-free, oral combination of dasatinib and prednisone appears promising induction efficacy in de novo Ph+ ALL; the high CR rates and acceptable molecular response rates were similar to results reported by GIMEMA LAL2116 study (Foà R, et al. N Engl J Med. 2020;383:1613-1623). However, two subsequent consolidation courses of the chemo-free regimen led to early molecular relapse with T315I mutations, which urged us to modify the study protocol such as strengthening consolidation therapy or incorporating promising drug blinatumomab. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment-Free Remission: the New Goal in CML Therapy.

Purpose of ReviewTreatment-free remission (TFR) is considered one of the main goals of therapy in patients with CML. Our goal in this paper is to review the current data on TFR, and discuss future directions.Recent FindingsMultiple studies have demonstrated that attempting a treatment-free remission is safe and effective in a select group of patients. More recent data suggested that undetectable BCR-ABL1 by digital PCR prior to discontinuation is highly predictive of successful TFR. However, some patients have a successful TFR with no evidence of clinical disease despite persistent detectable BCR-ABL1. Some recent studies have shed some more light on possible mechanisms for this phenomena. Some possible mechanisms include immune mechanism, BCR-ABL1 detected in the lymphoid component only, or stem cell exhaustion.SummaryTFR should be discussed with patients with CML. Patients who achieve a sustained deep molecular response may be eligible to attempt TFR, however, setting expectations that overall only 20% of patients with newly diagnosed CML will achieve a successful TFR. The importance of compliance to treatment early on cannot be overemphasized. Further studies using other drugs to get patients to a deeper remission in order to be eligible for TFR attempt, or attempting a second TFR in patients who had disease recurrence after first TFR attempt, are currently underway.

“Duration of Deep Molecular Response” Has Most Impact on the Success of Cessation of Tyrosine Kinase Inhibitor Treatment in Chronic Myeloid Leukemia - Results from the EURO-SKI Trial

“Duration of Deep Molecular Response” Has Most Impact on the Success of Cessation of Tyrosine Kinase Inhibitor Treatment in Chronic Myeloid Leukemia - Results from the EURO-SKI Trial

Persistence of Residual Circulating CD26+ Leukemia Stem Cells in Chronic Myeloid Leukemia Patients in Treatment Free Remission

Chronic myeloid leukemia (CML) patients achieving a deep and stable molecular response with tyrosine kinase inhibitors may discontinue TKI therapy and count on 50% about probability to maintain a “treatment free remission” (TFR) which is today the best surrogate for “cure” in CML. Albeit indispensable for attempting TFR, deepness and duration of molecular response are not the only criteria for a successful TFR as half of CML patients (pts) meeting those criteria still loose the response within 6-12 months after TKI discontinuation. Available data suggest that relapse after TKIs withdrawal is due to the persistence of leukemic stem cells (LSCs) intrinsically resistant to TKIs, however the presence and the impact of residual LSCs in CML patients in TFR has not been explored yet.CD34+/CD38-/Lin- CML LSCs specifically co-express dipeptidylpeptidase IV (CD26). We have recently demonstrated that CD26+ LSCs are easily detectable by flow-cytometry also in peripheral blood of CML pts both at diagnosis and during tyrosine kinase inhibitors (TKIs) treatment (Bocchia M, Blood 2016 128:942). The number of circulating residual CD26+LSCs appeared not correlating to the degree of molecular response, suggesting that residual TKI resistant quiescent LSCs may be transcriptionally low/silent and thus potentially not quantifiable by standard qRT-PCR analysis.We here evaluated the presence of circulating CD26+ LSCs in CML patients in TFR referring to several Italian hematology centers. During a follow-up visit, in which patients were checked for standard PB BCR-ABL1 molecular response, 3-6 mls of PB were collected in EDTA and sent to Siena Hematology Lab to detect CD34+/CD38-/CD26+ LSCs by multicolor flow cytometry. Cells were incubated with anti CD45, CD34 (581), CD38 (HIT2), CD26 (M-A261). Acquisition and analysis were performed by FACSCantoII using DIVA8 software.A total of 84 CML pts in TFR for a median of 31 months (range 2-192) were evaluated. All 84 patients were in first line treatment at the time of TKI withdrawal. Fifty-one out of 84 (60%) pts discontinued imatinib treatment and were in TFR for a median of 37 months (range 2-192); 14/84 (17%) pts discontinued dasatinib and were in TFR for a median 32 months (range 5-90) and 19/84 (23%) pts were in TFR for a median of 18 months (range 3-39) after nilotinib treatment.PB CD26+ CML LSCs were measurable in 54/84 (64%) pts with a median number of 0,0132 cells/µL (range 0,0053-1,77) while only 24/84 (28%) pts showed detectable BCR-ABL copies (median BCR-ABL/ABLIS ratio 0,0062 range 0,0032-0,493). Indeed, 18/84 (21%) pts showed both circulating CD26+ LSCs and detectable BCR-ABL, 26/84 (31%) pts scored negative for both determinations, while 36/84 (43%) pts showed detectable CD26+ LSCs and undetectable BCR-ABL. Only 4/84 (5%) pts showed measurable BCR-ABL copies with undetectable CD26+ LSCs.Kendall rank correlation coefficient, Mood test and bi-linear relation model of the whole cohort showed no correlation between BCR-ABL/ABLIS ratio and number of residual LSCs, while a significant linear inverse correlation was found between number of circulating CD26+ LSCs and TFR duration (r=-0.055 p <0.01 p=0.0063). Similar results were found also when considering only 51 pts in TFR after imatinib. A statistical analysis focusing on dasatinib and nilotinib subgroup of pts was not possible for due to the reduced sample size.This is the first time that residual circulating CML LSCs are detected in TFR CML pts even after long lasting TKI discontinuation and deep stable molecular response. Intriguingly, the evaluation of CD26+ LSCs appeared a more sensitive method than qRT-PCR to detect residual “quiescent” disease. The reason why CML LSCs may persist in pts who discontinued TKI, without resulting in an overt disease, it's still unknown but likely a CML LSCs specific immune control plays a crucial role. An ongoing prospective study, recruiting a consistent number of pts, monitoring the dynamics of circulating CD26+LSCs from TKI discontinuation may answer some still open questions. LSCs' clinical role, correlation with type and duration of previous TKI treatment as well interaction with active specific immune system will be investigated. DisclosuresBocchia:Roche: Other: Travel grant; Celgene: Other: Travel grant; Novartis: Other: Travel grant; Jansen: Other: Travel grant. Abruzzese:Novartis: Consultancy; Incyte: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Galimberti:Incyte: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau. Fava:Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Castagnetti:Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Iurlo:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Crugnola:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Rosti:Pfizer: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Comparation of Digital PCR and Quantitative Pcr for Molecular Monitorization of Chronic Myeloid Leukemia Patients

Introduction: Chronic myeloid leukemia (CML) is a neoplasm produced by the formation of the BCRABL fusion gene. Tyrosine kinase inhibitors (TKI) have changed dramatically the clinical course of the disease. Quantitative PCR (qPCR) is the reference technique for measuring response in CML patients. Droplet digital PCR (ddPCR) is, in brief, a technology based on partitioning of the sample in a high number of small fractions which, in a further step, are read as positive or negative by its fluorescence. ddPCR performs an absolute quantification of the number of transcripts and their results do not depend on PCR efficiency. In spite of these theoretical advantages over qPCR, ddPCR is not routinely applied for BCRABL monitoring. In this study, we sought to compare qPCR and ddPCR techniques in similar amplification conditions.Methods: We included samples from 43 CML patients with different levels of response determined by qPCR: 23 patients with BCRABL/ABL1 levels <15%, 7 patients with MR3.0, 10 patients with MR4.0 and 3 patients who showed MR4.5 (according to European LeukemiaNet guidelines). Additionally, 3 non-CML patients were included. High-Capacity cDNA Reverse Transcription Kit was employed, following manufacturer instructions, in a unique reaction for both subsequent technologies utilized. qPCR was carried out with primers and probes for BCRABL and ABL1 from Europe Against Cancer report with 5 microL of non-measured cDNA in duplicates. The standards were BCR-ABL P210 ELITe Standard (ELITech Group) and the conversion factor was obtained with the Philadelphia P210 RNA Reference (Elitech Group). qPCR assys were run in an ABI 7500 platform in standard quantitation mode. EUTOS recommendations for BCRABL quantification were followed.ddPCR reaction was performed with the same primers and probes. Median cDNA concentration was 4,485ng/µL (1,771-7,787ng/µL). In order to avoid saturation of the technology, 2microL of cDNA at a concentration of 600ng/microL (i.e. 1,200ng) were added and run in duplicates in a C1000 touch Thermal Cycler (Bio-RAD) with the same amplification conditions used in qPCR. The same RNA reference material was used to obtain the calibration factor for the ddPCR technique. Bio-Rad QX200 Droplet Digital PCR System was employed for analysis. We used the mode of analysis for rare event detection and thresholds were set individually in each reaction. Qualitative comparations were carried out using Kappa coefficient with quadratic weight. For quantitative comparations, we applied Pearson correlation, intraclass correlation coefficient for absolute agreement and Passing-Bablok tests. A subgroup analysis, including samples with MR3.0 or deeper, was also performed.Results: All the samples showed ≥10000 ABL copies per well by qPCR. More than 10000 events were analyzed in every ddPCR reaction well. Good replicates were observed in qPCR and ddPCR. Adequate separation of positive and negative events could be performed and no saturation was observed in the ddPCR reations. No signal from BCRABL was detected in non-CML patients in ddPCR. The Kappa coefficient between qPCR and ddPCR measurements was 0.76 (P< 0.001, 95% confidence interval (CI): 0.63-0.88). In the 20 samples with MR 3.0 or deeper, kappa=0.13 (P >0.05 ) .For quantitative comparison, 32 samples with detectable BCRABL were included. Pearson test yielded an R2=0.91 (P< 0.001). The intraclass correlation coefficient for absolute agreement showed a value of 0.96 (P <0.001). Passing-Bablok test provided a non significant constant difference (0.041, CI 95%: -0.04-0.05) and a significant proportional difference (0.8, CI 95%: 0.7-0.96) (i.e. ddPCR results= 0.041 + (0.8*qPCR results). There were only 9 samples with a BCRABL/ABL1 ratio and quantitative test were not performed.Conclusion: Although a global excelent qualitative correlation is observed, in patients with MR3.0 or deeper which are the more important to classify correctly, qualitative correlation is absent. The R2 and intraclass correlation coefficient for absolute agreement showed a high concordance but Passing-Bablok test yielded a significant proportional difference among qPCR and ddPCR. We sought to test both technologies in similar conditions but different ddPCR strategies could be implemented in order to improve reproducibility of both techniques: higher amount of replicates or, given the lack of saturation, a higher cDNA concentration in the reactions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Challenging the Prognostic Value of MRD: Sequential Tumor-Ablative Conditioning of FA5-Bucy for Ph-Positive Acute Lymphoblastic Leukemia

Background:Minimal residual disease (MRD) quantification prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a powerful predictor for post-transplant outcome. Thus, new strategies with modified transplantation procedures are needed to reverse the outcomes of that remain MRD-positive despite multiple induction attempts.OBJECTIVE:To determine the prognostic relevance of MRD status in patients with acute lymphoblastic leukemia (ALL), especially those with Philadelphia chromosome-positive ALL (Ph+ ALL), receiving the conditioning regimen of FA5-Bucy, registered on http:// ClinicalTrials.gov (NCT02328950).METHODS:We retrospectively reviewed our HSCT experience using FA5-Bucy, i.e., 5-day salvage chemotherapy (Fludarabine/Ara-C) and conditioning (Busulfan/ Cyclophosphamide) for patients with ALL. A total of 48 consecutive patients from 2013 to 2016 are analyzed. 11 out of 48 (7 males and 4 females) were diagnosed with Ph+ ALL with the induction treatments including a tyrosine kinase inhibitor (TKI). Median age was 30 years (range, 8-52 years). Among whom four (36.4%) achieved complete remission(CR)at transplantation, Six (54.5%) with detectable residual P190 BCR-ABL positive leukaemic cells, while 1 (9.1%) not in the status of remission with 39.5% blasts in the bone marrow (BM). The other 37 cases were Ph-negative (Ph-) ALL, with the median age of 21 years (range, 2-61 years). Among whom 25 (67.6%) patients were in CR, 6 (16.2%) MRD-positive and 6 (16.2%) with 23.5%-98% BM blasts. The MRD status was monitored by four-color FCM in Ph- group, and by RT-PCR to detect the chimeric bcr-abl mRNA transcript in Ph+ group. Patients were transplanted either from an HLA-identical sibling or haplo-identical related donors. The GVHD prophylaxis consisted of ATG, CsA, MMF and short-term MTX. None of these patients had severe infection or organ failure before HSCT.RESULTS:All patients achieved successful engraftment and full donor chimerism. Patients with Ph+ ALL seems have a better outcome than that with Ph- ALL (3-year OS of 54.5% vs 37.8%, P=0.245). With a median follow-up of 666 (83-1276) days, the 1y-OS and 1y-DFS were 72.7%, 3y-OS and 3y-DFS were 54.5% in Ph+ group, respectively, and non-relapse mortality and relapse incidence were 18.2% and 10%. MRD monitoring is an important tool for relapse prediction in Ph- ALL patients (3-year OS and DFS of MRD-positive vs MRD-negative =16.7% vs 56%, P=0.021).However, the profile of OS and DFS was similar in Ph+ patients with bcr-abl positive to that with bcr-abl negative (3-year OS and DFS of bcr-abl positive vs bcr-abl negative= 57.1% vs 50%, P=0.744). Severe acute-GvHD (aGVHD) occurred in 3/11 patients. Few patients experienced mild-to-moderate toxicity, and main causes of death were aGVHD.CONCLUSION:We concluded that allo-HSCT with the sequential tumor-ablative conditioning of FA5-Bucy offers great benefits for the patients with Ph+ ALL, with the potentially capability of reducing or overcoming the negative impacts of MRD. Thus creates exciting transplant opportunities for patients not in remission to be cured. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment Outcome of Dasatinib in Combination with Less-Intensive Induction, Homobarringtonie, Cytarabine, G-SCF in Newly Diagnosed Patients with Ph+ALL

Unprecedented achievement has been made in the treatment of childhood ALL within the past decades. However, how to improve the outcome of Ph+ALL remains a conundrum for the care providers towards the cure of the disease. Using Auto-hematopoietic stem cell transplant (HSCT), after a negative BCR-ABL, or allo-HSCT has become the curative options for Ph+ALL cases although either relapse or life threatening transplant related complications could lead to treatment failure.To determine the safety and efficiency of a dasatinib based mild induction with homobarringtonie, cytarabine, G-SCF (HAG) contained regimen, we enrolled 67 patients with de novo Ph+ALL from 2012-2017 for the trial. The medium follow-up time was 39 months (17 months to 65 months). In brief, the induction consisted of vincristine and prednisone, in addition to daily dasatinib (70mg; bid), followed by six cycles of HAG regimen (homobarringtonie 1mg/m2/days 1-14; G-CSF 5ug/kg on day one; Ara-C 10mg/q12h/days 1-14; every three month/cycle) plus daily dasatinib (70mg; bid). The maintenance included two cycles of HAG (every six months) in addition to daily dasatinib. The intrathecal injection was given 11 times along with the treatment to prevent CNS leukemia. A serial MRD detection of BCR-ABL by qPCR was performed every three months.There were four patients eligible for the study. Among the 63 evaluated patients, 55 (88%) achieved CR in induction. There was no treatment related mortality during the induction. Good prednisone response was indicated among 51 patients (81%) on day eight. The day 30 MRD detection for those in CR indicated 43 (68%) reached ≤5 *10-2 and four (7%) achieved 10-3. The MRD detection at month three and six suggested that 38 (60%) vs 33 (52%) patients were within the level of ≤5 *10-2 and six (10%) vs 10 (16%) achieved 10-3, respectively. Relapse-related death occurred in 11 of 63 patients (16%), and four patients (6%) experienced treatment-related mortality. Grade 3-4 adverse events were observed among 10 cases (30%). The main toxicities were liver dysfunction and coagulation abnormality which can be solved by close monitoring and multi-disciplinary care. Four-year probability of event-free survival (pEFS) was found to be 58%. Those who went with hematopoietic stem cell transplantation (HSCT) after chemotherapy achieved significantly better pEFS than only chemotherapy (65.7% vs 50.5%,p=0.03). Our protocol can be used as an effective and less toxic treatment option to improve the outcome of Ph+ALL with promising survival and acceptable adverse effects shedding new light on treating Ph+ALL. DisclosuresNo relevant conflicts of interest to declare.

Long-Term Follow-up of the ALLG CML8 Twister Study of Treatment-Free Remission (TFR) in Patients with Chronic Myeloid Leukemia (CML)

Long-Term Follow-up of the ALLG CML8 Twister Study of Treatment-Free Remission (TFR) in Patients with Chronic Myeloid Leukemia (CML)

Assessment of Outcomes After Stopping Tyrosine Kinase Inhibitors Among Patients With Chronic Myeloid Leukemia

Tyrosine kinase inhibitors (TKIs) have been associated with improved survival of patients with chronic myeloid leukemia (CML) but are also associated with adverse effects, especially fatigue and diarrhea. Discontinuation of TKIs is safe and is associated with the successful achievement of treatment-free remission (TFR) for some patients. To evaluate molecular recurrence (MRec) and patient-reported outcomes (PROs) after TKI discontinuation for US patients with CML. The Life After Stopping TKIs (LAST) study was a prospective single-group nonrandomized clinical trial that enrolled 172 patients from 14 US academic medical centers from December 18, 2014, to December 12, 2016, with a minimum follow-up of 3 years. Participants were adults with chronic-phase CML whose disease was well controlled with imatinib, dasatinib, nilotinib, or bosutinib. Statistical analysis was performed from August 13, 2019, to March 23, 2020. Discontinuation of TKIs. Molecular recurrence, defined as loss of major molecular response (BCR-ABL1 International Scale ratio >0.1%) by central laboratory testing, and PROs (Patient-Reported Outcomes Measurement Information System computerized adaptive tests) were monitored. Droplet digital polymerase chain reaction (ddPCR) was performed on samples with undetectable BCR-ABL1 by standard real-time quantitative polymerase chain reaction (RQ-PCR). Of 172 patients, 89 were women (51.7%), and the median age was 60 years (range, 21-86 years). Of 171 patients evaluable for molecular analysis, 112 (65.5%) stayed in major molecular response, and 104 (60.8%) achieved TFR. Undetectable BCR-ABL1 by either ddPCR or RQ-PCR at the time of TKI discontinuation (hazard ratio, 3.60; 95% CI, 1.99-6.50; P < .001) and at 3 months (hazard ratio, 5.86; 95% CI, 3.07-11.1; P < .001) was independently associated with MRec. Molecular recurrence for patients with detectable BCR-ABL1 by RQ-PCR was 50.0% (14 of 28), undetectable BCR-ABL1 by RQ-PCR but detectable by ddPCR was 64.3% (36 of 56), and undetectable BCR-ABL1 by both ddPCR and RQ-PCR was 10.3% (9 of 87) (P ≤ .001). Of the 112 patients in TFR at 12 months, 90 (80.4%) had a clinically meaningful improvement in fatigue, 39 (34.8%) had a clinically meaningful improvement in depression, 98 (87.5%) had a clinically meaningful improvement in diarrhea, 24 (21.4%) had a clinically meaningful improvement in sleep disturbance, and 5 (4.5%) had a clinically meaningful improvement in pain interference. Restarting a TKI resulted in worsening of PROs. In this study, TKI discontinuation was safe, and 60.8% of patients remained in TFR. Discontinuation of TKIs was associated with improvements in PROs. These findings should assist patients and physicians in their decision-making regarding discontinuation of TKIs. Detectable BCR-ABL1 by RQ-PCR or ddPCR at the time of TKI discontinuation was associated with higher risk of MRec; clinical application of this finding should be confirmed in other studies. ClinicalTrials.gov Identifier: NCT02269267.

Nilotinib Vs. Dasatinib in Achieving MR4.5 for Newly Diagnosed Chronic Myeloid Leukemia: Results of the Prospective Randomized Phase 3 Study, JALSG CML212

Nilotinib Vs. Dasatinib in Achieving MR4.5 for Newly Diagnosed Chronic Myeloid Leukemia: Results of the Prospective Randomized Phase 3 Study, JALSG CML212

Interim Results of the Phase I/II Study of the Ponatinib, Venetoclax and Dexamethasone for Patients with Relapsed or Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Interim Results of the Phase I/II Study of the Ponatinib, Venetoclax and Dexamethasone for Patients with Relapsed or Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

Allogeneic stem cell transplant (HSCT) for acute lymphoblastic leukaemia (ALL) using CD34 selected stem cells followed by prophylactic infusions of pathogen-specific and CD19 CAR T cells

Background & Aim Disease relapse, graft versus host disease (GVHD) and infection are responsible for the majority of deaths after HSCT. We assessed a cellular engineering approach to simultaneously reduce the incidence of these complications using CD34+ stem cell selection combined with post-transplant infusion of pathogen-specific and leukemia-specific T cells. Methods, Results & Conclusion METHODS We conducted a pilot study in which patients with ALL in remission were treated with a CD34-selected stem cell transplant followed by two planned prophylactic infusions of donor-derived T cells targeting opportunistic pathogens (CMV, EBV and Aspergillus), and leukaemia (CD19 CAR T cells). Cellular products were manufactured locally. Patients did not receive anti-thymocyte globulin or post-transplant GVHD prophylaxis. RESULTS We performed matched sibling donor transplants on two patients aged 45 and 27 years with ALL in morphological CR. Patient 1 had Ph1 positive ALL with detectable BCR-ABL at the time of transplant (0.001%). Patient 2 was Ph1 negative and had no detectable disease by flow cytometry pre-transplant. Conditioning was cyclophosphamide 120mg/kg and TBI 1200cGy. Patients received CD34 selected stem cells (total CD34+ doses 3.5 and 3.6 × 106/kg; total CD3+ cell doses 1.3 and 0.2 × 104/kg). Neutrophil engraftment (>0.5) occurred on days 11 and 12, platelet engraftment (>20) on days 8 and 13. Patients received infusions of pathogen specific T cells (day 21) and CD19 CAR T cells (days 27 and 21). CRS (grades 1 and 2) and neurotoxicity (grade 1) developed in both patients; treated with tocilizumab, patient 1 also received dexamethasone. CAR T cells proliferated in vivo despite low disease burden and persisted in blood for at least 6 weeks in both patients (Figure 1). Patient 1 received foscarnet for 9 days and a second pathogen-specific T cell infusion on day 47 post-transplant. Patient 2 developed asymptomatic reactivation of HHV6 and BK virus. At 364 and 350 days post-transplant, both patients are well, free of GVHD and remain in remission. CONCLUSION This is the first description of a strategy that combines prophylactic donor-derived CAR T cell and pathogen-specific T cell administration after HSCT in the context of CD34+ selection to minimise development of GVHD. Our results demonstrate absence of GVHD, minimal treatment for viral reactivation and remission at 12 months post-transplant suggesting that this approach has promise for improving GVHD relapse free survival following HSCT.

EFFICACY OF NILOTINIB IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

ABSTRACT:MATERIAL AND METHODS: This study was conducted on 92 diagnosed cases of chronic myeloid leukemia at Department of Oncology, Jinnah Hospital Lahore from August 2016 to January 2017. Patients from either gender, between the ages of 20 to 60 years were included in the study while patients having diabetes and end stage renal disease with glomerular filtration rate less than 15 ml/min were excluded. Nilotinib treatment with the standard dose (300 mg twice daily) was given to patients with chronic phase of chronic myeloid leukemia (CP-CML). Patients were monitored as recommended by the current treatment guidelines. Treatment outcome of CP-CML in terms of efficacy was assessed at the end of 6 months of treatment.OBJECTIVE: To determine the efficacy of nilotinib in patients of chronic myeloid leukemia, chronic phase, in terms of detection of BCR-ABL by FISH method. RESULT: The mean age of the patients was 38.84 ± 11.67 years, with male to female ratio of 1.04:1. The mean PH value of the patients was 17.05 ± 18.53 and efficacy was achieved in 36 (39.13%) patients.&#x0D; CONCLUSION: The efficacy of nilotinib was achieved in significant number of CML patients.

Lineage of measurable residual disease in patients with chronic myeloid leukemia in treatment-free remission.

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.

ASXL1 Mutations Detectable at Diagnosis May Predict Response to Imatinib in Patients with Chronic Myeloid Leukemia

Clinical resistance to tyrosine kinase inhibitors (TKI) remains a significant problem in the therapy of patients with chronic myeloid leukemia (CML). Although BCR-ABL1-kinase domain mutations are the major cause of resistance to TKI, some patients manifest primary or develop secondary resistance to TKI without detectable BCR-ABL1 mutations. We aimed to assess the prevalence of additional gene mutations in a group of 50 patients with primary (n = 26) or secondary resistance (n = 24) to TKI, in most cases to imatinib (n = 49) and in one to dasatinib. We employed ~1000 genes custom target enrichment kit and next-generation sequencing on Illumina platform, as well as Sanger sequencing. In 21 patients, we were able to match and analyze paired samples collected at diagnosis (before treatment) and at the time of resistance and in five other cases we analyzed additional samples collected later during TKI-resistant chronic phase or in blast crisis. The most frequent genetic aberrations detected at the time of TKI-resistance were mutations in ASXL1 and BCR-ABL1 kinase domain coding sequence, present in 28% (14/50) and 26% (13/50) of patients, respectively. Both genes were mutated in 12% of patients (6/50), while 30% (15/50) had either ASXL1 or BCR-ABL1 mutations alone. Non-recurrent genetic aberrations in other genes were also noted in single patients (e.g. DNMT3A mutations). According to COSMIC database, all but three ASXL1 mutations detected in this study were previously described in hematologic malignancies and all (including the novel ones) are predicted to introduce stop codons or cause frameshifts in codon range of 512-943, thus truncating the protein before the C-terminal PHD domain. Among patients with available paired samples three had more than one ASXL1 mutation at various time points and in all of them we observed dynamics of specific ASXL1 mutations, related partially to the leukemic cell content in the sample but also providing evidence of clonal evolution (Table 1). In most of patients with concurrent ASXL1 and BCR-ABL1 mutations, we observed that both mutations were present with similar variant allele frequency (VAF), suggesting that ASXL1 mutations occur in Philadelphia-positive leukemic clones (Table 2). ASXL1 mutation frequency was significantly higher in TKI-resistant patients, than in our previously characterized group of CML patients without resistance, who achieved major molecular response (28% ; 14/50 vs 5.6% ; 2/36, respectively, p = 0.0105, Fisher exact test). All ASXL1-mutated patients with secondary (n = 1) or primary resistance (n = 5) for whom diagnostic sample was available, carried ASXL1 mutation also at the time of diagnosis. No significant difference in mutation frequency was found between patients with primary and secondary resistance (ASXL1 mutation, 26.9% vs 29.2% ; BCR-ABL1 mutation, 15.4% vs 37.5%). Our results provide evidence that preexisting ASXL1 mutations in BCR-ABL1-positive leukemic clone present at diagnosis may have impact on clinical response to imatinib and may be useful in assessing the risk of treatment failure. Disclosures Niesiobedzka-Krezel: Novartis: Honoraria. Seferynska:Novartis: Honoraria. Gora Tybor:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sacha:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Optimum Imatinib Exposure Have Possibility of Leading to Appropriate Immune Response after Imatinib Discontinuation in CML Patients

Introduction: Imatinib, the first tyrosine kinase inhibitor (TKI), has dramatically improved the prognosis of chronic myeloid leukemia (CML) patients. Recently, many trials of TKI discontinuation revealed that approximately 40% to 60% of CML patients who treated long time TKI therapy reached the treatment free remission (TFR), thus now TFR is proposed as one of the goals in CML treatment. Achieving deep molecular response (DMR) by TKI therapy is a minimum requirement of challenge to TKI discontinuation in CML patient, actually CML patients with molecular residual disease (MRD) showed worse consequence than undetectable MRD (IJH 2017). On the other hand, it was known that some patients have continued TFR with detectable BCR-ABL fusion gene, these patients hadn't shown indubitable molecular relapse while BCR-ABL+ malignant cells continued to exist for prolonged time. We hypothesized that the malignant cells were eliminated by host immune systems in these fluctuated patients. Here, we focused on T-cell response, so we analyzed T-cell related markers to identify biomarkers that can predict patients which can continue TFR or not in Japanese CML patients. Furthermore, we confirmed the action of imatinib for T-cell response in vitro. Methods: Japanese CML patients treated with imatinib for at least three years and confirmed in DMR for at least two years were eligible. Patients who received other TKI or stem cell transplantations were excluded. Patients were re-confirmed in MR4.5 before discontinue imatinib and they were sampled peripheral blood at pre- and 1, 3 months after stopping imatinib (figure 1). Peripheral blood mononuclear cells (PBMCs) were subjected to staining with T-cell markers and analyzed by mass cytometry and flowcytometry. Plasma were subjected to detecting Imatinib trough concentrations. Purchased PBMCs of healthy individuals were cultured and analyzed by flowcytometry in vitro assay. Results: Samples of 68 CML patients were analyzed. We classified these CML patients into two groups (Non-retreatment and Retreatment groups) by clinical courses after stopping imatinib (figure 2). Frequency of CD4+ T cells and CD8+ T cells in CD3+ T cells were no difference between both groups. FoxP3+CD4+ regulatory T cells (Treg) were also no difference between both groups, but kinetics of Treg, especially Fraction II (Fr.II : FoxP3hiCD45RA-) of Treg from Pre-stopping imatinib to 1 month after stopping imatinib significantly increased in non-retreatment groups. Kinetics of Treg / CD8+ T cells ratio also significantly increased in non-retreatment groups, and predicted curve made by these kinetics of each groups were significant (figure 3). The expression of PD-1 or other suppressive co-stimulatory molecules in CD8+ T cells of non-retreatment groups at after stopping imatinib had tendency to decrease. Phosphorylated LCK in CD8+ T cells of non-retreatment groups at after stopping imatinib had tendency to increase. Next, we did in vitro assay to confirm the effect of pre-treatment of imatinib in imatinib free T cells. Pre-treatment of imatinib suppressed the proliferations of Treg Fr.II after TCR stimulation dose dependently, but not CD8+ T cells (figure 4). Frequency of phosphorylated LCK in Treg Fr.II increased after TCR stimulation even if pre-treated imatinib at reasonable dose, but didn't increased under the condition of high dose imatinib. Conclusion: Treg population and Treg / CD8+ T cells ratio in PBMCs elevated after stopping imatinib in non-retreatment groups of CML patients. Population of CD8+ T cells showed no differences in two groups but CD8+ T cells were tending to activate after stopping imatinib in non-retreatment groups. These data indicate that the kinetics of Treg after stopping imatinib connect with the immune response of imatinib discontinued CML patients. In vitro data indicate that Treg were more sensitive for imatinib treatment than CD8+ T cells, so kinetics of Treg may possibly become the biomarker of ability of immune responses. Our data suggested that optimum imatinib exposure induce appropriate immune responses leading good prognosis, and excess imatinib exposure induce exhaust immune responses leading poor prognosis. Disclosures Nishikawa: Taihou Pharmaceuticals: Research Funding; Kyowa Hakko Kirin: Research Funding; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding, Speakers Bureau; Chugai Pharmaceuticals: Research Funding, Speakers Bureau; Asahikasei Pharma: Research Funding; Sysmex: Research Funding; Daiichi Sankyo: Research Funding; Zennyaku: Research Funding. Takahashi:Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Chug Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Asahi Kasei Pharma: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Eisai Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding.

Conventional Real Time Quantitative Polymerase Chain Reaction Method Yields Similar Level of Sensitivity to Digital Droplet Polymerase Chain Reaction for Detection of BCR-ABL p210 Transcripts in Patients with Chronic Phase Chronic Myeloid Leukemia

Introduction: Several studies have investigated the utility of digital droplet polymerase chain reaction (ddPCR) in the detection of p210 BCR-ABL fusion transcripts in chronic myeloid leukemia (CML), and have suggested greater sensitivity compared to real-time quantitative PCR (RT-qPCR). There is limited data regarding the utility and sensitivity of ddPCR in patients with chronic phase CML (CP-CML) who are candidates for tyrosine kinase inhibitor (TKI) discontinuation. We aimed to investigate the capability of ddPCR to detect p210 BCR-ABL transcript levels in CP-CML patients and compare the sensitivity to RT-qPCR. Methods: We analyzed 103 different peripheral blood (n=98) and bone marrow (n=5) samples from 36 patients with CP-CML treated at Moffitt Cancer Center between 2013 and 2018. Samples were from patients who either met clinical criteria for TKI discontinuation or were enrolled in a phase I dose-escalation study evaluating combination therapy with nilotinib plus ruxolitinib who had at least a complete cytogenetic response (CCyR) to TKI therapy with detectable BCR-ABL transcripts by RT-PCR at the time of enrollment(1, 2). BCR-ABL levels were assessed by RT-qPCR at the time of collection with level of transcript reported as % International Standard (%IS). Samples collected from 2013 to 2014 were reported as p210 to ABL and converted to %IS per lab-specific conversion factor. Stored purified RNA samples were obtained retrospectively and ddPCR was performed using the QX-200 Droplet Digital PCR System and QXDx BCR-ABL %IS Kit (Bio-Rad). Droplets were analyzed by QXDx BCR-ABL reporter software to determine p210 transcript level (%IS). Analysis for RNA degradation of stored samples was performed via High Sensitivity RNA ScreenTape (Agilent). Samples that did not pass quality check upon performing ddPCR were excluded from further analysis. Correlation coefficient was calculated with p-value of &lt;0.05 being significant. Results: Of the 36 patients, 28 were in the TKI discontinuation cohort and 10 were enrolled in the clinical trial of nilotinib plus ruxolitinib. Two patients who participated in the clinical trial subsequently also met criteria for TKI discontinuation. In the TKI discontinuation cohort, 46% of patients (n=13) lost major molecular response (MMR) with a median time to loss of MMR of 88 days. We observed a statistically significant correlation between RT-qPCR and ddPCR levels when comparing %IS (correlation coefficient R2=0.42; p&lt;0.0001) as well as calculated molecular response (MR) logarithm values of all available samples (R2=0.67; p &lt;0.0001) (Figure 1). There was a weaker correlation between patients determined to have MMR by RT-qPCR (MR≥3.0) compared to ddPCR (R2=0.55; p &lt;0.0001) (Figure 2). A total of 91 different samples were consistent with MMR when tested by RT-PCR. Of these samples, 85.7% (n=78) were consistent with MMR when tested by ddPCR and the remaining 14.3% (n=13) appeared to have higher detectable levels of p210 transcripts (MR&lt;3.0). When investigating deeper molecular responses, 62 samples were consistent with MR≥4.0 by RT-PCR, and 20.9% (n=13) of these samples did not achieve a deep molecular response (MR&lt;4.0) when tested by ddPCR. Of note, 5 representative samples were also evaluated for potential RNA degradation and 3 were noted to have significant degradation present. Conclusion: Compared to RT-PCR, ddPCR represents a viable method for detection of p210 transcripts for patients with CP-CML. The two methods yield similar efficacy when detecting varying levels of molecular response, however further research is warranted for the ability to detect deeper molecular responses for patients in MMR. Our study is limited by inherent retrospective bias and quality of RNA in stored samples. 1. National Comprehensive Cancer Network. Chronic Myeloid Leukemia (Version 1.2019). 2. Sweet K, Hazlehurst L, Sahakian E, Powers J, Nodzon L, Kayali F, et al. A phase I clinical trial of ruxolitinib in combination with nilotinib in chronic myeloid leukemia patients with molecular evidence of disease. Leuk Res. 2018;74:89-96. Disclosures Talati: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Honoraria; Agios: Honoraria. Shelton:Bio-Rad: Employment. Nodzon:Pharmacyclics: Consultancy; Abbvie: Other: Speaker Fees; Genentech: Consultancy, Other: Speaker Fees; Pfizer: Consultancy. Sweet:Stemline: Consultancy; Pfizer: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Jazz: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees. Pinilla Ibarz:Novartis: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; TG Therapeutics: Consultancy; Teva: Consultancy; Sanofi: Speakers Bureau.