Successful Treatment-free Remission Research Articles

Advances in the Treatment of Chronic Myeloid Leukemia

Background: The treatment of chronic myeloid leukemia has progressed in recent decades, becoming a model for a disease whose pathogenesis is primarily based on a genetic mutation and has led to survivals comparable to those of the general population. Objective: This review aims to present recent therapeutic advances in this area. Methods: A mini-review was achieved using the articles published in Web of Science and Pub- Med between January 2021 - May 2022, and new patents were made in this field. Results: The three generations of tyrosine kinase inhibitors have transformed chronic myeloid leukemia into a manageable disorder and greatly improved the treatment results of the chronic phase, the prognosis, survival, and quality of life of patients. The therapeutic goals today include achieving a deep and lasting molecular response as soon as possible, successful treatment-free remission, and discovering and applying new therapeutic strategies to act on impaired immune modulation and dormant leukemic stem cells. The allosteric inhibitor asciminib targets the ABL myristoyl pocket, reduces Abl kinase activity, and is effective against most cells that have mutations in the ABL1 kinase domain. Progress and recommendations for achieving long-term treatment- free remission are set out. Nearly 50% of the patients who received first-line tyrosine kinase inhibitors required a change of treatment by 10 years due to intolerance or resistance to treatment. Their main side effects are presented. Conclusion: Obtaining a deep and persistent molecular response contributes to achieving longterm treatment-free remission.

How to improve treatment-free remission eligibility in chronic myeloid leukaemia?

The achievement of treatment-free remission (TFR) has become a significant clinical end-point in the management of patients with chronic myeloid leukaemia (CML), providing an opportunity to discontinue therapy with tyrosine kinase inhibitors (TKIs) while maintaining deep molecular response (DMR). Early studies, such as the French STIM trial, have demonstrated that a portion of patients can maintain DMR after treatment cessation, with rates ranging from 40% to 50%, and most relapses occurring within the first 6 months. Key prognostic factors for successful TFR, including treatment duration, duration of DMR, risk scores, and transcript type, have been identified. Optimal patient selection for TFR remains a challenge, but recent research provides insights into potential strategies to increase TFR eligibility. Evidence suggests that early intervention switching to achieve optimal response, treatment combinations, proactive switch in the case of absence of DMR, dose-optimization and induction-maintenance approach can improve molecular responses and, consequently, enhance TFR eligibility. In this review, we report and discuss all the potential therapeutic strategies that may enhance eligibility for a first attempt at TFR, with a particular emphasis on potential future approaches.

Improvement of Treatment-Free Remission Rate Following Discontinuation of BCR::ABL1 Tyrosine Kinase Inhibitors in Chronic Phase, Chronic Myeloid Leukemia

Backgrounds: Several studies have shown that approximately one-half of patients with chronic phase chronic myeloid leukemia (CP-CML) who receive sufficient tyrosine kinase inhibitors (TKIs) and achieve and maintain a deep molecular response (DMR) are able to successfully discontinue therapy. The main factors affecting Treatment free remission (TFR) are the duration of TKIs treatment and the duration of DMR maintenance during TKI treatment. Aims: To investigate impact of longer duration of DMR, we analyzed the outcome of patients with CP-CML who maintained DMR for more than 6 years during TKIs treatment and discontinued TKIs. The clinical and molecular immunological application criteria were also investigated for safer and more successful TFR. Methods: Among patients newly diagnosed with CP-CML and treated at least one of TKIs (Imatinib, Nilotinib, Dasatinib, Bosutinib, and Radotinib) as the first treatment, patients who maintained MR4.5 (BCR::ABL1 IS ≤0.0032%) continuously for at least 6 years through qRT-PCR tests were enrolled in this study. After discontinuation, molecular responses were monitored for up to six months per month, up to 12 months every two months, and every three months thereafter using the qRT-PCR method. In the case of relapse, defined as loss of major molecular response (MMR), the last TKI therapy was reintroduced and molecular responses after resumption were monitored monthly until achievement of MMR using qRT-PCR. Results: Altogether 116 patients (50 males and 66 females) discontinued TKI. Median age at diagnosis was 43.0 years (range, 40.8-45.3) and the percentages of patients with low, intermediate and high Sokal risk scores ware 58 (50.0%), 35 (30.2%) and 18 (15.5%) patients, respectively with unknown Sokal risk scores in 5 (4.3%) patients. Prior to discontinuation, all patients received TKIs for a median of 120.6 months (range, 113.9-127.3), and the duration of sustained MR4.5 was 75.0 months (range, 70.7-79.3). At the time of discontinuation, 93 patients (80.2%) were receiving their first TKI (70 imatinib, 13 nilotinib, 6 radotinib, and 4 dasatinib) while 13 (11.2 %) and 10 (8.6%) patients were receiving their second and more than three TKIs respectively. All patients who received more than one TKI had changed to adverse events. After a median follow-up from treatment discontinuation of 42.2 months (range, 35.3-49.1), 28 (24.1%) patients have lost their response at a median of 8.4 months (range, 4.9-11.9) after discontinuation; 18 (64.3%) patients had molecular relapse within 6 months of TKI discontinuation and 7 (25.0%) patients lost MMR after 12 months from discontinuation. TFR rates at 12, 24, and 48 months were 81.9%, 76.0%, and 71.0%, respectively. No progression toward advanced-phase CML occurred, and when 28 patients who lost MMR were retreated with same TKI, 26 patients (92.9%) re-achieved MMR with a median of 5.6 months (range, 4.0-7.2). By univariate analysis, a TKI treatment duration of 10 years or more was the only significant predictor for maintained response (P<0.048). Conclusion: Our result demonstrates that TFR rate can be improved in patients who received TKI treatment for approximately 10 years and maintained DMR for more than 6 years regardless of the type of TKI. Based on results, we can confirm one of important factors for safer TFR in CP-CML patients.

Successful Treatment-Free Remission in Low- and Middle-Income Countries

Introduction Discontinuation of tyrosine kinase inhibitors (TKIs) is feasible in a subset of chronic myeloid leukemia (CML) patients who have maintained a deep molecular response (MR) for ≥2 years. Numerous TKI discontinuation trials from high-income settings show that ~50% of patients relapse after discontinuing TKIs and need to restart treatment. However, most patients with CML worldwide are from low- and middle-income countries LMIC where TKI access and molecular testing make routine care and attempts of treatment-free remission (TFR) problematic. The successful implementation of TFR in LMICs would benefit both CML patients as well as the health economics of each country. This analysis describes the experience of attempting TKI discontinuation in the LMIC setting. Methods The Max Foundation's TFR Support Program provides financial and wrap-around support for patients attempting TFR in LMICs. The program operates in countries where The Max Foundation has existing partner institutions and physicians, imatinib and second line TKIs are available, and there is ≥1 reliable method of quantitative RT-PCR testing for BCR::ABL1. Eligible patients were >18 years old, diagnosed with chronic phase CML and treated with imatinib for ≥10 years with no history of resistance, and had a BCR-ABL level MR 4.5 or better (≤0.0032%) for ≥12 months (m) as demonstrated by 2 RT-PCR tests. Patients who consented were enrolled in the program and monitored by their treating physician. Upon starting TFR, patients' BCR::ABL1 levels were tested monthly for the first 6 m, then every 3 m for the following 6 m, then every 6 m. Patients who relapsed (BCR::ABL1 loss of MR 3 or >0.1%) after beginning TFR restarted imatinib and were tested approximately every 1-3 m until they achieved MR 3. We conducted a retrospective analysis on the outcomes of patients enrolled in The Max Foundation's TFR Support Program to better understand TFR outcomes in an LMIC setting. Results The molecular results pre- and post-TKI discontinuation are shown in Figure 1. In total, 39 patients from sites in 5 countries (Armenia, Honduras, India, Kenya, and Paraguay) were followed by 8 program physicians. The mean age of the patients was 51 years (range, 22-77), with 22 females and 17 males. Patients had been on imatinib for an average of 15 years (range, 10-20). At time of discontinuation, all but 1 patient was on 400 mg/day of imatinib (1 treated at 300 mg/day). Thirty-two patients had undetectable BCR::ABL1 for both pre-discontinuation tests, 6 had one test with a value of <0.01%, and 1 had both tests detectable at <0.01%. Of the 39 patients, 32 remain actively followed, with 14 patients completing >12 m of follow-up. Nine patients had a molecular relapse (23%) with a BCR::ABL1 >0.1% IS after a median of 2 m of discontinuation (range, 1-18 m). Of these, 2 patients have not had repeat testing to confirm molecular relapse, and 1 patient became undetectable on follow-up 1 m later. None of these 3 patients have restarted TKI therapy. Seven of 39 patients (18%) have restarted TKI treatment. Of these 7 patients, 3 were restarted on TKI, but have not had a follow-up BCR::ABL1 evaluation. Of the remaining 4 patients who restarted TKI, 2 had no detectable BCR::ABL1 at the 9 m and 12 m follow-up, and 2 remain with evidence of BCR::ABL1 at 12 m and 17 m follow-up. Conclusions The data suggest that under conditions that allow frequent follow-up examination and testing, patients attempting TFR in LMIC have similar outcomes to those patients described in controlled clinical trials from academic settings in high-income countries. Additional confirmation of these findings will likely impact both the health of CML patients and the health economics in these LMICs. Figure 1. BCR:ABL1 and outcomes following TKI discontinuation.

International, Prospective Study Comparing Nilotinib Versus Imatinib with Early Switch to Nilotinib to Obtain Sustained Treatment-Free Remission in Patients with Chronic Myeloid Leukemia (SUSTRENIM trial): Analysis of the Eligibility to Treatment Discontinuation

Background. Treatment free remission (TFR) is one of the most important aims of CML treatment but, so far, the best treatment strategy to obtain a durable deep molecular response (DMR) and the overall rate of patients able to achieve a successful TKI discontinuation based on intention-to-treat principle from CML diagnosis are still debated. In November 2016 we launched an international, prospective, randomized, two arms study to evaluate both the depth of the molecular response and the rate of TFR in newly diagnosed CP-CML patients treated with a second generation TKI (Nilotinib, NIL) or with imatinib (IM) followed by switching to NIL in absence of optimal response (Clinical Trial number 02602314). Patients were randomized 1:1 between NIL and IM, stratifying on the Sokal score. All the patients achieving a MR4.0 or better by three years and maintaining this level of response up to the end of the fourth year of therapy were qualified for the discontinuation phase. We recently reported that patients enrolled in the NIL arm had a rate of MR4.5 at two years (primary co-endpoint of the study), significantly higher than those in the IM arm (32 vs 22%, p = 0.023 Pane F. et al, EHA 2022). The aim of the present analysis is to assess, in both arms of the trial, the percentage of patients achieving the eligibility to treatment discontinuation, defined as a MR4 maintained in 4 consecutive MRD analysis within the fourth year of therapy. Methods. In this pilot analysis, we first examined the rate of patients eligible to discontinuation and their baseline features (i.e. age, gender, ELTS and Sokal risk, BCR::ABL1 isoform) compared to patients without sustained MR4, using Fisher's exact test or Wilcoxon according to variable type. In patients discontinuing TKIs at 48 months, the successful TFR probability at 12 months after TKI withdrawal was computed with the Kaplan Meier method. Molecular relapse, defined as the loss of MMR or confirmed loss of MR3.0, progression, or death were considering as event in the event-free survival curve. Results. Of the 448 randomized patients, 289 had at least 48-month observation at the time of the present analysis and were evaluated for TFR eligibility, 141 in the IM arm and 148 in the NIL arm, respectively. Ninety-three patients (32%) showed a sustained MR4 in the fourth year and were deemed eligible for treatment discontinuation, 45 in the IM arm (32%) and 48 in the NIL arm (32%). Of the remaining 196 patients, 59 did not have the criteria to discontinue treatment and 137 previously went off study for failure, progression, toxicity, death, or other causes (66 in the IM arm and 71 in the NIL arm, respectively). We found that sustained MR4 achievement was significantly associated with low disease risk, according to both ELTS (81.7% vs 47.2% p<0.0001) and Sokal scores (50.5% vs 34.2%, p=0.008). There is a weak correlation to the age, being those eligible younger (median 52.5 vs 55 years, p=0.037), while no differences in gender and BCR::ABL1 isoform distributions were observed. Within the IM arm, patients who switched to NIL were 60/141 (43%), 44 within the first 12 months and 16 after the first year; those patients had a lower probability of starting the TFR phase compared to patients continuing IM (22% vs 40%, p=0.025). The median follow-up of the 93 patients who attempted treatment discontinuation at 48 months is 9.9 months (IQR: 5.3-12.1). The percentages of those patients in TFR were 82.9% (95% CI:74.8-91.8) at 6 months and 74.3% (95% CI:63.9-86.4) at 12 months. The analysis per random was considered premature given the short follow-up and the proportion of patients not yet evaluable for the discontinuation. Conclusions Although the results are still preliminary to draw definitive conclusions on the rate of sustained DMR and stable TFR , our data indicates that despite higher 24-month rates of MR4.5 in the NIL arm, the probability of reaching criteria for TFR attempt may be not different in the two study arms: one third of patients attempted discontinuation and the majority of them appear to be free of relapse after a median of 10 months from the therapy discontinuation. Among patients of the IM arm, patients switching to NIL due to absence of optimal response had a lower probability to attempt discontinuation. Noteworthy, as for the MR4.5 rate at 24 mo. of therapy, patients at low ELTS risk score are those with the highest probability to achieve sustained MR4 and being eligible for the treatment discontinuation.

Retrospective Study to Evaluate Probability of MR4 Maintenance Following Frontline Nilotinib Dose De-Escalation after MR4 Achievement in the Patients with Chronic Myeloid Leukemia in Chronic Phase

Introduction: Nilotinib (NIL) is a potent second-generation tyrosine kinase inhibitor (TKI) approved for the treatment of newly diagnosed chronic myeloid leukemia (CML) patients (pts) in chronic phase, but reported to increase long-term toxicity such as cardiovascular (CV) risks. The ENESTfreedom study (Leukemia 2021) has reported 55% of treatment-free remission (TFR) rate after front line NIL discontinuation with a median 43.5 months of NIL treatment duration. Question still remains how to improve TFR rate after NIL frontline therapy. A potential solution is dose de-escalation (DESC) after MR4 achievement which could reduce the CV risk while extending MR4 duration prior to TFR attempt, thus increasing the chance of successful TFR attempt. DESTINY trial (Lancet Haematology 2019) had attempted TKI dose DESC to half of the standard dose for 12 months before stopping TKI therapy, reporting only 3 pts (2.4%) lost molecular response after TKI dose DESC in 125 pts who achieved molecular response with 4 log or deeper (MR4). However, 85% of this cohort is mainly treated with imatinib, limiting applying this experience in NIL treated pts. Accordingly, the present study attempted to evaluate the efficacy of NIL dose DESC following MR4 achievement based on real-world experience. Patients & Method: We have retrospectively analyzed the outcome of adult pts with CML in chronic phase treated with NIL frontline therapy and achieved sustained MR4 ( BCR::ABL1 transcript ≤0.01%IS). From the pts treated with NIL frontline therapy, we identified the cases attempted NIL de-scalation after achievement of MR4. The molecular response after NIL de-escalation was closely monitored every 3 months using BCR::ABL1 qPCR. Molecular recurrence was defined as an increase in BCR::ABL1 transcript levels above MR4 on at least two consecutive occasions or a single increase in BCR::ABL1 transcript level above MR3 ( BCR::ABL1 transcript ≤0.1%IS). At any time, pts who lost MR3 had returned Nilotinib dose to 300 mg BID. Primary endpoint of the study was event-free survival (EFS) following NIL de-escalation, which was defined from NIL DESC starts to molecular recurrence or death from any cause. Results: We have identified 52 pts who started NIL as first line therapy for CML-CP under clinical practice. With median follow-up duration of 9 years (range:2-12 years), 33 patient (64%) remains on NIL at the last follow-up visit. In 33 pts on NIL treatment, with a median follow up duration of 8 years (range;2-12), 25 (76%) had attempted NIL DESC at median of 6 years (range; 1-10 years) after frontline NIL therapy start, of whom the median time from NIL start to MR4 achievement was 12 months (range: 5-50 months). The median time from first achievement of MR4 to NIL DESC starts was 50 months (range: 3-105 months), while the median time from NIL start to NIL DESC was 77 months (range: 13-121 months). At the latest follow up, only one patient experienced molecular recurrence at 3 months after DESC but regained MR3 in 9 months after re-escalation of NIL to 300mg BID. One patient died of cardiac arrest at 80 months after NIL DESC with other comorbidities of coronary artery disease, hypertension and diabetes. One patient discontinued NIL at 51 months post DESC and has sustained MR4 till last follow up. In the DESC group, the EFS at 3 years, 95% (69.5-99.3%). There is another case developed myocardial infarction while on DESC dose of NIL, who switched the treatment to Asciminib. Conclusion: Achieving early and sustained MR4 with frontline NIL treatment facilitates DESC approach without compromising molecular response and might promote the TFR rate by extending MR4 duration. This DESC strategy can bridge CML pts on NIL frontline therapy prior to NIL discontinuation for TFR attempt.

Highly Sensitive Chip-Based Digital PCR Platform for Quantitative Detection of BCR::ABL1 Transcripts throughout CML Treatment

Introduction: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment-free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of the CML clone, it requires the development of more sensitive technologies. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and variations in sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods require the inclusion of one or more negative partitions to calculate the concentration using Poisson distribution. Consequently, this leads to a more than a 10-fold decrease in the maximum detectable concentration in a single test compared to conventional qPCR. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (so called HDR-drPCR) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10 -5 %IS with BCR::ABL1 negative HL60 cells. In HDR-drPCR, the GUSB gene was employed as a control gene to determine %IS values. For this purpose, the 1 st WHO international genetic reference panel was utilized to calculate the correction factor (CF). With the results from cell lines, we also evaluated equivalence between conventional qPCR and HDR-drPCR using 219 clinical specimens. Among them, 100 samples were determined as DMR (<0.01%IS) based on the qPCR test results In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. For HDR-drPCR (1-step reverse transcriptase PCR), 10 6 to 10 7 copies per reaction of GUSB gene were applied to disposable cartridges of Genotizer™ HDR BCR::ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). Based on the results, correlation analysis between qPCR and HDR-drPCR was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R 2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 10 1 to 10 -5(%). Notably, HDR-drPCR technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1A). The correlation analysis for 209 clinical samples showed that measured values from both qPCR and HDR-drPCR were closely correlated with the R 2 of 0.973 1 (Figure 1B) . The Bland-Altman plot showed a mean bias of 0.159, and the limit of agreement at the 95% confidence interval (LOA95) was calculated as -0.387 to 0.705. Furthermore, the concordance rate between the two methods was 96.17%, as measured by the proportion of samples in which the difference between the two methods was within a factor of 5. This indicates a strong concordance between the two methods. HDR-drPCR reliably detected BCR::ABL1 transcripts in all 90 clinical specimens measured below 0.01%IS (DMR) using qPCR. Additionally, HDR-drPCR detected trace amounts of BCR::ABL1 transcripts in 6 out of 10 specimens that were not detected by qPCR, showcasing its improved sensitivity compared to the conventional qPCR method. Conclusion: Taken together, we believe our high dynamic range real-time digital PCR(HDR-drPCR) may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for accurately assessing treatment in routine clinical practice and facilitating the application of treatment-free remission (TFR). We will include these results in further intensive studies focusing on DMR clinical specimens Study support: Funded by Korea Research Foundation

Outcomes of Adolescents and Young Adult Patients with Chronic Myeloid Leukemia in the Era of Second-Generation TKIs and Treatment-Free Remission

Background: While chronic myeloid leukemia (CML) is most often diagnosed in older adults, it is frequently diagnosed in adolescents and young adults (AYA). Defined by the NCI as individuals between the ages of 15-39 years old, AYA CML patients have been understudied by comparison to their older counterparts, with little known about their outcomes in the United States (US) in the era of second-generation tyrosine kinase inhibitors (TKIs) and treatment-free remission (TFR). We sought to determine the clinical outcomes of AYA patients with CML at our institution, a CML specialty center. Methods: We performed a single-center, retrospective cohort study of patients with CML who were diagnosed between the ages of 15-39 years old and seen in our adult hematology clinic between January 2012 and December 2022. Patients were identified by ICD-9 and ICD-10 codes from the electronic medical record and chart review. Descriptive statistics were used to summarize the data. Outcomes evaluating second-generation TKIs (2G-TKIs) started with patients diagnosed in June 2010. Outcomes evaluating molecular response included BCR-ABL1 PCR testing conducted after implementation of the International Scale (IS) in 2012. Molecular treatment milestones were defined per the 2023 NCCN guidelines for CML. Results: Of 396 CML patients evaluated at our institution, 136 (34.3%) were diagnosed as AYA at a median age of 31 years (range 15-39). The majority were male (62.5%; n=85/136) and English speaking (94.1%; n=128/136). The most frequently cited race/ethnicities were Caucasian (40.4%; n=55/136), Asian (20.6%; n=28/136), and Hispanic (19.1%; n=26/136). At time of diagnosis, 91.2% (n=124/136) were in chronic phase (CP), followed by 4.4% (n=6/136) in accelerated phase (AP) and 4.4% (n=6/136) in blast phase (BP). Of those diagnosed in advanced phase, 91.7% (n=11/12) were male and 66.7% (n=8/12) were Asian or Hispanic. Of CP patients evaluated, 75.8% (n=94/124) started treatment in the 2G-TKI era. Most CP patients were first prescribed dasatinib (61.7%; n=58/94), followed by imatinib (28.7%; n=27/94), and nilotinib (8.5%; n=8/94). Of 62 patients in CP with available molecular data after 2012, 64.5% (n=40/62) met a transcript level <10% at 3-months and 80.6% (n=50/62) met a transcript level <1% at 12-months. Major molecular response (MMR) was achieved and maintained in 59.7% (n=37/62) of these patients, with 25.8% (n=16/62) losing MMR and 14.5% (n=9/62) never achieving MMR. Reasons for loss of MMR included TFR attempts (31.3%; n=5/16), non-adherence from side effects (25%; n=4/16), lack of health insurance/inability to afford medication (18.8%; n=3/16), lack of response (18.8%; n=3/16), and pregnancy attempts (6.3%; n=1/16). Fertility was a notable issue for AYA patients, with 16 females (31.4%; n=16/51) and 18 patients overall (13.2%; n=18/136) interrupting or delaying start of TKI because of pregnancy/fertility. For patients who attempted TFR, 48.1% (n=13/27) remained off TKI in MMR at time of last follow-up. Of 130 AYA patients with CP or AP CML at diagnosis, 7.7% (n=10/130) experienced disease transformation, including 3.2% (n=3/94) of CP patients in the 2G-TKI era. Of 14 patients who presented or transformed into BP, 42.8% (n=6/14) underwent allogeneic stem cell transplantation and 64.3% (n=9/14) died. Of the entire cohort, 9.56% (n=13/136) patients died at a median age of 35 years old, including 5.4% who presented in CP (n=7/124) and 50% (n=6/12) of those who presented in AP/BP. Of the 13 who died, 11 (84.6%) died from CML-related complications. Conclusions: This represents the largest analysis of NCI-defined AYA CML patients in the US in the 2G-TKI era. Molecular response, progression and successful TFR rates in AYA CP CML patients appear to approximate those reported in prior large clinical trials that compared 2G-TKIs with imatinib. AYA patients can experience unique challenges that impact their management, including pregnancy/fertility and adherence, which can affect clinical outcomes. Patients may also present with aggressive advanced phase disease leading to mortality despite transplantation. Further study is warranted to better support the needs of this patient population. Evaluation of CML patients ≥ 40 years old at diagnosis at our institution is ongoing. A formal comparison between these groups will be presented.

In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

Minimal Residual Disease Detection at RNA and Leukemic Stem Cell (LSC) Levels: Comparison of RT-qPCR, d-PCR and CD26+ Stem Cell Measurements in Chronic Myeloid Leukemia (CML) Patients in Deep Molecular Response (DMR).

A Deep Molecular Response (DMR), defined as a BCR::ABL1 transcript at levels ≤ 0.01% by RT-qPCR, is the prerequisite for the successful interruption of treatment among patients with Chronic Myeloid Leukemia (CML). However, approximately 50% of patients in Treatment-Free Remission (TFR) studies had to resume therapy after their BCR::ABL1 transcript levels rose above the 0.1% threshold. To improve transcript detection sensitivity and accuracy, transcript levels can be analyzed using digital PCR (dPCR). dPCR increases BCR::ABL1 transcript detection sensitivity 10-100 fold; however, its ability to better select successful TFR patients remains unclear. Beyond the role of the immune system, relapses may be due to the presence of residual leukemic stem cells (LSCs) that are transcriptionally silent. Flow cytometry can be used to identify and quantify circulating bone marrow Ph+ LSCs CD34+/CD38- co-expressing CD26 (dipeptidylpeptidase-IV). To date, the significance of circulating Ph+ LSCs in TFR is unclear. The aim of this work is to compare and examine the values obtained using the three different methods of detecting minimal residual disease (MRD) in CML at RNA (RT-qPCR and dPCR) and LSC (flowcytometry) levels among patients in TFR or exhibiting a DMR. The twenty-seven patients enrolled received treatment with either imatinib (12), dasatinib (6), nilotinib (7), bosutinib (1), or interferon (1). Twelve patients were in TFR, while the rest exhibited a DMR. The TFR patients had stopped therapy for less than 1 year (3), <3 years (2), 6 years (6), and 17 years (1). Blood samples were collected and tested using the three methods at the same time. Both d-PCR and LSCs showed higher sensitivity than RT-qPCR, exhibiting positive results in samples that were undetectable using RT-qPCR (17/27). None of the patients tested negative with d-PCR; however, 23/27 were under the threshold of 0.468 copies/μL, corresponding to a stable DMR. The results were divided into quartiles, and the lowest quartiles defined the lowest MRD. These data were strongly correlated in 15/27 patients, corresponding to almost half of the TFR patients. Indeed, the TFR patients, some lasting up to 17 years, corresponded to the lowest detectable DMR categories. To the best of our knowledge, this is the first attempt to analyze and compare DMRs in a CML population using standard (RT-qPCR) and highly sensitive (dPCR and LSCs) methods.

Prospective monitoring of chronic myeloid leukemia patients from the time of TKI discontinuation: the fate of peripheral blood CD26+ leukemia stem cells.

Introduction: In chronic myeloid leukemia (CML), about half of the patients achieving a deep and stable molecular response with tyrosine kinase inhibitors (TKIs) may discontinue TKI treatment without disease recurrence. As such, treatment-free remission (TFR) has become an ambitious goal of treatment. Given the evidence that deepness and duration of molecular response are necessary but not sufficient requisites for a successful TFR, additional biological criteria are needed to identify CML patients suitable for efficacious discontinuation. Leukemia stem cells (LSCs) are supposed to be the reservoir of the disease. Previously, we demonstrated that residual circulating CD34+/CD38-/CD26+ LSCs were still detectable in a consistent number of CML patients during TFR. Methods: CML LSCs could be easily identified by flow-cytometry as they express the CD34+/CD38-/CD26+ phenotype. In this study, we explored the role of these cells and their correlation with molecular response in a cohort of 109 consecutive chronic phase CML patients prospectively monitored from the time of TKI discontinuation. Results: After a median observation time of 33months from TKI discontinuation, 38/109 (35%) patients failed TFR after a median time of 4months, while 71/109 (65%) patients are still in TFR. At TKI discontinuation, peripheral blood CD26+LSCs were undetectable in 48/109 (44%) patients and detectable in 61/109 (56%). No statistically significant correlation between detectable/undetectable CD26+LSCs and the rate of TFR loss was found (p = 0.616). The incidence of TFR loss based on the type of TKI treatment was statistically significant for imatinib treatment compared to that of nilotinib (p = 0.039). Exploring the behavior of CD26+LSCs during TFR, we observed fluctuating values that were very variable between patients, and they were not predictive of TFR loss. Discussion: Up to date, our results confirm that CD26+LSCs are detectable at the time of TKI discontinuation and during TFR. Moreover, at least for the observation median time of the study, the persistence of "fluctuating" values of residual CD26+LSCs does not hamper the possibility to maintain a stable TFR. On the contrary, even patients discontinuing TKI with undetectable CD26+LSCs could undergo TFR loss. Our results suggest that factors other than residual LSCs "burden" playing an active role in controlling disease recurrence. Additional studies evaluating CD26+LSCs' ability to modulate the immune system and their interaction in CML patients with very long stable TFR are ongoing.

Treatment discontinuation following low-dose TKIs in 248 chronic myeloid leukemia patients: Updated results from a campus CML real-life study.

TKIs long-term treatment in CML may lead to persistent adverse events (AEs) that can promote relevant morbidity and mortality. Consequently, TKIs dose reduction is often used to prevent AEs. However, data on its impact on successful treatment-free remission (TFR) are quite scarce. We conducted a retrospective study on the outcome of CML subjects who discontinued low-dose TKIs from 54 Italian hematology centers participating in the Campus CML network. Overall, 1.785 of 5.108 (35.0%) regularly followed CML patients were treated with low-dose TKIs, more frequently due to relevant comorbidities or AEs (1.288, 72.2%). TFR was attempted in 248 (13.9%) subjects, all but three while in deep molecular response (DMR). After a median follow-up of 24.9months, 172 (69.4%) patients were still in TFR. TFR outcome was not influenced by gender, Sokal/ELTS risk scores, prior interferon, number and last type of TKI used prior to treatment cessation, DMR degree, reason for dose reduction or median TKIs duration. Conversely, TFR probability was significantly better in the absence of resistance to any prior TKI. In addition, patients with a longer DMR duration before TKI discontinuation (i.e., >6.8years) and those with an e14a2 BCR::ABL1 transcript type showed a trend towards prolonged TFR. It should also be emphasized that only 30.6% of our cases suffered from molecular relapse, less than reported during full-dose TKI treatment. The use of low-dose TKIs does not appear to affect the likelihood of achieving a DMR and thus trying a treatment withdrawal, but might even promote the TFR rate.

Importance of TKI treatment duration in treatment-free remission of chronic myeloid leukemia: results of the D-FREE study

Treatment-free remission (TFR) is a new goal for patients with chronic myeloid leukemia in chronic phase (CML-CP) with a sustained deep molecular response (DMR) to treatment with tyrosine kinase inhibitors (TKIs). However, optimal conditions for successful TFR in patients treated with second-generation (2G)-TKIs are not fully defined. In this D-FREE study, treatment discontinuation was attempted in newly diagnosed CML-CP patients treated with the 2G-TKI dasatinib who achieved BCR-ABL1 levels of ≤ 0.0032% (MR4.5) on the international scale (BCR-ABL1IS) and maintained these levels for exactly 1 year. Of the 173 patients who received dasatinib induction therapy for up to 2 years, 123 completed and 60 (48.8%) reached MR 4.5. Among the first 21 patients who maintained MR4.5 for 1 year and discontinued dasatinib, 17 experienced molecular relapse defined as loss of major molecular response (BCR-ABL1IS > 0.1%) confirmed once, or loss of MR4 (BCR-ABL1IS > 0.01%) confirmed on 2 consecutive assessments. The estimated molecular relapse-free survival rate was 16.7% at 12 months. This study was prematurely terminated according to the protocol’s safety monitoring criteria. The conclusion was that sustained DMR for just 1 year is insufficient for TFR in CML-CP patients receiving dasatinib for less than a total of 3 years of treatment.

SOHO State of the Art Updates and Next Questions | Update on Treatment-Free Remission in Chronic Myeloid Leukemia (CML).

Tyrosine kinase inhibitor (TKI) discontinuation, also known as treatment-free remission (TFR) is currently one of the main goals of chronic myeloid leukemia (CML) therapy. TKI discontinuation should be considered in eligible patients for several reasons. Specifically, TKI therapy is associated with reduced quality of life, long-term side effects, and a heavy financial burden on both the patients and society. TKI discontinuation is a particularly important goal for younger patients diagnosed with CML because of the treatment's effects on their growth and development in addition to potential long-term side-effects. Numerous studies with thousands of patients have demonstrated the safety and feasibility of attempting TKI discontinuation in a select group of patients who have achieved a sustained deep molecular remission. With current TKIs, approximately 50% of patients will be eligible for attempting TFR of which only 50% will achieve a successful TFR. Therefore, in reality, only 20% of patients with newly diagnosed CML will achieve a successful TFR, and the majority of patients will need to continue TKI therapy indefinitely. However, several ongoing clinical trials are investigating treatment options for patients to achieve deeper remission with the ultimate goal of a cure, which is defined as being off drug with no evidence of disease.

Effect of BCR::ABL1 transcript type and droplet digital polymerase chain reaction on successful treatment-free remission in chronic myeloid leukemia patients who discontinued tyrosine kinase inhibitor.

Droplet digital polymerase chain reaction (ddPCR) is an exact method of measurement. We conducted this study to identify the prognostic factors for successful treatment-free remission in patients with chronic-phase chronic myeloid leukemia who discontinued tyrosine kinase inhibitors (TKIs). We also aimed to validate ddPCR for predicting molecular relapse. This is a prospective, multicenter study. We enrolled patients treated with TKIs for at least 3 years with a confirmed sustained deep molecular response (DMR) for at least 1 year. TKI was re-administered in patients who experienced the loss of major molecular response (MMR). A total of 66 patients from five institutions in South Korea were enrolled. During a median follow-up period of 16.5 months, 29/66 (43.9%) patients experienced molecular relapse; the probability of molecular relapse-free survival (RFS) at 6 or 12 months after TKI discontinuation was 65.6% or 57.8%, respectively, with most molecular relapses occurring within the first 7 months. All patients who lost MMR were re-treated with TKI, and all re-achieved MMR at a median of 2.8 months. E14a2 transcript type (p = 0.005) and longer DMR duration (⩾48 months) prior to TKI discontinuation (p = 0.002) were associated with prolonged molecular RFS and with sustained DMR. Patients with both e13a2 transcript type and detectable BCR::ABL1 (⩾MR5.0) by ddPCR at the time of TKI discontinuation showed shorter duration of molecular RFS (p = 0.015). Our data suggest that transcript type and BCR::ABL1 transcript levels on ddPCR should be taken into consideration when deciding whether to discontinue TKI therapy.

Treatment-Free Remission in Chronic Phase Chronic Myeloid Leukemia Patients Treated with Nilotinib Front-Line, in Real-Life Conditions: Influence of Switching to Another TKI Prior to Cessation.

Nilotinib (NIL) as front-line therapy in newly diagnosed chronic phase (CP) CML patients (pts) is able to induce high rates of sustained deep molecular response and high possibilities of treatment-free remission (TFR). However, the identification of factors influencing successful TFR in this population remains imprecise. This observational study retrospectively analyzed TFR rates, outcome, and tried to identify factors related to successful TFR in in- and out-study 221 pts treated in this setting with NIL 600 mg, in "real-life" conditions. All pts with newly diagnosed adult CP-CML receiving NIL 300 mg BID alone front-line between 10/2007 and 06/2022, were eligible. Data were retrospectively collected according to the national regulations with pts' information. All pts were assessed and followed according to ELN recommendations 2006→2020 along treatment and to the recommendations of the Fi-LMC (D. Rea et al., Cancer 2018, i. e. 2 years of sustained MR4.5) in case of TFR. In this regard, a TKI was resumed if loss of MMR on one instance. BCR::ABL1 assessments were performed in the 2 reference laboratories, standardised, and expressed in % (IS) with ≥32,000 copies of ABL1 as control. The primary endpoints were the rate of successful TFR on NIL first-line. Secondary endpoints were the kinetics of molecular response, factors influencing outcome, survival, and safety of NIL. Two hundred and one patients were enrolled with 56% males with a median age at diagnosis of 50 (17.5-83) years, and 26% of them had cardiovascular risk factors at onset (tobacco abuse 10%, hypercholesterolemia 9.55%, diabetes 1.36%, none with history of cardiovascular events [CVE]). ELTS scores were high in 13%, intermediate in 30% and low in 53% of pts, unknown in 4% of pts. Twenty-six (12%) pts harboured additional chromosomal abnormalities (ACA) at diagnosis. The median follow-up after NIL initiation was 85.16 (5.72-174) months. After a median of 37.7 months, 119 pts (58%) were not on NIL anymore for toxicities, resistance or TFR. Sixty-eight (31%) pts obtained the TFR criteria and stopped NIL after a median of 66.7 (27-132) months. The median time between first MR4.5 datapoint and TFR was 39 (15.31-120) months. With a median follow-up of 20.3 (0.03-108) months after NIL cessation, the survival without MMR loss after NIL cessation (figure 1.) is 64.38% (95% CI: 53.35- 77.69). Interestingly, no CVE occurred after NIL cessation, as well as no blast crisis. Two patients died in TFR from competitive events (2 solid tumours). We went on comparing the TFR group vs the non-TFR group. The median age at TFR was 56 (30-87) years with no difference at CML diagnosis in terms of age (49 vs 50 years, p=0.096), ELTS (p=0.276), ACA (15% vs 11%, p=0.483) between the 2 groups. In contrast, there was a difference in favour of female vs male gender in the TFR group (56%) vs non-TFR group (39%, p=0.031), and the median time to reach MMR was shorter in the TFR group 5.04 vs 10.2 months, p<0.001; as well as the median time to reach MR4.5 in the TFR group 18.2 vs 36.1 months, p<0.001. We finally compared the successful vs the unsuccessful TFR pts requiring TKI-rechallenge, in univariate analysis and multivariate analyses. The univariate analysis identified pts that have switched to another TKI before TFR for whatever reasons was a risk factor for subsequent loss of MMR [HR: 3.09 (95% CI: 1.30-7.34), Cox p-value=0.011]. This was confirmed by the log-rank survival curves of TFR in patients with or without switch (figure 2., log-rank p=0.008). Surprisingly, neither the duration of NIL before TFR [HR: 0.98 (0.96-1.00), Cox p-value=0.072], nor the duration of MR4.5 prior to NIL cessation [HR: 0.98 (0.95-1.01), Cox p-value=0.15] were found to have an impact. Additionally, the prognostic scores did not influence TFR success on NIL. The multivariate analysis confirmed that switching to another TKI was an unfavourable risk factor for molecular relapse [HR: 2.87 (1.20-6.84), p-value=0.018], and that the lack of impact of the duration of MR4.5 [HR: 0.98 (0.95-1.01), Cox p-value=0.218] had no significant influence. NIL first-line is able to induce high rates of successful TFR in newly diagnosed CP CML patients and switching to another TKI for toxicity and/or resistance, prior to TFR, represents an unfavourable risk factor for the success of TKI cessation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment-Free Remission Outcome in Patients with Chronic Myeloid Leukemia in Chronic Phase Following One Year of Nilotinib De-Escalation: 96-Week Update of Dante Study

Introduction: Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is demonstrated to be achievable and recommended for patients (pts) in sustained deep molecular response (sDMR) who can discontinue tyrosine kinase inhibitor (TKI) treatment while maintaining responses. Studies investigating TKI de-escalation before TFR, mainly in CML chronic phase (CP) imatinib-treated pts, suggested that this stepwise approach is feasible. Despite nilotinib (NIL) is the first and only TKI approved for stopping therapy, nilotinib-based TFR optimization strategy has not been formally studied. Our phase II, prospective, multicenter DANTE study (NCT03874858) aims to shed light on frontline NIL de-escalation and successful TFR in Italian CML-CP patients. Our previously interim analysis showed that 1 year of NIL de-escalation before TFR in patients with sDMR is safe and effective. Here we present 1 year follow up results of those 40 patients who successfully completed NIL de-escalation and entered in TFR. Methods: Adults patients with CML-CP treated with NIL 300 mg twice daily (bid) in first-line for ≥3 years who achieved sDMR for ≥1 year (≥MR 4.0; BCR-ABL level ≤0.01% IS) were enrolled in 27 centers. The study consisted of 4 phases: screening (week [wk] −4-0), consolidation (wk 0-48), TFR (wk 48-144), and follow-up (until wk 144). Ongoing treatment with ≥400 mg/day dose was allowed at study entry. During consolidation pts were treated with NIL 300 mg once daily (qd). At the end of consolidation phase, pts with sDMR entered TFR phase and NIL was discontinued. Patients with at least major molecular response (MMR; BCR-ABL ≤0.1% IS), but without sDMR, continued NIL 300 mg qd. At any time, pts with loss of MMR returned to NIL 300 mg bid. During TFR phase, BCR-ABL levels were monitored monthly from wk 52-96, and then every 3 months. Here we report updated results collected until the cut-off date of 8th February 2022, at which time all patients who entered the TFR phase had completed at least one year of TFR, or switched to full-dose phase, or entered in survival follow-up or discontinued the study. Results: Overall, 113 pts were screened and 107 entered in consolidation phase. The baseline characteristics and the clinical outcome of the 52 patients that reached the end of consolidation phase by the first data cut-off period (February 8, 2021) have been previously reported (Breccia et al., 2021). Specifically, 47 out of 52 pts completed the consolidation phase and 40 (76.9%) of them, with sDMR, entered in the TFR phase. At 1-year data cut off, with a median duration of 12.7 months (range 1.9 - 20.6), 27 out of 40 pts (67.5 %) who entered in the TFR phase remained in MMR or better while 13 pts (32.5%) had discontinued the TFR phase (11 pts due to loss of MMR and 2 pts for subject/guardian decision). The median time spent in TFR phase until loss of MMR was 3.6 months (range 2-7 months). The median time to loss of MMR in TFR phase was not reached (Fig 1). At the data cut off, all patients except one (10/11, 90%) who lost MMR, regained MMR after NIL resumption. Overall, among the 13 patients who restarted NIL after MMR loss (2 pts from consolidation phase and 11 from TFR phase), 12 of them (92.3%) regained MMR and MR4 after a median time of 2.6 months (range 0.9 - 5.2) and 4.2 months (range 2.7- 8.0), respectively. Notably, 22/52 (42%) pts remained in DMR from consolidation phase until week 96. During the TFR phase, all grade AEs were reported in 26/40 (65%) pts. One pt (2.5%) had serious AE (pneumonia). No deaths and disease progressions were observed. Conclusions: Our finding show that approximately 68% of pts remained disease-free at 1 year after stopping NIL, indicating that de-escalation of NIL before TFR attempt in CML-CP pts with sDMR can be a successful dose optimization strategy. Reference: Breccia et al., Blood (2021) 138 (Supplement 1): 1474. https://doi.org/10.1182/blood-2021-145411 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Free Remission after Combination Therapy with Asciminib (ABL001) Plus Imatinib in Chronic Phase Chronic Myeloid Leukemia (CP-CML) Patients Who Relapsed after a Prior Attempt at TKI Discontinuation-H Jean Khoury Cure CML Consortium Study

Successful tyrosine kinase inhibitor (TKI) discontinuation is currently one of the main goals of CML therapy. Patients who are in sustained deep molecular remission can attempt TKI discontinuation; approximately 50% of such patients will achieve a successful treatment free remission (TFR) and the other 50% will have molecular recurrence , warranting reinitiation of TKI therapy. Molecular recurrence is defined as loss of major molecular response (BCR-ABL1 International Scale ratio >0.1%) . With a goal to increase the ultimate fraction of successful TFR, inquiry has begun into the rate and optimal approach to TFR after a second attempt at TKI discontinuation in patients with molecular recurrence after their first attempt. Molecular recurrence, is suspected to be driven by CML progenitor element(s) capable of, permitted to, and/or signaled to proliferate; different approaches to eradicate such a compartment have been proposed. One such potential means may be through alternative and synergistic BCR::ABL1 inhibition with the allosteric inhibitor asciminib in combination with approved TKIs for CML. Asciminib is a potent BCR::ABL1 inhibitor with a novel, allosteric mode of action. The activity of asciminib is a result of its ability to bind a vacant pocket on the kinase domain that is normally occupied by the myristoylated N-terminus of ABL, a negative regulatory element lost upon the fusion of ABL to BCR. Asciminib has an excellent selectivity profile, is several-fold more potent than current approved TKIs such as nilotinib, and in vitro was active in the low nanomolar range against a spectrum of clinically observed ATP site mutations. Asciminib demonstrated efficacy in phase I studies in resistant/intolerant chronic and accelerated phase CML, including T315I mutation positive and ponatinib pretreated patients. In a phase III trial, asciminib was more active than bosutinib in a phase III trial in patients who had received 2 prior TKIs Our primary objective in this study is to determine the 1-year TFR rate after 12 months of combination therapy with imatinib plus asciminib, followed by treatment cessation, in subjects who have previously had molecular recurrence first TFR attempt. This is a single arm phase II study that will enroll a maximum of 51 patients (NCT04838041). All subjects will have a confirmed diagnosis of CML-CP. Each subject must have previously attempted to discontinue imatinib, and were restarted on imatinib at the time of molecular recurrence in order to be eligible for this trial. This trial will use asciminib 40 mg PO daily plus imatinib at a maximum dose of 400 mg PO once daily in the combination treatment phase of this trial. All eligible subjects will be treated with asciminib and imatinib on cycle 1 day 1 of the combination therapy phase. Patients will remain on imatinib for 12 cycles. RQ-PCR to measure BCR::ABL1 transcripts in the peripheral blood at screening and at specific time points during the first 12 cycles on trial (combination therapy phase). At the end of 12 cycles, asciminib and imatinib will be discontinued in any subject who has continued in stable deep remission and thus meets criteria for TFR. This study will assess a wide range of PROs before and after stopping imatinib. These will be done in conjunction with RQ-PCR testing, though at fewer time points, utilizing online and/or phone questionnaires. The alternative hypothesis of relapse rate after second discontinuation of TKI combination treatment is 35% will be tested against a one-sided alternative of historical control of 55% relapse rate. Simon's two-stage admissible design will be used. The null hypothesis that the true TFR rate (no loss of MMR) is 45% will be tested against a one-sided alternative. In the first stage 29 patients will be accrued. If there are 15 patients or fewer who maintain TFR in these 29 patients, the study will be stopped. Otherwise, 18 additional patients will be accrued for a total of 47. The null hypothesis will be rejected if 27 or more patients maintain TFR in 47 patients. This design yields a type I error rate of 5% and power of 85% when the true TFR rate is 65%. To account for a potential dropout rate of 8% due to adverse events or loss of response, 51 patients will be enrolled to achieve the 47 patients needed.

Chip-Based Digital PCR Platform, Lab on an Array, the Newly Developed Highly Accurate and Sensitive Method for the Detection of BCR-ABL1 Transcripts in Chronic Myeloid Leukemia

Introduction: With the development of various BCR-ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable hematologic malignancy and current therapeutic goal is treatment free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) has become one of the prerequisite requirements. Since undetectable BCR-ABL1 by conventional TaqmanTM-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of CML clone, more precise methods should be developed. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and various sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods such as nanofluidic or droplet dqPCR have been developed for accurate and precise detection of various genes and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods may have limitations due to partition-based amplification and end-point interpretation. Therefore the establishment of clinical guideline is crucial for wide application on TFR in CML. Therefore, we have developed a novel technology, a chip-based real-time dqPCR platform (LOAA; Lab On An Array; so called Dr. PCR™) to achieve more stable, accurate and precise monitoring of BCR-ABL1 in a timely manner. Methods: To assess the equivalence between Dr. PCR and conventional qPCR, we evaluate the efficacy of BCR-ABL1 detection using cell lines and clinical specimens. For linearity assessment, we used BCR-ABL1 positive (e14a2) K562 cell line diluted to 9 points by 10-fold serial dilutions from 100 to 10-8 with BCR-ABL1 negative HL60 cells. With the results from cell lines, we also evaluated linearities for both e13a2 and e14a2 major BCR-ABL transcripts using 7 clinical specimens obtained from Leukemia Omics Research Institute of Eulji University. In detail, conventional qPCR was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche, USA) for cDNA synthesis and Real-Q BCR-ABL Quantification Kit (Biosewoom, South Korea) for qPCR. Total 800ng of RNA per test was used for qPCR (QS5, Thermo Fisher, USA) with 8 replicates and duplicate for cell line and clinical specimens, respectively. For Dr. PCR (1-step reverse transcriptase PCR), 400ng (for cell lines) or 500ng (for clinical specimens) of RNA per test was applied to disposable cartridges of Dr. PCR™ BCR-ABL1 Major IS detection kit (Optolane Technologies, South Korea) with LOAA platform (Optolane Technologies, Korea). The copies of BCR-ABL1 and ABL1 were analyzed by LOAA analyzer S/W (Optolane Technologies, South Korea). Based on the results, correlation analysis between qPCR and Dr PCR was performed using correlation graphs and Blend-Altman plots. Results: Correlation analysis for cell line dilution assay showed that measured values from both qPCR and Dr. PCR were closely correlated with the R2 of 0.984. LOA95 (limit of agreement at 95% confidence interval) analyzed by Blend-Altman plot was -0.711 ~ 0.303 (mean = 0.02), indicating the two methods are closely correlated (Figure 1). The linearity assessment for e13a2 and e14a2 sub-genotypes using primary cells from 7 clinical specimens of CML patients again showed a close correlation with the R2 of 0.984 and 0.985, respectively. LOA95 values were -0.516 to 0.384 (mean = -0.066) and -0.407 to 0.361 (mean = -0.023), respectively. Interestingly, Dr PCR efficiently detected clinical specimens with low copy number of BCR-ABL1 compared to conventional qPCR, which is beneficial for the determination of MDR levels in patients especially those who are required to monitoring after cessation of TKIs. Comparison between conventional qPCR, droplet dqPCR and Dr PCR is listed (Table 1). Results with more specimens from CML patients and additional information will be updated at ASH presentation. Conclusion: Taken together, we believe our novel chip-based real-time dqPCR (Dr. PCR™) may minimize false positive results and maximize the analytical sensitivity of detecting BCR-ABL1 fusion transcripts and thus, would be useful for the selection of therapeutic option in routine clinical practice and TFR. We will include the result for further intensive study using more clinical specimens including samples of low copy number of BCR-ABL1. Study support: Funded by Korea Research FoundationFigure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunomodulatory Effects of IFNα on T and NK Cells in Chronic Myeloid Leukemia Patients in Deep Molecular Response Preparing for Treatment Discontinuation

A deep and stable molecular response (DMR) is a prerequisite for a successful treatment-free remission (TFR) in chronic myeloid leukemia (CML). In order to better identify and analyze potential candidates of successful TFR, we examined the phenotypic and functional host immune compartment in DMR patients who had received TKI treatment only (TKI-only) or had been previously treated with interferon-alpha (IFNα + TKI) or had received IFNα treatment only (IFNα-only). The T/NK-cell subset distribution, NK- and T-cell cytokine production, activation and maturation markers were measured in 44 patients in DMR treated with IFNα only (9), with IFNα + TKI (11) and with TKI-only (24). IFNα + TKI and TKI-only groups were eligible to TKI discontinuation according to the NCCN and ESMO guidelines (stable MR4 for more than two years). In IFNα-treated patients, we documented an increased number of lymphocytes capable of producing IFNγ and TNFα compared to the TKI-only group. In INFα + TKI patients, the percentage of NKG2C expression and its mean fluorescence intensity were significantly higher compared to the TKI-only group and to the INFα-only group in the CD56dim/CD16+ NK cell subsets (INFα + TKI vs. TKI-only p = 0.041, p = 0.037; INFα + TKI vs. INFα-only p = 0.03, p = 0.033, respectively). Furthermore, in INFα-only treated patients, we observed an increase of NKp46 MFI in the CD56bright/CD16- NK cell subset that becomes significant compared to the INFα + TKI group (p = 0.008). Our data indicate that a previous exposure to IFNα substantially and persistently modified the immune system of CML patients in memory T lymphocytes, differentiated NKG2C+ “long-lived” NK cells responses, even years after the last IFNα contact.