Treatment-free Remission In Chronic Myeloid Leukemia Research Articles

The Role of G0S2 in Lipid Metabolism Regulation in Chronic Myeloid Leukemia

Introduction: Chronic myeloid leukemia (CML) can be effectively treated with tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1, but resistance remains a clinical problem. TKIs do not target the quiescent CML leukemic stem cell (LSC), and many patients are treated for life at a high economic burden and sometimes with significant side effects. We recently published a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML. Our data showed that loss of G0S2 expression in CML promoted disease progression and drug resistance by disrupting glycerophospholipid metabolism (Gonzalezet al. Clin Transl Med, 2022). Interestingly, G0S2 is an estrogen-response gene that demonstrated anti-estrogenic and pro-migratory effects in ER+ versus ER- breast cancer. Additionally, deletion of G0S2 prevented obesity and insulin resistance in mouse models. Since sex-based differences in lipid metabolism and obesity are well documented, we hypothesized that the tumor suppressor role of G0S2 in CML would differ based on sex. Methods: We used peripheral blood from wild-type and G0s2 knockout ( G0s2 -/-) mice (both male and female) who were fed with a normal versus a high-fat diet. Resulting lineage-negative (Lin-) cells were cultured in recombinant murine cytokines ± granulocyte-colony stimulating factor (mG-CSF, 25 ng/ml, 7-10 days) in in vitro differentiation assays. Flow cytometry analysis was performed to measure CD11b+ neutrophils. Additionally, we transduced Lin- cells from wild-type or G0s2 knockout mice with BCR::ABL1, and assessed their ability to form a leukemia-like disease process in male versus female recipient mice. We also performed G0S2 ectopic expression and knockdown experiments in the K562 CML cell line and assessed the resulting cells by liquid chromatography (LC)/mass spectrometry (MS)-based lipidomics and proteomics analyses. Immunoblots were performed to assess the resulting cells for changes in the autophagy markers, LC3A/B I-II. Results: For the experiments conducted with mouse peripheral blood, RT-qPCR data showed high G0S2 mRNA expression upon neutrophil differentiation in wild-type male mice (n=5), but not in the wild-type female mice (n=5). Flow cytometry measuring CD11b markers showed that a high-fat diet-induced neutrophil production in wild-type male mice, but not in wild-type female mice or G0s2 -/- mice, suggesting hormonal regulation involving G0S2 and female mice. Consistent with this observation, forced BCR::ABL1 expression in G0s2 -/- bone marrow reduced overall survival in female (n=4/group) but not male (n=5/group) mice compared with wild-type controls. In mass spectrometry-based lipidomics analyses, G0S2 knockdown resulted in a substantial decrease of di- and triglycerides. G0S2 knockdown also resulted in substantial changes in phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Conversely, ectopic G0S2 expression promoted the accumulation of long- and very long-chain triglycerides and species of phosphatidylcholine and phosphatidylethanolamine in K562 cells. Proteomics analyses showed autophagy as one of the top pathways affected by altered G0S2 expression. When we measured LC3A/B I-II by immunoblot, we observed opposing effects comparing G0S2 ectopic expression versus knockdown. Conclusion: Altogether, these findings suggest sex-based differences in the role of G0S2 and lipid metabolism in CML stem and progenitor cell differentiation, survival, and autophagy, both in vitro and in vivo. Restoring G0S2 expression combined with BCR::ABL1 inhibition may be a novel clinical strategy to induce treatment-free remission in CML. However, further studies should assess whether this strategy will have similar effects in male versus female patients.

Assesment of Patient'S Perspective on Treatment Free Remission in CML: A Turkish Multicenter Cohort

Treatment free remission (TFR) is one of the main goals of therapy in Chronic Myeloid Leukemia (CML). Patient perspective and expectations have an indicative effect on discontinuation decisions. We analyzed 116 CML patients' perspective on TFR and factors that associated with willingness for tyrosine kinaz inhibitor (TKI) discontinuation from four different center in Turkey based on a national survey. Patients in our cohort had 7.8% awareness of TFR and after brief information willingness to attempt TFR has been reached to 58.9% in all population. Shorter disease duration was associated with enhanced willingness to attempt discontinuation in multivariate analyses (p=0.031). Most common patient related limitation for TKI discontinuation decision was anxiety about disease relaps and TKI resistance. Even disease relaps in TFR means molecular and detectable with close monitoring, patients still had considerable concerns about TKI discontinuation.

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

Update on Treatment-Free Remission in CML

Introduction Treatment-free remission (TFR) is one of the main goals of chronic myeloid leukemia (CML) therapy. Tyrosine kinase inhibitor (TKI) discontinuation should be considered in eligible patients for several reasons. Patients with CML on TKI have a reduced quality of life.1 TKIs are associated with long-term side effects, such as cardiovascular and renal complications.2 TKIs are associated with a heavy financial burden, both to patients and society. Numerous studies with thousands of patients have demonstrated the safety and feasibility of attempting TKI discontinuation.3–7 Finally, TKI discontinuation is an important goal for children diagnosed with CML due to long-term side effects and effects on growth and development.8 With current TKIs, approximately 50% of patients will be eligible for attempting TFR. Of those, only 50% will achieve a successful TFR. So, 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.

In Pursuit of Surrogate Markers for Treatment-Free Remission in Patients with Chronic Myeloid Leukemia from Argentina Stop Trial

INTRODUCTION: Long-term survival of patients with chronic myeloid leukemia (CML) has significantly improved since the introduction of BCR-ABL1 tyrosine kinase inhibitors (TKIs). Several considerations about the side effects, risks and cost associated with the lifetime treatment, have led patients and physicians to explore the possibility of TKI discontinuation after achievement of a sustained deep molecular response (DMR), so-called treatment-free remission (TFR). Several clinical trials show that approximately half of patients who achieve a sustained DMR during TKI treatment maintain molecular remission after suspension of TKIs. There is currently no biomarker that reliably predicts TFR in CML, mainly due to different study designs that have generated inconsistent data. Thus, further investigations are needed to identify factors that consistently favor achievement of TFR. With the aim of developing a biomarker for TFR prediction we analyzed the phenotype of Natural Killer (NK) cells and their relation to successful TKI cessation. METHODS: This analysis was conducted as a substudy of the Argentina Stop Trial. Altogether, 50 consecutive chronic phase CML patients who participated in the clinical trial were recruited from 7 Argentinian centers. Peripheral blood samples were collected before stopping TKI treatment, at month 3, 12 and at any time when MR3.0 was lost. Freshly isolated mononuclear cells from 46 patients were immunophenotyped by staining with CD3, CD16, CD25, CD56, CD57, CD158, NKp30, NKp44, NKp46, NKG2A, NKG2C, NKG2D and PD-1 antibodies and NK cells subpopulations were analyzed by flow cytometry (BD FACS Canto™II). Molecular recurrence-free survival was estimated by the Kaplan-Meier method and compared within groups by the log-rank test. The cutoffs of the numerical variables were optimized according to the log-rank test. Quantitative variables were dichotomized according to receiver operating characteristics (ROC) curves in order to describe sensibility and specificity. Multivariate analysis was performed through Cox proportional hazards model. Main results are provided with hazard ratio (HR) at 6 months and 95% confidence intervals (95% CI). RESULTS: At the time of discontinuation the median proportion of NK cells (CD3-CD56+) among lymphocytes was significantly increased in patients compared with controls (15% vs 9%, P = 0.0016). A significant difference between molecular relapsed vs no-relapsed patients was observed when optimal cutoff (0.43) for CD56bright low and high was determined (at 6 months 74% vs 100% respectively, log rank test, p=0.023). At this time of follow up, no significant difference was observed for CD56dim NK cells. Phenotypic markers for adaptive-like NK cells were analyzed, however, no significant differences were observed between the non-relapsing and relapsing groups. Nevertheless, molecular non-relapsing patients had significantly higher frequencies of PD-1+ NK cells as compared with molecular relapsing patients (at 6 months 85% vs 64%, Log Rank test, P=0.009). Based on the ROC and Youden Index analysis, at 6 months the 1.2 cutoff shows an 80% specificity and 50% sensitivity. Moreover, after multivariable Cox proportional analysis, including age, time of treatment, deep molecular response time, Sokal risk, NK cells and PD-1+ NK cells, the last subpopulation was identified as an independent prognostic factor for molecular-relapse-free survival (Hazard ratio = 3.63; 95% CI 1.3 - 10.1; P=0.014). CONCLUSIONS: The clinical impact of NK cells in patients who have discontinued TKIs is controversial. The effects of TKIs against immune cells, including NK cell subsets, could depend on the type of TKIs; this aspect is particularly relevant in Argentinian treated patients real world, since many different copies of TKIs are routinely used in the clinical setting. Our study is the first, to our knowledge, to report a significant increase in PD-1 expression in NK cells at TKI cessation in patients who do not relapse. Accordingly to recent reports, PD-1 expression is more abundant on NK cells with an activated and more responsive phenotype and does not mark NK cells with an exhausted phenotype. To fully understand how PD-1 on NK cells modulates immune responses we are planning to carry out functional studies. In the future, we are also planning a comprehensive study of immune suppressors, including regulatory T cells and myeloid-derived suppressor cells. Disclosures Moiraghi: Novartis: Speakers Bureau; BMS: Speakers Bureau. Varela:Novartis: Consultancy, Speakers Bureau. Pavlovsky:Varifarma: Speakers Bureau; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel grants, Speakers Bureau. Pavlovsky:Pint Pharma: Speakers Bureau; Pfizer: Speakers Bureau; BMS: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Treatment-Free Remission in CML: the US Perspective.

Chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKI) have near-normal life expectancy. However, lifelong TKI therapy is associated with reduced quality of life and significant economic burden. Currently, the management of CML is shifting from continuous TKI therapy towards the goal of TKI cessation which is discussed in this review. Several studies in the last decade have demonstrated the feasibility and safety of TKI discontinuation in selected patients with CML who achieve deep and sustained molecular response with TKI. This has moved prime-time into clinical practice although open questions remain in terms of understanding the disease biology that leads to successful TKI cessation in some patients while not in others. Cessation of TKI for CML patients is a feasible approach. Ongoing research aims to find out optimal strategies to sustain ongoing treatment-free remission (TFR) and increase the number of patients who achieve TFR.

Treatment-free remission in CML: who, how, and why?

Chronic myeloid leukemia (CML) is the best example of successful targeted therapy. Today, the overall survival of patients with CML treated by using tyrosine kinase inhibitors (TKIs) is very close to that of the healthy population. The current question is: how can we further ameliorate the clinical outcome of patients with CML? Clinical trials have shown that some patients with CML in the chronic phase who achieve sustained deep molecular responses on TKI therapy can safely suspend therapy with no evidence of relapse. The long follow-up studies and the number of eligible patients have now validated the concept of treatment-free remission (ie, the ability to maintain a molecular response after stopping therapy). It should be considered as the future criterion to evaluate the success of clinical trials, especially if we want to take into account the quality of life of patients in addition to the economic aspect. Because post-TKI discontinuation follow-ups have been increasing over time with no evidence of relapse in some patients, the next step for the coming decade will be to address the topic of CML cure.

Two-Year Consolidation By Nilotinib Is Associated with Successful Treatment Free Remission in Chronic Myeloid Leukemia with MR4.5: Subgroup Analysis from STAT2 Trial in Japan

▪Background Nilotinib (NIL) is a second-generation tyrosine kinase inhibitor (TKI) that exhibits significant efficacy as first- or second-line treatment in patients with chronic myeloid leukemia (CML). Superior rates of deeper molecular responses (DMR) were achieved with NIL vs. imatinib (IM) in patients newly diagnosed with CML in chronic phase (CML-CP) in the ENESTnd trial. In addition, the ENESTcmr study demonstrated that switching to NIL after a minimum of 2 years on IM led to increased rates of DMR vs. remaining on IM. Switching to NIL treatment for 2 years safely led to MR4,5 (BCR-ABLIS…0.0032%) in 47.5% of patients with major molecular response (MMR) on long-term IM therapy in our STAT1 trial. Recently, treatment free remission (TFR) was proposed as one of the goals in CML treatment. Indeed, prospective trials suggest that IM therapy may be safely and successfully discontinued in 40% of CML patients with MR4.5. STAT2 is the first study to evaluate the efficacy of two-year consolidation by NIL for successful TFR in patients with CML-CP who had achieved MR4.5. Before enrolling in STAT2, some patients were treated by not only IM but also NIL because of MMR but no MR4.5 after IM therapy, and some patients changed over from STAT1 to STAT2. Here, we present the results of the subgroup analysis from STAT2 based on the prior treatments at the time of entry into the study.Methods In the STAT2 trial, patients who achieved MR4.5 on IM front line therapy (subgroup 1; SG1) or NIL second line therapy after IM therapy (subgroup 2; SG2) were eligible and NIL was given twice daily at the dose of 600 mg/day for 2 years in consolidation phase. The primary endpoint of STAT2 was the proportion of patients with successful TFR, defined as no confirmed loss of MR4.5 (2 consecutive IS RQ-PCR tests), within the first 12 months of TFR phase. Thirty-five institutions in STAT study group participated. The study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was signed by all patients according to institutional guidelines. The study was approved by all institutional review boards and registered with UMIN-CTR (000005904).Results Between July 2011 and December 2012, 96 patients were enrolled in STAT2. Among 96 patients, 50 patients were treated by IM first line only as prior therapy (SG1). On the other hand, 40 patients were treated by IM first line and NIL second line including 21 patients who changed over from STAT1 to STAT2 because they achieved MR4.5 (SG2). Six patients were excluded in this analysis because second generation TKIs were taken as a first line therapy. Among patients treated by NIL for 2 years in this study, 40/50 (80%; 95% CI, 68.4%-88.7%) in SG1 and 33/40 (82.5%; 95% CI, 69.6%-91.5%) in SG2 entered the TFR phase, respectively. The median age was 54.5 years in SG1 and 56.0 years in SG2. The ratio of men to women was 26:14 in SG1 and 18:15 in SG2. The total duration of TKI treatment was 110 months for the SG1 with a median of 86 months of IM, and 24 months of NIL, and 93 months in SG2 with a median of 62 months of IM, and 31 months of NIL, respectively. All patients achieved MR4.5 at the time of entry into the study and the median time to MR4.5 was 47 months in SG1 and 60 months in SG2.The proportion of patients who maintained TFR at 12 months after stopping NIL was similar across the 2 subgroups: 25/40 (62.5%; 95% CI, 48.3%-77.3%) in SG1, and 23/33 (69.7%; 95% CI, 54.0%-82.5%) in SG2. The Kaplan-Meier (KM) analysis of TFR survival showed that in the 2 subgroups, the majority of events occurred within the first 6 months after stopping NIL (Figure 1). There were no significant differences between these 2 subgroups.Conclusion After two-year consolidation by NIL of CML-CP patients who achieved MR4.5, the TFR rate was 67.9% (90%CI: 58.2% to 76.6%) at 12 months in the STAT2 trial. In the present analysis looking at the prior TKI exposure, the TFR rate was similar in patients treated with IM first line only or who switched from IM to NIL before entering the study, despite the fact that the treatment duration of switched patients was slightly shorter. These findings suggest that two-year consolidation by NIL is associated with successful TFR in CML with MR4.5 that was achieved with IM alone or after switching to NIL. [Display omitted] DisclosuresTakahashi:PFIZER: Honoraria, Research Funding; BMS: Honoraria; NOVARTIS PHARMA: Honoraria, Research Funding. Nakaseko:BMS: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; NOVARTIS: Honoraria. Nishiwaki:Novartis PHARMA: Research Funding.