Treatment-free Remission In Patients Research Articles

Single-Cell Analysis of Immune Recognition in Chronic Myeloid Leukemia Patients Following Tyrosine Kinase Inhibitor Discontinuation

Background: Modern treatment goal for patients with chronic myeloid leukemia (CML) is to maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. The control of residual leukemia cells may occur with immunological mechanisms as several studies have shown that a higher proportion of CD56 dim NK cells before TKI discontinuation is associated with a better probability of TFR. In addition, CD8+ T cell abundances and phenotypes have been associated with responses to TKIs. However, the more detailed phenotypes and mechanisms and how to translate these findings to increased TFR rates have remained unknown. Methods: We analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced (scRNA+TCRαβ-seq) samples from patients with CML ( n=13, N=25), other cancers (AML n=11, CLL n=13, solid tumors n=4), and healthy ( n=7). Additionally, we profiled 90 CML and 786 healthy TCRβ-seq samples, and sorted T cells specific to leukemia-associated antigen PR1 and matched these back to the scRNA+TCRαβ-seq and TCRβ-seq data. To validate and refine our findings, we performed co-culture of primary and expanded NK cells with CML cell lines (K562 and LAMA84) with a multiplexed scRNA-seq readout. Results: In the pan-cancer scRNA+TCRαβ-seq comparison of >250,000 cells, CML was separated from other cancers and healthy by the high number and active phenotype NK cells. The other phenotype more pronounced in CML was CD8+ T EM/T EMRA phenotype. Most NK cells in CML had an active CD56 dim phenotype with high expression of GZMA/B, PRF1, CCL3/4 and IFNG. In the off-TKI samples from patients with TFR, these NK cells upregulated NK cell activation genes such as TNFRSF4/OX40, TNFRSF9/4-1BB, and TNFRSF18/GITR. The most important predicted interactions between NK cells and CD34+ leukemic cells were inhibitory LGALS9- TIM3 and PVR- TIGIT interactions. Following TKI discontinuation, the inhibitory markers HAVCR2/TIM3 and TIGIT decreased in patients with TFR, which was not seen in patients with relapse following TFR. Accordingly, in our co-culture data of NK cells with CML cell lines, we noted activation of NK cells to a similar phenotype as noted in ex vivo off-TKI samples. In co-culture with CML target cells, NK cells gained an activation by upregulation of genes like TNFRSF4/OX40, TNFRSF9/4-1BB, TNFRSF18/GITR and the inhibitory markers HAVCR2/TIM3 and TIGIT. In co-culture with NK cells, CML target cells upregulated the IFN-γ response pathway including elevated HLA-expression and accordingly, upregulation of LGALS9 and PVR. The CD8+ T EM/EMRA cells that were expanded in CML diagnosis were suppressed following TKI treatment and decreased following TKI discontinuation. To track anti-CML T cells, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 from the TCRb-sequenced samples. As a validation of our classifier, we noticed that anti-PR1 T cells were more prevalent in CML than in healthy ( P adj<0.0001) or melanoma ( P adj<0.0001), more expanded than similar anti-viral T cells ( P adj<0.0001) and enriched in bone marrow samples compared to peripheral blood ( P adj<0.0001). From different TKIs, dasatinib expanded anti-PR1 T cells the most ( P adj<0.01). In the scRNA+TCRαβ-seq data, anti-PR1 T cells were enriched in the mature, cytotoxic CD8+ T EMRA phenotype ( P adj<0.0001). In a patient maintaining TFR, anti-PR1 T cells had a highly cytotoxic profile ( GZMB, PRF1) throughout the therapy with upregulated NK receptors like FCGR3A/CD16 and KIR2DL2, even more so than T cells targeting other targets e.g., different viruses. In comparison, the anti-PR1 T cells in a patient with early relapse had an exhausted profile with upregulation of memory marker GZMK and exhaustion markers like LAG3 and HAVCR2/TIM3. Conclusions: With our in-depth analysis of cellular and molecular immune responses in CML, we identified the active NK cells and anti-PR1 T cells that could help maintain TFR in patients discontinuing TKI treatment. Different immune-checkpoint blockade therapies such as anti-TIM3 could be considered as future strategies to improve the TFR rates.

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.

A long-term follow-up of observation in treatment-free remission in patients with chronic myeloid leukemia

Introduction. The option of observation without therapy with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) patients is already included in Russian and international clinical guidelines. Evaluation of long-term follow-up results of treatment free remission (TFR) in CML patients is relevant for the introduction of this approach into routine clinical practice. Aim — to demonstrate the outcomes in a long-term follow-up of CML patients who discontinued TKI therapy in the RU-SKI trial. Patients and methods. The prospective study included 98 CML patients with TKI therapy duration ≥ 3 years and a deep molecular response (DMR, BCR::ABL1 ≤ 0.01 %) duration ≥ 2 years. TKI therapy was resumed with the loss of a major MR (MMR, BCR::ABL1 > 0,1 %). Results. Median time of follow-up after TKI discontinuation was 64 months (range of 51–86 months). Survival without MMR loss at 3 and 5 years after TKI discontinuation was 51 % (CI 41–61 %) and 46 % (CI 36–57 %) respectively. From 3 to 5 years of follow-up without therapy, the loss of MMR occurred in 2 (4 %) patients. There was no MMR loss observed after 5 years of follow-up. In patients with first and second treatment discontinuation, survival without MMR loss was 50 % versus 12,5 %(р = 0,039). All 50 patients with molecular relapses regained MMR and MR4 after TKI therapy resumption. BCR::ABL1 level fluctuations 0,01–0,1 % were in 62 % (n = 29) patients, who were in TFR at the time of analysis. Loss of MR4 was observed in 38 (42 %) from 90 patients with first TKI discontinuation. Survival without MMR loss from MO4 loss was 24 % at 5 years after TKI discontinuation. Loss of MO4 in the first 3 months after TKI cessation was associated with a high probability of further MMR loss (8 % versus 54 % in patients with loss of MO4 for > 3 months, p = 0.00015). Conclusion. The low frequency of late relapses (4 % after 3 years of follow-up) and the possibility of long-term persistence of minimal residual disease (MRD) after discontinuation of therapy determine the need to optimize the timing of molecular monitoring, taking into account the MRD status of patients.

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.

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.

Feasibility of treatment-free remission with generic imatinib: Results of generic imatinib-free trial-in-chronic myeloid leukaemia-chronic phase study.

Both innovator and generic imatinib are approved for the treatment of Chronic Myeloid Leukaemia-Chronic phase (CML-CP). Currently, there are no studies on the feasibility of treatment-free remission (TFR) with generic imatinib. This study attempted to determine the feasibility and efficacy of TFR in patients on generic Imatinib. In this single-centre prospective Generic Imatinib-Free Trial-in-CML-CP study, twenty six patients on generic imatinib for ≥3 yr and in sustained deep molecular response (BCR ABLIS ≤0.01% for more than two years) were included. After treatment discontinuation, patients were monitored with complete blood count and BCR ABLIS by real-time quantitative PCR monthly for one year and three monthly thereafter. Generic imatinib was restarted at single documented loss of major molecular response (BCR ABLIS>0.1%). At a median follow up of 33 months (interquartile range 18.7-35), 42.3 per cent patients (n=11) continued to be in TFR. Estimated TFR at one year was 44 per cent. All patients restarted on generic imatinib regained major molecular response. On multivariate analysis, attainment of molecularly undetectable leukaemia (>MR5) prior to TFR was predictive of TFR [P=0.022, HR 0.284 (0.096-0.837)]. The study adds to the growing literature that generic imatinib is effective and can be safely discontinued in CML-CP patients who are in deep molecular remission.

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

Retrospective Study of Treatment Free Remission in Patients with Chronic Myeloid Leukaemia(CML) Treated at Tertiary Care Oncology Centre from India

Background Tyrosine kinase inhibitors (TKI) have dramatically improved outcomes in Chronic Myeloid Leukemia(CML) patients, but lifelong TKI therapy can negatively impact patients' quality of life due to chronic adverse events. Treatment free remission(TFR) is now the new goal of CML therapy. Studies have shown this is feasible in developed countries. Literature on discontinuation of TKI therapy in resource limited countries such as India is scarce. The purpose of this study was to investigate whether some chronic myeloid leukemia patients with excellent and sustained molecular responses to tyrosine kinase inhibitor treatment can gradually de-escalate TKI therapy and stop therapy in Indian setting. Methods We included all patients above the age of 18years with diagnosis of CML in first Chronic Phase (CP) on TKI therapy of minimum 3 years duration with major molecular response (MMR i.e., <0.1% BCR-ABL1 transcript) of minimum of 2 years duration, enrolled for treatment free remission between January 2018 to December 2021. Starting dose of imatinib was 400mg once daily, dasatinib 100mg once daily and nilotinib 300mg twice a day. In this study the dose of these drugs was reduced by 25% every 6 months . This dose reduction was done with 2 monthly outpatient clinic visits and 2 monthly BCR-ABL1 quantitative PCR monitoring. Patients who maintained MMR during 6 months of dose de-escalation qualified for next level of dose de-escalation. Eventually treatment was stopped completely and monitoring continued. This was retrospective medical record based study done at a tertiary care centre in North India. Results Between Jan 2018 to Dec 2021, 29 patients were enrolled for dose de-escalation and treatment cessation. Among these 29 patients, 19 were on imatinib, 2 on dasatinib and 8 patients on nilotinib. Median age was 39 years( range 21 years to 67 years). Twelve patients were male and 17 female. Median duration of TKI therapy prior to de-escalation was 8 years (range 4years-14 years). Median duration of major molecular response prior to dose reduction was 6 years( range 3years-12 years). In our study 17 of 29 patients had an outcome, while 12 patients are still on de-escalation phase with maintained MMR and are not included in the outcome analysis. Eight patient had loss of MMR(47.05%), 6 while on dose de-escalation and 2 after cessation of TKI. All eight patients achieved MMR after re-initiation of the prior dose, out of these 6 patients had sustained MMR(>6months) at reduced TKI dosing compared to conventional dose. Nine patients (52.9%) have successfully stopped TKI for median duration of 11months (range 6 to 19 months). Conclusion TKI de-escalation was safe for most patients with excellent prior response to TKI therapy. This way of dose reduction , ensured motivation and commitment of patients by doing regular OPD follow ups and BCR-ABL1 PCR testing. In our study 52.9% patient could successfully maintain TFR at a median follow up of 11 months of stopping the drug. We also identified that a subgroup of patients(35.29%) who were unsuccessful in complete cessation of therapy but maintained their molecular response at reduce dose of TKI . These findings show that lower TKI doses might maintain molecular responses in these patients, implying that such patients could be overtreated with routine dosing. More studies of dose de-escalation are warranted.

Clonal Hematopoiesis Detected at the Time of Tyrosine Kinase Inhibitor Cessation Is Associated with Delayed Molecular Recurrence after Treatment-Free Remission in Patients with CML

Background Treatment-free remission (TFR) is a goal of therapy. However, ~50% of patients who attempt TFR will relapse, which mostly occurs by 6 months. The acquisition of somatic mutations in hematopoietic cells is common with age (clonal hematopoiesis, CH). Through next-generation sequencing studies we identified CH in patients in deep molecular remission. CH variants provide a selective growth advantage to cells, driving their clonal expansion. The clones also give rise to mutated immune effector cells with a proinflammatory profile that exacerbate diseases with a chronic inflammatory component, such as atherosclerosis. CH variants could potentially influence TFR, either by outcompeting an emerging residual CML clone or by altering immune function, which is known to play an important role in TFR. Aim To determine whether CH variants present at the time of therapy cessation could influence TFR. Methods An RNA-based hybridization capture sequencing method targeting 17 of the most frequently mutated CH genes was developed and applied to samples of 138 patients who attempted TFR. CH variants met strict criteria for pathogenicity based on their potential to confer growth and survival advantages. The CH status was confirmed by repeat testing of samples of 28 patients. The method reproducibly detected variants with a variant allele frequency (VAF) of ≥0.4%. Results The median follow-up for the 138 patients was 66 months from the TFR attempt. Molecular recurrence was defined as loss of major molecular response (MMR, BCR::ABL1 ≤0.1%). The probability of TFR was 64.5% at 6 months, 59.4% at 12 months and 51.0% at 84 months. CH variants were detected in 61/138 patients (44%): 107 variants in 12 genes. Multiple variants, range 2 - 7, were detected in 26 patients (19%). The median VAF was 0.9%, range 0.4 - 44%. Variants most frequently occurred in TET2 (44% of variants), DNMT3A (25%), PPM1D (7.4%) and ASXL1 (6.5%). The average age at cessation was 59.6 years, range 33 - 85. The average age of patients with CH was higher than those without CH: 64.7 versus 55.5 years, P < .0001. Only 3/13 patients (15%) aged ≤40 had CH compared with 6/7 patients (86%) ≥80. CH was significantly associated with TFR to 36 months after cessation. The optimal criteria for TFR prediction were at least one CH variant of VAF >2% or >2 variants (CH >2%/>2). Thirty-one of 138 patients (22%) had CH of VAF >2% (n = 24) or >2 variants (n = 7). The probability of TFR for these 31 patients was significantly higher than for the remaining patients when assessed at 6, 12, 24 and 36 months after cessation, Figure. The probability of TFR at 6 months was 90.3% for patients with CH >2%/>2 versus 57.0% for the remaining patients, P = .001. The probability of TFR at 36 months was 65.9% versus 51.2%, P = .049. A landmark analysis was performed at 12 months for the 74 patients who maintained TFR at 12 months. MMR was subsequently lost in 8/74 patients and the probability of TFR at 84 months was 86.9%. CH >2%/>2 occurred for 23/74 patients (31%). The probability of late molecular recurrence was higher among the patients with CH >2%/>2: 27.3% versus 5.5% for the remaining patients, P = .009. Overall, molecular recurrence occurred for 12/31 patients (39%) with CH >2%/>2 and 53/107 of remaining patients (50%). Time to molecular recurrence after cessation was longer for patients with CH >2%/>2 than the remaining patients: median 12.5 months versus 3.5 months, P < .0001. The longer time to molecular recurrence was linked to a different pattern of loss of MMR. The median time between first loss of MR4 (BCR::ABL1 ≤0.01%) and loss of MMR was 145 days for patients with CH >2%/>2 and 28 days for remaining patients, P = .0007. BCR::ABL1 values more frequently fluctuated before loss of MMR in patients with CH >2%/>2: 8/12 patients (67%, fluctuation over 120-1335 days) versus 6/53 (11%, fluctuation over 85-715 days) in the remaining patients, P =.016. Conclusion CH may improve the probability and duration of TFR, delaying the time to molecular recurrence, which has implications for patient selection and monitoring. Patients with CH who are eligible for therapy cessation have an excellent prospect of remaining drug-free for 1-2 years but their long term prospects of sustained TFR may be less certain compared to other patients, given the slower dynamics of relapse. Delayed relapse could be due to competition for clonal dominance between residual leukemic cells and larger CH clones, or modulation of the immune microenvironment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase II, Single-Arm Study of De-Escalation and Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Treated with Nilotinib in First-Line Therapy Followed By a Second Attempt after Nilotinib and Asciminib Combination: Dante Study

Introduction: Treatment-free remission (TFR) is considered one of the main goals of therapy in patients with chronic myeloid leukemia (CML). Several studies have shown that treatment discontinuation can be successfully achieved in roughly 50% of patients with a sustained deep molecular response (sDMR). However, the remaining 50% require reintroduction of tyrosine kinase inhibitor (TKI) therapy. Although about 90% of patients who experience molecular relapse regain a major molecular response (MMR) after restarting TKI therapy, due to poorly understood mechanism(s), the probability for those patients of failing a second attempt to discontinue TKI (TFR2) is higher compared to the first attempt. The above-mentioned data implies that, to aim to a second TFR, a different therapeutic strategy might be needed. Asciminib (ASC), unlike all approved TKIs that bind to the ATP site of the BCR-ABL1 oncoprotein, is an allosteric, first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor with a new mechanism of action that has shown efficacy in heavily pre-treated patients after two or more TKIs (Rea et al. 2021). An ongoing phase I dose-finding study (NCT02081378) has shown that ASC combined with TKI is both effective and well tolerated in heavily pretreated CML patients, suggesting that combination treatment between an allosteric and an ATP-competitive TKI may deepen clinical responses without serious safety concerns (Mauro et al. 2019). The changes introduced in the DANTE study (NCT03874858) by the amendment 4 aim to assess whether ASC in combination with nilotinib (NIL) can lead to higher and more durable TFR2 rates than those reported in other studies. Methods: After providing written informed consent, the TFR2 phase will enroll - from 29 Italian centers, approximately 60-70 adult CML patients in chronic phase, who have been (i) treated for at least 3 calendar years with NIL, (ii) failed a first TFR attempt, (iii) restarted NIL for at least 1 year and (iv) fulfill all inclusion/exclusion criteria. The study consists of 4 phases: screening, re-induction (week 0-96), TFR2 (week 96-144) and follow up phase. In the re-induction phase, patients will be treated with ASC 40 mg BID and NIL 300 mg BID for 96 weeks and will have scheduled biweekly/monthly visits until week 12, and then every 12 weeks. NIL dose may be less than 300 mg BID if appropriate by clinical judgement but must be at least 300 mg daily. During re-induction, sDMR is defined as no loss of MR4.0 (BCR-ABL level ≤ 0.01% IS) in any BCR-ABL RQ-PCR assessment. Patients with sDMR at the end of re-induction will enter in TFR2 and ASC plus NIL will be discontinued. If two or more consecutive quarterly BCR-ABL RQ-PCR assessments are not performed during re-induction or results are not available, the patient will not be eligible for TFR2 and will be treated with NIL 300 mg BID until the end of the TFR2 stage (week 144). Patients not eligible for TFR2 but with a response more than an MMR will continue treatment with ASC and NIL until the end of re-induction (week 96) and then continue NIL monotherapy at 300 mg BID until the end of the TFR2 stage (week 144). Patients with loss of MMR at any time during re-induction or during NIL monotherapy will be discontinued from the study. If MMR loss occurs during TFR2, NIL 300 mg BID will be restarted and administered until end of study (Fig1). Endpoints: The primary endpoint is the percentage of patients in TFR 48 weeks after stopping NIL and ASC combination. Key secondary endpoints are: (i) percentage of patients eligible for TFR2, (ii) the number of patients with loss of MMR in TFR2 and regain MMR/DMR by the end of the TFR2 stage (iii) BCR-ABL kinetics in the different study phases (iv), OS, TFS and PFS and (v) the safety of the combination. The TFR2 stage is descriptive and no formal statistical testing is necessary or applicable. Conclusions: The expected results from this phase II interventional study, will potentially represent a milestone in treatment strategy for those patients who aim to discontinue NIL for the second time by providing them with several benefits including an improved quality of life.

Observational study of chronic myeloid leukemia Chinese patients who discontinued tyrosine kinase inhibitors in the real-world

Objective: This study aimed to observe whether the treatment-free remission (TFR) of second-generation tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) is better than imatinib (IM) . Methods: The clinical data of 274 CML patients who discontinued treatment and with complete clinical data were retrospectively studied from June 2013 to March 2021. Using both univariate and multivariate Cox proportional hazards regression models, risk factors influencing TFR outcomes after drug withdrawal in CML patients were assessed. Results: A total of 274 patients were enrolled, 140 patients were women (51.1%) , with a median age of 48 (9-84) years at the time of TKI discontinuation. Prior to TKI discontinuation, 172 (62.8%) patients were treated with IM, and 102 (37.2%) had received second-generation TKI treatment, including 73 patients who had shifted from IM to a second-generation TKI and 29 patients who used second-generation TKI as the first-line treatment. The rationale for converting to a second-generation TKI are as follows: 37 patients aimed deep molecular response (DMR) to achieve TFR, seven patients changed due to IM intolerance, and 29 patients changed because of failure to achieve the optimal treatment response. The use of the last type of TKI included 96 patients (94.1%) with nilotinib, three patients (2.9%) with dasatinib, and two patients (2%) with flumatinib, including one patient who changed to IM due to second-generation TKI intolerance. No statistical differences were found in the median age at diagnosis and TKI discontinuation, sex, Sokal score, IFN treatment before TKI, median time of TKI treatment to achieve DMR, and the reasons for TKI discontinuation between the second TKI and IM (P>0.05) .The median cumulative treatment time of TKI (71.5 months vs 88 months, P<0.001) , the last TKI median treatment time (60 months vs 88 months, P<0.001) , and the median duration of DMR (58 months vs 66 months, P=0.002) were significantly shorter in the second-generation TKI compared with IM. In the median follow-up of 22 (6-118) months after TKI discontinuation, 88 patients (32.1%) had lost their MMR at a median of 6 (1-91) months; of the 53 patients (60.2%) who lost MMR within 6 months, the overall TFR rate was 67.9%, and the cumulative TFR rates at 12 and 24 months were 70.5% and 67.5%, respectively. Withdrawal syndrome occurred in 26 patients (9.5%) . For patients who restarted TKI treatment, 72 patients (83.7%) achieved DMR again at a median treatment of 4 (1 to 18) months. The univariate analysis showed that the TFR rate of patients treated with second-generation TKI was significantly higher than those who were treated with IM (77.5% vs 62.2%, P=0.041) . A further subgroup analysis found that the TFR rate of the second-generation TKI patients was significantly higher than those treated with IM (80.8% vs 62.2%, P=0.026) . No significant difference was found in the second-generation TKI used as the first line treatment compared with those who were treated with IM (69.0% vs 62.2%, P=0.599) . The multivariate analysis results showed that second-generation TKI treatment was an independent prognostic factor affecting TFR in patients who discontinued TKI (RR=1.827, 95%CI 1.015-3.288, P=0.044) . Conclusion: In the clinical setting, more CML patients rapidly achieved TFR using second-generation TKI than IM treatment.

Analysis the influence factors of treatment free remission outcome with chronic myeloid leukemia patients who discontinued tyrosine kinase inhibitors

Objective: To explore the related factors affecting the outcome of treatment free remission (TFR) in patients with chronic myeloid leukemia (CML). Methods: Clinical data of CML patients with automatic discontinuation of tyrosine kinase inhibitor (TKI) from the CML cooperative organization of Henan province between June 2, 2013 to March 27, 2021 and the follow-up time was ≥ 6 months were retrospectively analyzed. Log-rank test was used for univariate analysis and Cox proportional risk regression model was used for multivariate analysis. Results: A total of 135 patients were enrolled, and 69 patients (51.1%) were femal and 66 patients (48.9%)were male. Median age was[M(Q1,Q3)] 49 years (38, 58)at discontinuation.Before discontinuation, 72 patients (53.3%) were on treatment with second-generation TKI, 63 patients (46.7%) were on treatment with IM, 17patients (12.6%) had a history of TKI reduction/withdrawal;median duration of treatment was months 84 (68, 108) for all patients;median time of TKI treatment to DMR was months 12(8, 26);median duration of DMR was months 65 (54, 84), and 9 patients (6.7%) had unsustained DMR.The median follow-up time was months 16(6-96), 35 patients (25.9%) lost MMR at a median months 3(1-22), overall estimated TFR was 74.1%.The univariate analysis results showed that:second-generation TKI was used, the time of TKI treatment to DMR was ≤12 months, DMR duration time ≥48 months, had sustained DMR, without TKI reduction/withdrawal history were favorable factors affecting of TFR in patients with TKI discontinuation (all P<0.05).The TFR rate of the second-generation TKI therapy group was significantly higher than the IM therapy group (81.9% vs 65.1%, P=0.019).The multivariate analysis results showed that second-generation TKI treatment[RR=0.451, 95%CI (0.227-0.896), P=0.023] and had sustained DMR [RR=0.120, 95%CI (0.053-0.271), P<0.001] were the protective factors of TFR in patients with TKI discontinuation. Conclusions: Treated with second-generation TKI and had sustained DMR are the protective factors of TFR in patients with TKI discontinuation.The CML patients who had sustained DMR more≥48 months before TKI discontinuation showed a better TFR.

Non-albuminuric chronic kidney disease in diabetes: A different approach?

Background & objectives: Both innovator and generic imatinib are approved for the treatment of Chronic Myeloid Leukaemia-Chronic phase (CML-CP). Currently, there are no studies on the feasibility of treatment-free remission (TFR) with generic imatinib. This study attempted to determine the feasibility and efficacy of TFR in patients on generic Imatinib. Methods: In this single-centre prospective Generic Imatinib-Free Trial-in-CML-CP study, twenty six patients on generic imatinib for ≥3 yr and in sustained deep molecular response (BCR ABLIS ≤0.01% for more than two years) were included. After treatment discontinuation, patients were monitored with complete blood count and BCR ABLIS by real-time quantitative PCR monthly for one year and three monthly thereafter. Generic imatinib was restarted at single documented loss of major molecular response (BCR ABLIS>0.1%). Results: At a median follow up of 33 months (interquartile range 18.7-35), 42.3 per cent patients (n=11) continued to be in TFR. Estimated TFR at one year was 44 per cent. All patients restarted on generic imatinib regained major molecular response. On multivariate analysis, attainment of molecularly undetectable leukaemia (>MR5) prior to TFR was predictive of TFR [P=0.022, HR 0.284 (0.096-0.837)]. Interpretation & conclusions: The study adds to the growing literature that generic imatinib is effective and can be safely discontinued in CML-CP patients who are in deep molecular remission.

Regulatory T Cell as a Biomarker of Treatment-Free Remission in Patients with Chronic Myeloid Leukemia.

Simple SummaryTyrosine kinase inhibitors (TKIs) have dramatically improved the treatment of chronic myeloid leukemia (CML). Recently, TKIs were discontinued in patients with CML with deep molecular remission, and some patients have been reported to be able to maintain long-term treatment-free remission (TFR). However, there is no certainty regarding which patients can maintain TFR. We focused on immunity in the TFR phase and investigated the immunological mechanism of continuous TFR or recurrence. Our results suggest that the group that maintains the TFR is immunologically activated. In addition, regulatory T cells can be used as a biomarker. These results may have important implications for future strategies for maintaining TFR in CML treatment. Treatment-free remission (TFR) has become a therapeutic goal in chronic myeloid leukemia (CML), and approximately half of the patients with chronic phase-CML (CML-CP) with deep molecular remission (DMR) by tyrosine-kinase inhibitors (TKIs) have achieved TFR. However, the mechanism of continuous TFR is still unclear, as there are “fluctuate” patients who have BCR–ABL-positive leukemia cells but do not observe obvious relapse. We focused on the immune response and conducted an immune analysis using clinical samples from the imatinib discontinuation study, JALSG-STIM213. The results showed that, in the group that maintained TFR for 3 years, changes in regulatory T (Treg) cells were observed early after stopping imatinib treatment. The effector Treg (eTreg) cells increased transiently at 1 month after stopping imatinib and then returned to baseline at 3 months after stopping imatinib treatment. There was no difference in the Treg phenotype, and CD8+ T cells in the TFR group were relatively activated. High concentrations of imatinib before stopping were negatively correlated with eTreg cells after stopping imatinib. These data suggest immunological involvement in the maintenance of the TFR, and that Treg cells after stopping imatinib might be a biomarker for TFR. Furthermore, high imatinib exposure may have a negative immunological impact on the continuous TFR.

Treatment Free Remission in Patients with Chronic Phase CML: A Single Center Experience

Treatment Free Remission in Patients with Chronic Phase CML: A Single Center Experience

Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Following the Discontinuation of Tyrosine Kinase Inhibitors

Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Following the Discontinuation of Tyrosine Kinase Inhibitors

First Interim Analysis of the Italian Dante Study: De-Escalation before Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Treated with First-Line Nilotinib

First Interim Analysis of the Italian Dante Study: De-Escalation before Treatment-Free Remission in Patients with Chronic Myeloid Leukemia Treated with First-Line Nilotinib

Treatment-free remission in patients with chronic myeloid leukemia: recommendations of the LALNET expert panel

AbstractTyrosine kinase inhibitors (TKIs) have dramatically changed the survival of chronic myeloid leukemia (CML) patients, and treatment-free remission (TFR) has recently emerged as a new goal of CML treatment. The aim of this work was to develop recommendations for TKI discontinuation in Latin America (LA), outside of clinical trials. A working group of CML experts from LA discussed 22 questions regarding TFR and reached a consensus for TFR recommendations in the region. TFR is indicated in patients in first chronic phase, with typical BCR-ABL transcripts, under TKI treatment of a minimum of 5 years, in sustained deep molecular response (DMR; molecular response 4.5 [MR4.5]) for 2 years. Sustained DMR must be demonstrated on at least 4 international reporting scale quantitative polymerase chain reaction (PCR) tests, separated by at least 3 months, in the immediate prior 2 years. After second-line therapy, TFR is indicated in previously intolerant, not resistant, patients. Molecular monitoring is recommended monthly for the first 6 months, every 2 to 3 months from months 7 to 12, and every 3 months during the second year, indefinitely. Treatment should be reintroduced if major molecular response is lost. Monitoring of withdrawal syndrome, glucose levels, and lipid profile is recommended after discontinuation. After TKI reintroduction, molecular monitoring is indicated every 2 to 3 months until MR4.0 achievement; later, every 3 to 6 months. For the TFR attempt, having standardized and reliable BCR-ABL PCR tests is mandatory. These recommendations will be useful for safe discontinuation in daily practice and will benefit patients who wish to stop treatment in emergent regions, in particular, with TKI-related chronic adverse events.

ABL001 with Catalytic-Site Inhibitors Limit the CML Immature Cell Population: Re-Differentiation of CML-iPSCs Study

Background:There is growing evidence that chronic myeloid leukemia stem cells (CML-SCs) use survival signals distinct from the BCR-ABL1 kinase to maintain their viability in the presence of ABL-tyrosine kinase inhibitors (TKIs). Understanding the mechanisms through which CML-SCs are resistant to ABL TKIs would help in designing future therapies leading to complete eradication. Induced pluripotent stem cells (iPSCs) are promising for studying stem cell signaling pathways. When reprogramming is performed with CML cells, CML-derived iPSCs offer a unique system for modeling CML pathogenesis and developing high throughput drug screening. ABL001(asciminib) is a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development including CML. In the present study, we investigated the molecular mechanisms by which ABL001 and ponatinib regulate the CML-SCs developing from CML-derived iPSCs.Methods:We generated iPSCs from bone marrow mononuclear cells of three CML patients including CML-CP and CML-AP. Karyotyping analysis revealed that CML-derived iPSCs have Philadelphia chromosome (Ph). In vitro re-differentiation of CML-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokine (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4).Results:We generated the phenotypically immature cell population (CD34+CD38-CD90+CD45+) and mature cell population (CD34-CD45+) from CML-derived iPSCs. The single treatment of ABL001 or ponatinib significantly reduced the mature cell population (CD34-CD45+) from CML-derived iPSCs. Although the single treatment of ABL001 or ponatinib alone have minimum effect on immature cell population (CD34+CD38-CD90+CD45+), co-treatment with ABL001 and ponatinib significantly reduce the cell population (CD34+CD38- CD90+CD45+) (p<0.001). Also co-treatment with ABL001 and ponatinib significantly reduced the colony formations of mature erythroid, granulocyte-macrophade, and mixed of these hematopoietic cells derived from CML-iPSCs for CFC assay. Further, NOD/SCID mice were injected with CML-iPSCs, and then treated with ABL001 and ponatinib on day 21 for 7 days. Co-treatments with ABL001 and ponatinib significantly reduced the population of CD34+CD38-CD90+ cells compared with ABL001 or ponatinib as single agents. To identify the mechanisms that limit the CML immature cell population by ABL001 and ponatinib, CD34+CD38-CD90+CD45+ cell fractions from CML-iPSCs were cultured with ABL001 and or ponatinib for 72 hrs. Co-treatment with ABL001 and ponatinib reduced the expression of BMI1, c-Myc, Oct4 and Nanog and induced the expression of p21Cip1. Finally, we investigate the engraftment potential of CML-derived iPSCs treated with ABL001 and ponatinib. One million of CD34+ cells treated with ABL001 and ponatinib were intravenously transplanted into NOD/SCID mouse. Transient engraft was observed from control mice, however, co-treatment of ABL001 and ponatinib never shows the engraftment.Conclusion:Our preclinical results indicate that combination with ABL001 and ponatinib have potential as an important option for controlling the immature CML cell population. It is expected that ABL001 with catalytic-site inhibitors may become extremely useful therapeutic interventions to achieve treatment-free remission in patients with CML. DisclosuresTauchi:Otsuka pharmaceutical KK: Speakers Bureau; Phizer KK: Research Funding, Speakers Bureau; Celegen KK: Speakers Bureau; Bristol-Myers Squibb KK: Speakers Bureau; Novartis pharma KK: Research Funding, Speakers Bureau. Ohyashiki:Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Chugai pharmaceutical KK: Research Funding; Otsuka pharmaceutical KK: Honoraria; Alexion: Honoraria; Taiho pharmaceutical KK: Honoraria; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; MSD KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding; MSD KK: Honoraria; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau.