• KIR3DL1 and its cognate ligand for HLA-Bw status is associated with favorable achievement of TFR in patients with CML.

  • KIR/HLA status may reflect the strength of NK cell immune responses in patients with CML.

Abstract

Achievement of treatment-free remission (TFR) after tyrosine kinase inhibitor (TKI) discontinuation in patients who show a durable deep molecular response (DMR) during TKI treatment of chronic myeloid leukemia in chronic phase (CML-CP) is a therapeutic goal; however, the prognostic factors that predict successful achievement of TFR are unclear. Previously, we reported that killer immunoglobulin-like receptor (KIR) and HLA polymorphisms are associated with achievement of a DMR. Here, we investigated the association between KIR and HLA polymorphisms and TFR. We conducted the POKSTIC (POlymorphisms of Killer immunoglobulin-like receptor, which affect Stop Tyrosine kinase Inhibitor in patients with Chronic myeloid leukemia) trial, a multicenter collaborative observational study that enrolled 76 patients with CML-CP. The median age was 63 years (interquartile range [IQR], 49-70). Of 76 patients, 42 (56.6%; 95% confidence interval [CI], 47.7-66.8 at 6 months) discontinued TKIs without molecular relapse; the median follow-up time for TFR was 24 months (IQR, 16-64). KIR genotyping and allele typing did not identify risk factors for molecular relapse; however, univariate and multivariate analysis identified the combination of KIR3DL1-HLA-Bw4 (an HLA-B allele) as an independent factor for a higher risk of molecular relapse (hazard ratio, 2.206; 95% CI, 1.112-4.376; P = .024). Notably, patients at higher risk of relapse had a significantly lower number of natural killer (NK) cells at TKI discontinuation than the other patients (CD16+/CD56+ NK cells: median 499.63 cells per μL vs 629.17 cells per μL, respectively; P = .049). Thus, KIR3DL1-HLA-Bw status reflects NK cell responses and is associated with TFR. The study is registered with the UMIN Clinical Trials Registry as #UMIN000041798.

Survival outcomes of patients with chronic myeloid leukemia in chronic phase (CML-CP) have improved profoundly after the introduction of ABL1 tyrosine kinase inhibitors (TKIs), resulting in a life expectancy close to that of the general population.1 Several clinical trials show that approximately one half of patients with CML-CP who achieve a durable deep molecular response (DMR) after TKI treatment remain in molecular remission even after TKI discontinuation.2-4 Three TKIs discontinuation studies, the DOMEST trial (UMIN000012472, imatinib stop study),5 the DADI trial (UMIN000005130, second-line dasatinib stop study),6 and the first-line DADI trial (UMIN000011099, first-line dasatinib stop study),7 confirmed that TKI discontinuation is a feasible treatment strategy; indeed, it is a therapeutic goal for patients with CML-CP. However, no biomarkers for treatment-free remission (TFR) success have been identified. To establish effective prognostic factors for TFR, we need to identify patients who are at high risk of molecular relapse.8 Several lines of the evidence demonstrate that CML is responsive to immunotherapy (eg, treatment with interferon alfa, allogeneic hematopoietic stem cell transplantation, or donor lymphocyte infusion)9; thus, cancer immunosurveillance against CML is important for maintaining molecular remission in patients with CML-CP.9 

Despite advances in development of ABL1 TKIs, patients with CML-CP harbor residual leukemic cells originating from leukemic stem cells, which are resistant to TKIs.10 These residual cells remain even after TKI discontinuation; therefore, the host immune system must prevent molecular relapse.11 The importance of natural killer (NK) cell responses against CML cells is supported by several lines of evidence; indeed,9 the magnitude of the NK cell response determines whether a patient achieves TFR.6,12,13 NK cell activity is determined by the balance between activating and inhibitory signals elicited by molecules expressed on the cell surface.14 The killer immunoglobulin-like receptor (KIR) is a representative NK cell surface molecule involved in recognition, binding, and/or adhesion to HLA class I molecules expressed by target cells; this molecule has abundant structural and functional polymorphisms, which endows NK cells with functional diversity. Poor interactions between inhibitory KIRs and their cognate ligands may be associated with weak inhibition of NK cell immune responses, leading to activated NK cell functions.15,16 

Previously, we used next-generation sequencing to show that allelic polymorphisms of KIRs and HLA molecules are associated with achievement of a DMR,17 and that favorable HLA genotypes are associated with successful achievement of TFR in patients with CML-CP.18 Higher CD16+/56+ NK cell counts and lower CD4+ T-cell counts at the time of TKI discontinuation are also favorable prognostic factors for TFR.6,7 However, no comprehensive study has examined polymorphisms in KIR/HLA, as well as immune cell populations (eg, NK cells and T cells), in patients with CML-CP after discontinuation of TKIs.

The KIR3DL1 gene harbors abundant allelic polymorphisms, and expression of KIR3DL1 protein (high, low, or null) is determined by its alleles. KIR3DL1 binds the HLA-Bw4 epitope, in which the amino acid at position 80 (80Ile [isoleucine] or 80Thr [threonine]) determines binding affinity. Allelic combinations of KIR3DL1 (high, low, or null) and HLA-Bw (Bw6, Bw4-80Ile, or Bw4-80Thr) determine the avidity of the interaction (eg, KIR3DL1high binds HLA-Bw4-80Ile more strongly than Bw4-80Thr), leading to strong or weak NK cell inhibition. Hence, the combination of KIR3DL1 and HLA-Bw determines the clinical outcome of patients infected with HIV,19 patients with AML who received allogeneic stem cell transplantation,15 and others.16 

HLA-A∗24:02, which harbors the HLA-Bw4 epitope (Bw4-80Ile), is relatively predominant in Japanese individuals, and we reported previously that HLA-A∗24:02 is associated with successful achievement of TFR in patients with CML-CP.18 Although the avidity of the KIR3DL1 and HLA-Bw interaction is determined by levels of the KIR3DL1 protein and the HLA-Bw4 epitope, HLA-Bw4-80Ile (HLA-A∗24:02) weakly inhibits KIR3DL1-expressing NK cells compared with HLA-Bw4-80Ile (a HLA-B allele) regardless of the level of KIR3DL1 protein expression.20 Moreover, patients with HLA-A alleles bearing the Bw4 motif were excluded from studies investigating links between the KIR3DL1-HLA-Bw interaction and clinical outcome.15,16,19 Thus, further modifications may be needed to allow assessment of NK cell immune responses defined by KIR3DL1 and HLA-Bw combination, including the Bw4 motif on HLA-A alleles. Moreover, activating KIRs have extracellular domains that are highly homologous to those of inhibitory KIRs. Although the function of activating KIRs are not well defined, they may be associated with the clinical outcome in patients with several cancers.9 

Here, we report the results of the POKSTIC (POlymorphisms of Killer immunoglobulin-like receptor, which affect Stop Tyrosine kinase Inhibitor in patients with Chronic myeloid leukemia) trial, which is a multicenter retrospective observational study (UMIN Clinical Trials Registry [UMIN]000041798).

Study design and patients

The POKSTIC trial was an observational substudy of the DOMEST trial (UMIN000012472, imatinib stop study),5 the DADI trial (UMIN000005130, second-line dasatinib stop study),6 and the first-line DADI trial (UMIN000011099, first-line dasatinib stop study)7 performed at 18 Japanese hospitals. Patients who agreed to participate in the POKSTIC trial after completion of the 3 TKI discontinuation trials were enrolled. The clinical trial was approved by the institutional review board of each participating hospital (UMIN000041798). All procedures involving human participants were undertaken in accordance with the principles of the Declaration of Helsinki, and all participants provided written informed consent.

Procedures

For the POKSCTIC trial, peripheral blood was collected once at any time point after enrollment for KIR and HLA genotyping. Patient DNA was purified from whole blood using the QIAamp DNA Blood Mini kit (Qiagen, Germany) and stored at −20°C. Allelic genotyping of KIR genes was performed by deep-target KIR sequencing using a capture method (STAR Methods), and allelic genotyping of HLA genes was performed using Luminex-based technology, as previously described.21,22,KIR haplotypes are classified as group A or group B (eg, AA or Bx),9 and the ligands for inhibitory KIRs and KIR2DS1 have been identified (eg, KIR2DS1 is the ligand for HLA-C2). HLA-B alleles, and some HLA-A alleles, can be divided into Bw4 or Bw6 subtypes depending on their serological epitopes. HLA-Bw4 is further subdivided into Bw4-80Ile and Bw4-80Thr subtypes depending on an amino acid dimorphism (isoleucine or threonine at position 80). Boudreau et al15 defined the interaction avidity between allelic combinations of KIR3DL1 and HLA-Bw. Genotyping of KIRs and HLAs was performed by GenoDive Pharma Inc (Kanagawa, Japan). Two-color flow cytometry (FACSCalibur cytometer and BD CellQuest software, version 3.3; BD Biosciences, Franklin Lakes, NJ) was undertaken to examine T-cell and NK cell profiles before TKIs discontinuation. Real-time quantitative reverse transcription polymerase chain reaction analyses were performed at a single central laboratory (BML Inc, Bio Medical Laboratories, Tokyo, Japan) to detect molecular relapse, as previously described.5-7 According to normalized procedures, BCR::ABL1/ABL1 mRNA levels were converted using the laboratory’s conversion factor (0.87).23 

Statistical analysis

All variables (continuous variables were dichotomized based on median values) that affected TFR were assessed using a Cox proportional hazards model and the log-rank test. Significant differences between the 2 groups were established using Student t test. P < .05 was considered significant. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Tochigi, Japan), a graphical user interface for R.24 The study is registered with the UMIN Clinical Trials Registry (UMIN000041798).

Patient characteristics

Between 19 October 2020 and 7 September 2022, 76 patients who were enrolled in the DOMEST (n = 28), DADI (n = 15), and first-line DADI (n = 33) trials agreed to participate in the POKSTIC trial. The median age was 63 years (interquartile range [IQR], 49-70); 41 patients were male and 35 were female; and 50, 18, and 6 patients had low, intermediate, and high Sokal scores, respectively (2 patients had missing data). Nine patients had been treated previously with interferon-α. The TKI administered before discontinuation was imatinib in 28 cases, and dasatinib in 48 cases. The median TKI treatment duration and duration of DMR were 55.0 months (IQR, 39.0-94.5) and 28.0 months (IQR, 16.0-36.0), respectively. Of 76 patients with CML-CP, 42 (56.6%; 95% confidence interval [CI], 47.7-66.8 at 6 months) discontinued TKIs without molecular relapse, and the median follow-up time for TFR was 24 months (IQR, 16-64). Patient characteristics are summarized in Table 1. Inhibitory KIR alleles KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3 exhibited diverse polymorphisms, a finding consistent with a previous report of KIR alleles in Japanese individuals (supplemental Table 1).17,25 The KIR haplotype, and the HLA-Bw, HLA-C and KIR3DL1/HLA-Bw subtype combinations, of the enrolled patients are listed in supplemental Table 2.

Table 1.

Patient characteristics

N = 76n, median%, 95% CI, IQR
Age, y 63 49-70 
Sex   
Male 41 53.9% 
Female 35 46.1% 
Sokal risk score   
Low 50 65.8% 
Intermediate 18 23.7% 
High 7.9% 
Missing 2.6% 
Previous interferon treatment   
Yes 11.8% 
No 67 88.2% 
TFR at 6 mo 56.6% 47.7%-66.8% 
TFR at 12 mo 53.3% 43.4%-65.6% 
Median DMR time (mo) 27 16-36 
Median TKI treatment time (mo) 55 39-94 
Median follow-up time (mo) 24 14-64 
Clinical trial   
DOMEST 28 36.9% 
DADI 15 19.7% 
First-line DADI 33 43.4% 
TKI before discontinuation   
Imatinib 28 36.8% 
Dasatinib 48 63.2% 
N = 76n, median%, 95% CI, IQR
Age, y 63 49-70 
Sex   
Male 41 53.9% 
Female 35 46.1% 
Sokal risk score   
Low 50 65.8% 
Intermediate 18 23.7% 
High 7.9% 
Missing 2.6% 
Previous interferon treatment   
Yes 11.8% 
No 67 88.2% 
TFR at 6 mo 56.6% 47.7%-66.8% 
TFR at 12 mo 53.3% 43.4%-65.6% 
Median DMR time (mo) 27 16-36 
Median TKI treatment time (mo) 55 39-94 
Median follow-up time (mo) 24 14-64 
Clinical trial   
DOMEST 28 36.9% 
DADI 15 19.7% 
First-line DADI 33 43.4% 
TKI before discontinuation   
Imatinib 28 36.8% 
Dasatinib 48 63.2% 

Long-term TKI treatment (>55 months) may be a favorable prognostic factor for TFR

Univariate analysis revealed that long-term TKI treatment (>55 months) was associated with a lower risk of molecular relapse (hazard ratio [HR], 0.476; 95% CI, 0.239-0.947; P = .034; supplemental Table 3), whereas KIR genotype, the combination of KIR and its cognate ligand, the KIR3DL1/HLA-Bw interaction according to Boudreau et al,15 HLA-Bw or C genotype, and differences of T-cell and NK cell fractions did not affect the risk of molecular relapse (supplemental Tables 3 and 4). Although we further investigated whether allelic polymorphisms of KIR genes were associated with the risk of molecular relapse, this was not shown by univariate analysis (supplemental Table 5).

The KIR3DL1-HLA-Bw interaction, including HLA-Bw4 (an HLA-A allele), predicts TFR outcome in patients with CML-CP

Although we examined the KIR3DL1/HLA-Bw interaction according to Boudreau et al,15 patients with HLA-Bw4 (a HLA-A allele) were excluded from studies that investigated the association between the KIR3DL1-HLA-Bw interaction and clinical outcome.15,16,19 The HLA-A∗24:02 allele is common in Japanese individuals; indeed, the majority of patients (47 of 76 patients; 61.8%) in this study had the HLA-A∗24:02 allele. Furthermore, the affinity of KIR3DL1 for its cognate ligands HLA-Bw4 80Ile (an HLA-A allele) and HLA-Bw4 80Ile (an HLA-B allele) may be different20,26-28; therefore, further modification of the KIR3DL1 and HLA-Bw combination may be needed, including HLA-Bw4 (an HLA-A allele). Therefore, we evaluated the impact on TFR by subdividing HLA-Bw4-80Ile alleles into HLA-Bw4 80Ile HLA-A, which harbors the HLA-A allele only, and HLA-Bw4 80Ile HLA-B (HLA-B allele with/without HLA-A allele). Patients with HLA-Bw4-80Ile (HLA-B) tended to have a less favorable TFR prognosis at 6 months than those with HLA-Bw4-80Ile (HLA-A), whereas the prognosis was similar for patients with HLA-Bw4-80Thr (Bw6 [n = 15]; 73.3% [43.6-89.1] vs Bw4-80Ile [HLA-A; n = 32]; 59.4% [40.5-74.0] vs Bw4-80Ile [HLA-B; n = 24]; 41.7% [22.2-60.1] vs Bw4-80Thr [n = 5]; 60.0% [12.6-88.2]; P = .211; Table 2; Figure 1A). Furthermore, unlike Boudreau et al,15 we defined 3 KIR3DL1/HLA-Bw interaction groups to validate the risk of molecular relapse in patients with CML: a non–KIR3DL1-Bw interaction group (lacking KIR3DL1 or Bw6 with KIR3DL1), a weak–KIR3DL1-Bw interaction group (KIR3DL1 with Bw4 (HLA-A)), and a strong–KIR3DL1-Bw interaction group (KIR3DL1 with Bw4-80Ile [HLA-B]/80Thr). The strong–KIR3DL1-Bw interaction group had a higher risk of molecular relapse than the other 2 groups (HR, 1.986; 95% CI, 1.02-3.868; P = .044; Table 2; Figure 1B). Multivariate analysis also identified a strong KIR3DL1-Bw interaction as an independent factor for a higher risk of molecular relapse (HR, 2.206; 95% CI, 1.112-4.376; P = .024; Table 3).

Table 2.

Univariate analysis of polymorphisms of HLA-Bw or KIR3DL1/HLA-Bw that affect achievement of TFR

VariableHR95% CIP value
HLA-Bw4-80Ile (HLA-B) Ref   
HLA-Bw4-80Ile (HLA-A) 0.624 0.297-1.313 .214 
HLA-Bw4-80Thr 0.961 0.278-3.321 .950 
HLA-Bw6 0.373 0.124-1.126 .080 
Non–KIR3DL1-Bw interaction Ref   
Weak KIR3DL1-Bw interaction 1.343 0.488-3.696 .568 
Strong KIR3DL1-Bw interaction 2.364 0.943-5.928 .067 
Non/Weak KIR3DL1-Bw interaction Ref   
Strong KIR3DL1-Bw interaction 1.969 1.020-3.868 .044 
VariableHR95% CIP value
HLA-Bw4-80Ile (HLA-B) Ref   
HLA-Bw4-80Ile (HLA-A) 0.624 0.297-1.313 .214 
HLA-Bw4-80Thr 0.961 0.278-3.321 .950 
HLA-Bw6 0.373 0.124-1.126 .080 
Non–KIR3DL1-Bw interaction Ref   
Weak KIR3DL1-Bw interaction 1.343 0.488-3.696 .568 
Strong KIR3DL1-Bw interaction 2.364 0.943-5.928 .067 
Non/Weak KIR3DL1-Bw interaction Ref   
Strong KIR3DL1-Bw interaction 1.969 1.020-3.868 .044 

Ref, reference.

Figure 1.

Treatment free remission in patients with chronic myeloid leukemia after tyrosine kinase inhibitor discontinuation. TFR according to (A) HLA-Bw genotype; and (B) KIR3DL1 and HLA-Bw genotype.

Figure 1.

Treatment free remission in patients with chronic myeloid leukemia after tyrosine kinase inhibitor discontinuation. TFR according to (A) HLA-Bw genotype; and (B) KIR3DL1 and HLA-Bw genotype.

Close modal
Table 3.

Multivariate analysis of factors that affect achievement of TFR

VariableHR95% CIP value
KIR3DL1/Bw combination Strong KIR3DL1-Bw interaction 2.206 1.112-4.376 .024 
TKI treatment duration >55 mo 0.494 0.120-1.221 .127 
Age >63 y 0.748 0.372-1.504 .415 
Previous interferon treatment Yes 0.458 0.097-2.169 .325 
Sex Male 1.255 0.635-2.482 .514 
TKI at the time of discontinuation Imatinib 1.206 0.489-2.974 .685 
VariableHR95% CIP value
KIR3DL1/Bw combination Strong KIR3DL1-Bw interaction 2.206 1.112-4.376 .024 
TKI treatment duration >55 mo 0.494 0.120-1.221 .127 
Age >63 y 0.748 0.372-1.504 .415 
Previous interferon treatment Yes 0.458 0.097-2.169 .325 
Sex Male 1.255 0.635-2.482 .514 
TKI at the time of discontinuation Imatinib 1.206 0.489-2.974 .685 

Patients with a strong KIR3DL1-Bw interaction have a lower number of NK cells at the time of TKI discontinuation

Next, we evaluated the T-cell or NK cell counts before TKI discontinuation according to KIR3DL1-HLA-Bw status to see whether KIR/HLA genotyping reflected immune cell fractions. Notably, patients with a strong KIR3DL1-Bw interaction had a significantly lower number of NK cells at TKI discontinuation than the other 2 groups (CD16+/CD56+ cells: median, 499.63 cells per μL vs 629.17 cells per μL; P = .049; Table 4), indicating that KIR3DL1-HLA-Bw status may reflect NK cell immune responses. Because inhibitory KIRs have an extracellular domain that is highly homologous with that of activating KIRs, we evaluated the combined effect of the activating KIR gene, KIR3DS1. The non–/weak– KIR3DL1-HLA-Bw group harboring KIR3DS1 was the most likely to achieve TFR (76.5% [95% CI, 48.8-90.4]; P = .032; supplemental Table 6; supplemental Figure 1). These results suggest that the balance between activating and inhibitory signals sensed by NK cells might be important, and that KIR and HLA genotyping reflects NK cell immune responses and predicts the outcome of TFR in patients with CML.

Table 4.

Percentages of T cells or NK cells at the time of tyrosine kinase discontinuation according to KIR and human leukocyte antigen haplotype

Median (IQR)Non/weak KIR3DL1-Bw interactionStrong KIR3DL1-Bw interactionP value
CD3+/CD8+ (/mL) 444.08 (335.05-898.14) 389.03 (251.80-637.37) .227 
CD3−/CD56+ (/mL) 679.93 (484.87-1755.35) 494.04 (332.24-680.76) .048 
CD3+/CD57+ (/mL) 378.18 (216.79-723.29) 267.56 (70.54-406.70) .142 
CD16+/CD56+ (/mL) 629.17 (495.55-1645.73) 499.63 (324.16-673.62) .049 
CD56+/CD57+ (/mL) 615.71 (437.74-1486.91) 384.03 (276.53-618.24) .052 
Median (IQR)Non/weak KIR3DL1-Bw interactionStrong KIR3DL1-Bw interactionP value
CD3+/CD8+ (/mL) 444.08 (335.05-898.14) 389.03 (251.80-637.37) .227 
CD3−/CD56+ (/mL) 679.93 (484.87-1755.35) 494.04 (332.24-680.76) .048 
CD3+/CD57+ (/mL) 378.18 (216.79-723.29) 267.56 (70.54-406.70) .142 
CD16+/CD56+ (/mL) 629.17 (495.55-1645.73) 499.63 (324.16-673.62) .049 
CD56+/CD57+ (/mL) 615.71 (437.74-1486.91) 384.03 (276.53-618.24) .052 

In this study, we provide evidence that KIR3DL1 and its cognate ligand HLA-Bw are associated with successful achievement of TFR after TKI discontinuation in patients with CML-CP. The study is, to our knowledge, the largest comprehensive analysis of the effect of KIR/HLA genotyping, as well as the T-cell and NK cell fractions, on achievement of TFR by patients with CML-CP. The data show that inhibitory or activating KIR alleles exhibit diverse polymorphisms (consistent with a previous study of KIR alleles in Japanese individuals), suggesting that there may be no dominant KIR genotype involved in development of CML.17,25 

KIR genotype, KIR allele type, the combination of KIR and its cognate ligand HLA-Bw, C genotype, and differences in T-cell and NK cell fractions were not associated with the risk of molecular relapse, whereas KIR3DL1 and its cognate ligand HLA-Bw were associated with successful achievement of TFR, indicating that the KIR/HLA combination may be important for regulation of NK cell immune responses.

Neither this nor our previous study show that the allelic polymorphism in KIR3DL1/HLA-Bw identified by Boudreau et al predicts TFR outcomes in patients with CML-CP.18 The algorithms determined the strength of the KIR3DL1/HLA-Bw interaction according to KIR3DL1 protein expression levels (high, low, and null) and different HLA-Bw epitope combinations (Bw6, Bw4-80Ile, and Bw4-80Thr). Patients with HLA-Bw4 (an HLA-A allele) were excluded from North American and European studies investigating associations between the KIR3DL1–HLA-Bw interaction and clinical outcomes because the number of individuals with HLA-Bw4 (an HLA-A allele) was low.15,16,19 Furthermore, HLA-Bw4-80Ile (an HLA-A allele) binds weakly to KIR3DL1 alleles, regardless of KIR3DL1 protein expression levels, whereas HLA-Bw4-80Ile (an HLA-B allele) binds strongly.26-28 KIR3DL1+ NK cells are strongly inhibited by HLA-B∗38/B∗52 or HLA-A∗32 but weakly inhibited by HLA-A∗24, thereby leading to different clinical outcomes in patients with AML20 and suggesting that HLA-Bw4-80Ile (an HLA-A allele) and HLA-Bw4-80Ile (an HLA-B allele) inhibit KIR3DL1+ NK cells to different extents. The HLA class I α2 helix determines its capacity to bind KIR3DL1.28 

Our data also suggest that the TFR outcomes of patients with the KIR3DL1Bw4-80Ile (an HLA-B allele) combination are inferior to those of patients with the Bw4-80Ile (an HLA-A allele), possibly because of differences in affinity. Furthermore, patients with the KIR3DL1 and Bw4-80Ile (an HLA-B allele) combination had lower NK cell counts at the time of TKI discontinuation. Strong inhibitory KIR-mediated signals may result in impaired NK cell activity, suggesting that KIR3DL1/HLA-Bw status affects NK cell immune responses. Although the biological avidity of KIR3DL1 for HLA-Bw is not clear, our data suggest that genotyping of KIR3DL1/HLA-Bw may be associated with TFR outcomes in patients with CML-CP, reflecting the strength of the NK cell immune response.

HLA-A∗24:02 acts as an HLA-Bw4-80Ile motif and is relatively predominant in East Asian populations, including Japanese individuals; however, it is uncommon in Europeans and Americans.

Indeed, the majority of patients (47 of 76 patients; 61.8%) in this study had the HLA-A∗24:02 allele. HLA-A∗24:02 is a poor ligand for KIR3DL1,27,28 and patients with HLA-Bw4-80Ile (an HLA-A allele) and HLA-Bw4-80Ile (an HLA-B allele) exhibit different clinical outcomes after monoclonal antibody therapy (based on the different avidity of these alleles for KIR3DL1).29 Notably, we reported previously that HLA-A∗24:02 is a favorable predictor of TFR in patients with CML-CP, possibly because HLA-A∗24:02 is a poor ligand for KIR3DL1.18,20 HLA-Bw4-80Ile exhibits diverse allelic polymorphisms according to race; therefore, further investigation of allelic polymorphisms of KIR3DL1/HLA-Bw, and their interactions in vitro, is needed to clarify whether NK cell immune responses in CML are affected by allelic polymorphism of KIR/HLA.

Patients with AML receiving hematopoietic stem cell transplantation and who do not have a cognate ligand for KIR2DS1 (HLA-C2/C2) benefit from a donor with KIR2DS1.30 Patients with HLA-C2/C2 are at high risk of childhood acute lymphoblastic leukemia relapse31; however, the percentage of Japanese individuals with HLA-C2/C2 is very small32 (none were enrolled in this study), indicating that racial differences should also be considered with respect to the combination of KIR2DS1-HLA-C.

Unlike inhibitory KIRs, activating KIRs are not well characterized. Because activating KIRs have an extracellular domain (eg, KIR3DL1 and KIR3DS1) that is highly homologous with that of the inhibitory counterparts, coupled with a lower affinity for their cognate ligand, it is difficult to distinguish their functions and ligands from those of inhibitory KIRs. However, several activating KIR alleles (eg, KIR2DS1 for AML, and KIR2DS1 and KIR3DS1 for CML) are protective in patients with hematological malignancies,9 suggesting that stimulatory signals induced by activating KIRs may be necessary to regulate these diseases. Here, we identified the inhibitory KIR3DL1/HLA-Bw combination plus activating KIR3DS1 as the most favorable inhibitory/activating KIR combination in patients with CML-CP who discontinued TKIs. These results suggest that missing or weak inhibitory signals have the potential to activate NK cells, whereas strong activating signals have greater potential for NK cell activation upon interaction between KIR and HLA. KIR/HLA genotype–informed risk assessment may lead to development of personalized treatments that can be administered to patients at high risk of high molecular relapse before TKI discontinuation. Each TKI has a different effect on the immunomodulatory function of NK cells.33 The combination of interferon-α plus TKIs that increase NK cell–mediated cytotoxicity34 and deplete CML stem cells35 may be a candidate therapy for these patients who are at high risk.

NK cells are divided into 2 subpopulations based on surface expression of the CD56 molecule. The CD56bright NK cell subset has an immature phenotype, whereas the CD56dim NK cell subset is considered to be a mature phenotype that releases cytotoxic granules. An increase in the mature CD56dim NK subset is associated with successful TFR in patients with CML.12,13 

Several predictors of a successful TFR have been reported for CML-CP. These include longer TKI treatment duration, DMR status at TKI discontinuation, e14a2 BCR-ABL1 transcript type, a lower Sokal risk score, male sex, higher/lower NK cell counts, lower numbers of CD4+ or regulatory T cells, and prior treatment with interferon-α.9 Among these, the longer DMR time may be an important factor that determines the optimal duration of DMR time before TKI discontinuation (generally 2 years).4,36 Although different study designs generate inconsistent data, we found that long-term TKI treatment and the magnitude of NK cell immune responses may be favorable predictors of TFR in this study, indicating that our patients were a representative cohort.

The study has several limitations. First, it was retrospective in nature and enrolled only 76 subjects who agreed to participate during enrollment on larger clinical trials; this is a small number compared with other TKI stop studies (eg, EURO-SKI).4 Second, we did not undertake in vitro examination of the biological effects of KIR/HLA polymorphisms in patients with CML-CP. Third, the enrolled patients were all Japanese; thus the data may not be generalizable to other ethnic groups.

In conclusion, we found that the status of KIR3DL1 and its cognate ligand HLA-Bw reflects the strength of NK cell immune responses and predicts the outcome of TFR in patients with CML.

The authors thank Nobuyo Yawata (Kyushu University), Hidenori Tanaka (HLA Foundation, Japan), and Saji Hiroh (HLA Foundation) for critical discussion.

The study was supported mainly by Bristol Myers Squibb (S.K.), and partially by research grants from the Japan Society for the Promotion of Science grants-in-aid for scientific research (21K16245 to H.U.), the Shinnihon Foundation of Advanced Medical Treatment Research (H.U.), the Takeda Science Foundation (H.U.), the Medical Research Encouragement Prize of The Japan Medical Association (H.U.), a Japanese Society of Hematology research grant (H.U.), and the Japanese Foundation for Multidisciplinary Treatment of Cancer (H.U.).

The funder of the study reviewed the report before submission but played no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Contribution: H.U. and S. Kimura made substantial contributions to study conception, design, and data analysis and interpretation; Y.U., S.F., K.U., H.T., M.O., S. Kowata, K.M., A.H., M.S., T. Kumagai, T.T., S.I., J.I., T.N, T. Kobayashi, E.K., H.O., T.I., and S. Kimura enrolled patients and collected data; H.U., K. Kamachi, K. Kidoguchi, T.S., and S. Kimura wrote the manuscript; A.K. performed statistical analyses; H.U. and S. Kimura critically reviewed the draft manuscripts; and all authors approved the final version.

Conflict-of-interest disclosure: Y.U. received honoraria from Sanofi. S.F. received honoraria from Bristol Myers Squibb, Nippon Shinyaku, Otsuka Pharmaceuticals, Pfizer, Novartis, MSD, Sanofi, Janssen Pharmaceuticals, SymBio Pharmaceuticals, Kyowa Hakko Kirin, AstraZeneca, CSL Behring, Meiji Seika Pharma, AbbVie, Takeda Pharmaceuticals, and Chugai Pharmaceuticals; and received research funding from Shionogi, Kyowa Hakko Kirin, Chugai Pharma, Otsuka Pharmaceuticals, Asahi-Kasei Pharma, and Daiichi Sankyo. K.U. received research funding from Astellas, AbbVie, Bristol Myers Squibb, Janssen Pharmaceuticals, Ono Pharmaceuticals, Otsuka Pharmaceuticals, Chugai Pharmaceuticals, Apellis Pharmaceuticals, Yakult Pharmaceutical Industry, MSD, Amgen, Alexion Pharmaceuticals, Incyte, Eisai, Kyowa Kirin, Sanofi, SymBio Pharmaceuticals, Celgene, Daichi Sankyo, Dainippon Sumitomo Pharma, Nippon Shinyaku, Novartis, Mundipharma, and Takeda Pharmaceuticals. H.T. received honoraria from Novartis, AbbVie, Alexion Pharmaceuticals, Incyte, Ono Pharmaceuticals, Kyowa-Kirin, Sanofi, Takeda Pharmaceuticals, Nippon Shinyaku, Pfizer, and Bristol Myers Squibb. M.O. received honoraria from Astellas, Amgen, Alnylam Japan, Alexion Pharmaceuticals, Eisai, Otsuka Pharmaceuticals, Ohara Pharmaceuticals, Kyowa-Kirin, Sanofi, Sandoz, SymBio Pharmaceuticals, Takeda Pharmaceuticals, Chugai Pharmaceuticals, and Nippon Shinyaku. K.M. received honoraria from Bristol Myers Squibb, Otsuka Pharmaceuticals, Chugai Pharmaceutical, Ono Pharmaceuticals, Novartis, Pfizer, Takeda Pharmaceuticals, Sanofi, and Meiji Seika Pharma. T. Kumagai received honoraria from Bristol Myers Squibb, Novartis, Pfizer, and Otsuka Pharmaceuticals. S.I. received honoraria from Alexion Pharmaceuticals, Astellas, CSL Behring, Daiichi Sankyo, Otsuka Pharmaceuticals, Meiji Pharma, Nippon Shinyaku, Novartis, Otsuka, Sanofi, and SymBio Pharmaceuticals; and reports research funding from Alexion Pharmaceuticals, MSD, Otsuka, Sanofi, and SymBio Pharmaceuticals. J.I. received honoraria from Bristol Myers Squibb and Novartis. T.I. received honoraria from Asahi-Kasei Pharma and Nippon Shinyaku; and received research funding from Asahi-Kasei Pharma, Nippon Shinyaku, Astellas, AbbVie, Otsuka Pharmaceuticals, Takeda Pharmaceuticals, Novartis, and Janssen Pharmaceuticals. S.K. received honoraria from Bristol Myers Squibb, Novartis, Pfizer, and Otsuka Pharmaceuticals; and received research funding from Bristol Myers Squibb, Pfizer, and Ohara Pharmaceuticals. The remaining authors declare no competing financial interests.

Correspondence: Hiroshi Ureshino, Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan;; and Shinya Kimura, Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan; email: shkimu@cc.saga-u.ac.jp.

1.
Bower
H
,
Björkholm
M
,
Dickman
PW
, et al
.
Life expectancy of patients with chronic myeloid leukemia approaches the life expectancy of the general population
.
J Clin Oncol
.
2016
;
34
(
24
):
2851
-
2857
.
2.
Mahon
F-X
,
Réa
D
,
Guilhot
J
, et al
.
Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial
.
Lancet Oncol
.
2010
;
11
(
11
):
1029
-
1035
.
3.
Hochhaus
A
,
Masszi
T
,
Giles
FJ
, et al
.
Treatment-free remission following frontline nilotinib in patients with chronic myeloid leukemia in chronic phase: results from the ENESTfreedom study
.
Leukemia
.
2017
;
31
(
7
):
1525
-
1531
.
4.
Saussele
S
,
Richter
J
,
Guilhot
J
, et al
.
Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial
.
Lancet Oncol
.
2018
;
19
(
6
):
747
-
757
.
5.
Fujisawa
S
,
Ueda
Y
,
Usuki
K
, et al
.
Feasibility of the imatinib stop study in the Japanese clinical setting: delightedly overcome CML expert stop TKI trial (DOMEST Trial)
.
Int J Clin Oncol
.
2019
;
24
(
4
):
445
-
453
.
6.
Imagawa
J
,
Tanaka
H
,
Okada
M
, et al
.
Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial
.
Lancet Haematol
.
2015
;
2
(
12
):
e528
-
e535
.
7.
Kimura
S
,
Imagawa
J
,
Murai
K
, et al
.
Treatment-free remission after first-line dasatinib discontinuation in patients with chronic myeloid leukaemia (first-line DADI trial): a single-arm, multicentre, phase 2 trial
.
Lancet Haematol
.
2020
;
7
(
3
):
e218
-
e225
.
8.
Hochhaus
A
,
Baccarani
M
,
Silver
RT
, et al
.
European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia
.
Leukemia
.
2020
;
34
(
4
):
966
-
984
.
9.
Ureshino
H
,
Shindo
T
,
Kimura
S
.
Role of cancer immunology in chronic myelogenous leukemia
.
Leuk Res
.
2020
;
88
:
106273
.
10.
Hamilton
A
,
Helgason
GV
,
Schemionek
M
, et al
.
Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival
.
Blood
.
2012
;
119
(
6
):
1501
-
1510
.
11.
Ross
DM
,
Branford
S
,
Seymour
JF
, et al
.
Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study
.
Blood
.
2013
;
122
(
4
):
515
-
522
.
12.
Rea
D
,
Henry
G
,
Khaznadar
Z
, et al
.
Natural killer-cell counts are associated with molecular relapse-free survival after imatinib discontinuation in chronic myeloid leukemia: the IMMUNOSTIM study
.
Haematologica
.
2017
;
102
(
8
):
1368
-
1377
.
13.
Ilander
M
,
Olsson-Strömberg
U
,
Schlums
H
, et al
.
Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia
.
Leukemia
.
2017
;
31
(
5
):
1108
-
1116
.
14.
Vivier
E
,
Raulet
DH
,
Moretta
A
, et al
.
Innate or adaptive immunity? The example of natural killer cells
.
Science
.
2011
;
331
(
6013
):
44
-
49
.
15.
Boudreau
JE
,
Giglio
F
,
Gooley
TA
, et al
.
KIR3DL1/ HLA-B subtypes govern acute myelogenous leukemia relapse after hematopoietic cell transplantation
.
J Clin Oncol
.
2017
;
35
(
20
):
2268
-
2278
.
16.
Forlenza
CJ
,
Boudreau
JE
,
Zheng
J
, et al
.
KIR3DL1 allelic polymorphism and HLA-B epitopes modulate response to Anti-GD2 monoclonal antibody in patients with neuroblastoma
.
J Clin Oncol
.
2016
;
34
(
21
):
2443
-
2451
.
17.
Ureshino
H
,
Shindo
T
,
Kojima
H
, et al
.
Allelic polymorphisms of KIR s and HLA s predict favorable responses to tyrosine kinase inhibitors in CML
.
Cancer Immunol Res
.
2018
;
6
(
6
):
745
-
754
.
18.
Ureshino
H
,
Shindo
T
,
Tanaka
H
,
Saji
H
,
Kimura
S
.
HLA polymorphisms are associated with treatment-free remission following discontinuation of tyrosine kinase inhibitors in chronic myeloid leukemia
.
Mol Cancer Ther
.
2021
;
20
(
1
):
142
-
149
.
19.
Martin
MP
,
Qi
Y
,
Gao
X
, et al
.
Innate partnership of HLA-B and KIR3DL1 subtypes against HIV-1
.
Nat Genet
.
2007
;
39
(
6
):
733
-
740
.
20.
van der Ploeg
K
,
Le Luduec
JB
,
Stevenson
PA
, et al
.
HLA-A alleles influencing NK cell function impact AML relapse following allogeneic hematopoietic cell transplantation
.
Blood Adv
.
2020
;
4
(
19
):
4955
-
4964
.
21.
Sakaue
S
,
Hosomichi
K
,
Hirata
J
, et al
.
Decoding the diversity of killer immunoglobulin-like receptors by deep sequencing and a high-resolution imputation method
.
Cell Genomics
.
2022
;
2
(
3
):
100101
.
22.
Hashimoto
S
,
Nakajima
F
,
Imanishi
T
, et al
.
Implications of HLA diversity among regions for bone marrow donor searches in Japan
.
HLA
.
2020
;
96
(
1
):
24
-
42
.
23.
Yoshida
C
,
Fletcher
L
,
Ohashi
K
, et al
.
Harmonization of molecular monitoring of chronic myeloid leukemia therapy in Japan
.
Int J Clin Oncol
.
2012
;
17
(
6
):
584
-
589
.
24.
Kanda
Y
.
Investigation of the freely available easy-to-use software “EZR” for medical statistics
.
Bone Marrow Transplant
.
2013
;
48
(
3
):
452
-
458
.
25.
Yawata
M
,
Yawata
N
,
Draghi
M
, et al
.
Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function
.
J Exp Med
.
2006
;
203
(
3
):
633
-
645
.
26.
Boudreau
JE
,
Mulrooney
TJ
,
Le Luduec
J-B
,
Barker
E
,
Hsu
KC
.
KIR3DL1 and HLA-B density and binding calibrate NK education and response to HIV
.
J Immunol
.
2016
;
196
(
8
):
3398
-
3410
.
27.
Saunders
PM
,
Pymm
P
,
Pietra
G
, et al
.
Killer cell immunoglobulin-like receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition
.
J Exp Med
.
2016
;
213
(
5
):
791
-
807
.
28.
Saunders
PM
,
MacLachlan
BJ
,
Widjaja
J
, et al
.
The role of the HLA class I α2 helix in determining ligand hierarchy for the killer cell Ig-like receptor 3DL1
.
J Immunol
.
2021
;
206
(
4
):
849
-
860
.
29.
Erbe
AK
,
Wang
W
,
Reville
PK
, et al
.
HLA-Bw4-I-80 isoform differentially influences clinical outcome as compared to HLA-Bw4-T-80 and HLA-A-Bw4 isoforms in rituximab or dinutuximab-based cancer immunotherapy
.
Front Immunol
.
2017
;
8
:
1
-
9
.
30.
Venstrom
JM
,
Pittari
G
,
Gooley
TA
, et al
.
HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1
.
N Engl J Med
.
2012
;
367
(
9
):
805
-
816
.
31.
Babor
F
,
Manser
AR
,
Fischer
JC
, et al
.
KIR ligand C2 is associated with increased susceptibility to childhood ALL and confers an elevated risk for late relapse
.
Blood
.
2014
;
124
(
14
):
2248
-
2251
.
32.
Hsu
KC
,
Gooley
T
,
Malkki
M
, et al
.
KIR ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy
.
Biol Blood Marrow Transplant
.
2006
;
12
(
8
):
828
-
836
.
33.
Salih
J
,
Hilpert
J
,
Placke
T
, et al
.
The BCR/ABL-inhibitors imatinib, nilotinib and dasatinib differentially affect NK cell reactivity
.
Int J Cancer
.
2010
;
127
(
9
):
2119
-
2128
.
34.
Takahashi
N
,
Kyo
T
,
Maeda
Y
, et al
.
Discontinuation of imatinib in Japanese patients with chronic myeloid leukemia
.
Haematologica
.
2012
;
97
(
6
):
903
-
906
.
35.
Talpaz
M
,
Mercer
J
,
Hehlmann
R
.
The interferon-alpha revival in CML
.
Ann Hematol
.
2015
;
94
(
2
):
195
-
207
.
36.
Haddad
FG
,
Sasaki
K
,
Issa
GC
, et al
.
Treatment-free remission in patients with chronic myeloid leukemia following the discontinuation of tyrosine kinase inhibitors
.
Am J Hematol
.
2022
;
97
(
7
):
856
-
864
.

Author notes

Data are available upon reasonable request from the corresponding author, Hiroshi Ureshino (hureshin@hiroshima-u.ac.jp/sr0795@cc.saga-u.ac.jp).

The online version of this article contains a data supplement.

Supplemental data