REGULAR ARTICLE

Prospective evaluation of prognostic impact of KIT mutations on acute

myeloid leukemia with RUNX1-RUNX1T1 and CBFB-MYH11

Yuichi Ishikawa,1,* Naomi Kawashima,1,* Yoshiko Atsuta,2 Isamu Sugiura,3 Masashi Sawa,4 Nobuaki Dobashi,5 Hisayuki Yokoyama,6
Noriko Doki,7 Akihiro Tomita,8 Toru Kiguchi,9 Shiro Koh,10 Heiwa Kanamori,11 Noriyoshi Iriyama,12 Akio Kohno,13 Yukiyoshi Moriuchi,14
Noboru Asada,15 Daiki Hirano,16 Kazuto Togitani,17 Toru Sakura,18 Maki Hagihara,19 Tatsuki Tomikawa,20 Yasuhisa Yokoyama,21
Norio Asou,22 Shigeki Ohtake,23 Itaru Matsumura,24 Yasushi Miyazaki,25 Tomoki Naoe,16 and Hitoshi Kiyoi,1 for the Japan Adult Leukemia
Study Group
1
Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Japanese Data Center for Hematopoietic Cell Transplantation,
Nagoya, Japan; 3Division of Hematology and Oncology, Toyohashi Municipal Hospital, Toyohashi, Japan; 4Department of Hematology and Oncology, Anjo Kosei Hospital, Anjo,
Japan; 5Division of Clinical Oncology and Hematology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan; 6Department of Hematology,
National Hospital Organization Sendai Medical Center, Sendai, Japan; 7Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital,
Tokyo, Japan; 8Department of Hematology, Fujita Health University School of Medicine, Toyoake, Japan; 9Department of Hematology, Chugoku Central Hospital, Fukuyama,
Japan; 10Department of Hematology, Fuchu Hospital, Izumi, Japan; 11Department of Hematology, Kanagawa Cancer Center, Yokohama, Japan; 12Division of Hematology and
Rheumatology, Nihon University School of Medicine, Tokyo, Japan; 13Department of Hematology and Oncology, JA Aichi Konan Kosei Hospital, Konan, Japan; 14Department of
Hematology, Sasebo City General Hospital, Sasebo, Japan; 15Department of Hematology and Oncology, Okayama University Hospital, Okayama, Japan; 16Department of
Hematology, National Hospital Organization Nagoya Medical Center, Nagoya, Japan; 17Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi,
Japan; 18Leukemia Research Center, Saiseikai Maebashi Hospital, Maebashi, Japan; 19Department of Hematology and Clinical Immunology, Yokohama City University Hospital,
Japan; 20Department of Hematology, Saitama Medical Center, Saitama Medical University, Kawagoe, Japan; 21Department of Hematology, Faculty of Medicine, University of
Tsukuba, Tsukuba, Japan; 22Department of Hematology, International Medical Center, Saitama Medical University, Hidaka, Japan; 23Kanazawa University, Kanazawa, Japan;
24
Department of Hematology and Rheumatology, Kindai University Faculty of Medicine, Osaka, Japan; and 25Department of Hematology, Atomic Bomb Disease Institute,
Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

The prognostic impact of KIT mutation on core-binding factor acute myeloid leukemia (CBF-
Key Points
AML) remains controversial. We registered 199 newly diagnosed de novo CBF-AML patients,
• KIT exon 17 mutation is aged 16 to 64 years, who achieved complete remission. They received 3 courses of high-dose
a poor prognostic fac- cytarabine therapy and no further treatment until hematological relapse. Mutations in
tor in AML patients with
exons 8, 10-11, and 17 of the KIT gene were analyzed. Furthermore, we analyzed mutations
RUNX1-RUNX1T1, but
in 56 genes that are frequently identified in myeloid malignancies and evaluated minimal
not in those with
residual disease (MRD). The primary end point was relapse-free survival (RFS) according to
CBFB-MYH11.
KIT mutations. The RFS in KIT-mutated patients was inferior to that in unmutated patients
• NRAS mutation is (hazard ratio, 1.92; 95% confidence interval, 1.23-3.00; P 5 .003). Based on subgroup
a poor prognostic fac-
analysis, KIT mutations had a prognostic impact in patients with RUNX1-RUNX1T1, but not
tor in AML patients with
in those with CBFB-MYH11, and only exon 17 mutation had a significant prognostic impact.
CBFB-MYH11.
Multivariate Cox regression analysis with stepwise selection revealed that the KIT exon 17
mutation and the presence of extramedullary tumors in patients with RUNX1-RUNX1T1, and
loss of chromosome X or Y and NRAS mutation in patients with CBFB-MYH11 were poor
prognostic factors for RFS. MRD was evaluated in 112 patients, and it was associated with
a poorer RFS in the patients with CBFB-MYH11, but not in those with RUNX1-RUNX1T1.
These results suggested that it is necessary to separately evaluate AML with
RUNX1-RUNX1T1 or CBFB-MYH11 according to appropriate prognostic factors. This
study was registered at www.umin.ac.jp/ctr/ as #UMIN000003434.

Submitted 15 July 2019; accepted 7 November 2019; published online 3 January Send data sharing requests to the corresponding author at kiyoi@med.nagoya-u.ac.jp.
2020. DOI 10.1182/bloodadvances.2019000709. The full-text version of this article contains a data supplement.
*Y.I. and N.K. contributed equally to this study as first authors. © 2020 by The American Society of Hematology
Presented in part at the 60th annual meeting of the American Society of Hematology,
San Diego, CA, 2 December 2018.

66 14 JANUARY 2020 x VOLUME 4, NUMBER 1

Introduction those with a complete left bundle branch block, those with 2 branch
blocks, those with ventricular or atrial tachyarrhythmia requiring
treatment, those with a digestive tract ulcer of A2 stage or higher,
Acute myeloid leukemia (AML) with RUNX1-RUNX1T1 or CBFB- those with uncontrolled diabetes mellitus, those with a fasting
MYH11 is categorized into a favorable cytogenetic risk group, and blood glucose level #200 that could not be maintained by insulin
allogeneic hematopoietic stem cell transplantation (HSCT) is not administration, and those with active uncontrolled infections.
generally recommended during the first complete remission (CR).1 Written informed consent was received from all patients. The protocol
However, several prognostic factors, including genetic alterations, was approved by the ethics committees of all participating institutions.
have been demonstrated.2-8 In particular, KIT mutation has been This study was registered in the UMIN Clinical Trials Registry
suggested to be associated with a poor prognosis in AML patients (UMIN000003434, http://www.umin.ac.jp/ctr/).
with RUNX1-RUNX1T1 or CBFB-MYH11.3,5,9-12 Many types of
KIT mutations have been identified in cancer cells, but there are Treatments
3 mutation hot-spots (exon 8, exon 10-11, and exon 17) in
All patients received 3 courses of HiDAC therapy (2 g/m2 by 3-hour
AML.3,10,12-14 Several groups previously reported that KIT mutation
infusion every 12 hours for 5 days), as previously reported.20,21 For
was a poor prognostic factor for overall survival (OS), event-free-
patients older than 60 years of age, 1 dose of cytarabine was reduced
survival, and/or relapse-free-survival (RFS) in AML with RUNX1-RUNX1T1
to 1.5 g/m2. We recommended that patients be hospitalized in
or CBFB-MYH11.3,10,12,15,16 On the other, some groups reported
the lower than NASA Class 10 000 clean room during treatment.
that KIT mutation was not associated with the long-term prognosis.1,17-19
Best supportive care, including administration of antibiotics and
This controversy may be caused by several study limitations such as
platelet transfusion, was performed if indicated. When patients
the prognostic relevance being mostly evaluated retrospectively,
had life-threatening documented infections during neutropenia,
not all types of KIT mutations being evaluated, and the analyzed
the use of granulocyte colony-stimulating factor was permitted.
patient number being insufficient for statistical power. Furthermore,
Bone marrow (BM) examination was performed to confirm CR
the prognostic impact of recently identified recurrent mutations,
before each course and at the end of the last course. After the
such as ASXL1, ASXL2, and ZBTB7A, on AML with RUNX1-RUNX1T1
completion of 3 courses of HiDAC therapy, patients did not
or CBFB-MYH11 remains unclear. We therefore conducted a pro-
receive further chemotherapy, immunotherapy, or HSCT until hema-
spective, multicenter cooperative study (Japan Adult Leukemia Study
tological relapse was observed. If patients developed hematological
Group [JALSG] core-binding factor [CBF]-AML209-KIT) to evaluate
relapse, the best treatment, including HSCT, was applied at each
the prognostic impact of KIT mutation in AML patients with
institute.
RUNX1-RUNX1T1 or CBFB-MYH11 who were treated using
the same high-dose cytarabine (HiDAC) regimen. Furthermore, Cytogenetic and molecular analyses
we evaluated the frequency and clinical relevance of other gene
mutations and prognostic impact of minimal residual disease (MRD). Cytogenetic G-banding analysis was performed using standard
methods at each institute. Chimeric gene transcripts of RUNX1-
Methods RUNX1T1 and CBFB-MYH11 were centrally quantified by the
real-time quantitative polymerase chain reaction (RT-qPCR) method
Patients
using BM or peripheral blood samples at diagnosis according to
Patients aged 16 to 64 years old with newly diagnosed de novo a previous report.22 FLT3-ITD mutation was centrally examined by
AML according to the World Health Organization 2008 classifica- the PCR method; genomic PCR was performed and the amplified
tion, and an Eastern Cooperative Oncology Group performance products were subjected to agarose gel electrophoresis as
status of 2 or lower were eligible for enrollment if they had a previously reported. 23 These results were immediately reported
RUNX1-RUNX1T1 or CBFB-MYH11 chimeric transcript and achieved to each institute. Residual DNA and RNA samples were
CR within 2 courses of standard induction therapy. All patients in preserved at the JALSG sample storage center. Mutations in
this study were registered in the JALSG registration study after exons 8, 10, 11, and 17 in the KIT gene were analyzed using the
being diagnosed with AML and were treated using a standard dose preserved DNA extracted from AML cells at diagnosis, as
of idarubicin 1 cytarabine or daunorubicin 1 cytarabine for induction previously reported. 24 We also analyzed mutations in FLT3,
therapy, as shown in supplemental Table 1. Other inclusion criteria NPM1, CEBPA, NRAS, TP53, WT1, and IDH1 genes, and partial
were: serum alanine aminotransferase or serum aspartate aminotrans- tandem duplication of the KMT2A gene (KMT2A-PTD) in 198
ferase level up to 2.5 times the institutional upper limit of normal; serum patients (supplemental Table 2). In addition, we analyzed mutations
bilirubin up to 2.0 mg/dL; serum creatinine level up to 1.5 times the in 49 other genes in 170 patients using the TruSight Myeloid
institutional upper limit of normal; left ventricular ejection fraction Sequencing Panel (Illumina, San Diego, CA), as previously reported
greater than 50% on ultrasound echocardiography; or PaO2 greater (supplemental Table 2).8,25
than 60 Torr or SpO2 greater than 90% under room air. We excluded
patients with secondary AML, those with a history of hematological Assessment of MRD
abnormalities before registration, those with other types of malignant
The chimeric transcript level of RUNX1-RUNX1T1 or CBFB-MYH11
tumors, those with a history of craniotomy, and those with a history
using BM samples was evaluated after the hematological recovery
of receiving whole brain radiation to have a more uniform patient
from the third course of HiDAC therapy by RT-qPCR, as previously
background and to exclude safety concerns associated with
reported.22 Because the lower detection limit of RUNX1-RUNX1T1
previous treatments.
and CBFB-MYH11 transcripts was 50 copies/mg RNA in our
We also excluded patients with cardiac dysfunction corresponding system, we defined MRD as positive if each transcript was
to either of the following: Patients who need to use cardiac pacemakers, $50 copies/mg RNA. To avoid interfering with protocol treatment,

14 JANUARY 2020 x VOLUME 4, NUMBER 1 PROGNOSTIC IMPACT OF KIT MUTATIONS ON CBF-AML 67

the results of gene mutations and MRD levels were not disclosed to Results
institutes until 2 years after registration.
Enrollment
Definitions and study end points Between May 2010 and September 2014, 203 patients from
Relapse after CR was defined as the presence of at least 1 of the 85 institutes were enrolled. Four patients were excluded: 3 did not
following: reappearance of leukemic blasts in the peripheral blood, fulfill the eligibility criteria and 1 received other treatment (Figure 1).
recurrence of more than 5% blasts in BM not attributable to any Thus, 199 patients consisting of 132 (66.3%) patients with
other cause, such as BM regeneration after chemotherapy, and RUNX1-RUNX1T1 and 67 (33.7%) with CBFB-MYH11 were
development of extramedullary leukemia. We did not include included in the primary analysis.
chimeric transcript levels of RUNX1-RUNX1T1 or CBFB-MYH11
KIT mutations and patient characteristics
in the definition of CR or relapse. OS was defined as the time
from the start date of induction therapy to death from any KIT mutations were identified in 63 of the 199 patients (31.7%): 42
cause or last follow-up. RFS was defined as the time from of 132 (31.8%) and 21 of 67 (31.3%) patients with RUNX1-RUNX1T1
the date of CR to relapse or death of any cause or the last and CBFB-MYH11, respectively (Table 1; supplemental Table 3).
follow-up. A total of 68 mutations were identified in the 63 patients with KIT
mutations and mutation in exon 17 was the most frequently
The primary end point was RFS in AML patients with RUNX1- identified (50/68, 73.5%), followed by that in exon 8 (14/68,
RUNX1T1 or CBFB-MYH11 according to KIT mutations. 20.6%) and in exons 10-11 (4/68, 5.9%) (supplemental Table 4).
Secondary end points were OS according to KIT mutations, KIT mutation in exon 8 was more frequent in AML with CBFB-
clinical relevance of the genetic alterations, prognostic impact MYH11 (9/24, 37.5%) than in that with RUNX1-RUNX1T1 (5/44,
of known prognostic factors, and MRD levels after the completion 11.4%) (P 5 .014). Although mutation at the N822 residue in exon
of therapy. 17 was identified in 13 of 44 (29.5%) KIT mutations of the patients
with RUNX1-RUNX1T1, no patient with CBFB-MYH11 had this
Statistical analysis mutation (P 5 .008); however, mutation at the D816 residue was
This study was prospectively powered to demonstrate a lower equally identified in patients with RUNX1-RUNX1T1 (21/44, 47.7%)
RFS of AML patients with RUNX1-RUNX1T1 or CBFB-MYH11 and CBFB-MYH11 (13/24, 54.1%). Patient characteristics accord-
harboring KIT mutations than in those without them. With a sample ing to KIT mutation are presented in Table 1 and supplemental
size of 175 patients, the study had a power .90% at a 5% level of Table 4. The median BM blast percentage and white blood cell
significance by the log-rank test if the incidence of KIT mutation in (WBC) index27 in the patients with RUNX1-RUNX1T1 were higher
AML with RUNX1-RUNX1T1 or CBFB-MYH11 was 25%, and the in the KIT-mutated patients than in the unmutated patients.
2-year RFS of patients with and without KIT mutations was 35% G-banding karyotype analysis was performed on 197 patients. An
and 60%, respectively, according to previous reports.9,10,15,26 additional cytogenetic abnormality was observed in 126 patients.
The RFS and OS were estimated by the Kaplan-Meier method; However, there was no significant difference in additional cytoge-
differences in survival distributions were evaluated using the log- netic abnormalities between KIT-mutated and unmutated patients
rank test. Differences in continuous variables were analyzed by (supplemental Table 5).
the Mann-Whitney U test for distribution between 2 groups.
Analysis of frequencies was performed using Fisher’s exact test Landscape of gene mutations in CBF-AML
for 2 3 2 tables or Pearson’s x2 test for larger tables. The Identified gene mutations in analyzed AML patients are shown in
prognostic significance of the clinical variables was assessed Figure 2A. KIT mutation (31.7%) was the most frequently identified,
using the Cox proportional hazards model. We also attempted to followed by NRAS (21.7%), FLT3 (12.1%), and ASXL2 (11.8%)
construct a prognostic factor model in CBF-AML by adding mutations in AML with RUNX1-RUNX1T1 or CBFB-MYH11; however,
a molecular type that stratifies the prognosis of CBF-AML. the mutation status was different between AML with RUNX1-RUNX1T1
Factors that independently affect disease-free survival were and CBFB-MYH11 (Figure 2B). ASXL2, ASXL1, RAD21, and
narrowed down using the stepwise method and multivariate ZBTB7A mutations were more frequent in AML with RUNX1-
analysis using the Cox proportional hazard model. Factors that RUNX1T1 than in that with CBFB-MYH11. In contrast, NRAS,
were candidates for model construction were factors whose P , .1 KRAS, and FLT3-TKD mutations were more frequent in AML
in the univariate analysis. Two-sided P , .05 was considered with CBFB-MYH11 than in that with RUNX1-RUNX1T1 (Figure 2B).
significant. Analyses were performed using Stata version 13×1 Significantly overlapping mutations were observed between
(StataCorp, College Station, TX). KIT and ASXL2, NRAS and KRAS, and CSF3R and ASXL1.
One planned interim analysis for the primary end point was to be Mutually exclusive mutations were observed between KIT and
performed 1 year after the 100th patient was enrolled; this analysis NRAS and KIT and ZBTB7A (Figure 2C-E; supplemental
took place independently of the study secretariat in March 2014, Figure 1).
and the JALSG data and safety monitoring board made the decision
to continue this study. Significance for the primary end point Prognostic impact of KIT mutation
followed the O’Brien-Fleming method to maintain a 5% level of The median follow-up period was 1566 days (range, 356-2453),
significance. Allogeneic transplantation was performed during and the 2-year RFS and OS in the entire cohort were 61.31% (95%
the first remission period for 3 patients, and they were treated as confidence interval [95% CI]: 54.11-67.72) and 85.79% (95% CI:
deviations and censored at the time of transplantation in RFS 80.09-89.97), respectively. By chimeric transcripts, the RFS and
analysis, including the multivariate analysis. OS of patients with RUNX1-RUNX1T1 were not significantly different

68 ISHIKAWA et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1

Figure 1. CONSORT flow diagram. The primary end
point, relapse-free survival, was evaluated in 199 eligible 203 patients enrolled
patients. Prognostic analysis of MRD was performed on 112
3 excluded
patients whose samples were collected.
3 did not meet eligibility criteria

200 analyzed for KIT mutations

1 excluded
1 recieved the other treatment

199 patients eligible for the primary endopoint analysis

170 analyzed for 56 genes mutations

28 analyzed for 11 genes mutations
1 not available the sample

87 not collected the sample for MRD

5 relapsed during the treatment
5 died during the treatment
7 changed or stpped the treatment
70 unknown reason

112 patients analyzed for MRD

from those of patients with CBFB-MYH11: the 2-year RFS rates Prognostic factors in CBF-AML
were 62.3% (95% CI, 53.3-70.0) and 59.6% (95% CI, 46.8-70.2)
We examined prognostic factors for RFS in 199 patients who were
for RUNX1-RUNX1T1 and CBFB-MYH11, respectively (P 5 .88)
eligible for analysis of the primary end point. Multivariate Cox
(supplemental Figure 4).
regression analysis with stepwise selection demonstrated that
The 2-year RFS rates were 48.6% (95% CI, 35.7-60.3) and 67.1% only the KIT exon 17 mutation was an independent poor
(95% CI, 58.5-74.4) in KIT-mutated and unmutated patients, prognostic factor for RFS in CBF-AML patients (HR, 2.42; 95% CI,
respectively (hazard ratio [HR], 1.92; 95% CI, 1.23-3.00; P 5 .003 1.52-3.85; P , .001).
by log-rank test) (Figure 3A). Among the 3 types of KIT mutations,
By CBF-subtype, KIT exon 17 mutation (HR, 4.17; 95% CI,
only the mutation in exon 17 had a lower prognostic impact on the
2.38-7.34; P , .001) and the presence of extramedullary tumors
RFS of CBF-AML patients (HR, 2.30; 95% CI, 1.45-3.64; P , .001)
(HR, 3.85; 95% CI, 1.35-10.9; P 5 .011) in patients with
(Figure 3B). Furthermore, mutations at D816 and N822 residues
RUNX1-RUNX1T1, and loss of chromosome X or Y (HR, 5.79;
had a significant prognostic impact, whereas the prognostic impact
95% CI, 1.21-27.6; P 5 .03) and NRAS mutation (HR, 2.38;
of other mutations in exon 17 was unclear because of the small
95% CI, 1.03-5.53; P 5 .04) in those with CBFB-MYH11 were
number of patients (supplemental Figure 2).
identified as poor prognostic factors for RFS by multivariate
Although there was no significant difference in RFS between the analysis (Table 2).
patients with RUNX1-RUNX1T1 and CBFB-MYH11 (supplemental
Figure 3), based on subgroup analysis, KIT mutations had a We also analyzed the prognostic impact of gene mutation in
prognostic impact on RFS only in patients with RUNX1-RUNX1T1: 170 patients in whom 56 gene mutations were examined. By
the 2-year RFS rates were 39.5% (95% CI, 24.7-53.9) and multivariate analysis, KIT mutation (HR, 3.56; 95% CI, 1.97-6.44;
72.8% (95% CI, 62.2-80.9) in KIT-mutated and unmutated P , .001) and TET2 mutation (HR, 2.53; 95% CI, 1.37-11.5; P 5 .01)
patients, respectively (HR, 3.27; 95% CI, 1.90-5.64; P , .001) in patients with RUNX1-RUNX1T1, and NRAS mutation (HR, 2.36;
(Figure 3C). Furthermore, only the KIT exon 17 mutation had 95% CI, 1.00-5.58; P 5 .05) in patients with CBFB-MYH11
a lower prognostic impact on the RFS of AML patients with were found to be poor prognostic factors for RFS (supplemental
RUNX1-RUNX1T1 (HR, 3.82; 95% CI, 2.21-6.60; P , .001) Table 5).
(Figure 3D). In contrast, no KIT mutations affected the RFS of
patients with CBFB-MYH11 (Figure 3E, F). KIT mutation was MRD analysis
also associated with a poorer OS for AML with RUNX1-RUNX1T1, We evaluated the MRD level after the completion of 3 courses
but not for that with CBFB-MYH11 (supplemental Figure 4), and of HiDAC therapy in 112 patients. MRD was positive in 32 of
the prognostic impact of each KIT mutation on OS was the same as 75 (42.7%) and 16 of 37 (43.2%) patients with RUNX1-RUNX1T1
that on RFS. and CBFB-MYH11, respectively (Figure 4A). The RFS of patients

14 JANUARY 2020 x VOLUME 4, NUMBER 1 PROGNOSTIC IMPACT OF KIT MUTATIONS ON CBF-AML 69

Table 1. Patient characteristics according to KIT mutation
Characteristic All patients (n 5 199) KIT unmutated (n 5 136) KIT mutated (n 5 63) P

Chimera transcript type, n (%) 1.00

RUNX1-RUNX1T1 132 (66) 90 (66) 42 (67)

CBFB-MYH11 67 (34) 46 (34) 21 (33)

Sex, n (%) .53

Male 125 (63) 81 (61) 42 (67)

Female 74 (37) 55 (39) 21 (33)

Age, y .72

Median 41 41 41
Range 16-64 17-64 16-64
WBC, 3109/L .12

Median 9.6 8.5 12.1

Range 0.80-287.0 0.80-287.0 1.84-192.4

BM blasts, % ,.001
Median 61.2 53.8 73.5

Range 10.8-97.0 10.8-96.5 26.1-97.0

WBC index* .002

Median 4.73 3.35 6.11

Range 0.35-41.3 0.35-41.3 0.93-29.6

Extramedullary tumor, n (%) 20 (10) 13 (10) 7 (11) .80

CD19 expression, n/N (%) 68/192 (35) 49/129 (38) 19/63 (30) .34
CD56 expression, n/N (%) 91/193 (47) 56/130 (43) 35/63 (56) .13

Induction therapy, n (%) .27

Daunorubicin base 68 (34) 43 (32) 25 (40)

Idarubicin base 131 (66) 93 (68) 38 (60)

Induction cycle, n (%) 1.00

1 course 187 (94) 128 (94) 59 (93)

2 courses 12 (6) 8 (6) 4 (6)

Additional cytogenetic abnormalities (n 5 197), n/N (%)

Loss of X/Y 78/197 (40) 55/135 (40) 23/62 (38) .64
Trisomy 8 7/197 (4) 4/135 (3) 3/62 (5) .68

Trisomy 22 18/197 (9) 11/135 (8) 7/62 (11) .60

del(9q) 14/197 (7) 12/135 (9) 2/62 (3) .23

del(7q)/-7 4/197 (2) 3/135 (2) 1/62 (2) 1.00

Complex 14/197 (7) 7/135 (5) 7/62 (11) .14

*WBC index calculated in patients with RUNX1-RUNX1T1.

with MRD was lower than that of those without MRD (HR, 2.39; were poor prognostic factors in patients with RUNX1-RUNX1T1
95% CI, 1.24-4.61; P 5 .009) (Figure 4B). Of note, the presence of (supplemental Table 7).
MRD was associated with a poorer RFS in patients with CBFB-
MYH11 (HR, 4.55; 95% CI, 1.20-17.2; P 5 .03), but not in those
with RUNX1-RUNX1T1 (P 5 .11) (Figure 4C-D). The presence Discussion
of MRD was significantly associated with KIT exon 17 mutation The prognostic impact of KIT mutation is a major clinical
in the patients with RUNX1-RUNX1T1 (supplemental Table 6). concern in AML patients with RUNX1-RUNX1T1 and CBFB-MYH11
Multivariate analysis of 112 patients demonstrated the presence of because controversial results were reported by several groups. In
MRD (HR, 5.49; 95% CI, 1.43-21.0; P 5 .01) and NRAS mutation this large prospective study, we demonstrated that the adverse
(HR, 3.93; 95% CI, 1.03-15.0; P 5 .05) to be poor prognostic effects of KIT mutation were observed only in AML patients with
factors for RFS in patients with CBFB-MYH11. In contrast, WBC RUNX1-RUNX1T1 and not in AML patients with CBFB-MYH11,
count (.50 3 109/L) (HR, 5.57; 95% CI, 1.24-15.0; P 5 .03), although our study included a small number of patients with CBFB-
KIT exon17 mutation (HR, 3.71; 95% CI, 1.60-8.64; P 5 .002), MYH11 compared with the previous study.3 Furthermore, there was
and FLT3-TKD mutation (HR, 3.39; 95% CI, 1.13-10.2; P 5 .03) no significant difference in the RFS or OS between patients with

70 ISHIKAWA et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1

Figure 2. Mutation landscape of AML with
RUNX1-RUNX1T1 or CBFB-MYH11. (A) Identified A
mutations in analyzed patients are shown. Gray RUNX1-RUNX1T1 CBFB-MYH11
CBF-type
boxes indicate the patients whose samples were not KIT
FLT3
analyzed. (B) The frequency of recurrently mutated NRAS
Activating KRAS
genes by CBF-AML fusion type is shown. (C) Circos kinase PTPN11
CSF3R
plots illustrate the association of mutated genes in JAK2
CBL
AML with RUNX1-RUNX1T1 or CBFB-MYH11. ASXL1
ASXL2
(D-E) Circos plots illustrate the association of EZH2
Chromatin
KDM6A
mutated genes in AML with RUNX1-RUNX1T1 and modifier
KMT2A
AML with CBFB-MYH11. The width of the arches BCORL1
ZBTB7A
Transcription
indicates the percentage of mutations. factor
RUNX1
ETV6
Cohesin RAD21
complex SMC1A
SMC3
DNA TET2
methylation DNMT3A
Tumor TP53
suppressor WT1
CUX1
Spicesome SRSF2
–x/–Y
del(7)
Chromosome del(9)
abnormalities Trisomy 8
Trisomy 22
complex

B
(%)
30
CBFB-MYH11
RUNX1-RUNX1T1
20

10

0
KIT
NRAS
ASXL2
FLT3-TKD
ASXL1
RAD21
ZBTB7A
EZH2
KDM6A
KRAS
WT1
CSF3R
FLT3-ITD
TET2
SMC1A
SMC3
PTPN11
RUNX1
JAK2
CBL
DNMT3A
TP53
BCORL1
KMT2A
ETV6
SRSF2
C D E
Total
RUNX1-RUNX1T1 CBFB-MYH11
T1 2 PTPN11 N RAS
S R SF KRAS
X1 D2
1 KR
UN RA AS
CB

-R
X1
FB

N
RU 1A
-M

C
Y

SM
H

KI
11

T
KI
T
C3
SM
PT
PN

X1
1

11

CU
F2
AD2
CUX1 SMC1A SRS

3
TP5
SMC3 R

N RAS

JAK2
N MT3A WT1

FLT3
KRAS
A TP53v

N RAS
TET2 D

FLT3
WT1

CSF3
MT3

R
T2
DN

X L1
TE

CB
KI T

AS
1

L
XL

ZB
AS

TB
7A
2

T1

XL
W
R

AS JA U
K2 NX
2 1
ZH FLT L2 ET
L1E 2A 3 AS
X V6
OR
MT R Z BTB7A ETV6 B
C CSF3 KD
M6 6A PTP
A K A KDM N11
DM6 BC O
R U NX1 K CB L E Z H2 R L1 R U NX1

RUNX1-RUNX1T1 and CBFB-MYH11 in this study. Because the mutation did not affect the RFS of patients with RUNX1-RUNX1T1
results of mutation analysis were not reported to each institute until (Table 2). The fusion transcripts RUNX1-RUNX1T1 and CBFB-
the completion of the protocol therapy and any further intervention MYH11 are not sufficient for leukemia development and addi-
was prohibited until hematological relapse, the present results are tional driver mutations, such as KIT, FLT3, and RAS mutations, are
sufficient to evaluate the clinical relevance of KIT mutations and required for its onset.28 However, the present study suggested
other molecular abnormalities in adult patients with CBF-AML that the prognostic impact of these driver mutations differs between
treated using HiDAC. Moreover, in the patients with CBFB-MYH11, patients with RUNX1-RUNX1T1 and CBFB-MYH11.
NRAS mutation was preferentially identified in KIT-unmutated Several groups previously reported that the MRD level examined by
patients, and had an adverse effect on RFS, whereas NRAS the chimeric transcripts using RT-qPCR was useful for predicting

14 JANUARY 2020 x VOLUME 4, NUMBER 1 PROGNOSTIC IMPACT OF KIT MUTATIONS ON CBF-AML 71

 A B
Total Total
KIT wild type

Probability of relapse-free survival

Probability of relapse-free survival
1.00 KIT wild type 1.00 KIT exon 8 mt.
KIT exon10-11 mt.
KIT mutation
KIT exon17 mt.
0.75 0.75

0.50 0.50

0.25 0.25
P=0.0033
P=0.004
Hazard ratio, 1.92 (95%CI, 1.23 - 3.00) 0.00
0.00
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Years Years
No. at risk
No. at risk
wild type 136 104 85 72 53 30 11
wild type 136 104 85 72 53 30 11
ex. 8 mt. 11 7 7 6 5 2 0
mutation 63 31 30 24 16 7 4 ex. 10-11 mt. 3 2 2 0 0 0 0
ex. 17 mt. 49 22 21 18 11 5 4

C D
RUNX1-RUNX1T1 RUNX1-RUNX1T1
KIT wild type
KIT exon 8 mt.
Probability of relapse-free survival

1.00 KIT wild type

Probability of relapse-free survival 1.00 KIT exon10-11 mt.
KIT mutation KIT exon17 mt.

0.75 0.75

0.50 0.50

0.25 0.25
P<0.001
Hazard ratio, 3.27 (95%CI, 1.90 - 5.64) P<0.001
0.00 0.00
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Years Years
No. at risk No. at risk
wild type 90 74 62 52 40 22 8 wild type 90 74 62 52 40 22 8
mutation 42 17 16 12 6 2 1 ex. 8 mt. 4 3 3 2 1 0 0
ex. 10-11 mt. 2 1 1 0 0 0 0
ex. 17 mt. 36 13 12 10 5 2 1

E F
CBFB-MYH11 CBFB-MYH11
KIT wild type
KIT exon 8 mt.
KIT exon10-11 mt.
Probability of relapse-free survival

Probability of relapse-free survival

1.00 KIT wild type 1.00 KIT exon17 mt.

KIT mutation
0.75 0.75

0.50 0.50

0.25 0.25
P=0.35
Hazard ratio, 0.67 (95%CI, 0.28 - 1.57) P=0.72
0.00 0.00
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Years Years
No. at risk No. at risk
wild type 46 30 23 20 13 8 3
wild type 46 30 23 20 13 8 3
ex. 8 mt. 7 4 4 4 4 2 0
mutation 21 14 14 12 10 5 3
ex. 10-11 mt. 1 1 1 0 0 0 0
ex. 17 mt. 13 9 9 8 6 3 3

Figure 3. RFS according to KIT mutation. (A) Kaplan-Meier estimates of RFS according to KIT mutation in 199 CBF-AML patients. Kaplan-Meier estimates of RFS in
patients with (C) RUNX1-RUNX1T1 or (E) CBFB-MYH11. (B,D,F) Kaplan-Meier estimates of RFS according to the KIT mutation type.

72 ISHIKAWA et al 14 JANUARY 2020 x VOLUME 4, NUMBER 1

Table 2. Multivariate analysis for RFFS the present study evaluated the clinical significance of MRD
RUNX1-RUNX1T1 CBFB-MYH11 in patients who were able to maintain CR during consolida-
Variables HR 95% CI P HR 95% CI P
tion therapy. Although further studies are required to clarify
when MRD should be evaluated, our study demonstrated the
KIT exon17 mutation 4.17 2.38-7.34 ,.001
importance of MRD for evaluating the prognosis of AML
Extramedullary tumor 3.85 1.35-10.93 .011 patients with CBFB-MYH11.
Loss of X/Y 5.79 1.21-27.6 .03
In conclusion, we clarified the prognostic impact of KIT mutation
NRAS mutation 2.38 1.03-5.53 .04
and the MRD status in adult AML patients with RUNX1-RUNX1T1
or CBFB-MYH11 who were treated using HiDAC, refining the
the long-term prognosis of CBF-AML patients; however, there are concept of risk stratification of AML patients with RUNX1-RUNX1T1
several opinions regarding thresholds and time points for MRD and CBFB-MYH11. Other treatment strategies, including alloge-
assessment.1,19,29-32 Although we evaluated MRD after complet- neic HSCT during the first remission, or addition of gemtuzumab or
ing the 3-course consolidation therapy, MRD samples were not ozogamicin to chemotherapy, should be considered for patients
collected from 87 patients for several reasons, including disease with a high risk of relapse identified by this study.33 The molecular
progression (Figure 1). The RFS was significantly lower in the risk groups presented in this study are amenable to routine
sample-uncollected patients than the collected patients among diagnostic assessment, and provide a foundation for future clinical
those with either RUNX1-RUNX1T1 or CBFB-MYH11. Therefore, trials and research.

A B
104
Total
Probability of relapse-free survival 1.00
Copy number of fusion transcripts

0.75
103
0.50
MRD  50 copies/PgRNA
MRD ! 50 copies/PgRNA
0.25
102
Hazard ratio, 2.39 (95%CI, 1.24 - 4.61), P=0.009
0.00
0 1 2 3 4 5 6
Years
RUNX1-RUNX1T1 CBFB-MYH11 No. at risk
MRD  50 64 56 52 43 30 18 5
MRD ! 50 48 34 25 19 18 9 5

C D
RUNX1-RUNX1T1 CBFB-MYH11
1.00 1.00
Probability of relapse-free survival

Probability of relapse-free survival

0.75 0.75

0.50 0.50

MRD  50 copies/PgRNA MRD  50 copies/PgRNA

0.25 MRD ! 50 copies/PgRNA 0.25 MRD ! 50 copies/PgRNA

Hazard ratio, 1.89 (95%CI, 0.87 - 4.08), P=0.11 Hazard ratio, 4.55 (95%CI, 1.20 - 17.22), P=0.03
0.00 0.00
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Years Years
No. at risk No. at risk
MRD  50 43 36 34 26 18 12 4 MRD  50 21 20 18 17 12 6 1
MRD ! 50 32 23 17 13 12 5 2 MRD ! 50 16 11 8 6 6 4 3

Figure 4. RFS according to the MRD level. (A) The RUNX1-RUNX1T1 or CBFB-MYH11 chimeric transcript level in each patient after the completion of 3 courses of
HiDAC therapy is shown. (B) Kaplan-Meier estimates of RFS according to the MRD status in 112 CBF-AML patients. Kaplan-Meier estimates of RFS in patients with (C)
RUNX1-RUNX1T1 or (D) CBFB-MYH11.

14 JANUARY 2020 x VOLUME 4, NUMBER 1 PROGNOSTIC IMPACT OF KIT MUTATIONS ON CBF-AML 73

Acknowledgments Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. Ltd.,
Sanofi K.K., and Celgene Corporation; consulting fees from Astellas
The authors thank the clinicians and leaders of the 85 institutions Pharma Inc., Amgen Astellas BioPharma K.K., and Daiichi Sankyo
who entered their patients into the JALSG CBF-AML209-KIT study Co. Ltd.; honoraria from Bristol-Myers Squibb, Astellas Pharma Inc.,
and provided the necessary data to make this study possible. and Novartis Pharma K.K. N. Dobashi received research funding
This study was supported by a grant from the Practical Research from Pfizer Japan Inc., Chugai Pharmaceutical Co. Ltd., Astellas
for Innovative Cancer Control from Japan Agency for Medical Pharma Inc., Kyowa Hakko Kirin Co. Ltd., Zenyaku Kogyo Co. Ltd.,
Research and Development, AMED (17ck0106251) (H. Kiyoi). Eisai Co. Ltd., Otsuka Pharmaceutical Co. Ltd., Celgene Co., and
Authorship Sysmex Co. N. Asou received research funding from Chugai Phar-
maceutical Co. Ltd. and Toyama Chemical Co., Ltd., and consulting
Contribution: H. Kiyoi was the chief investigator of the trial; H. Kiyoi, fees from SRL Inc. The remaining authors declare to competing
Y.I., N.K., Y.A., I.M., Y. Miyazaki, and T.N. were involved in concep- financial interests.
tion and study design; I.S., M.S., N. Dobashi, H.Y., N. Doki, A.T., T.K.,
S.K., H. Kanamori, N.I., A.K., Y. Moriuchi, N. Asada, D.H., K.T., T.S., A list of participating investigators and institutes of Japan Adult
M.H., T.T., Y.Y., and S.O. were involved in patient accrual and data Leukemia Study Group, in addition to the authors, appears in the
acquisition; H. Kiyoi, Y.I., N.K., and N. Asou performed laboratory supplemental appendix.
experiments and analysis; Y.I. and Y.A. performed the statistical
analysis; H. Kiyoi, Y.I., N.K., S.O., Y. Miyazaki, and Y.A. were re- ORCID profiles: Y.I., 0000-0001-6024-6617; I.S., 0000-0002-
sponsible for data analysis and interpretation; H. Kiyoi, Y.I., and N.K. 4430-7544; H.Y., 0000-0001-7658-0687; S.K., 0000-0002-1732-
were responsible for the preparation and writing of the manuscript; 2220; N.I., 0000-0001-9176-1988; Y.Y., 0000-0003-0366-4147;
and all authors contributed to and approved the final manuscript. S.O., 0000-0003-0112-6564; I.M., 0000-0003-2818-4270; H.K.,
0000-0001-6382-9498.
Conflict-of interest disclosure: H. Kiyoi received research
funding from Chugai Pharmaceutical Co. Ltd., Kyowa Hakko Kirin Correspondence: Hitoshi Kiyoi, Department of Hematology
Co. Ltd., Zenyaku Kogyo Co. Ltd., FUJIFILM Corporation, Astellas and Oncology, Nagoya University Graduate School of Medicine,
Pharma Inc., Otsuka Pharmaceutical Co. Ltd., Nippon Shinyaku Co. Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan; e-mail:
Ltd., Eisai Co. Ltd., Pfizer Japan Inc., Takeda Pharmaceutical Co. kiyoi@med.nagoya-u.ac.jp.

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