Integrative Genomic Analysis Reveals Cancer-Associated Gene Mutations in Chronic Myeloid Leukemia Patients with Resistance or Intolerance to Tyrosine Kinase Inhibitor

Figures & data

Table 1 Clinical Data of the Patients

		TKIs-Resistant	TKIs-Intolerant	P value
Number		42	21
Gender	(Male/Female)	26/16	11/10	Χ^2=0.524 P=0.589^a
Age		38.5 (19, 66)	49.0 (31, 65)	P=0.068^b
Disease staging				Χ^2=0.554 P=0.768^a
	CP	29	16
	AP/BC	13	5
Clinical data at diagnosis	WBC (×10^9/L)	201 (32.95, 406.54)	232.03 (37.02, 508)	P=0.212^b
	HGB(g/L)	100.1(66, 140)	82.36(225, 1790)	F=6.426 P=0.017^c
	PLT (×10^9/L)	572.84(49, 1197)	732.18(225, 1790)	F=1.272 P=0.269^c
	BM blast (%)	1.0(0, 17.0)	3.0(0.5, 7.5)	P=0.085^b
	Spleen size (subcostal length/cm)	4.8(0, 23)	4(0, 23)	P=0.506^b
Sokal		0.98(0.22,1.56)	0.98(0.11,2.32)	P=0.484^b
	Low	16(38.1%)	5(23.8%)
	Medium	16(38.1%)	12(57.1%)
	High	10(23.8%)	4(19%)
EUTOS		66.5(7, 112)	56.4(11.4, 101)	P=0.743^b
	Low	25(59.5%)	16(76.2%)
	High	15(35.7%)	5(23.8%
Clinical data at detection	WBC (×10^9/L)	5.54(1.93;149.87)	4.6(1.48;11.8)	P=0.035 ^b
	HGB(g/L)	111(49;165)	108.5(43;131)	P=0.236 ^b
	PLT (×10^9/L)	156(12;1126)	34(6;75)	P=0.000 ^b
	EOS (%)	0.4(0;9.7)	0.35(0;2.9)	P=0.704 ^b
	BASO (%)	1.2(0;18)	0.75(0;8.9)	P=0.470 ^b
Additional chromosomal aberrations*				P=1.000^d
Yes	Major route	4(12.12%)	3(20%)
	Minor route	4(12.12%)	1(6.67%)
No		25(75.76%)	11(73.33%)
Achieved MMR
	Yes	1(2.94%)	1(5.00%)
	No	33(97.06%)	19(95.00%)

Notes: ^aChi-square test. ^bWilcoxon rank sum test. ^ct-test. ^dFisher’s exact test. *Only 48 patients with karyotype analysis.

Abbreviations: TKIs, tyrosine kinase inhibitors; WBC, white blood cell; HGB, hemoglobin; PLT, platelets; BM, bone marrow; EOS, eosinophil; BASO, basophil; MMR, major molecular remission.

Table 2 Detected Genes

Pathway	Genes
Methylation	DNMT3A, TET2, IDH1, IDH2, TPMT, MLL, EZH2, SETD2, NPM1
Tumor suppressor	WT1, TP53, MPL, APC, CBLB, CBLC, MDM2, CDKN2A
Transcription factor	CEBPA, AML1, RUNX1, GATA1, GATA2, GATA3, ETV6, IKZF1, CREBBP, CUX1, ERG, MEF2B, RB1, STAT5B, STAT5A, TCF3, GFI1
Spliceosome and RNA metabolism	SF3B1, SRSF2, U2AF1, ZRSR2, PRPF8, DDX41
Chromatin modifiers	ASXL1, KDM6A, BCORL1, BCOR, KMT2C, ID3, SETBP1, PHF6, EP300, BLM
Activated Signaling	CBL,NRAS,KRAS,HRAS,PTPN11,KIT,CSF1R,CSF3R,JAK1,JAK2,JAK3,SH2B3,NTRK1,NTRK2,ABL,CRLF2,AKT2,AKT3,AMER1,ATM,ATRX,BRAF,EGFR,IL7R,MAP2K4,MAP3K7,NOTCH1,NOTCH2,PIK3CA,PTEN,PDGFRA,SMAD4,SYK,TRAF3,NF1,ETNK1,STAT3,MYD88,FLT3-ITD,FLT3
Cell Metabolism	GSTP1, NT5C2, NQO1, PIGA, MTHFR, ELA2, CYP2C19, CYP3A4, GSTM1
Adhesion and cohesion complex	RAD21, STAG2, SMC1A, SMC3, FAT1
Others	ABCB1, ABCC3, BCL2, CACNA1E, CARD11, CCND1, CD79B, CDA, DIS3, ERCC1, CTLA4, FAM46C, FBXW7, GNAS, MLH1, NF2, TERC, XRCC1, CALR, BIRC3, SRP72, HAX1, TERT, DNAH9, DKC1

Figure 1 Summary of mutations (A) Most of the 68 individual somatic mutations were missense mutations, while multiple hit mutations accounted for 38% of KMT2C mutations, and frameshift mutations also accounted for 46.67% of ASXL1. Among the total mutation rates, the highest was ABL1 KD (31%), followed by that of KMT2C, FAT1 and ASXL1, which were 31%, 25% and 22%, respectively. (B) The coexistence relationship among genes is complicated. (C) The TKIs-resistant group accounted for the majority of the mutations in most genes, while the number of mutations in the TKIs-intolerant group in the CUX1, GATA2 and KIT genes was higher than those in the TKIs resistant group.

Abbreviations: KD, kinase domain; TKIs, tyrosine kinase inhibitors.

Table 3 Mutations Rates of Genes Related to MDS

	AMER1	CUX1	KIT	GATA2	ASXL1	RUNX1
TKIs-resistant	7/44 (15.90%)	1/44 (2.27%)	1/44 (2.27%)	3/44 (6.82%)	9/44 (20.45%)	6/44 (13.64%)
TKIs-intolerant	0/23 (0.000%)	4/23 (17.39%)	4/23 (17.39%)	8/23 (34.78%)	6/23 (26.09%)	4/23 (17.39%)
P value	0.044	0.044	0.044	0.006	0.408	0.470

Abbreviation: MDS, myelodysplastic syndrome.

Figure 2 Mutation rates of MDS related genes. Mutation rates of 20 genes related to MDS; Mutation rates of KIT, GATA2 and CUX1 in patients with TKIs intolerance were significantly higher than in TKIs-resistant group.

Abbreviation: MDS, myelodysplastic syndrome.

Figure 3 Mutation rates of genes with different functions. According to the function of genes, they were divided into chromatin modification (86.57%, 58/67), activated signaling (58.70%, 40/67), transcription factor (47.76%, 32/67), methylation (44.78%, 30/67), other (34.33%, 23/67), adhesion and cohesion complex (26.87%, 18/67), cell metabolism (16.42%, 11/67), tumor suppressor (8.96%, 6/67), spliceosome and RNA metabolism (5.97%, 4/67). It can be seen that the mutations rate in transcription factor is higher in patients intolerant to TKIs (82.60% vs 27.27%, P=0.016).

Table 4 Statistics on Mutation Rates of Additional Genes Associated with ABL KD

	ASXL1	FAT1	RUNX1	KIT	SETP1B	IKZF1
ABL1 KD positive	8/21(38.1%)	8/21(38.1%)	5/21(23.81%)	0/21(0%)	5/21(23.81%)	0/21(0.00%)
ABL1 KD negative	7/46(15.21%)	9/46(19.56%)	5/46(10.87%)	5/46(10.87%)	7/46(15.22%)	1/46(2.17%)
P value	0.041	0.096	0.156	0.142	0.299	0.687

Abbreviation: KD, kinase domain.

Table 5 Relationship Between ASXL1 Mutation and Clinical Features at Initial Diagnosis

	HB (g/L)	WBC (×10^9/L)	PLT (×10^9/L)	BASO(%)	Spleen Size (Subcostal Length/cm)	Sokal	EUTOS
ASXL1 positive	91 (80, 100)	232.0 (108.4;406.5)	663(616;845)	4.6(3.5;15.25)	7.3(5.1;23)	0.98(0.82;1.17)	67.5 (45.9;91.7)
ASXL1 negative	97(72;105)	203.2 (110.9;273.0)	538(307;813)	3 (1.75;4.2)	3.95(0;6.67)	0.98(0.63;1.2)	61.85(42;88.2)
P value	0.777	0.914	0.065	0.031	0.041	0.99	0.995

Figure 4 EFS of patients with/without TET2 mutation. In Kaplan-Meier analysis, the EFS of patients with TET2 mutation was significantly lower than those without (P=0.039).