Expression characteristic of 4Ig B7-H3 and 2Ig B7-H3 in acute myeloid leukemia

Figures & data

Table 1. Primer sequences for B7-H3 isoforms

Primers	Products	Sequences (5ʹ-3ʹ)
Primer 1	B7-H3 (134bp) [32]	F:5ʹ-CTCTGCCTTCTCACCTCTTTG-3ʹ R:5ʹ-CCTTGAGGGAGGAACTTTATC-3’
Primer 2	4Ig B7-H3 (380bp) [25]	F:5ʹ-CTCACGAAGCAGGTGAAGCTGCC-3ʹ R:5ʹ-ACCTACAGCTGCTGGTGCGCAA-3’
Primer 3	B7-H3 isoforms (1444bp and 790bp) [5]	F:5ʹ-CACAGGAAGATGCTGCGTCG-3ʹ R:5ʹ-CAATGAGACAGACAGACAGC-3’
Primer 4	B7-H3 isoforms (1572bp and 918bp) [6]	F:5ʹ-CAGCCCTGGCATGGGTGTGCAT-3ʹ R:5ʹ-CCATCATCTTCTTTGCTGTCAGAG-3’
Primer 5	β-actin (205bp) [33]	F:5ʹ-TGACGTGGACATCCGCAAAG-3ʹ R:5ʹ-CTGGAAGGTGGACAGCGAGG-3’

Figure 1. The mRNA expression of B7-H3 isoforms in 8 AML cell lines. (a-e) RT-PCR was used to analyze mRNA expression with various B7-H3-specific primers. The ordinate in B-E represents gray values of B7-H3, 4Ig B7-H3, B7-H3 isoforms (detected by B7-H3-specific primer 3), and B7-H3 isoforms (detected by B7-H3-specific primer 4)/gray value of β-actin, respectively. The data is gained from no less than 3 individual experiments

Figure 2. RT-PCR analysis of THP-1 and SKM-1 with B7-H3-specific primers by changing PCR conditions. (a) RT-PCR using B7-H3-specific primers in THP-1 and SKM-1 by increasing annealing temperature. The annealing temperature of primer 3 (up) and primer 4 (down) was 58.1°C, 61.3°C, 63.1°C, 66.4°C, 69°C and 59.9°C, 61.4°C, 62.5°C, 64.8°C, 68.9°C from left to right lanes, respectively. (b) RT-PCR using B7-H3-specific primers in THP-1 and SKM-1 by reducing cycles. The cycles were 35, 33, and 30 cycles from left to right lanes. The data is gained from no less than 3 individual experiments

Figure 3. The mRNA expression of B7-H3 isoforms in de novo AML patients and controls. RT-PCR with B7-H3-specific primer3 was used to detect mRNA expression of B7-H3 isoforms in AML patients and controls followed by analysis by ROC curve. (a-b) The ordinate represents gray values of 4Ig B7-H3 and 2Ig B7-H3/gray value of β-actin, respectively. (c-d) The ROC curve analysis of 4Ig B7-H3 and 2Ig B7-H3 mRNA expression. AUC, area under the ROC curve

Table 2. Comparison of clinical manifestations and laboratory features between AML patients with low and high mRNA expression of 2Ig B7-H3 (2Ig)

Patient’s parameters	The whole AML patients
	2Ig^low (n = 32)	2Ig^high (n = 36)	P
Sex, male/female	19/13	17/19	0.316
Median age, years (range)	55 (15–80)	66 (20–93)	0.026
Median WBC, ×10^9 /L (range)	28.2 (0.8–136.1)	18.7 (0.8–207.5)	0.468
Median Hb, g/L (range)	88 (45–126)	86 (49–141)	0.672
Median Plt, ×10^9 /L (range)	31 (3–192)	41 (5–382)	0.555
BM blasts, % (range)	50.0 (3.0*-94.0)	61.3 (16.5*-93.5)	0.414
FAB classification			0.371
M0	0 (0.0%)	2 (5.6%)
M1	1 (3.1%)	2 (5.6%)
M2	13 (40.6%)	15 (41.7%)
M3	5 (15.6%)	2 (5.6%)
M4	9 (28.1%)	6 (16.7%)
M5	4 (12.5%)	8 (22.2%)
M6	0 (0.0%)	1 (2.8%)
Karyotype			0.361
Normal	12 (37.5%)	13 (36.1%)
t(8;21)	2 (6.3%)	3 (8.3%)
t(16;16)	2 (6.3%)	0 (0.0%)
t(15;17)	5 (15.6%)	2 (5.6%)
+8	1 (3.1%)	2 (5.6%)
−5/5q-	0 (0.0%)	2 (5.6%)
−7/7q-	0 (0.0%)	1 (2.8%)
t(9;22)	0 (0.0%)	1 (2.8%)
Complex	3 (9.4%)	6 (16.7%)
Others	3 (9.4%)	5 (13.9%)
Not available	4 (12.5%)	1 (2.8%)
Karyotype classification			0.056
Favorable	9 (28.1%)	5 (13.9%)
Intermediate	16 (50.0%)	19 (52.8%)
Poor	3 (9.4%)	11 (30.6%)
Not available	4 (12.5%)	1 (2.8%)
Gene Mutation
NPM1 (+/-)	9/21	2/31	0.012
CEBPA (+/-)	8/22	5/28	0.259
FLT3-ITD (+/-)	3/27	10/23	0.047
c-KIT (+/-)	1/29	2/31	1.000
N/K-RAS (+/-)	4/26	6/27	0.857
IDH1/2 (+/-)	2/28	5/28	0.504
DNMT3A (+/-)	3/27	3/30	1.000
U2AF1 (+/-)	3/27	1/32	0.538
SRSF2 (+/-)	3/27	2/31	0.912
SETBP1 (+/-)	2/28	1/32	0.933
TP53 (+/-)	1/29	3/30	0.675
CR (+/-)	19/12	12/23	0.028

*:Diagnosis was done in regards to the World Health Organization criteria for patients with low percentage blasts (< 20%) in BM. The cytogenetic aberrations t (15; 17) (q22; q12), were also detected in these patients. Abbreviations: AML = acute myeloid leukemia; WBC = white blood cells; Hb = hemoglobin; Plt = platelets; BM = bone marrow; CR = complete remission. P values were determined using χ² test for the sex, karyotype, common gene mutations and CR rates after standard induction therapy for 2 cycles, and applying Mann-Whitney’s U test for the age, WBC, Hb, Plt and BM blasts.

Figure 4. B7-H3 isoforms in AML cell line membrane protein expression. (a) The histograms revealed the expression of B7-H3 isoforms, gating with isotype controls. The red and green lines in each histogram represent isotype control and B7-H3 isoforms, respectively. The antibody 2E6 bound both 4Ig B7-H3 and 2Ig B7-H3, while the antibody 3H4 specifically bound 4Ig B7-H3. (b-c) The mean fluorescence intensity (MFI) of total B7-H3 isoforms and 4Ig B7-H3 in AML cell lines. The data is gained from no less than 3 individual experiments

Figure 5. The membrane protein expression of B7-H3 isoforms in de novo AML patients and controls. (a-b) The expression rates of total B7-H3 isoforms and 4Ig on blast cells in AML patients and controls. (c-d) The mean fluorescence intensity ratio of blast cells and lymphocytes (MFI ratio) of total B7-H3 isoforms and 4Ig B7-H3 in AML patients and controls

Figure 6. The ROC curve analysis of B7-H3 isoforms membrane protein expression in de novo AML patients and controls. (a-b) The expression rates of total B7-H3 isoforms and 4Ig on blast cells. (c-d) The mean fluorescence intensity ratio of blast cells and lymphocytes (MFI ratio) of total B7-H3 isoforms and 4Ig B7-H3. AUC, area under the ROC curve

Table 3. Evaluation of clinical manifestations and laboratory features amongst AML patients with low and high membrane protein expression of total B7-H3 isoforms

Patient’s parameters	The whole AML patients
	B7-H3^low (n = 34)	B7-H3^high (n = 28)	P
Sex, male/female	18/16	16/12	0.741
Median age, years (range)	54.5 (15–93)	66 (32–90)	0.077
Median WBC, ×10^9 /L (range)	18.6 (0.8–207.5)	33.3 (0.8–158.7)	0.581
Median Hb, g/L (range)	83 (45–141)	88 (50–138)	0.445
Median Plt, ×10^9 /L (range)	34 (5–192)	35 (3–382)	0.983
BM blasts, % (range)	50 (3.0*-94.0)	64 (16.5*-93.5)	0.471
FAB classification			0.345
M0	2 (5.9%)	0 (0.0%)
M1	1 (2.9%)	2 (7.1%)
M2	16 (47.1%)	9 (32.1%)
M3	5 (14.7%)	2 (7.1%)
M4	6 (17.6%)	8 (28.6%)
M5	4 (11.8%)	6 (21.4%)
M6	0 (0.0%)	1 (3.6%)
Karyotype			0.419
Normal	14 (41.2%)	10 (35.7%)
t(8;21)	2 (5.9%)	2 (7.1%)
t(16;16)	2 (5.9%)	0 (0.0%)
t(15;17)	5 (14.7%)	2 (7.1%)
+8	1 (2.9%)	1 (3.6%)
−5/5q-	1 (2.9%)	1 (3.6%)
−7/7q-	0 (0.0%)	1 (3.6%)
t(9;22)	1 (2.9%)	0 (0.0%)
Complex	2 (5.9%)	7 (25.0%)
Others	4 (11.8%)	1 (3.6%)
Not available	2 (5.9%)	3 (10.7%)
Karyotype classification			0.165
Favorable	9 (26.5%)	4 (14.3%)
Intermediate	19 (55.9%)	12 (42.9%)
Poor	4 (11.8%)	9 (32.1%)
Not available	2 (5.9%)	3 (10.7%)
Gene Mutation
NPM1 (+/-)	7/25	3/22	0.534
CEBPA (+/-)	8/24	3/22	0.370
FLT3-ITD (+/-)	4/28	7/18	0.257
c-KIT (+/-)	0/32	2/23	0.188
N/K-RAS (+/-)	6/26	3/22	0.743
IDH1/2 (+/-)	5/27	1/24	0.325
DNMT3A (+/-)	4/28	2/23	0.909
U2AF1 (+/-)	4/28	0/25	0.190
SRSF2 (+/-)	2/30	3/22	0.772
SETBP1 (+/-)	2/30	1/24	1.000
TP53 (+/-)	2/30	2/23	1.000
CR (+/-)	20/14	11/17	0.126

*:Diagnosis was done in regards to the World Health Organization criteria for patients with low percentage blasts (< 20%) in BM. The cytogenetic aberrations t (15;17) (q22; q12), were also detected in these patients. Abbreviations: AML = acute myeloid leukemia; WBC = white blood cells; Hb = hemoglobin; Plt = platelets; BM = bone marrow; CR = complete remission. P values were determined using χ² test for the sex, karyotype, common gene mutations and CR rates after standard induction therapy for 2 cycles, and utilizing Mann-Whitney’s U test for the age, WBC, Hb, Plt and BM blasts.

Figure 7. The Kaplan–Meier analysis of B7-H3 isoforms expression in de novo AML patients. (a-b) The influence of 2Ig B7-H3 mRNA expression on OS in the whole AML and non-M3 AML patients, respectively. (c-d) The influence of total B7-H3 isoforms membrane protein expression on OS in the whole AML and non-M3 AML patients, respectively. Non-M3 AML, non-acute promyelocytic leukemia in AML; OS, overall survival

Table 4. Univariate and multivariate analysis of prognostic factors for overall survival in de novo AML patients

	Whole AML patients		Non-M3 AML patients
	Hazard ratio (95%CI)	P	Hazard ratio (95%CI)	P
Univariate analysis
Age(<60y/≥60y)	3.242 (1.663–6.308)	0.001	3.268 (1.590–6.718)	0.001
WBC(<30 × 10^9/L /≥30 × 10^9/L)	2.216 (1.187–4.139)	0.013	2.242 (1.171–4.293)	0.015
Karyotypic classifications
(Favorable/Intermediate)	3.127 (1.071–9.130)	0.037	2.681 (0.628–11.452)	0.183
(Favorable/Poor)	8.836 (2.737–28.528)	0.000	7.604 (1.670–34.625)	0.009
(Favorable and intermediate/Poor)	3.705(1.775–7.736)	0.000	3.257 (1.555–6.821)	0.002
2Ig B7-H3 mRNA expression (Low/High)	2.352 (1.245–4.444)	0.008	1.894 (0.989–3.625)	0.054
B7-H3 membrane protein expression (Low/High)	1.776 (0.957–3.296)	0.069	1.975 (1.037–3.764)	0.038
NPM1 mutation (Wild-type/Mutant)	0.321 (0.113–0.915)	0.033	0.261 (0.091–0.753)	0.013
FLT3-ITD mutation (Wild-type/Mutant)	1.973 (0.926–4.205)	0.078	1.956 (0.884–4.326)	0.098
TP53 mutation (Wild-type/Mutant)	6.346 (2.042–19.716)	0.001	6.204 (1.972–19.516)	0.002
CR (No/Yes)	0.146 (0.068–0.314)	0.000	0.180 (0.082–0.395)	0.000
Multivariate
2Ig B7-H3 mRNA expression (Low/High)	1.581 (0.660–3.785)	0.304	1.724 (0.702–4.236)	0.235
Total B7-H3 membrane protein expression (Low/High)	1.896 (0.858–4.191)	0.114	1.334 (0.557–3.193)	0.518
TP53 mutation (Wild-type/Mutant)	9.291 (1.994–43.298)	0.005	8.027 (1.615–39.896)	0.011
CR (No/Yes)	0.124 (0.044–0.350)	0.000	0.162 (0.057–0.457)	0.001

Abbreviations: AML = acute myeloid leukemia; non-M3 AML = non-acute promyelocytic leukemia in AML; WBC = white blood cells; CR = complete remission.

Figure 8. B7-H3 unmethylation level and its correlation with the mRNA expression of B7-H3 isoforms in de novo AML patients. (a) B7-H3 unmethylation level in AML patients and controls. (b-c) Correlation between B7-H3 promoter unmethylation level and the mRNA expression of 4Ig B7-H3 and 2Ig B7-H3 in AML patients

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