HLA-DQB1 expression on tumor cells is a novel favorable prognostic factor for relapse in early-stage lung adenocarcinoma

Figures & data

Figure 1 Workflow of the screening and validation procedures used in this study. Abbreviations: LUAD, lung adenocarcinoma; RFS, recurrence-free survival.

Table 1 Clinical characteristics of the Daping and TCGA discovery sets

	Daping discovery set n=14			TCGA discovery set n=37
	RFS >3 years (n=7)	RFS <1 years (n=7)	P-value	RFS >3 years (n=22)	RFS <1 years (n=15)	P-value
Age at surgery			0.592			0.286
Mean ± SD	54.86±8.87	61.14±10.58		74.72±10.68	63.87±6.23
<60	5(71.43%)	3(42.86%)		9(40.91%)	3(20%)
≥60	2(28.57%)	4(57.14%)		13(59.09%)	12(80%)
Sex			1			0.514
Male	3(42.86%)	4(57.14%)		10 (45.45%)	5(33.33%)
Female	4(57.14%)	3(42.86%)		12 (54.55%)	10(66.67%)
Smoking history			0.559			1
Non-smoker	6(85.71%)	4(57.14%)		8(36.36%)	6(40%)
Smoker	1(14.29%)	3(42.86%)		14(63.64%)	9(60%)
Tumor size			1			0.594
T1	3(42.86%)	3(42.86%)		11(50%)	7(46.67%)
T2	4(57.14%)	4(57.14%)		11(50%)	8(53.33%)
Adjuvant chemotherapy			0.462			–
Yes	5(71.43%)	7(100.00%)		0(0.00%)	5(33.33%)
No	2(28.57%)	0(0.00%)		0(0.00%)	2(13.33%)
Unknown	0(0.00%)	0(0.00%)		22(100.00%)	8(53.34%)
Anatomic subdivision			1			1
Left	3(42.86%)	3(42.86%)		10 (45.45%)	4(26.67%)
Right	4(57.14%)	4(57.14%)		12 (54.55%)	11(73.33%)

Abbreviation: RFS, recurrence-free survival.

Table 2 Clinical characteristics of the Daping and TCGA validation sets

	Daping validation set	TCGA validation set
	n=165	n=233
Age (mean ± SD)	59.81±9.03	65.40±9.74
Sex
Male	86(55.13%)	98 (42.06%)
Female	79(44.87%)	135 (57.94%)
Smoking history
Non-smoker	102(61.82%)	92(39.48%)
Smoker	63(38.18%)	130(55.79%)
Unknown	0(0%)	11(4.73%)
Stage
I	131(79.40%)	137(58.80%)
II	30(18.18%)	62(26.61%)
IIIA	4(2.42%)	34(14.59%)
Adjuvant chemotherapy
Yes	127(76.97%)	40(17.17%)
No	30(18.18%)	48(20.60%)
Unknown	8(4.85%)	145(62.23%)
Recurrence or metastasis
Yes	80(48.48%)	91(39.06%)
No	85(51.52%)	142(60.94%)
Median time to recurrence (days)
	1,975	1,433
Mean follow-up time (days)
	1,814(153–3,034)	923(60–6,812)

Table 3 Overlapping genes in the Daping and TCGA DEG lists

Gene Symbol	Description	Daping		TCGA
		GeneFC	P-value	GeneFC	P-value
(3 y vs 1 y)	(3 y vs 1 y)
SFTPB	Surfactant protein B	17.89	0.0134	2.39	0.0325
SFTPD	Surfactant protein D	2.88	0.0168	2.39	0.0234
SFTA1P	Surfactant associated 1	1.98	0.0165	2.13	0.0181
HLA-DQB1	Major histocompatibility complex, class II, DQ beta 1	1.61	0.028	1.55	0.0024
ATP13A4	ATPase type 13A4	−1.53	0.0081	1.63	0.0407
ITGB8	Integrin, beta 8	−2.58	0.0017	−1.81	0.0155
ANLN	Anillin, actin-binding protein	−3.24	0.0237	−1.59	0.0006
LRRN1	Leucine rich repeat neuronal 1	−4.07	0.0194	−1.83	0.0152

Table 4 patients

Variables	TCGA validation set (Stage I n=137)
Univariate analysis		Multivariate analysis
	P-value	HR (95% CI)	P-value	HR (95% CI)
Age	0.444	–	0.092	–
Sex	0.601	–	0.694	–
Smoking status	0.837	–	0.526	–
Stage	–	–	–	–
SFTPB	2.133E-04	0.844(0.771–0.923)	0.512	–
SFTPD	0.003	0.866(0.788–0.952)	0.414	–
SFTA1P	0.001	0.821(0.730–0.925)	0.545	–
HLA-DQB1	2.518E-04	0.715(0.602–0.850)	0.002	0.686(0.542–0.868)
ITGB8	0.019	1.194(1.029–1.386)	0.588	–
ANLN	0.000	1.519(1.214–1.900)	0.258	–
LRRN1	0.016	1.202(1.035–1.395)	0.089	–

Abbreviation: RFS, recurrence-free survival.

Table 5 patients

Variables	TCGA validation set (Stage I-IIIA n=233)
Univariate		Multivariate
	P-value	HR (95% CI)	P-value	HR (95% CI)
Age	0.746	–	0.184	–
Sex	0.728	–	0.972	–
Smoking status	0.717	–	0.760	–
Stage	0.003	1.483(1.142–1.925)	0.020	–
SFTPB	0.007	0.906(0.843–0.973)	0.176	–
SFTPD	0.042	0.927(0.862–0.997)	0.687	–
SFTA1P	0.057	–	0.838	–
HLA-DQB1	0.005	0.857(0.770–0.954)	0.044	0.875(0.768–0.996)
ITGB8	0.552	–	0.427	–
ANLN	0.004	1.252(1.072–1.462)	0.324	–
LRRN1	0.180	–	0.059	–

Table 6 Correlation between HLA-DQB1 expression and clinical characteristics in the Daping and TCGA validation sets

HLA-DQB1 Level	Daping validation set (n=165)			TCGA validation set (n=233) cutoff =8,817
	High (n=98)	Low (n=67)	P-value	High (n=64)	Low (n=169)	P-value
Age			0.051			0.604
Mean ± SD	61.00±7.99	58.00±10.01		66.59±10.57	65.12±10.39
<60	39(39.80%)	37(55.23%)		16(25.00%)	48(28.40%)
≥60	59(60.20%)	30(44.77%)		48(75.00%)	121(71.60%)
Sex			0.004			0.785
Male	42(42.86%)	44(65.67%)		26(40.63%)	72(42.61%)
Female	56(57.14%)	23(34.33%)		38(59.37%)	97(57.39%)
Smoking status			0.328			0.826
Non-smoker	64(65.31%)	38(56.72%)		26(40.63%)	66(39.05%)
Smoker	34(34.69%)	29(43.28%)		35(54.69%)	95(56.21%)
Unknown	0(0.00%)	0(0.00%)		3(4.68%)	8(4.74%)
P Stage			0.019			0.912
I	84(85.71%)	47(70.15%)		38(59.38%)	99(58.58%)
II-IIIA	14(14.29%)	20(29.85%)		26(40.62%)	70(41.42%)
Recurrence or metastasis			2E-05			4E-05
Yes	34(34.69%)	46(68.66%)		11(17.19%)	80(47.34%)
No	64(65.31%)	21(31.34%)		53(82.81%)	89(52.66%)

Figure 2 Correlation between HLA-DQB1 mRNA level and recurrence rate and RFS in the TCGA LUAD cohort. Notes: (A) The difference in HLA-DQB1 mRNA levels between tumor and adjacent normal tissues in LUAD (***P<0.001). (B) Recurrence rate in the HLA-DQB1 high-expression group and low-expression group. (C) Kaplan-Meier plots depicting the RFS of patients stratified by HLA-DQB1 mRNA expression in stage I LUAD in the TCGA validation set (P<0.0001, HR=0.2127, 95% CI, 0.1108–0.4081). (D) Kaplan-Meier plots depicting the RFS of patients stratified by HLA-DQB1 mRNA expression in early-stage LUAD (P=0.0008, HR=0.4012, 95% CI, 0.2574–0.6255). Abbreviations: LUAD, lung adenocarcinoma; RFS, recurrence-free survival.

Figure 3 (A) Representative examples of HLA-DQB1 expression in tumor cells a: score 3, b: score 2, c: score 1, d: score 0 (100×). (B) Patient recurrence status in the high- and low-HLA-DQB1 expression subgroups. The recurrence rate was significantly different in the HLA-DQB1-high (34/98) and HLA-DQB1-low (46/67) subgroups (P<0.0001). (C) Kaplan-Meier plots depicting the RFS of early-stage LUAD patients stratified by tumor HLA-DQB1 staining scores. (D) Kaplan-Meier plots depicting the RFS of early-stage LUAD patients stratified by HLA-DQB1 expression level. (E) Kaplan-Meier plots depicting the RFS of stage I LUAD patients stratified by HLA-DQB1 expression level. RFS was significantly worse in patients with low levels of HLA-DQB1 expression (P<0.0001) in the Daping validation set. Abbreviations: LUAD, lung adenocarcinoma; RFS, recurrence-free survival.

Figure 4 Correlation between HLA-DQB1 mRNA level and antitumor immune activity. Notes: Patients with high levels of HLA-DQB1 expression showed more lymphocytic infiltration in tumors (A). In addition, mRNA levels of CD4 (B), CD8 (C), GZMK (D), GZMH (E) and the immune score (F) were positively correlated with HLA-DQB1 (***P<0.001).

Figure 5 Correlation between HLA-DQB1 protein level and antitumor immune activity. Notes: (A) Up to 69% of LUAD patients in the high-HLA-DQB1 group had a CD4-positive T cell infiltration rate greater than 10%, with only 43% in the low-HLA-DQB1 group. (B) Approximately one-third of high-HLA-DQB1 patients had a CD8-positive T cell infiltration rate of greater than 10%, with only 8% among low-HLA-DQB1 patients. (C) Representative examples of HLA-DQB1, CD4 and CD8 staining that demonstrated more CD4- or CD8-positive T cell infiltration in tumors with high expression of HLA-DQB1 (40×). Abbreviation: LUAD, lung adenocarcinoma.

Figure S1 (A) Representative examples of interstitial cell HLA-DQB1 expression patterns (100X). a: Both tumor and interstitial cells expressed HLA-DQB1. b: Tumor cells did not express HLA-DQB1, but interstitial cells did. c: Tumor cells expressed HLA-DQB1, and interstitial cells did not. d: Neither tumor nor interstitial cells expressed HLA-DQB1. (B) Kaplan-Meier plots depicting RFS of Daping LUAD patients stratified by interstitial cell HLA-DQB1 expression. HLA-DQB1 expression in interstitial cells had no significant correlation with RFS in LUAD patients (P=0.4096). Abbreviations: LUAD, lung adenocarcinoma; RFS, recurrence-free survival.

Figure S2 (A) Kaplan-Meier plots depicting RFS of early-stage LUAD patients without adjuvant chemotherapy stratified by HLA-DQB1 level (P=0.0027). (B) Kaplan-Meier plots depicting RFS of early-stage LUAD patients with adjuvant chemotherapy stratified by HLA-DQB1 level (P<0.0001). (C) Kaplan-Meier plots depicting the OS of early-stage LUAD patients stratified by tumor HLA-DQB1 staining scores (P=0.0005). (D) Kaplan-Meier plots depicting the OS of early-stage LUAD patients stratified by tumor HLA-DQB1 level (P<0.0001). Abbreviations: LUAD, lung adenocarcinoma; RFS, recurrence-free survival.