Overexpression of PD-L2 is associated with shorter relapse-free survival in patients with malignant salivary gland tumors

Figures & data

Table 1 Baseline characteristics for all patients with salivary gland tumors

Parameters	N	%
Sex
Male	30	42.9
Female	40	57.1
Age, median (range)	54 (11–84)
Primary site
Parotid gland	37	52.9
Submandibular gland	9	12.9
Sublingual gland	1	1.4
Hard palate	5	7.1
Soft palate	1	1.4
Nasal cavity	5	7.1
Maxilla	4	5.7
Oral cavity	8	11.4
Histology
Adenoid cystic carcinoma	15	21.4
Salivary duct carcinoma	11	15.7
Mucoepidermoid carcinoma	27	38.6
Acinic cell carcinoma	7	10
Adenocarcinoma	2	2.9
Carcinoma ex-pleomorphic adenoma	2	2.9
Squamous cell carcinoma	3	4.3
Epithelial-myoepithelial carcinoma	2	2.9
Undifferentiated carcinoma	1	1.4
T stage
1	28	40
2	18	25.7
3	14	20
4	10	14.3
N stage
0	61	87.1
1	6	8.6
2	3	4.3
M stage
0	68	97.1
1	4	5.7
Initial stage
1	28	40
2	19	27.1
3	12	17.1
4	13	18.6
Treatment
Surgery	45	64.3
Surgery + adjuvant RT	18	25.7
Surgery + adjuvant CCRT	2	2.9
CCRT	1	1.4
RT alone	3	4.3
No treatment	1	1.4
Recurrence
No	53	75.7
Yes	17	24.3

Abbreviations: CCRT, concurrent chemoradiotherapy; T, tumor; N, lymph nodes; M, metastases; RT, radiotherapy.

Figure 1 Representative examples of hematoxylin and eosin staining and immunohistochemical staining for PD-L1, PD-L2, PD-1, CD8⁺ tumor-infiltrating lymphocytes, and CTLA-4 in malignant salivary gland tumors (×200).

Notes: Low PD-L1 (A, B, acinic cell carcinoma), high PD-L1 (C, D, mucoepidermoid carcinoma), low PD-L2 (E, F, acinic cell carcinoma), high PD-L2 (G, H, acinic cell carcinoma), low PD-1 (I, J, adenoid cystic carcinoma), high PD-1 (K, L, salivary duct carcinoma), low CD8⁺ tumor-infiltrating lymphocytes (M, N, acinic cell carcinoma), high CD8⁺ tumor-infiltrating lymphocytes (O, P, epithelial-myoepithelial carcinoma), low CTLA-4 (Q, R, adenoid cystic carcinoma), and high CTLA-4 expression (S, T, adenoid cystic carcinoma). Scale bars at the bottom of the figure indicate 200 μm.

Figure 2 The proportion of staining intensity of PD-L1, PD-L2, and CTLA-4 expression in adenoid cystic carcinoma and salivary duct carcinoma.

Notes: (A) Adenoid cystic carcinoma; (B) salivary duct carcinoma.

Table 2 Molecular marker expression in adenoid cystic carcinoma, salivary duct carcinoma, and mucoepidermoid carcinoma

H-score	Adenoid cystic carcinoma (N=15)		Salivary duct carcinoma (N=11)		Mucoepidermoid carcinoma (N=27)
PD-L1
<1	11	73.3	6	54.5	20	74.1
≥1	4	26.7	5	45.5	7	25.9
PD-L2
<1	2	13.3	4	36.4	10	37
≥1	13	86.7	7	63.6	17	63
CTLA-4
<1	4	26.7	2	18.2	7	25.9
≥1	11	73.3	9	81.8	20	74.1
PD-1
<1	12	80	3	27.3	5	18.5
≥1	3	20	8	72.7	22	81.5
CD8^+ TILs
<1	9	60	2	18.2	2	7.4
≥1	6	40	9	81.8	25	92.6

Note: H-score, percentage of positive cells multiplied by intensity.

Abbreviations: H-score, Histoscore; TILs, tumor-infiltrating lymphocytes.

Table 3 Associations between the clinicopathological variables and immune-related proteins in all patients

Clinicopathological variables	Total cases (n=70)	PD-L1		P-value^*	PD-L2		P-value^*	CTLA-4		P-value^*	PD-1		P-value^*	CD8		P-value^*
		H-score <1	H-score ≥1		H-score <1	H-score ≥1		H-score <1	H-score ≥1		H-score <1	H-score ≥1		H-score <1	H-score ≥1
Tumor size				0.017^*			0.885			0.459			0.424			0.001^*
<4 cm	62	48 (68.6)	14 (20.0)		17 (24.3)	45 (64.3)		15 (21.4)	47 (67.1)		22 (31.4)	40 (57.1)		5 (7.1)	57 (81.4)
≥4 cm	8	3 (4.3)	5 (7.1)		2 (2.9)	6 (8.6)		1 (1.4)	7 (10.0)		4 (5.7)	4 (5.7)		4 (5.7)	4 (5.7)
N stage				0.261			0.905			0.217			0.543			0.467
0	61	46 (65.7)	15 (21.4)		16 (22.9)	45 (64.3)		16 (22.9)	45 (64.3)		24 (34.3)	37 (52.9)		9 (12.9)	52 (74.3)
1	6	4 (5.7)	2 (2.9)		2 (2.9)	4 (5.7)		0	6 (8.6)		1 (1.4)	5 (7.1)		0	6 (8.6)
2	3	1 (1.4)	2 (2.9)		1 (1.4)	2 (2.9)		0	3 (4.3)		1 (1.4)	2 (2.9)		0	3 (4.3)
M stage				0.290			0.209			0.916			0.584			0.429
0	66	49 (70.0)	17 (24.3)		19 (27.1)	47 (67.1)		15 (21.4)	51 (72.9)		24 (34.3)	42 (60.0)		9 (12.9)	57 (81.4)
1	4	2 (2.9)	2 (2.9)		0	4 (5.7)		1 (1.4)	3 (4.3)		2 (2.9)	2 (2.9)		0	4 (5.7)
Initial stage				0.150			0.398			0.337			0.253			0.948
1	28	24 (34.3)	4 (5.7)		10 (14.3)	18 (25.7)		4 (5.7)	24 (34.3)		8 (11.4)	20 (28.6)		3 (4.3)	25 (35.7)
2	17	11 (15.7)	6 (8.6)		3 (4.3)	14 (20.0)		6 (8.6)	11 (15.7)		5 (7.1)	12 (17.1)		2 (2.9)	15 (21.4)
3	12	9 (12.9)	3 (4.3)		4 (5.7)	8 (11.4)		2 (2.9)	10 (14.3)		7 (10.0)	5 (7.1)		2 (2.9)	10 (14.3)
4	13	7 (10.0)	6 (8.6)		2 (2.9)	11 (15.7)		4 (5.7)	9 (12.9)		6 (8.6)	7 (10.0)		2 (2.9)	11 (15.7)
Vascular emboli				0.389			0.357			0.021			0.254			0.083
No	51	35 (50.0)	16 (22.9)		16 (22.9)	35 (50.0)		16 (22.9)	35 (50.0)		15 (22.9)	35 (50.0)		4 (5.7)	47 (67.1)
Yes	9	8 (11.4)	1 (1.4)		2 (2.9)	7 (10.0)		0	9 (12.9)		5 (7.1)	4 (5.7)		3 (4.3)	6 (8.6)
Unknown	10	8 (11.4)	2 (2.9)		1 (1.4)	9 (12.9)		0	10 (14.3)		5 (7.1)	5 (7.1)		2 (2.9)	8 (11.4)
Perineural invasion				0.860			0.307			0.165			0.619			0.350
No	39	28 (40.0)	11 (15.7)		13 (18.6)	26 (37.1)		11 (15.7)	28 (40.0)		13 (18.6)	26 (37.1)		3 (4.3)	36 (51.4)
Yes	21	15 (21.4)	6 (8.6)		5 (7.1)	16 (22.9)		5 (7.1)	16 (22.9)		8 (11.4)	13 (18.6)		4 (2.7)	17 (24.3)
Unknown	10	8 (11.4)	2 (2.9)		1 (1.4)	9 (12.9)		0	10 (14.3)		5 (7.1)	5 (7.1)		2 (2.9)	8 (11.4)

Notes: Data presented as n (%). Statistically significant P-values are indicated in bold. H-score, percentage of positive cells multiplied by intensity.

Abbreviations: H-score, Histoscore; T, tumor; N, lymph nodes; M, metastases.

Figure 3 Relapse-free survival curve for all salivary gland tumors according to PD-L2 expression.

Abbreviation: H-score, Histoscore.

Table 4 Univariate analysis for relapse-free survival and overall survival in all patients

	RFS		OS
	95% CI	P-value	95% CI	P-value
PD-L1 (H-score <1 vs ≥1)	25.9 vs 20.9	0.644	75.9 vs 73.1	0.853
PD-L2 (H-score <1 vs ≥1)	10.0 vs 26.0	0.004	63.5 vs 77.3	0.782
CTLA-4 (H-score <1 vs ≥1)	15.1 vs 22.4	0.26	– vs 75.9	0.782
PD-1 (H-score <1 vs ≥1)	26.4 vs 21.3	0.044	73.1 vs 75.9	0.772
CD8^+ (H-score <1 vs ≥1)	29.2 vs 21.3	0.037	75.9 vs 73.1	0.731

Note: H-score, percentage of positive cells multiplied by intensity.

Abbreviations: CI, confidence interval; H-score, Histoscore; RFS, relapse-free survival; OS, overall survival.

Table 5 Multivariate analysis for relapse-free survival in all patients

	RFS
	HR	95% CI	P-value
PD-L2 (H-score <1 vs ≥1)	6.514	1.173–36.186	0.032
PD-1 (H-score <1 vs ≥1)	0.706	0.184–2.716	0.613
CD8 (H-score vs ≥1)	0.242	0.046–1.263	0.092

Notes: Statistically significant P-values are indicated in bold. H-score, percentage of positive cells multiplied by intensity.

Abbreviations: HR, hazard ratio; H-score, Histoscore; RFS, relapse-free survival; CI, confidence interval.

Table 6 Expression of PD-L1, PD-L2, CTLA-4, PD-1, and CD8⁺ tumor-infiltrating lymphocytes in primary and recurrent tumors

Patient	PD-L1 (H-score)		PD-L2 (H-score)		CTLA-4 (H-score)		PD-1 (H-score)		CD8 (H-score)
	Primary site	Site of recurrence	Primary site	Site of recurrence	Primary site	Site of recurrence	Primary site	Site of recurrence	Primary site	Site of recurrence
A	6.6	0	100.6	58.6	1.1	0	0.5	0	0.9	0
B	0	1.7	37.7	63.9	30.6	5.2	0.8	0.8	3	5.2
C	0	7.1	48.7	37.7	41.5	3	2.2	5.2	22.5	3
D	0	0	131.4	89.5	0	3	0	0	1	3
E	15.3	0	230.2	60.3	0	5.4	0.5	5	12.3	5.4
F	3	0	44.3	41.2	81.5	76.5	3.8	0.9	27.3	76.5
G	0	3.5	78.5	68.5	48.5	9.3	3.4	1.5	5.6	9.3
P-value	0.753		0.091		0.5		0.735		0.735

Note: H-score, percentage of positive cells multiplied by intensity.

Abbreviation: H-score, Histoscore.