Interactions between hematological biomarkers of virus infection and immune cells in mediating distant metastasis in nasopharyngeal carcinoma: insights into prognosis and induction chemotherapy administration

Shuqi Li; Biyun Chen; Di Cao; Chao Luo; Zhiying Liang; Ian Kou; Ge Ren; Wenjie Huang; Guangying Ruan; Lizhi Liu; Haojiang Li; Siyu Zhu; Ai Fei

doi:10.2478/raon-2026-0005

Figures & Tables

Patient enrolment and study flowchart. (A) Patient enrolment flowchart from the two hospitals. (B) Statistical analysis flowchart.
Note 1. The time involved in the exclusion criteria is set at the time before treatment. As all enrolled patients in Hospital 1 had data from routine blood examinations, biochemical tests, and HBsAg tests, no other patients were further excluded from Hospital 1.
Note 2. Complete MRI included scanning of the nasopharynx and neck regions, which can be used for restaging assessment of the 8th TNM staging system.
Note 3. Low ALC indicated an absolute lymphocyte count ≤ 1.9 ×109/L. High EBV indicated plasma EBV DNA level > 4 ×103 copies/mL.
ALC = absolute lymphocyte count; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interaction between HBsAg (+) and low ALC; IC = induction chemotherapy; MRI = magnetic resonance imaging; OS = overall survival; non_IC = treated without induction chemotherapy; + = positive; - = negative — Patient enrolment and study flowchart. (A) Patient enrolment flowchart from the two hospitals. (B) Statistical analysis flowchart. Note 1. The time involved in the exclusion criteria is set at the time before treatment. As all enrolled patients in Hospital 1 had data from routine blood examinations, biochemical tests, and HBsAg tests, no other patients were further excluded from Hospital 1. Note 2. Complete MRI included scanning of the nasopharynx and neck regions, which can be used for restaging assessment of the 8th TNM staging system. Note 3. Low ALC indicated an absolute lymphocyte count ≤ 1.9 ×109/L. High EBV indicated plasma EBV DNA level > 4 ×103 copies/mL. ALC = absolute lymphocyte count; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interaction between HBsAg (+) and low ALC; IC = induction chemotherapy; MRI = magnetic resonance imaging; OS = overall survival; non_IC = treated without induction chemotherapy; + = positive; - = negative

Association rules, directed acyclic graph, and prognostic value of HBsAg, EBV, and ALC on DMFS. (A) The top 10 association rules related to HBsAg (+) demonstrated strong associations among high EBV, low ALC, and HBsAg (+) with distant metastasis in patients with NPC. (B) The directed acyclic graph (DAG) shows the associations among variables. Significant interactions were observed between HBsAg and plasma EBV DNA load, HBsAg, and ALC, defined as HE and HL. In the mediation analysis, EBV and HE mediated the effect of N staging on distant metastasis, and HL mediated the effect of T staging on distant metastasis. After 1:1 random matched analysis (C–E), HBsAg (+), HL (+), and HE (+) were the independent adverse prognosticators of DMFS in patients with NPC.
Note 1. The width of the curves represents the occurrence frequency of each association rule. X-axis: the number of the position indicates the number of variables involved in the rule. Rhs was set as distant metastasis. The plasma EBV DNA load classification is divided by the cut-off of 4 ×103 copies/mL. The classification of ALC and PNI is divided by the cut-off with its median value. The original rules are presented in Supplementary Table 1.
Note 2. Supplementary Tables 2 and 4 show the interaction and mediation analyses, respectively.
Note 3. The 1:1 random matched survival analyses were performed with the match factors of T and N stages, plasma EBV DNA, and age group. The P values were calculated using a log-rank test.
ALC = absolute lymphocyte count; DMFS = distant metastasis-free survival; EBV = pre-treatment plasma EBV DNA level; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interaction between HBsAg (+) and low ALC; lhs = left-hand side; PNI = prognostic nutritional index; rhs = right-hand side; WBC = white blood cell; +, positive; -, negative — Association rules, directed acyclic graph, and prognostic value of HBsAg, EBV, and ALC on DMFS. (A) The top 10 association rules related to HBsAg (+) demonstrated strong associations among high EBV, low ALC, and HBsAg (+) with distant metastasis in patients with NPC. (B) The directed acyclic graph (DAG) shows the associations among variables. Significant interactions were observed between HBsAg and plasma EBV DNA load, HBsAg, and ALC, defined as HE and HL. In the mediation analysis, EBV and HE mediated the effect of N staging on distant metastasis, and HL mediated the effect of T staging on distant metastasis. After 1:1 random matched analysis (C–E), HBsAg (+), HL (+), and HE (+) were the independent adverse prognosticators of DMFS in patients with NPC. Note 1. The width of the curves represents the occurrence frequency of each association rule. X-axis: the number of the position indicates the number of variables involved in the rule. Rhs was set as distant metastasis. The plasma EBV DNA load classification is divided by the cut-off of 4 ×103 copies/mL. The classification of ALC and PNI is divided by the cut-off with its median value. The original rules are presented in Supplementary Table 1. Note 2. Supplementary Tables 2 and 4 show the interaction and mediation analyses, respectively. Note 3. The 1:1 random matched survival analyses were performed with the match factors of T and N stages, plasma EBV DNA, and age group. The P values were calculated using a log-rank test. ALC = absolute lymphocyte count; DMFS = distant metastasis-free survival; EBV = pre-treatment plasma EBV DNA level; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interaction between HBsAg (+) and low ALC; lhs = left-hand side; PNI = prognostic nutritional index; rhs = right-hand side; WBC = white blood cell; +, positive; -, negative

The 5-year DMFS among patients treated with and without induction chemotherapy.
Note 1. The 1:1 random matched survival analysis was performed among stage III–IVa patients, with the match factors of T and N stages, plasma EBV DNA, and age group. The P values were calculated using a log-rank test.
DMFS = distant metastasis-free survival; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interactions between HBsAg (+) and low ALC; IC = induction chemotherapy; non_IC = treated without induction chemotherapy; + = positive; - = negative — The 5-year DMFS among patients treated with and without induction chemotherapy. Note 1. The 1:1 random matched survival analysis was performed among stage III–IVa patients, with the match factors of T and N stages, plasma EBV DNA, and age group. The P values were calculated using a log-rank test. DMFS = distant metastasis-free survival; HBsAg = hepatitis B surface antigen; HE = interaction between HBsAg (+) and high EBV; HL = interactions between HBsAg (+) and low ALC; IC = induction chemotherapy; non_IC = treated without induction chemotherapy; + = positive; - = negative

Consideration of interaction effect of HL and HE in distant-metastasis-free survival (DMFS) prediction

Variables	DMFS		Variables	DMFS
Variables	HR (95% CI)	P	Variables	HR (95% CI)	P
Step 1:Without interaction item			Step 2:Interaction consideration
NA			HBsAg (+) × Low ALC (HL)	2.56 (1.14–5.76)	0.023
NA			HBsAg (+) × High EBV (HE)	2.16 (1.02–4.58)	0.044
HBsAg (-/+)	1.12 (0.79–1.6)	0.524	HBsAg (-/+)	0.38 (0.17–0.88)	0.024
NA			ALC (≤ 1.9 vs. > 1.9)*	0.94 (0.69–1.27)	0.692
EBV (≤ 4 vs > 4)^#	1.56 (1.16–2.08)	0.003	EBV (≤ 4 vs. > 4)^#	1.34 (0.97–1.84)	0.075
T stage			T stage
T1	1 (reference)		T1	1 (reference)
T2	1.61 (0.94–2.76)	0.084	T2	1.66 (0.97–2.84)	0.066
T3	1.62 (1.04–2.52)	0.032	T3	1.62 (1.04–2.52)	0.034
T4	2.62 (1.67–4.1)	< 0.001	T4	2.63 (1.68,4.13)	< 0.001
N stage			N stage
N0	1 (reference)		N0	1 (reference)
N1	2.01 (1.18–3.43)	0.010	N1	2.01 (1.18–3.43)	0.010
N2	3.25 (1.83–5.8)	< 0.001	N2	3.34 (1.87–5.95)	< 0.001
N3	5.09 (2.69–9.61)	< 0.001	N3	5.28 (2.8–9.97)	< 0.001

The changes in the hazard ratio of tumor staging when HL and HE are considered as mediators in the multivariate Cox regression model on distant-metastasis-free survival (DMFS)

Mediator	Exposure	Unadjusted for mediator		Adjusted for mediator		Mediation proportion %
Mediator	Exposure	HR (95%CI)	P	HR (95% CI)	P	HR (95%CI)	P
HL (+)	T stage
	T1-2	1 (Reference)	0.004	1 (Reference)	0.006
	T3-4	1.59 (1.16-2.18)		1.56 (1.14-2.15)		7.58 (0.538-26)	0.032
EBV	N stage
	N0-1	1 (Reference)	< 0.001	1 (Reference)	< 0.001
	N1-2	2.35 (1.78-3.11)		2.01 (1.51-2.69)		19.3 (7.28-36)	0.004
HE (+)	N stage
	N0-1	1 (Reference)	< 0.001	1 (Reference)	< 0.001
	N1-2	2.01 (1.51-2.69)		2.05 (1.53-2.74)		5.39 (0.37-14.0)	0.032

Basic patient characteristics and univariate analysis

Variables	Total	HBsAg (-)	HBsAg (+)	Chi-square	DMFS
Variables	(n = 1650)	(n = 1365)	(n = 285)	P value	5-year	Log-Rank P
Age group				< 0.001		0.548
≤ 45	821 (49.8%)	651 (47.7%)	170 (59.6%)		87.06
> 45	829 (50.2%)	714 (52.3%)	115 (40.4%)		85.87
Sex				0.035		0.424
Male	1221 (74.0%)	996 (73.0%)	225 (78.9%)		86.05
Female	429 (26.0%)	369 (27.0%)	60 (21.1%)		87.64
Plasma EBV DNA load (copies/ml)			0.536			< 0.001
≤ 4×10³	1071 (64.9%)	891 (65.3%)	180 (63.2%)		90.49
> 4×10³	579 (35.1%)	474 (34.7%)	105 (36.8%)		78.83
Histologic type^a				0.391		0.463
WHO type 1/2	63 (3.8%)	55 (4.0%)	8 (2.8%)		82.71
WHO type 3	1587 (96.2%)	1310 (96.0%)	277 (97.2%)		86.63
T stage^b				0.186		< 0.001
T1	415 (25.2%)	350 (25.6%)	65 (22.8%)		93.54
T2	201 (12.2%)	172 (12.6%)	29 (10.2%)		85.97
T3	649 (39.3%)	521 (38.2%)	128 (44.9%)		86.49
T4	385 (23.3%)	322 (23.6%)	63 (22.1%)		78.71
N stage^b				0.962		< 0.001
N0	331 (20.1%)	272 (19.9%)	59 (20.7%)		95.22
N1	934 (56.6%)	774 (56.7%)	160 (56.1%)		87.63
N2	277 (16.8%)	231 (16.9%)	46 (16.1%)		79.05
N3	108 (6.5%)	88 (6.4%)	20 (7.0%)		66.79
Stage^b				0.590		< 0.001
I	139 (8.4%)	119 (8.7%)	20 (7.0%)		97.55
II	361 (21.9%)	302 (22.1%)	59 (20.7%)		93.05
III	677 (41%)	551 (40.4%)	126 (44.2%)		86.81
IVa	473 (28.7%)	393 (28.8%)	80 (28.1%)		77.13
IC treatment				0.188		< 0.001
non_IC	809 (49.0%)	659 (48.3%)	150 (52.6%)		90.10
IC	841 (51.0%)	706 (51.7%)	135 (47.4%)		82.91
HBsAg status				< 0.001		0.612
HBsAg (-)	1365 (82.7%)	1365 (100.0%)	0		86.60
HBsAg (+)	285 (17.3%)	0	285 (100.0%)		85.87
ALC^c (10⁹/L)	1650 (100%)	1.9 (1.5–2.3)	1.9 (1.5–2.4)	0.514	-	0.663
ALC group (10⁹/L)				0.694		0.223
> 1.9	742 (45.0%)	617 (45.2%)	125 (43.9%)	160 (56.1%)	87.58
≤ 1.9	908 (55.0%)	748 (54.8%)			85.55
ALT^c (U/L)	1650 (100.0%)	19.9 (16–24.9)	24 (18.9–34)	< 0.001	-	0.256
AST^c (U/L)	1650 (100.0%)	20 (16.2–26)	24.2 (20–36.5)	< 0.001	-	0.273

Interactions between hematological biomarkers of virus infection and immune cells in mediating distant metastasis in nasopharyngeal carcinoma: insights into prognosis and induction chemotherapy administration

Figures & Tables

Figure 1.

Figure 2.

FIGURE 3.

Consideration of interaction effect of HL and HE in distant-metastasis-free survival (DMFS) prediction

The changes in the hazard ratio of tumor staging when HL and HE are considered as mediators in the multivariate Cox regression model on distant-metastasis-free survival (DMFS)

Basic patient characteristics and univariate analysis

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