Expression and clinical significance of neuropilin-1 in Epstein-Barr virus-associated lymphomas

Abstract

BACKGROUND:

The expression of neuropilin-1 (NRP-1) in Epstein-Barr virus (EBV)-associated lymphomas and its relationships with clinicopathological parameters was investigated.

METHODS:

The researchers compared 111 cases of patients with lymphoma to 20 cases of reactive lymphoid hyperplasia. In situ hybridization was applied to observe the expression of EBV-encoded RNA (EBER) in lymphomas, and immunohistochemistry was used to detect the NRP-1 expression in lymphoma tissues and lymph node tissues with reactive hyperplasia.

RESULTS:

In these 111 cases, the EBER of 62 cases (55.9%) appeared positive. NRP-1 was relatively highly expressed in lymphomas ( $P=$ 0.019). Further, NRP-1 showed higher expression in lymphomas with positive EBER than in negative ones. A comprehensive analysis revealed that NRP-1 was differently expressed in NK/T-cell lymphoma, Hodgkin’s lymphoma, diffuse large B-cell lymphoma, and anaplastic large cell lymphoma ( $P=$ 0.027). Moreover, highly expressed NRP-1 was found to be a useful independent prognostic factor in assessing overall survival and progression-free survival rates in cases of non-Hodgkin’s lymphoma (NHL).

CONCLUSIONS:

NRP-1 exhibited higher expression in lymphomas, and it was positively expressed in EBV-positive lymphomas. Moreover, highly expressed NRP-1 can be used as an undesirable independent prognostic factor in NHL.

Keywords

Neuropilin-1 Epstein-Barr virus lymphoma clinical parameters prognosis

1. Introduction

Lymphoma is a type of malignancy that develops in the lymph nodes and/or extranodal lymphoid tissues. The two main types of lymphoma include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) [1]. It has been suggested that the morbidity rate of NHL ranked seventh among all malignancies, with its mortality rate ranking ninth [2]. Recently, researchers have focused their studies on the treatment of lymphomas [3, 4].

The Epstein-Barr virus (EBV) consists of a double helix of DNA and has a high infection rate (approximately 90%) in humans. Once activated, this type of virus (which often lies dormant) causes various cancers such as nasopharyngeal carcinoma, lymphoma, gastric cancer, etc. [5, 6, 7, 8, 9]. The viral proteins produced by EBV include latent membrane proteins (LMP-1, LMP-2A, and LMP-2B) and nuclear antigens (EBNA1, EBNA2, EBNA3a, EBNA3b, EBNA3c, and EBNA-LP). Moreover, EBV produces viral RNAs (EBER-1 and EBER-2). LMP-1 has been confirmed as a carcinogen involved in the initiation and progression of many tumors [10, 11, 12]. Since the mechanism by which EBV leads to lymphomas is thus far not very well understood, it is worth exploring [13, 14, 15].

Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is versatile and cell-surface expressed [16, 17]. NRP-1 has been found expressed in vascular endothelial cells [18, 19], and its overexpression has also been detected in neuroglioma, gastric cancer, breast cancer, and lung cancer tumors, which has been regarded as an unwanted prognosis [20, 21, 22, 23, 24]. Currently, there are only a limited number of studies on the associations between NRP-1 and lymphomas. Vadasz et al. employed the immunohistochemistry (IHC) method to detect the expression of NRP-1 in HL tissues [25]. Huang et al. applied gene expression profiling and microarray analysis to observe the highly expressed NRP-1 in EBV-positive NK/T-cell lymphoma (NKTCL) tissue microarray, and the high expression correlated with angiogenesis [26]. Wang et al. discovered that in nasopharyngeal carcinoma, NRP-1 could directly interact with glycoprotein B (gB ${}^{23-431}$ ) and EBV (via NRP-1-mediated assimilation, integration pinocytosis, and lipid-mediated endocytosis), enter nasopharyngeal epithelial cells, and activate EGFR/RAS/ERK signaling and receptor tyrosine kinase signaling to promote the EBV infection [27]. Despite these studies, we still question whether NRP-1 in lymphomas promotes the EBV infection and angiogenesis to regulate the proliferation, apoptosis, invasion, and metastasis of tumor cells.

Only a few studies have explored NRP-1 in lymphomas, and no reports have been found on NRP-1 expression and its role in EBV-associated lymphoma. In this research, in situ hybridization was applied to observe the expression of EBV in NKTCL, HL, diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL) tissues. Moreover, immunohistochemistry was used to detect the expression of NRP-1 in lymphoma tissues [28, 29]. We examined the different expressions of NRP-1 in lymphomas with positive and negative EBV, then we analyzed the relationships between NRP-1 and clinicopathological parameters and investigated the prognostic significance of NRP-1. In addition, we explored the possibility that NRP-1 could be used to indicate the clinical course and the prognosis of EBV-associated lymphoma.

2. Materials and methods

2.1 Tissue samples

A total of 111 cases of NKTCL, HL, DLBCL, and ALCL were collected from the Department of Pathology of the First Hospital Affiliated of Guangxi Medical University during the period of March 2012 to May 2016, and 20 cases of normal tissues of reactive lymphoid hyperplasia were selected as controls. None of the cases involved had undergone chemotherapy, radiotherapy, or other tumor treatments before operation and biopsy, and these cases had not been diagnosed with diseases of the immune system, tumors other than lymphoma, or connective tissue diseases. The present study was approved by the Research Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Nanning, China), and written informed consent was obtained from all patients.

2.2 Immunohistochemistry

The main reagents used for the immunohistochemistry included anti-NRP-1 antibody (sc-5307, mouse monoclonal antibody purchased from Santa Cruz Biotechnology), anti-VEGF165 antibody (ab46154, rabbit polyclonal antibody purchased from Abcam), VEGFR-2 antibody (55B11, rabbit monoclonal antibody purchased from Cell Signaling Technology), LMP-1 antibody (CS1-4, mouse monoclonal antibody), CD34 antibody (kit-0004, mouse monoclonal antibody purchased from Maxim Biotechnologies, Fuzhou), Ki67 antibody (ZA-0502, rabbit monoclonal antibody), 3,3’-diaminobenzidine kits (purchased from Zhong Shan Golden Bridge Biotechnology, Beijing), and rabbit/mouse horse radish peroxidase conjugated secondary antibody (purchased from Shanghai Long Island Biotech. Co., Ltd). The streptavidin-peroxidase method of immunohistochemistry was employed to detect the protein expressions of NRP-1, VEGF165, VEGFR-2, LMP-1, Ki67, and CD34. Paraffin sections 4 $\mu$ m in size were prepared, and then the sections were dewaxed with xylene and ethanol, and finally the retrieval of heat-induced antigens was accomplished. Thereafter, IHC staining was conducted; the color was stained by 3,3’-diaminobenzidine tetrahydrochloride and controlled under microscope. The positive tissues were used as a positive control, and the primary antibody diluent, or phosphate-buffered saline, was used as a negative (blank) control.

Figure 1.

Hematoxylin-eosin staining of reactive hyperplasia of lymph node and lymphoma tissues. (A) Reactive hyperplasia of lymph node tissues. (B) NK/T-cell lymphoma. (C) Hodgkin’s lymphoma. (D) Diffuse large B-cell lymphoma. (E) Anaplastic large cell lymphoma.

2.3 Determination

Two pathologists who did not participate in the experiment observed the sections independently. Through in situ hybridization, when granules of brown-yellow or brown showed up in the nuclei of tumor cells, a tumor appeared positive, while a tumor was negative if blue or light purple cell nuclei were seen. IHC staining of NRP-1, VEGF165, and VEGFR-2 was scored based on the intensity of the staining and the percentage of positive cells. Based on the staining intensity, the colorless cells were graded 0, light yellow 1, brown-yellow 2, and brown 3. According to the percentage of positive cells, 0 was assigned for 0%, 1 for 1%–25%, 2 for 26%–50%, 3 for 51%–75%, and 4 for $>$ 75%. If the staining intensity score multiplied by that of the percentage was in the range of 0–4, it was considered negative ( $-$ ), 5–8 was positive ( $+$ ), and 9–12 was strongly positive. Both positive and strongly positive were regarded as highly expressed. In HL tumor cells, LMP-1 was determined to be positive when its staining was brown-yellow. In NKTCL, DLBCL, and ALCL, over 5% staining of tumor cells was considered positive. Ki67 was positive if it was discovered to carry brown-yellow granules in no less than 60% of the cell nuclei. Each section had three average microvessel count values, and CD34 was considered highly expressed if it exceeded or equaled the median of the average value.

2.4 Statistical analysis

SPSS 23.0 was used to carry out the statistical analysis. The median value of the CD34 staining microvessel count was mean $\pm$ SD ( $\bar{x}\pm s$ ). The chi-squared test ( $\chi^{2}$ ) or Fisher’s exact test was used to calculate the positive expression rates of VEGF165, VEGFR-2, LMP-1, Ki67, and CD34 in lymphomas and to analyze their associations with clinicopathological parameters. Spearman’s rank correlation coefficient was employed to investigate the correlations between the clinicopathological parameters and expressions of NRP-1, VEGF165, VEGFR-2, LMP-1, Ki67, and CD34. The Kaplan-Meier estimator was used for survival analysis, and the log-rank test was used for comparisons. In addition, Cox proportional-hazards regression was utilized for the multiple factor analysis of prognosis. $P$ values of less than 0.05 were considered statistically significant.

3. Results

3.1 Clinicopathological features

This research involved 111 cases of lymphoma, of which 40 were NKTCL, 29 were HL, 31 were DLBCL, and 11 were ALCL. Among these 111 cases, 71 patients were male (64%) and 40 were female (36%), with an average age of 40.7 years and a median age of 42 years (age range 3–89 years) (Table 1).

Table 1
Expression of neuropilin-1 protein in lymphoma and its relationship with clinicopathological parameters

Clinicopathological parameters	N	NRP-1		$\chi^{2}$	$P$ value	$r$	$P$ value
		$+$	$-$
Sex
Male	71	38	33	0.371	0.542	0.058	0.547
Female	40	19	21
Age
$\geqslant$ 60	25	13	12	0.005	0.941	0.007	0.942
$<$ 60	86	44	42
Race
Ethnic han	74	38	36	0	1	0	1
Other	37	19	18
Tumor size (cm)
$\geqslant$ 5	23	13	10	0.128	0.721	0.049	0.727
$<$ 5	31	16	15
Extranodal metastasis
$\geqslant$ 2	37	14	23	4.365	0.037 ${}^{*}$	$-$ 0.199	0.037 ${}^{*}$
$<$ 2	73	43	30
Clinical stage
III–IV	39	20	19	0	0.991	$-$ 0.001	0.992
I–II	72	37	35
LDH level
High	50	24	26	0.378	0.539	$-$ 0.069	0.545
Normal	29	16	13
ECOG score
$\geqslant$ 2	18	6	12	2.792	0.095	$-$ 0.159	0.096
$<$ 2	93	51	42
IPI score
3–5	36	15	21	2.209	0.137	$-$ 0.142	0.14
0–2	74	42	32
B symptoms
Yes	63	32	31	0.018	0.893	$-$ 0.013	0.894
No	48	25	23
Hemoglobin
Low	74	38	36	0	1	0	1
Normal	37	19	18
Albumin
Low	78	38	40	0.628	0.428	$-$ 0.078	0.433
Normal	26	15	11
Lymphocytes
High/low	45	25	20	0.537	0.464	0.072	0.468
Normal	60	29	31
White blood cells
High/low	33	17	16	0	0.99	0.001	0.991
Normal	72	37	35
Erythrocyte sedimentation rate
High	18	10	8	–	1	0.051	0.803
Normal	8	4	4
$\beta$ 2-microglobulin
High	14	7	7	–	1	0.072	0.717
Normal	14	6	8
Diseases
NKTCL	40	23	17	9.161	0.027 ${}^{*}$	0.162	0.09
HL	29	19	10
DLBCL	31	9	22
ALCL	11	6	5
EBER
$+$	62	38	24	5.554	0.018 ${}^{*}$	0.224	0.018 ${}^{*}$
$-$	49	19	30
LMP-1
$+$	50	28	22	0.787	0.375	0.084	0.38
$-$	61	29	32

Table 1, continued
Clinicopathological parameters	N	NRP-1		$\chi^{2}$	$P$ value	$r$	$P$ value
		$+$	$-$
VEGF165
$+$	76	46	30	8.122	0.004 ${}^{*}$	0.27	0.004 ${}^{*}$
$-$	35	11	24
VEGFR-2
$+$	63	40	23	8.596	0.003 ${}^{*}$	0.278	0.003 ${}^{*}$
$-$	48	17	31
Ki67
$+$	73	36	37	0.354	0.552	$-$ 0.056	0.556
$-$	38	21	17
CD34
High	54	32	22	3.496	0.062	0.184	0.062
Low	49	20	29

LDH, Lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; NRP-1, Neuropilin-1; IPI, International prognostic index; NKTCL, NK/T-cell lymphoma; HL, Hodgkin’s lymphoma; DLBCL, Diffuse large B-cell lymphoma; ALCL, Anaplastic large cell lymphoma; EBER, EBV-encoded RNA; LMP-1, Latent membrane protein 1; VEGF165, Vascular endothelial growth factor 165; VEGFR-2, VEGF receptor 2; ${}^{*}P<$ 0.05.

3.2 Histopathological morphology

Under the microscope, despite having a normal structure, the reactive lymphoid hyperplasia carried increasing numbers of lymphoid follicles, its size increased with an amplified germinal center or widened deep cortex, and the number of vessels increased; however, the NKTCL, HL, DLBCL, and ALCL structures were damaged (Fig. 1).

3.3 Results of in situ hybridization

We examined the expression of EBER in these 111 cases and found that 62 cases had positive expressions of EBER, comprising 55.9% (62/111). In terms of NKTCL, EBER was positively expressed in all 40 cases, with a positivity rate of 100%. In the HL cases, 75.9% of patients (22/29) showed a positive expression of EBER. In contrast, a negative expression of EBER was observed in the DLBCL and ALCL cases (Fig. 2).

Figure 2.

In situ hybridization of EBV-encoded RNA of lymphoma tissues. (A) NK/T-cell lymphoma. (B) Hodgkin’s lymphoma. (C) Diffuse large B-cell lymphoma. (D) Anaplastic large cell lymphoma.

Figure 3.

Immunohistochemistry of neuropilin-1 of reactive hyperplasia of lymph node and lymphoma tissues. (A) Reactive hyperplasia of lymph node. (B) NK/T-cell lymphoma. (C) Hodgkin’s lymphoma. (D) Diffuse large B-cell lymphoma. (E) Anaplastic large cell lymphoma.

Figure 4.

Immunohistochemistry of VEGF165, VEGFR-2, LMP-1, Ki67, and CD34 of lymphoma tissues. (A) VEGF165. (B) VEGFR-2. (C) LMP-1. (D) Ki67. (E) CD34.

3.4 Immunohistochemistry results

3.4.1 The IHC of NRP-1

The positivity rate of NRP-1 was relatively higher in the 111 cases (51.4%) than in the 20 control cases (20%), with statistical significance ( $\chi^{2}=$ 5.494, $P=$ 0.019). A positive expression of NRP-1 was seen in 61.3% (38/62) of cases of lymphomas with positively expressed EBER, whereas only 38.8% (19/49) of cases of lymphomas with negatively expressed EBER had a positive expression of NRP-1, which indicated that the positive expression of NPR-1 was comparatively higher in lymphomas with positive EBER expression than negative ( $P=$ 0.018). The percentage of positively expressed NRP-1 in all cases was 57.5% (NKTCL), 65.5% (HL), 29% (DLBCL), and 54.5% (ALCL) (Fig. 3).

3.4.2 The IHC of VEGF165, VEGFR-2, LMP-1, Ki67, and CD34

Among the 111 cases, 76 (68.5%) had a positive expression of VEGF165; 63 (56.8%) had a positive expression of VEGFR-2; 50 (45%) had a positive expression of LMP-1; and 73 (65.8%) had a positive expression of Ki67. In 103 cases of lymphoma, 54 (52.4%) had a positive expression of CD34 (Fig. 4).

3.5 The relationships between NRP-1 and the clinicopathological parameters of 111 cases

It was discovered that NRP-1 was differently expressed in NKTCL, HL, DLBCL, and ALCL, with statistical significance ( $P=$ 0.027). In all the cases, NRP-1 was overexpressed and EBER was positively expressed. The high expression of VEGF165 was positively associated with the high expression of VEGFR-2, while the high expression of VEGF165 had a negative relationship with multiple extranodal metastases, and statistical significance was observed ( $P<$ 0.05) (Table 1).

Table 2
Univariate analysis of clinical pathological parameters and prognosis in lymphoma

Risk factor	N	OS (%)	$P$ value	PFS (%)	$P$ value
Sex
Male	71	57.4	0.761	52.5	0.870
Female	40	55.1		50.8
Age
$\geqslant$ 60	25	32.4	0.010 ${}^{*}$	30.4	0.068
$<$ 60	86	62.2		56.9
Race
Ethnic han	74	55.9	0.268	50.6	0.155
Other	37	59.3		54.6
Tumor size (cm)
$\geqslant$ 5	23	50.0	0.443	41.6	0.182
$<$ 5	31	62.6		63.5
Extranodal metastasis
$\geqslant$ 2	37	42.7	0.004 ${}^{*}$	38.9	0.005 ${}^{*}$
$<$ 2	73	63.5		58.0
Clinical stage
III–IV	39	39.7	0.001 ${}^{*}$	28.4	$<$ 0.001 ${}^{*}$
I–II	72	69.5		64.9
LDH level
High	50	45.7	0.016 ${}^{*}$	36.9	0.003 ${}^{*}$
Normal	29	76.9		77.9
ECOG score
$\geqslant$ 2	18	47.3	0.409	48.0	0.794
$<$ 2	93	58.7		52.9
IPI score
3–5	36	24.8	0.001 ${}^{*}$	26.0	0.006 ${}^{*}$
0–2	74	68.2		61.1
B symptoms
Yes	63	51.7	0.052	45.9	0.084
No	48	63.9		60.4
Hemoglobin
Low	74	54.0	0.383	49.5	0.535
Normal	37	63.0		57.6
Albumin
Low	78	57.6	0.760	52.6	0.916
Normal	26	61.5		54.5
Lymphocyte
High/low	45	41.7	0.003 ${}^{*}$	37.0	0.013 ${}^{*}$
Normal	60	69.1		63.2
White blood cells
High/low	33	43.8	0.024 ${}^{*}$	35.2	0.036 ${}^{*}$
Normal	72	63.4		59.0
Erythrocyte sedimentation rate
High	18	51.6	0.486	47.6	0.655
Normal	8	75.0		63.5
$\beta$ 2-microglobulin
High	14	50.6	0.251	48.2	0.166
Normal	14	79.6		78.6
Diseases
NKTCL	40	63.4	0.005 ${}^{*}$	59.3	0.001 ${}^{*}$
HL	29	65.7		53.5
DLBCL	31	48.8		49.8
ALCL	11	0.0		24.2
EBER
$+$	62	65.2	0.066	56.9	0.192
$-$	49	61.0		44.5
LMP-1
$+$	50	63.6	0.429	56.5	0.371
$-$	61	51.7		48.8

Table 2, continued
Risk factor	N	OS (%)	$P$ value	PFS (%)	$P$ value
NRP-1
$+$	57	51.6	0.356	46.9	0.559
$-$	54	61.3		55.9
VEGF165
$+$	76	57.0	0.615	54.1	0.941
$-$	35	58.6		50.2
VEGFR-2
$+$	63	63.1	0.408	58.9	0.315
$-$	48	46.8		42.4
Ki67
$+$	73	57.9	0.997	55.8	0.705
$-$	38	55.7		45.9
CD34
High	54	64.8	0.145	60.3	0.323
Low	49	50.3		46.3
Chemotherapy
Yes	57	62.8	0.392	55.1	0.558
No	54	51.0		50.7
Radiotherapy
Yes	20	80.7	0.030 ${}^{*}$	74.4	0.093
No	91	51.4		46.8
Chemotherapy $+$ radiotherapy
Yes	17	83.7	0.028 ${}^{*}$	76.0	0.120
No	94	52.0		47.6

OS, Overall survival; PFS, Progression-free survival; LDH, Lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; NRP-1, Neuropilin-1; IPI, International prognostic index; NKTCL, NK/T-cell lymphoma; HL, Hodgkin’s lymphoma; DLBCL, Diffuse large B-cell lymphoma; ALCL, Anaplastic large cell lymphoma; EBER, EBV-encoded RNA; LMP-1, Latent membrane protein 1; VEGF165, Vascular endothelial growth factor 165; VEGFR-2, VEGF receptor 2; ${}^{*}P<$ 0.05.

3.6 The relationships between EBV and the clinicopathological parameters of 111 cases

The analysis indicated that the positive expression of EBER in lymphomas positively correlated with male patients ( $\chi^{2}=$ 13.835, $P<$ 0.001; $r=$ 0.353, $P<$ 0.001), highly expressed LMP-1 ( $\chi^{2}=$ 65.538, $P<$ 0.001; $r=$ 0.768, $P<$ 0.001), high expression of VEGFR-2 ( $\chi^{2}=$ 20.766, $P<$ 0.001; $r=$ 0.433, $P<$ 0.001), and overexpression of CD34 ( $\chi^{2}=$ 4.919, $P=$ 0.027; $r=$ 0.219, $P=$ 0.027), with statistical significance. By contrast, positively expressed EBER had a negative association with clinical stages III–IV ( $\chi^{2}=$ 5.363, $P=$ 0.021; $r=-$ 0.220, $P=$ 0.020) and high Eastern Cooperative Oncology Group (ECOG) scores ( $\chi^{2}=$ 4.420, $P=$ 0.036; $rt=-$ 0.200, $P=$ 0.036), with statistical significance.

3.7 The prognostic analysis of 111 cases

By the end of the follow-up period, 40 of the 111 patients (36%) had died. For the remaining 71 cases, the patients’ average survival period was 36.5 months, with the median survival period being 45 months (0.3–54.1 months). The univariate analysis of overall survival (OS) suggested that patients with the following characteristics were more likely to face a bad prognosis: advanced age ( $\geqslant$ 60 years), two or more extranodal metastases, clinical stages III–IV, high level of lactate dehydrogenase (LDH), unsatisfactory International Prognostic Index (IPI) score, increased or decreased lymphocytes and leucocytes, and no radiotherapy or radio-chemotherapy ( $P<$ 0.05). Moreover, positively expressed NRP-1 was a sign of a bad prognosis, but it was not statistically significant ( $P=$ 0.356). The univariate analysis of progression-free survival (PFS) also showed that patients with the following features had an undesirable survival outcome: two or more extranodal metastases, clinical stages III–IV, high levels of LDH, unsatisfactory IPI scores, and increased or decreased lymphocytes and leucocytes ( $P<$ 0.05). Moreover, the positive expression of NRP-1 was linked with an undesirable prognosis, but no statistical significance was observed ( $P=$ 0.559) (Fig. 4, Table 2).

The multivariate Cox regression analysis of OS revealed that the IPI score was considered an independent prognostic factor for lymphomas, with a risk ratio (RR) $=$ 4.252 (95% CI: 1.811–9.980, $P=$ 0.001). Further, the multivariate Cox regression of PFS indicated that clinical stage was an independent prognostic factor (RR $=$ 3.260, 95% CI: 1.564–6.794, $P=$ 0.002) (Table 3).

Table 3
Multivariate analysis of prognosis of lymphoma

Category	N	Risk factor	OS				PFS
			RR (95% CI)		$P$ value		RR (95% CI)		$P$ value
All lymphomas	111
		IPI score	4.252	(1.811–9.980)		0.001	–
		Clinical stage	–				3.260	(1.564–6.794)		0.002
NKTCL	40
		Age	18.944	(3.863–92.903)	$<$	0.001	5.561	(1.810–17.087)		0.003
		Radiotherapy	0.127	(0.024–0.678)		0.016	–
		CD34	0.217	(0.069–0.684)		0.009	0.321	(0.115–0.896)		0.03
DLBCL	31
		Extranodal metastasis	–				5.291	(1.273–22.002)		0.022
ALCL	11
		Lymphocyte	–				12.939	(1.470–113.864)		0.021
NHL	82
		NRP-1	3.005	(1.229–7.348)		0.016	2.440	(1.068–5.572)		0.034
		IPI score	6.36	(2.461–16.435)	$<$	0.001	6.733	(2.770–16.367)	$<$	0.001

OS, Overall survival; PFS, Progression-free survival; NRP-1, Neuropilin-1; IPI, International prognostic index; NKTCL, NK/T-cell lymphoma; HL, Hodgkin’s lymphoma; DLBCL, Diffuse large B-cell lymphoma; ALCL, Anaplastic large cell lymphoma; NHL, Non-Hodgkin’s lymphoma.

Table 4

Univariate analysis of clinicopathological parameters and prognosis in Hodgkin’s lymphoma and non-Hodgkin’s lymphoma

Risk factor	NHL					HL
	N	OS (%)	$P$ value	PFS (%)	$P$ value	N	OS (%)	$P$ value	PFS (%)	$P$ value
Sex
Male	51	54.5	0.679	53.5	0.861	20	63.9	0.655	51	0.637
Female	31	51.5		48.4		9	66.7		58.3
Age
$\geqslant$ 60	18	20.8	0.007 ${}^{*}$	20.8	0.073	7	68.6	0.449	57.1	0.545
$<$ 60	64	60.9		58.2		22	65.5		52.5
Tumor size (cm)
$\geqslant$ 5	17	48.1	0.336	50.4	0.332	6	55.6	0.827	0	0.167
$<$ 5	18	75.8		67.9		13	56.4		64.1
Extranodal metastasis
$\geqslant$ 2	29	32	0.002 ${}^{*}$	30.4	0.001 ${}^{*}$	8	87.5	0.642	75	0.531
$<$ 2	53	66.9		64.3		20	56		44.1
Clinical stage
III–IV	25	19.6	0.001 ${}^{*}$	18.5	$<$ 0.001 ${}^{*}$	14	65.5	0.633	51.4	0.563
I–II	57	69.5		66.9		15	70		56.9
LDH level
High	34	30.4	0.007 ${}^{*}$	29.7	0.005 ${}^{*}$	16	79.1	0.882	56.3	0.299
Normal	23	75		75.9		6	83.3		83.3
ECOG score
$\geqslant$ 2	17	50.8	0.757	51.8	0.884	1	0	0.006 ${}^{*}$	0	0.095
$<$ 2	65	54.5		51.8		28	68.1		55.4
IPI score
3–5	21	0	$<$ 0.001 ${}^{*}$	0	$<$ 0.001 ${}^{*}$	15	72	0.624	73.3	0.28
0–2	61	69.8		67.4		13	62.3		32.3
B symptoms
Yes	46	45.6	0.103	41.4	0.071	17	74.5	0.403	64.7	0.739
No	36	64.1		66.1		12	64.2		51.4
EBER
$+$	40	63.4	0.142	59.3	0.173	22	67.7	0.805	51.9	0.346
$-$	42	39.4		41.7		7	64.3		64.3
LMP-1
$+$	32	54.8	0.853	53.7	0.581	18	80.8	0.468	63.2	0.782
$-$	50	53.7		51.2		11	42		43.6

Table 4, continued
Risk factor	NHL					HL
	N	OS (%)	$P$ value	PFS (%)	$P$ value	N	OS (%)	$P$ value	PFS (%)	$P$ value
NRP-1
$+$	38	45.5	0.12	46	0.304	19	63.7	0.825	47.4	0.959
$-$	44	60.3		56.2		10	66.7		56
VEGF165
$+$	62	53.3	0.807	52.1	0.823	14	72.4	0.725	65.5	0.229
$-$	20	57.2		52.6		15	51.1		42.9
VEGFR-2
$+$	49	59.9	0.349	58.2	0.311	14	77.9	0.858	64.3	0.909
$-$	33	41.4		38.7		15	58.8		51.9
Ki67
$+$	54	57.3	0.89	59	0.576	19	52.7	0.34	42.6	0.465
$-$	28	44.4		37		10	78.8		70
CD34
High	41	67.6	0.102	64.3	0.407	13	54.4	0.677	42.7	0.921
Normal	35	42.3		43.3		14	70.3		55.1
Lymphocyte							–
High/low	32	36.7	0.001 ${}^{*}$	31.9	0.002 ${}^{*}$
Normal	46	65.9		66.2
White blood cells							–
High/low	21	44.1	0.021 ${}^{*}$	32	0.015 ${}^{*}$
Normal	57	57.5		57.8
Diseases							–
NKTCL	40	63.4	0.011 ${}^{*}$	59.3	0.002 ${}^{*}$
DLBCL	31	48.8		49.8
ALCL	11	0		24.2
Chemotherapy							–
Yes	40	58.2	0.445	56	0.821
No	42	50.3		49.8
Radiotherapy							–
Yes	18	78.7	0.036 ${}^{*}$	71.4	0.152
No	64	46.8		46.1
Chemotherapy $+$ radiotherapy							–
Yes	15	81.7	0.036 ${}^{*}$	72.7	0.2
No	67	47.7		47.1

OS, Overall survival; PFS, Progression-free survival; LDH, Lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; NRP-1, Neuropilin-1; IPI, International prognostic index; NKTCL, NK/T-cell lymphoma; HL, Hodgkin’s lymphoma; NHL, Non-Hodgkin’s lymphoma; DLBCL, Diffuse large B-cell lymphoma; ALCL, Anaplastic large cell lymphoma; EBER, EBV-encoded RNA; LMP-1, Latent membrane protein 1; VEGF165, Vascular endothelial growth factor 165; VEGFR-2, VEGF receptor 2; ${}^{*}P<$ 0.05.

Figure 5.

Univariate survival curves of the influences of neuropilin-1 protein expression on the overall survival (OS) and progression-free survival (PFS) of lymphoma patients. (A) OS of 111 cases of lymphoma patients. (B) PFS of 111 cases of lymphoma patients. (C) OS of Hodgkin’s lymphoma (HL) patients. (D) PFS of HL patients. (E) OS of non-Hodgkin’s lymphoma (NHL) patients. (F) PFS of NHL patients.

3.8 The prognostic analysis of patients with HL or NHL

Analyses of lymphomas were conducted on the two main categories: HL and NHL (NKTCL, DLBCL, and ALCL). When the follow-up was completed, 9 of the 29 patients with HL had died. The remaining 20 patients had an average survival period of 35.6 months, with a median survival period of 45 months (3.5–45 months). The univariate analysis of OS demonstrated that patients who had a disappointing ECOG score were prone to a bad prognosis ( $P=$ 0.006); however, statistical significance was not observed in the relationship between the positive expression of NRP-1 and the prognosis ( $P=$ 0.825). The univariate analysis of PFS also showed that statistical significance did not exist in the association between positively expressed NRP-1 and the prognosis ( $P=$ 0.959). The multivariate Cox regression analyses of OS and PFS failed to discover an independent prognostic indicator (Fig. 5, Table 4).

By the completion of follow-up, 31 of the 82 patients with NHL had died. The 51 remaining patients experienced an average survival period of 35.6 months (0.3–54.1). We conducted a univariate analysis of OS and found that patients with the following clinicopathological parameters were closely linked with a bad prognosis: senior age, two or more extranodal metastases, clinical stages III–IV, high levels of LDH, undesirable IPI scores, increased or decreased lymphocytes and leucocytes, and no radiotherapy or radio-chemotherapy ( $P<$ 0.05). The positive expression of NRP-1 might be connected with an unwanted prognosis, but disappointingly, statistical significance was not achieved ( $P=$ 0.120). Using a univariate analysis of PFS, we discovered that poor medical outcomes were more likely to be related to $\geqslant$ 2 extranodal metastases, clinical stages III–IV, high levels of LDH, undesirable IPI scores, and increased or decreased lymphocytes and leucocytes ( $P<$ 0.05). Positively expressed NRP-1 was likely to result in a poor prognosis, nonetheless, with statistical significance ( $P=$ 0.304). In addition, we also performed univariate analysis of OS and FPS for NHL subtypes (including NKTCL, DLBCL and ALCL). The analysis results show that senior age, overexpressions of CD34 and disappointing IPI scores ( $\geqslant$ 3) were correlated with poor prognosis of NKTCL ( $P<$ 0.05); Patients with DLBCL with the following characteristics have a bad prognosis: Extranodal metastases ( $\geqslant$ 2), clinical stages III–IV, high levels of LDH, disappointing IPI scores ( $\geqslant$ 3), and increased or decreased lymphocytes ( $P<$ 0.05); overexpressions of VEGFR-2, and increased or decreased lymphocytes are associated with unfavourable prognosis in ALCL patients ( $P<$ 0.05) (Tables S1–S3). However, no statistical significance was observed in the relationship between positive expression of NRP-1 and prognosis ( $P>$ 0.05) (Fig. S1).

Subsequently, a multivariate Cox regression analysis of OS was conducted to confirm that the independent prognostic factors for NHL included NRP-1 (RR $=$ 3.005, 95% CI: 1.229–7.384, $P=$ 0.016) and the IPI score (RR $=$ 6.360, 95% CI: 2.461–16.435, $P<$ 0.001). In addition, a multivariate Cox regression analysis of PFS suggested that independent prognostic factors for NHL involved NRP-1 (RR $=$ 2.440, 95% CI: 1.068–5.572, $P=$ 0.034) and the IPI score (RR $=$ 6.733, 95% CI: 2.770–16.367, $P<$ 0.001) (Fig. 5, Table 4).

4. Discussion

EBV’s genome could encode multiple types of anti-apoptotic proteins and cytokines that lead to infection, activation, and proliferation. People with weak immune systems are more prone to suffer from lymphomas or EBV-related diseases, including nasopharynx cancer, gastric carcinoma, etc. [30, 31, 32, 33, 34]. Despite numerous studies on EBV, its specific roles and mechanism in lymphomas remain unclear.

It has been revealed that NRP-1 is overexpressed in neuroglioma, gastric, breast, and lung cancers, and thereby plays a boosting role in tumor cells [18, 20, 21, 23, 35, 36, 37, 38]. Vadasz et al. detected NRP-1 expression in 92% of HL tissues, while its expression was not observed in tissues of reactive lymphoid hyperplasia [25]. In addition, by gene expression profiling and microarray analysis, Huang et al. discovered overexpressed NRP-1 in NKTCL tissues with positive EBV expression, which was associated with angiogenesis [26]. Overall, the results indicated that aberrant expression of NRP-1 exists in lymphomas. Based on in situ hybridization, this research examined the expression of EBV in NKTCL, HL, DLBCL, and ALCL. Moreover, IHC aided our investigation of the expressions of NRP-1, VEGF165, VEGFR-2, LMP-1, CD34, and Ki67 in the four types of lymphoma. Further, we analyzed the relationships of EBV and NRP-1 with individual clinicopathological parameters and prognoses.

The in situ hybridization revealed that EBER was positively expressed in NKTCL, which was consistent with the results of Mo et al. [39] and Chen et al. [40], thereby suggesting that patients with NKTCL have higher EBV infection rates. In HL, the rate of EBER positivity reached 75.9%. The detection rate of EBV in HL appeared higher than that which has been found in previous studies [41], which was explained by the small sample size or false positives in the results. However, EBER was negatively expressed in DLBCL and ALCL, thereby indicating that DLBCL and ALCL have no relationship with EBV infection.

The expression of NRP-1 was obviously higher in lymphomas than in normal reactive lymphoid hyperplasia, with declining expression in EBV-positive lymphomas, EBV-negative lymphomas, and normal reactive lymphoid hyperplasia. Statistical significance was observed, which indicated that NPR-1 played a cancer-promoting role in lymphomas. We categorized the lymphomas into two types (HL and NHL) and conducted comprehensive analyses, finding that the overexpression of NRP-1 was positively associated with EBV-positive high expressions of VEGF165, VEGFR-2, and CD34, which suggests that NRP-1 correlates with EBV in lymphomas, and NRP-1 might accelerate the angiogenesis by interacting with VEGF165 and VEGFR-2. Nevertheless, its specific mechanism needs further confirmation. Numerous studies have demonstrated that in nasopharynx epithelial cells, NRP-1 can promote EBV infection, and the infection could be inhibited by downregulating NRP-1 [27]. Zhou et al. verified that in pancreatic cancer, the increased expression of NRP-1 could promote the expression of VEGFR-2, therefore inducing the growth of vascular endothelial cells; further, the suppression of interactions between NRP-1 and VEGF165 could deactivate the VEGFR signaling pathways [42]. Xu et al. found that in liver cancer, downregulating NRP-1 could reduce the expression of CD34 and then suppress the growth of cancer cells [43].

In this research, it was confirmed that EBER-positive was positively associated with a positive expression of LMP-1, male patients, and overexpressions of VEGFR-2 and CD34, thereby indicating that males were more likely to contract EBV-associated lymphomas, and EBV might promote the formation of tumor vessels. However, the specific mechanism required further studies for confirmation. In all these cases, EBER positivity was negatively associated with clinical stages III–IV, high ECOG scores, disappointing IPI scores ( $\geqslant$ 3), and high expressions of Ki67, thereby suggesting that EBV in lymphomas might predict a relatively better clinical course.

The OS and PFS of patients with lymphomas were also analyzed in this research. The analysis of OS showed that patients with the following features were more likely to face a bad prognosis: senior age, $\geqslant$ 2 extranodal metastases, clinical stages III–IV, high levels of LDH, disappointing ECOG scores ( $\geqslant$ 2), unsatisfactory IPI scores, increased or decreased lymphocytes and leucocytes, and no radiotherapy or radio-chemotherapy. Among them, senior age and unsatisfactory IPI scores were considered independent prognostic factors. By investigating the PFS, we found that patients with the following characteristics tended to have shorter progress-free survival periods: senior age, $\geqslant$ 2 extranodal metastases, clinical stages III–IV, high levels of LDH, unsatisfactory IPI scores, and increased or decreased lymphocytes and leucocytes. Furthermore, the above parameters could function as independent factors to predict the progress status of lymphoma patients, which was consistent with the results of Guo et al. [44], Ma et al. [45], Yin et al. [41], and Myriam et al. [46]. Hence, the validity of this research had been enhanced. Moreover, in NHL, NRP-1 could be used as an independent indicator to predict medical outcomes and determine the progression of tumors. NRP-1 has been confirmed as an independent risk factor in the prognosis of certain types of tumors, including lung, breast, liver, gastric, and bile duct cancers [18, 21, 23, 24, 47, 48, 49, 50]. Thus, it can be said that this research further elaborates on the clinical significance of NRP-1.

5. Conclusions

This study found that NRP-1 was overexpressed in lymphomas, particularly in EBV-positive lymphomas, which might be closely correlated with the infection of EBV and angiogenesis. EBV was much more easily detected in male patients and was positively related to the expression of VEGFR-2. EBV positivity could predict a better clinical course and improved prognosis for lymphoma patients. The analysis of prognoses revealed that senior age, clinical stages III–IV, and undesirable IPI scores could be utilized as independent factors of a poor prognosis. In addition, the high expression of NRP-1 could be used as an independent factor of a poor prognosis for NHL patients. In conclusion, NRP-1 has the potential to become the prognostic indicator for EBV-associated lymphomas.

Footnotes

Acknowledgments

This study was supported by the Open Fund of Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research (GXBMR20 1601), the Guangxi Zhuang Autonomous Region Health, Family Planning Commission Self-Financed Scientific Research Project (Z20170556), the Natural Science Foundation of Guangxi in China (2017GXNS FAA198107, 2015GXNSFDA139028), and the Guang-xi Education Department Research Project (ZD20140 33).

Conflict of interest

The authors declare no conflict of interest.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-192437.

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Expression and clinical significance of neuropilin-1 in Epstein-Barr virus-associated lymphomas

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Tissue samples

2.2 Immunohistochemistry

2.4 Statistical analysis

3. Results

3.1 Clinicopathological features

Table 1 Expression of neuropilin-1 protein in lymphoma and its relationship with clinicopathological parameters

3.3 Results of in situ hybridization

3.4.1 The IHC of NRP-1

3.4.2 The IHC of VEGF165, VEGFR-2, LMP-1, Ki67, and CD34

3.5 The relationships between NRP-1 and the clinicopathological parameters of 111 cases

Table 2 Univariate analysis of clinical pathological parameters and prognosis in lymphoma

3.7 The prognostic analysis of 111 cases

Table 3 Multivariate analysis of prognosis of lymphoma

4. Discussion

5. Conclusions

Footnotes

Acknowledgments

Conflict of interest

Supplementary data

References

Table 1
Expression of neuropilin-1 protein in lymphoma and its relationship with clinicopathological parameters

Table 2
Univariate analysis of clinical pathological parameters and prognosis in lymphoma

Table 3
Multivariate analysis of prognosis of lymphoma