Abstract

The aim of the present study was to evaluate the relationship of Epstein–Barr virus (EBV) infection and multiple myeloma (MM) and its impact on clinical characteristics and prognosis. Fresh peripheral blood mononuclear cells (PBMCs) from 139 MM patients who had been diagnosed and treated from January 2010 to May 2018 and 50 PBMC samples from healthy donors were obtained. PCR was carried out for detection of EBV-DNA. The results indicated a significantly higher EBV-DNA concentration among 139 MM patients compared with healthy controls (P<0.05). Correlation analysis showed that the expression of EBV-DNA was positively correlated with the serum free light chain ratio (sFLCR) and progressive disease (PD)/relapse (P<0.05). Especially, in EBV-DNA high-expression MM patients, EBV-DNA concentration for patients with sFLCR ≥100 was higher than that of patients with sFLCR <100. EBV-DNA concentration was higher in patients with disease PD/relapse than those without disease PD/relapse. In univariate analysis, the progress free survival (PFS) was inferior in MM patients with high expression of EBV-DNA, high lactate dehydrogenase (LDH), and high-risk according to mSMART and International Myeloma Working Group (IMWG), stage III according to R-ISS staging, extramedullary lesions, and genetic changes (P<0.05). However, in multivariate analysis, LDH, poor karyotype, R-ISS staging, and mSMART were independent prognostic factors for PFS. Taken together, our studies suggest that an association exists between EBV infection and clinical characteristics of MM patients, and EBV infection appears to have a slight impact on the prognosis of MM. However, the results require further validation in other independent prospective MM cohorts.

Introduction

Multiple myeloma (MM) is the second most common hematological malignancy characterized by monoclonal proliferation of plasma cells that are derived from mature, terminally differentiated B cells [1]. MM patients frequently have depressed levels of polyclonal immunoglobulins and exhibit severe-to-moderate humoral immunodeficiency [2]. Moreover, immune suppressive drug-use during the treatment is another important factor of depressed immunity and subsequent-induced infection [3]. Especially, identification of various infectious factors associated with the progression of MM is crucial in determining disease prognosis and selecting individualized treatment options.

Epstein–Barr virus (EBV) is a member of the Herpesviridae family, and EBV infection occurs worldwide. By adulthood, more than 90% individuals are infected by EBV [4]. Individuals in the Chinese population infected with EBV are younger than those in the Western countries [5]. Thus, it is more important to study the relationship between EBV infection and tumor in China. Previous studies showed that EBV interferes with cellular DNA repair mechanisms and could lead to genetic changes in the infected cells [6,7]. EBV, as well as the other herpes viruses, have been associated with different types of B-cell-derived lymphoid malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma, and diffuse large B-cell lymphoma [8]. The B-cell lymphoid malignancies can initiate from a clone of EBV-infected B cells; furthermore, there is evidence that persistent EBV infection may induce disease progression [9]. The correlation between EBV infection and MM is still controversial [10]. Further studies are required to verify the relationship between EBV infection and MM.

Choosing appropriate clinical specimens and laboratory test method is very important for the diagnosis of different EBV infection-related diseases. Real-time PCR (RT-PCR) has the advantages of fast operation and low risk of laboratory pollution [11]. EBV-DNA loads are the most common specimens and have been widely applied in EBV-related disease diagnosis, treatment effect, and prognostic evaluation [12].

In the present study, peripheral blood mononuclear cells (PBMCs) from 139 MM patients were detected by real-time quantitative PCR and 50 healthy donors were selected as control. We evaluated the potential relationship of EBV infection and MM, and its impact on clinical characteristics and prognosis.

Materials and methods

Patients

We obtained fresh peripheral blood and isolated mononuclear cells from 139 MM patients who had been diagnosed and treated from January 2010 to May 2018. In addition, our study included 50 fresh peripheral blood samples of age and sex-matched healthy donors that represented the control samples. All patients were staged before treatment using both DS staging system and R-ISS staging system. MM patients were not routinely screened for EBV-DNA at diagnosis in China.

DNA extraction and PCR

Mononuclear cells from fresh peripheral blood were extracted by lymphocyte isolation fluid (Solarbio, China). EBV nucleic acid amplification fluorescence detection kit was purchased from Da An Gene Co., Ltd. of Sun Yat-Sen University, and it contained the critical positive quality control product, positive product, negative quality control product, and a PCR reaction tube. PCR products were amplified using specific primers (upstream primer, 5′-GTAGAAGGCCATTTTTCCAC-3′; downstream primer, 5′-TTTCTACGTGACTCCTAGCC-3′) and a double fluorescent-labeled probe (5′-(FAM)ACCACCGTGGCCCAGATGG(TAMRA)-3′). The PCR cycling parameters were set as follows: 93°C for 2 min with 1 cycle, 93°C for 45 s and 55°C for 60 s with 60 cycles, followed by 30 cycles of PCR reaction at 93°C for 30 s, and 55°C for 45 s. The reactions were performed in the Bio-Rad CM9600 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The detection methods, results analysis and quality control methods followed the company’s reagent instructions. EBV-DNA was divided into high expression (>5 × 103 copies/ml) and low expression (<5 × 103 copies/ml) according to the copy number. All PCR reactions were repeated thrice.

Treatment and follow-up

The diagnosis and therapeutic criteria of MM were identified in accordance with the NCCN guidelines [13]. Follow-up began in January 2010. During induction and consolidation therapy, each course of treatment was followed-up. During the maintenance therapy, the follow-up with the patients was every 3 months. progress free survival (PFS) was measured from the date of diagnosis to disease progression, disease relapse, or to the date of the final follow-up.

Statistical analysis

The results of EBV-DNA expression level are presented as the mean ± S.D. An unpaired t test was used to find the EBV-DNA expression level. Correlation analysis between EBV-DNA expression level and clinical characteristics were analyzed by Spearman’s test. PFS rate was calculated by the Kaplan–Meier method and multivariate survival analysis was performed using the Cox regression model. P<0.05 was considered statistically significant. All statistical analyses were evaluated using SPSS24.0 (IBM Corporation, Armonk, NY, U.S.A.).

Results

Clinical characteristics

A total of 139 cases were identified. Patients had a median age of 60 years (range: 41–82 years). The group of patients included in the study consisted of 69 men and 70 women, median follow-up was 76 (0–100) months, median PFS was 70 months, all patients were alive and the 5-year PFS was 62.6%. In the MM group, there were 139 patients, including 59 (42.4%) patients with IgG type, 31 (22.3%) patients with IgA type, 28 (20.1%) patients with light chain type, 11 (7.9%) patients with non-secretory type, and 10 (7.3%) patients with IgD type. Fifty-eight cases were treated with bortezomib (41.7%), 23 cases were treated with thalidomine (16.5%), 47 cases were treated with both bortezomib and thalidomine (33.8%), 11 cases were treated without bortezomib and thalidomine (8.0%). Fifteen cases accepted autologous stem cell transplantation (ASCT) as consolidation therapy. Twenty cases were EBV-DNA high-expression patients, 119 cases were EBV-DNA low-expression patients. The clinical features of the EBV-DNA high-expression and EBV-DNA low-expression patients are summarized in Tables 1 and 3. Among 20 cases with EBV-DNA high-expression patients, the median age was 61 years (range: 46–82 years), median PFS was 24 months (3–70), the 5-year PFS was 32.8%, including eight (40.0%) patients with IgG type, two (10.0%) patients with IgA type, four (20.0%) patients with light chain type, two (10.0%) patients with non-secretory type, and four (20.0%) patients with IgD type; ten (50.0%) patients with serum free light chain ratio (sFLCR) ≥100; five (25.0%) patients with extramedullary lesions (adrenal gland, skin, liver, pancreas, and pleura, respectively); among 12 patients (60.0%) with genetic change, four patients with del(17p), one patient with del(17p), lq21amplification and del(13q4), one patient with t(4; 14), one patient with t(14;16), one patient with t(11;14), two patients with del(13q4), two patients with lq21amplification. One patient accepted ASCT as consolidation therapy.

Table 1
Clinical features of patients with high expression level of EBV-DNA patients
Age (year)/sex Classification DS/R-ISS stage Msmart/ IMWG sFLCR EBV-DNA (copies/ml) Extramedullary lesions Genetics change Theraputic Therapeutic evaluation PFS (months) 
82/F IgG-k III/II MR/LR <100 6.0 × 103 No No PR 60 
69/M IgG-k II/I MR/LR <100 8.4 × 103 Yes No PR 30 
60/F IgG-λ I/II MR/MR <100 1.0 × 104 Yes Yes SD 16 
69/M IgD-λ III/II MR/LR ≥100 1.4 × 104 No No PD 
46/M II/II LR/LR <100 5.0 × 103 No No B + T CR 12 
59/M — II/I MR/MR <100 8.1 × 103 No Yes VGPR 
61/F λ III/III MR/LR <100 1.1 × 104 No No PD 22 
67/F IgG-λ III/II MR/MR ≥100 6.8 × 103 Yes Yes PD 12 
56/F IgG-k III/II MR/MR ≥100 1.2 × 104 No Yes PD 
10 68/F IgD-λ III/III HR/HR ≥100 1.3 × 104 No Yes Replase 10 
11 68/F IgD-λ III/III HR/HR ≥100 1.8 × 104 No Yes Replase 24 
12 53/F IgA-λ III/III HR/HR <100 9.7 × 103 No Yes PD 12 
13 67/F IgA-k III/III HR/MR ≥100 1.2 × 104 No Yes Without B + T Replase 36 
14 61/M IgG-k II/II MR/MR ≥100 8.3 × 103 No Yes T + ASCT Replase 70 
15 58/M IgG-λ III/III HR/HR <100 7.2 × 103 No Yes PR 12 
16 72/M III/II MR/LR ≥100 5.1 × 103 No Yes B + T SD 18 
17 67/M IgG-λ III/III HR/HR ≥100 9.0 × 103 No Yes PD 
18 61/M λ III/II LR/MR <100 6.1 × 103 No No B + T CR 18 
19 61/M — I/I MR/LR <100 5.0 × 103 Yes No PR 15 
20 53/M IgD-λ III/II LR/LR <100 1.2 × 104 Yes No VGPR 30 
Age (year)/sex Classification DS/R-ISS stage Msmart/ IMWG sFLCR EBV-DNA (copies/ml) Extramedullary lesions Genetics change Theraputic Therapeutic evaluation PFS (months) 
82/F IgG-k III/II MR/LR <100 6.0 × 103 No No PR 60 
69/M IgG-k II/I MR/LR <100 8.4 × 103 Yes No PR 30 
60/F IgG-λ I/II MR/MR <100 1.0 × 104 Yes Yes SD 16 
69/M IgD-λ III/II MR/LR ≥100 1.4 × 104 No No PD 
46/M II/II LR/LR <100 5.0 × 103 No No B + T CR 12 
59/M — II/I MR/MR <100 8.1 × 103 No Yes VGPR 
61/F λ III/III MR/LR <100 1.1 × 104 No No PD 22 
67/F IgG-λ III/II MR/MR ≥100 6.8 × 103 Yes Yes PD 12 
56/F IgG-k III/II MR/MR ≥100 1.2 × 104 No Yes PD 
10 68/F IgD-λ III/III HR/HR ≥100 1.3 × 104 No Yes Replase 10 
11 68/F IgD-λ III/III HR/HR ≥100 1.8 × 104 No Yes Replase 24 
12 53/F IgA-λ III/III HR/HR <100 9.7 × 103 No Yes PD 12 
13 67/F IgA-k III/III HR/MR ≥100 1.2 × 104 No Yes Without B + T Replase 36 
14 61/M IgG-k II/II MR/MR ≥100 8.3 × 103 No Yes T + ASCT Replase 70 
15 58/M IgG-λ III/III HR/HR <100 7.2 × 103 No Yes PR 12 
16 72/M III/II MR/LR ≥100 5.1 × 103 No Yes B + T SD 18 
17 67/M IgG-λ III/III HR/HR ≥100 9.0 × 103 No Yes PD 
18 61/M λ III/II LR/MR <100 6.1 × 103 No No B + T CR 18 
19 61/M — I/I MR/LR <100 5.0 × 103 Yes No PR 15 
20 53/M IgD-λ III/II LR/LR <100 1.2 × 104 Yes No VGPR 30 

Abbreviations: CR: complete remission; HR: high risk; IMWG: International Myeloma Working Group; LR: low risk; MR: minimal response; PD: progressive disease; PR: Partial response; VGPR: very good partial response.

DNA levels of EBV in MM patients and healthy individuals in control group

The results of real-time PCR showed that the expression level of EBV-DNA in MM patients is higher than the expression level of EBV-DNA in the healthy control group (2823 ± 272.8 copies vs 1561 ± 214.9 copies, P=0.008) (Figure 1).

The expression level of EBV-DNA between healthy controls and MM patients

Figure 1
The expression level of EBV-DNA between healthy controls and MM patients
Figure 1
The expression level of EBV-DNA between healthy controls and MM patients

Correlation analysis between EBV-DNA expression level and clinical characteristics in MM patients

Correlation analysis showed that sFLCR (rs=0.237; P=0.029) and progressive disease (PD)/relapse (rs=0.285; P=0.008) positively correlated with EBV-DNA expression (Table 2). Other clinical characteristics including age, gender, staging, risk stratification, genetic change, extramedullary lesions, therapeutic, the level of lactate dehydrogenase (LDH), albumin, β2-MG, HGB, and ASCT were not correlated with EBV-DNA expression. Meanwhile, among the high-expression EBV-DNA group, sFLCR ≥100 and PD/relapse patients had higher EBV-DNA expression level compared with sFLCR <100 (P=0.043) and non-PD/relapse (P=0.021) patients (Figure 2).

The expression level of EBV-DNA between different subgroups of MM patients

Figure 2
The expression level of EBV-DNA between different subgroups of MM patients

(A) Among high EBV-DNA expression level group, sFLCR ≥100 patients have higher EBV-DNA expression level compared with sFLCR <100 patients; (B) PD/relapse patients have higher EBV-DNA expression level compared with non-PD/relapse patients.

Figure 2
The expression level of EBV-DNA between different subgroups of MM patients

(A) Among high EBV-DNA expression level group, sFLCR ≥100 patients have higher EBV-DNA expression level compared with sFLCR <100 patients; (B) PD/relapse patients have higher EBV-DNA expression level compared with non-PD/relapse patients.

Table 2
Correlation analysis between EBV-DNA expression level and clinical characteristics in MM patients
Factors EBV-DNA expression rs P-value 
 Low (%) High (%)   
Gender   −0.017 0.842 
Male 60 (50.4%) 9 (45.0%)   
Female 59 (49.6%) 11 (55.0%)   
Age (years)   0.079 0.353 
<60 58 (48.7%) 8 (40.0%)   
≥60 61 (51.3%) 12 (60.0%)   
HGB (g/l)   0.029 0.731 
<115 75 (63.0%) 13 (65.0%)   
≥115 44 (37.0%) 7 (35.0%)   
sFLCR   0.237 0.029 
<100 104 (87.4%) 10 (50.0%)   
≥100 15 (12.6%) 10 (50.0%)   
Serum creatinine (umol/l)   0.064 0.454 
<177 98 (82.4%) 16 (80.0%)   
≥177 21 (17.6%) 4 (20.0%)   
LDH (U/l)   −0.010 0.910 
<248 101 (84.9%) 18 (90.0%)   
≥248 18 (15.1%) 2 (10.0%)   
β2-MG (mg/l)   −0.023 0.786 
<5.5 75 (63.0%) 14 (70.0%)   
≥5.5 44 (37.0%) 6 (30.0%)   
Albumin (g/l)   0.064 0.454 
<35 30 (25.2%) 7 (35.0%)   
≥35 89 (74.8%) 13 (65.0%)   
Genetics change   −0.019 0.827 
No 42 (35.3%) 8 (40.0%)   
Yes 77 (64.7%) 12 (60.0%)   
Extramedullary lesions   −0.013 0.876 
No 89 (74.8%) 15 (75.0%)   
Yes 30 (25.2%) 5 (25.0%)   
DS stage   0.037 0.665 
stage I 19 (16.0%) 2 (10.0%)   
stage II 16 (13.4%) 4 (20.0%)   
stage III 84 (70.6%) 14 (70.0%)   
R-ISS stage   0.007 0.936 
stage I 30 (25.2%) 3 (15.0%)   
stage II 55 (46.2%) 13 (65.0%)   
stage III 34 (28.6%) 4 (20.0%)   
mSMART   0.019 0.823 
Low 30 (41.1) 5 (41.7%)   
Middle 24 (32.9%) 4 (33.3%)   
High 22 (26.0) 3 (25.0%)   
IMWG   0.089 0.299 
Low 21 (17.6%) 3 (15.0%)   
Middle 72 (60.5%) 11 (55.0%)   
High 26 (21.9%) 6 (30.0%)   
Theraputic  −0.071 0.405 
50 (42.0%) 8 (40.0%)   
17 (14.3%) 6 (30.0%)   
B + T 42 (35.3%) 5 (25.0%)   
without B and T 10 (8.4%) 1 (5.0%)   
ASCT   0.112 0.188 
No 108 (88.3%) 19 (95.0%)   
Yes 14 (11.7%) 1 (5.0%)   
Therapeutic evaluation   0.076 0.376 
CR/nCR 35 (29.7%) 7 (35.0%)   
VGPR 8 (6.8%) 2 (10.0%)   
PR/SD/PD 75 (63.6%) 11 (55.0%)   
Follow-up   0.285 0.008 
Alive 99 (83.2%) 10 (50.0%)   
Alive with progression 15 (12.6%) 6 (30.0%)   
Alive with relapse 5 (4.2%) 4 (20.0%))   
Factors EBV-DNA expression rs P-value 
 Low (%) High (%)   
Gender   −0.017 0.842 
Male 60 (50.4%) 9 (45.0%)   
Female 59 (49.6%) 11 (55.0%)   
Age (years)   0.079 0.353 
<60 58 (48.7%) 8 (40.0%)   
≥60 61 (51.3%) 12 (60.0%)   
HGB (g/l)   0.029 0.731 
<115 75 (63.0%) 13 (65.0%)   
≥115 44 (37.0%) 7 (35.0%)   
sFLCR   0.237 0.029 
<100 104 (87.4%) 10 (50.0%)   
≥100 15 (12.6%) 10 (50.0%)   
Serum creatinine (umol/l)   0.064 0.454 
<177 98 (82.4%) 16 (80.0%)   
≥177 21 (17.6%) 4 (20.0%)   
LDH (U/l)   −0.010 0.910 
<248 101 (84.9%) 18 (90.0%)   
≥248 18 (15.1%) 2 (10.0%)   
β2-MG (mg/l)   −0.023 0.786 
<5.5 75 (63.0%) 14 (70.0%)   
≥5.5 44 (37.0%) 6 (30.0%)   
Albumin (g/l)   0.064 0.454 
<35 30 (25.2%) 7 (35.0%)   
≥35 89 (74.8%) 13 (65.0%)   
Genetics change   −0.019 0.827 
No 42 (35.3%) 8 (40.0%)   
Yes 77 (64.7%) 12 (60.0%)   
Extramedullary lesions   −0.013 0.876 
No 89 (74.8%) 15 (75.0%)   
Yes 30 (25.2%) 5 (25.0%)   
DS stage   0.037 0.665 
stage I 19 (16.0%) 2 (10.0%)   
stage II 16 (13.4%) 4 (20.0%)   
stage III 84 (70.6%) 14 (70.0%)   
R-ISS stage   0.007 0.936 
stage I 30 (25.2%) 3 (15.0%)   
stage II 55 (46.2%) 13 (65.0%)   
stage III 34 (28.6%) 4 (20.0%)   
mSMART   0.019 0.823 
Low 30 (41.1) 5 (41.7%)   
Middle 24 (32.9%) 4 (33.3%)   
High 22 (26.0) 3 (25.0%)   
IMWG   0.089 0.299 
Low 21 (17.6%) 3 (15.0%)   
Middle 72 (60.5%) 11 (55.0%)   
High 26 (21.9%) 6 (30.0%)   
Theraputic  −0.071 0.405 
50 (42.0%) 8 (40.0%)   
17 (14.3%) 6 (30.0%)   
B + T 42 (35.3%) 5 (25.0%)   
without B and T 10 (8.4%) 1 (5.0%)   
ASCT   0.112 0.188 
No 108 (88.3%) 19 (95.0%)   
Yes 14 (11.7%) 1 (5.0%)   
Therapeutic evaluation   0.076 0.376 
CR/nCR 35 (29.7%) 7 (35.0%)   
VGPR 8 (6.8%) 2 (10.0%)   
PR/SD/PD 75 (63.6%) 11 (55.0%)   
Follow-up   0.285 0.008 
Alive 99 (83.2%) 10 (50.0%)   
Alive with progression 15 (12.6%) 6 (30.0%)   
Alive with relapse 5 (4.2%) 4 (20.0%))   

Note: *B was bortezomib group; T was thalidomine group; B+T was bortezomib and thalidomine.Abbreviation: IMWG: International Myeloma Working Group.

Survival analysis

In univariate analysis (Figure 3), compared with the low-expression EBV-DNA group, the high-expression EBV-DNA group had lower PFS (P=0.046). In addition, the PFS rates were inferior in MM patients with high LDH (P=0.014), high-risk by mSMART (P=0.003), and International Myeloma Working Group (IMWG) (P=0.001), stage III by R-ISS staging (P=0.048), extramedullary lesions (P=0.035), and genetic change (P=0.001). In multivariate analysis, LDH (hazard ratio [HR]: 0.404; 95%CI: 0.146–1.116; P=0.048), genetic change (HR: 0.295; 95% CI: 0.105–0.825; P=0.020), R-ISS staging (HR: 0.462; 95% CI: 0.192–1.111; P=0.045), and mSMART (HR: 2.478; 95% CI: 1.006–6.103; P=0.049) were independent predictors for PFS (Table 3).

Univariate analysis for survival of MM patients

Figure 3
Univariate analysis for survival of MM patients

(A) The expression level of EBV-DNA for PFS in MM patients; (B) extramedullary lesions for PFS in MM patients; (C) genetic change for PFS in MM patients; (D) R-ISS staging for PFS in MM patients; (E) IMWG for PFS in MM patients; (F) mSMART for PFS in MM patients.

Figure 3
Univariate analysis for survival of MM patients

(A) The expression level of EBV-DNA for PFS in MM patients; (B) extramedullary lesions for PFS in MM patients; (C) genetic change for PFS in MM patients; (D) R-ISS staging for PFS in MM patients; (E) IMWG for PFS in MM patients; (F) mSMART for PFS in MM patients.

Table 3
Cox multivariate regression analysis for PFS in MM patients
Factors PFS 
 HRs (95% CI) P-value 
The expression level of EBV-DNA 0.583 (0.241–1.407) 0.230 
LDH 0.404 (0.146–1.116) 0.048 
R-ISS staging 0.462 (0.192–1.111) 0.045 
Extramedullary lesions 0.409 (0.097–1.722) 0.223 
Genetics change 0.295 (0.105–0.825) 0.020 
mSMART 2.478 (1.006–6.103) 0.049 
IMWG 1.080 (0.417–2.798) 0.874 
Factors PFS 
 HRs (95% CI) P-value 
The expression level of EBV-DNA 0.583 (0.241–1.407) 0.230 
LDH 0.404 (0.146–1.116) 0.048 
R-ISS staging 0.462 (0.192–1.111) 0.045 
Extramedullary lesions 0.409 (0.097–1.722) 0.223 
Genetics change 0.295 (0.105–0.825) 0.020 
mSMART 2.478 (1.006–6.103) 0.049 
IMWG 1.080 (0.417–2.798) 0.874 

Discussion

Several case reports have consistently demonstrated an association between EBV and MM. As early as 1986, there was a case report of EBV positivity after kidney transplantation in patients with secondary MM [14]. In 1995, a case was reported of an AIDS patient who developed MM with a particularly aggressive course that suggested that in the presence of EBV RNA in the plasma cells, EBV plays an etiological role in the development of MM [15]. Another case was described of a patient who developed MM after renal transplantation. EBV-RNA was demonstrated in the neoplastic cells implicating this virus genome in the pathogenesis of the post-transplantation lymphoproliferative disorder [16]. However, most reported patients with EBV-positive plasmacytoma are in a state of immunosuppression. Whether EBV infection promoted the occurrence and progress of MM is controversial. The finding that EBV-DNA concentrations of PBMC in patients with MM were significantly higher than that in individuals in the general population suggests that an association exists. However, our data indicated that the positive rate of EBV-DNA was not statistically significant between patients and the normal controls. Immunodeficient patients, such as those with an EBV infection and those undergoing immunosuppressive drug therapy for transplantation immunosuppressive drug therapies or transplantations, have a slightly increased risk of developing EBV-associated plasmacytoma, and immune regulatory events triggered by the monoclonal myeloma Ig establish and maintain the immunodeficiency in MM patients [17]. The latent infection rate of EBV is generally higher in healthy people [18].

Our research also indicated that the EBV-DNA concentration of PBMC in MM patients was significantly correlated with disease progression/relapse (P<0.05). Likewise, in MM patients with EBV high expression, EBV-DNA concentration was higher in patients with progression/relapse than those without progression/relapse. Similarly, Yan et al. [19] investigated the correlation between EBV infection and solitary plasmacytoma (SP) in 46 patients. The results indicated that EBER-positive patients were more likely to show disease progression (relapse/progression to MM) than EBER-negative patients. Furthermore, we found that EBV-DNA was clearly related to sFLCR, and in the EBV high-expression group, EBV-DNA concentration in cases where sFLCR >100 was higher than that in the sFLCR <100 cases. The sFLCR at initial diagnosis has been widely accepted and applied as a new parameter for the prognosis of patients with myeloma [20]. Paiva et al. [21] found that patients who obtained stringent complete response (sCR) with normal sFLCR had longer PFS than those with abnormal sFLCR, suggesting that sFLCR is an independent prognostic factor of MM. But the related mechanisms remain to be further investigated.

Follow-up data is available for 139 MM patients, all of whom were alive during the last follow-up. The results showed that compared with the low-expression EBV-DNA group, the high-expression EBV-DNA group has lower PFS (P=0.047), but multivariate analysis indicated that EBV-DNA expression was not the independent prognostic factor. A previous study has suggested that EBV positivity is of importance when the differential diagnosis exists between MM and plasmablastic lymphoma (PBL), which is an aggressive malignant B-cell neoplasm. PBL patients (60–75%) have EBV infections and usually die within 1 year [22]. However, the clinical course of MM is indolent. To our knowledge, this is the first study that suggests the role of EBV infection in affecting prognosis of MM. More definitive results need to be confirmed by large sample prospective studies. Similar to previous studies, the present study also showed LDH, genetic change, and R-ISS staging. mSMART and IMWG were independent predictors of PFS [23].

Conclusion

In conclusion, the current study suggests that an association may indeed exist between EBV infection and MM. In addition, our results also indicate that EBV infection is closely related to the clinical characteristics and prognosis of MM patients.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Funding

This work was supported by grants from the National Natural Science Foundation of China [grant numbers 81600163, 81570201].

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Author Contribution

B.X. and X.W. contributed to the manuscript preparation; X.W. and M.L. helped to finish the statistical analyses; R.Y., K.Y., L.R., S.L., S.W., and Y.Z. provided essential reagents, clinical samples, information, and intellectual support; B.X., X.W., and Y.Z. designed the experiments, interpreted the data, and wrote the manuscript.

Abbreviations

     
  • ASCT

    autologous stem cell transplantation

  •  
  • DNA

    deoxyribonucleic acid

  •  
  • EBV

    Epstein–Barr virus

  •  
  • FLC

    free light chain

  •  
  • HR

    hazard ratio

  •  
  • IMWG

    International Myeloma Working Group

  •  
  • LDH

    lactate dehydrogenase

  •  
  • MM

    multiple myeloma

  •  
  • NCCN

    The National Comprehensive Cancer Network

  •  
  • PBL

    plasmablastic lymphoma

  •  
  • PBMCs

    peripheral blood mononuclear cell

  •  
  • PD

    progressive disease

  •  
  • PFS

    progress free survival

  •  
  • RNA

    ribonucleic acid

  •  
  • RT-PCR

    real-time PCR

  •  
  • sCR

    stringent complete response

  •  
  • sFLCR

    serum free light chain ratio

References

References
1.
Guillerey
C.
,
Nakamura
K.
,
Vuckovic
S.
,
Hill
G.R.
and
Smyth
M.J.
(
2016
)
Immune responses in multiple myeloma: role of the natural immune surveillance and potential of immunotherapies
.
Cell. Mol. Life Sci.
73
,
1569
1589
[PubMed]
2.
Sørrig
R.
,
Klausen
T.W.
,
Salomo
M.
,
Vangsted
A.J.
,
Frølund
U.C.
,
Andersen
K.T.
et al.
(
2017
)
Immunoparesis in newly diagnosed multiple myeloma patients: effects on overall survival and progression freeg survival in the Danish population
.
PLoS ONE
12
,
e0188988
[PubMed]
3.
Smirnova
O.V.
,
Manchuk
V.T.
and
Agilova
Y.N.
(
2015
)
Clinical and immunological features of infectious complications in patients with multiple myeloma
.
Vestn. Ross. Akad. Med. Nauk.
5
,
534
540
4.
Ali
A.S.
,
Al-Shraim
M.
,
Al-Hakami
A.M.
and
Jones
I.M.
(
2015
)
Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis
.
Open Virol. J.
9
,
7
28
[PubMed]
5.
Sawada
A.
,
Croom-Carter
D.
,
Kondo
O.
,
Yasui
M.
,
Koyama-Sato
M.
,
Inoue
M.
et al.
(
2011
)
Epstein-Barr virus latent gene sequences as geographical markers of viral origin: unique EBNA3 gene signatures identify Japanese viruses as distinct members of the Asian virus family
.
J Gen. Virol.
92
,
1032
1043
6.
Welch
J.J.G.
,
Schwartz
C.L.
,
Higman
M.
,
Chen
L.
,
Buxton
A.
,
Kanakry
J.A.
et al.
(
2017
)
Epstein-Barr virus DNA in serum as an early prognostic marker in children and adolescents with Hodgkin lymphoma
.
Blood Adv.
1
,
681
684
[PubMed]
7.
Vrzalikova
K.
,
Ibrahim
M.
,
Nagy
E.
,
Vockerodt
M.
,
Perry
T.
,
Wei
W.
et al.
(
2018
)
Co-Expression of the Epstein-Barr virus-encoded latent membrane proteins and the pathogenesis of classic Hodgkin lymphoma
.
Cancers
10
,
pii: E285
[PubMed]
8.
Tracy
S.I.
,
Habermann
T.M.
,
Feldman
A.L.
,
Maurer
M.J.
,
Dogan
A.
,
Perepu
U.S.
et al.
(
2018
)
Outcomes among North American patients with diffuse large B-cell lymphoma are independent of tumor Epstein-Barr virus positivity or immunosuppression
.
Haematologica
103
,
297
303
[PubMed]
9.
Lara
J.
,
Cohen
M.
,
De Matteo
E.
,
Aversa
L.
,
Preciado
M.V.
and
Chabay
P.
(
2014
)
Epstein-Barr virus (EBV) association and latency profile in pediatric Burkitt’s lymphoma: experience of a single institution in Argentina
.
J. Med. Virol.
86
,
845
850
[PubMed]
10.
Ohashi
A.
,
Kato
S.
,
Okamoto
A.
,
Inaguma
Y.
,
Satou
A.
1
,
Tsuzuki
T.
et al.
(
2017
)
Reappraisal of Epstein-Barr virus (EBV) in diffuse large B-cell lymphoma (DLBCL): comparative analysis between EBV-positive and EBV-negative DLBCL with EBV-positive bystander cells
.
Histopathology
71
,
89
97
[PubMed]
11.
Hill
J.A.
,
Mayer
B.T.
,
Xie
H.
,
Leisenring
W.M.
,
Huang
M.L.
,
Stevens-Ayers
T.
et al.
(
2017
)
The cumulative burden of double-stranded DNA virus detection after allogeneic HCT is associated with increased mortality
.
Blood
129
,
2316
2325
12.
Park
J.H.
,
Yoon
D.H.
,
Kim
S.
,
Park
J.S.
,
Park
C.S.
,
Sung
H.
et al.
(
2016
)
Pretreatment whole blood Epstein-Barr virus-DNA is a significant prognostic marker in patients with Hodgkin lymphoma
.
Ann. Hematol.
95
,
801
808
[PubMed]
13.
Anderson
K.C.
,
Alsina
M.
,
Atanackovic
D.
,
Biermann
J.S.
,
Chandler
J.C.
,
Costello
C.
et al.
(
2016
)
NCCN Guidelines insights: Multiple Myeloma, Version 3.2016
.
J. Natl. Compr. Canc. Netw.
14
,
389
400
,
PMCID: PMC6016087
[PubMed]
14.
Waller
E.K.
,
Ziemianska
M.
,
Bangs
C.D.
,
Cleary
M.
,
Weissman
I.
and
Kamel
O.W.
(
1993
)
Characterization of posttransplant lymphomas that express T-cell-associated markers: immunophenotypes, molecular genetics, cytogenetics, and heterotransplantation in severe combined immunodeficient mice
.
Blood
82
,
247
261
15.
Ventura
G.
,
Lucia
M.B.
,
Damiano
F.
,
Cauda
R.
and
Larocca
L.M.
(
1995
)
Multiple myeloma associated with Epstein-Barr virus in an AIDS patient: a case report
.
Eur. J. Haematol.
55
,
332
334
[PubMed]
16.
García-Álvarez
K.G.
,
Garibaldi-Covarrubias
R.
,
Flores-Márquez
M.R.
and
Ortiz-Hidalgo
C.
(
2012
)
Plasma cell myeloma associated with Epstein-Barr virus infection in an 11-year-old girl
.
Pediatr. Dev. Pathol.
15
,
339
342
[PubMed]
17.
Sørrig
R.
,
Klausen
T.W.
,
Salomo
M.
,
Vangsted
A.J.
,
Østergaard
B.
and
Gregersen
H.
(
2016
)
Smolderingmultiple myeloma risk factors for progression: a Danish population-based cohort study
.
Eur. J. Haematol.
97
,
303
309
[PubMed]
18.
Farge
D.
,
Hervé
R.
,
Mikol
J.
,
Sauvaget
F.
,
Ingrand
D.
and
Singer
B.
(
1994
)
Simultaneous progressive multifocal leukoencephalopathy, Epstein-Barr virus (EBV) latent infection and cerebral parenchymal infiltration during chronic lymphocytic
.
Leukemia
8
,
318
321
[PubMed]
19.
Yan
J.
,
Wang
J.
,
Zhang
W.
,
Chen
M.
,
Chen
J.
and
Liu
W.
(
2017
)
Solitary plasmacytoma associated with Epstein-Barr virus: a clinicopathologic, cytogenetic study and literature review
.
Ann. Diagn. Pathol.
27
,
1
6
[PubMed]
20.
Larsen
J.T.
,
Kumar
S.K.
,
Dispenzieri
A.
,
Kyle
R.A.
,
Katzmann
J.A.
and
Rajkumar
S.V.
(
2013
)
Serum free light chain ratio as a biomarker for high-risk smoldering multiple myeloma
.
Leukemia
27
,
941
946
[PubMed]
21.
Paiva
B.
,
Martinez-Lopez
J.
,
Vidriales
M.B.
,
Mateos
M.V.
,
Montalban
M.A.
and
Fernandez-Redondo
E.
(
2011
)
Comparison of immunofixation, serum free light chain, and immunophenotyping for response evaluation and prognostication in multiple myeloma
.
J. Clin. Oncol.
29
,
1627
1633
[PubMed]
22.
Koizumi
Y.
,
Imadome
K.I.
,
Ota
Y.
,
Minamiguchi
H.
,
Kodama
Y.
,
Watanabe
D.
et al.
(
2018
)
Dual threat of Epstein-Barr virus: an autopsy case report of HIV-positive plasmablastic lymphoma complicating EBV-associated hemophagocytic lymphohistiocytosis
.
J. Clin. Immunol.
38
,
478
483
[PubMed]
23.
Dimopoulos
M.A.
,
Goldschmidt
H.
,
Niesvizky
R.
,
Joshua
D.
,
Chng
W.J.
,
Oriol
A.
et al.
(
2017
)
Carfilzomib or bortezomib in relapsed or refractory multiple myeloma (ENDEAVOR): an interim overall survival analysis of an open-label, randomised, phase 3 trial
.
Lancet Oncol.
18
,
1327
1337

Author notes

*

These authors are co-first authors and contributed equally to this work.

This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).