Sociodemographic characteristics
A total of 305 study participants from both prospective and retrospective studies were recruited. Of these, 133 samples were retrospectively collected FFPE lymphoma blocks, of which 120 were confirmed lymphoma cases by an independent hematopathologist review. Fifty-two study participants were lymphoma suspected patients. The study participants' male to female ratio was1.8 to 1. The age of the participants ranged from 3 to 85 years with a mean age of 38+17.5 SD, and majority of study participants 69 (22.6%) were categorized within the age group of between 41 to 50 years. The sociodemographic and clinical profiles of the study participants are provided in Table 1.
Table 1: Demographic characteristics of the study participants
Characteristics
|
Number
|
Percent
|
Study participants (n = 305)
|
|
|
FFPE blocks
|
133
|
43.6
|
Lymphoma confirmed subjects
|
120
|
39.3
|
Lymphoma suspected subjects
|
52
|
17
|
Sex
|
|
|
Male
|
197
|
64.6
|
Female
|
108
|
35.4
|
Age at diagnosis
|
|
|
<20
|
50
|
16.4
|
21-30
|
58
|
19.0
|
31-40
|
59
|
19.3
|
41-50
|
69
|
22.6
|
51-60
|
34
|
11.1
|
>61
|
32
|
10.5
|
Not applicable
|
3
|
1
|
HIV status(n=172)
|
|
|
Positive
|
29
|
16.9
|
Negative
|
122
|
70.9
|
Unknown
|
21
|
12.2
|
Lymphoma types(n=305)
|
|
|
Hodgkin lymphoma
|
91
|
29.8
|
Non-Hodgkin lymphoma
|
197
|
64.6
|
Non-lymphoma (from suspected patients)
|
17
|
5.6
|
Histopathological profiles
Of the total of 305 study participants, 288 had a confirmed diagnosis of lymphoma. Of the 52 fresh lymph node specimens, 17 resulted in non-lymphoma diagnoses. From these 288 lymphoma patients, 91(31.6%) were classified as Hodgkin lymphoma (HL) and 197(69.4%) of the patients were Non-Hodgkin lymphoma (NHL). Of the 197 NHL patients, 49 (24.9%) were small lymphocytic lymphoma (SLL), 37 (18.8%) Diffuse Large B Cell Lymphoma (DLBCL), 10(5.1%) Burkitt Lymphoma (BL), 5(2.5%) were Follicular Lymphoma (FL), 2(1%) Mantle Cell Lymphoma (MCL), 1(0.5%) Extranodal Natural Killer/T-cell lymphoma (ENKTCL) and the rest were NHL, not classified otherwise (Figure 1).
Both HL and NHL were more predominant in male patients. Of 91 HL patients, 59(64.4%) and out of 197 NHL patients, 132(67%) were male. HL was also more predominant within the age group younger than 20 years old (60.4%). Whereas higher NHL patients were found within the age group of 51-60 years old (91%) (Figure 2). n=9 (9.9%) HL and n=20(8.6%) of NHL patients were HIV positive. Out of the 20 NHL patients who were HIV positive, n=9(45%) was DLBCL patients.
EBV Serology
We collected 164 serum samples from both suspected and confirmed lymphoma patients and tested for EBV viral capsid antigen antibody test (VCA IgG). One hundred fifty samples were from lymphoma patients, and from these we detected EBV in n=144 (96%) of the study participants. The remaining n=5 (3.3%) and n=1(0.07%) was tested negative and intermediate for EBV IgG antibody, respectively. EBV IgG was detected in the serum of =48 (98%) of n=49 HL, and n=96/101 (95%) of NHL patients respectively. 95.8% of male and 96.3% of female participants were EBV seropositive. Meanwhile, all n=26 (100%) HIV-positive patients were also EBV seropositive, and 94.3% of HIV negative patients were EBV seropositive. HIV status was significantly associated with EBV positivity with a p value of less than 0.01.
EBV DNA quantification using real-time qPCR.
Detection of the EBV EBNA1 open reading frame using real time qPCR was performed and EBV DNA was detected in 302 of a total of 305 (99%) study participants with the number of EBV particles/ml ranging from 102 EBV particles/ml to 126e9 EBV particles/ml with the median viral load count was around 11,700 EBV particles/ml. We have divided the EBV viral copy number into three groups based on their EBV particles/ml counts (high, low and very low) based on the limit of the detection of the assay on counting the negative control.
Out of the n=288 lymphoma patients, a High (more than 10,000 EBV particles/ml) EBV viral load was detected in n=162 (56.3%) of the study participants, n=46 (16%) had a low EBV viral load (between 5,000-10,000 EBV particles/ml) and n=80 (27.8%) had a very low EBV viral load (less than 5,000 EBV particles/ml).
High EBV viral load was quantified in n=54 (59.3%) and n=108 (54.8%) HL and NHL patients, respectively. Additionally, DLBCL patients had a higher EBV viral load prevalence n=25/37 (67.6%) from NHL patients followed by BL n=5/10 (50%), SLL n=24/52 (44.9%), and FL n= 2/6(33.4%). No significant association between lymphoma types and EBV viral load was observed (p=0.11).
Based on the study participants, tissue samples from suspected lymphoma patients had a higher EBV particles/ml compared to retrospectively collected FFPE blocks and confirmed lymphoma patients n=33/35 (94.3%). But only n=87/120(72.5%) of the PBMC samples from confirmed lymphoma patients and n=42/133(31.6%) FFPE lymphoma blocks had an EBV viral copy number of more than 10,000 EBV particles/ml. There was a significant association between the sample from these study participants and EBV viral load with a p-value of <0.001, in which lymph node biopsy had a higher EBV viral copy number detection compared to blood and FFPE samples.
High EBV viral copy number were detected in 56% of male and 56.7% of female participants. The age group between 21 and 30 had higher EBV particles/ml compared to other groups (73.5%). However, we couldn’t find any significant association between gender with EBV viral load and age groups with EBV viral load. In addition, of the 28 HIV-positive lymphoma patients n=24 (85.7%) had an EBV copy number greater than 10,000 particles/ml however from 109 HIV negative lymphoma patients n=80 (73.4%) had a high EBV copy number. HIV positivity was significantly associated with the EBV viral load with a p-value of <0.01 (Table 2).
Table 2: Distribution of EBV viral copy number within the sociodemographic and clinical characteristics (n=288)
Characteristics
|
<5000 EBV particles/ml
|
5000-10,000 EBV particles/ml
|
>10,000 EBV particles/ml
|
p-value
|
Total count (n=288)
|
Study participants
|
FFPE blocks
|
77
|
14
|
42
|
<0.01
|
133
|
Lymphoma confirmed subjects
|
3
|
30
|
87
|
120
|
Lymphoma suspected subjects
|
0
|
2
|
33
|
35
|
Age group
|
<20
|
18
|
9
|
21
|
0.077
|
48
|
21-30
|
8
|
6
|
39
|
53
|
31-40
|
15
|
11
|
27
|
53
|
41-50
|
16
|
7
|
44
|
67
|
51-60
|
12
|
7
|
14
|
33
|
>61
|
10
|
6
|
15
|
31
|
NA
|
1
|
0
|
2
|
3
|
Gender
|
Male
|
53
|
31
|
107
|
0.986
|
191
|
Female
|
27
|
15
|
55
|
97
|
Lymphoma types
|
HL
|
22
|
15
|
54
|
0.648
|
91
|
NHL
|
58
|
31
|
108
|
197
|
HIV status
|
Positive
|
0
|
4
|
24
|
<0.01
|
28
|
Negative
|
3
|
26
|
80
|
109
|
Unknown
|
77
|
16
|
58
|
151
|