Abstract
Epstein Barr virus (EBV) associated Hodgkin lymphoma (HL) has been defined as cases with clonal EBV infection, EBV genome and gene products in the Reed Sternberg cells. We evaluated the prevalence and clinico-pathological association of EBV in North Indian HL patients. Eighty-eight cases of histologically confirmed classic HL were evaluated for EBV by both IHC expression of LMP1 and real time PCR on formalin fixed lymph node tissue. The expression pattern was analyzed for any association with clinical and histomorphological parameters. Nodular sclerosis subtype was seen in 79.5% patients and mixed cellularity was seen in the remaining patients. Ninety percent of the cases were positive for EBV. The detection rate of EBV by IHC was higher. The EBV positive cases presented with higher disease stage (p < 0.05). The presence of histomorphological features like granuloma formation (5/5), atypical lymphocytes (8/8), histiocyte clusters (26/28), large area of necrosis (11/12), less prominent inflammatory response (25/27) was associated with EBV positivity (p > 0.05). In our study population a high proportion of HL cases showed positivity for EBV indicating a pathogenic role. The positivity was independent of age, gender and histological subtype. Further evaluation of EBV positivity in modulation of tumor immunity may provide insights into variable treatment outcome in EBV positive cases.
Keywords: Epstein–Barr virus, EBV, Hodgkin lymphoma, Nodular sclerosis Hodgkin lymphoma, Mixed cellularity Hodgkin lymphoma
Introduction
The infectious etiology for Hodgkin lymphoma (HL) has been under investigation. Epstein Barr virus (EBV) in HL has several reports with plausible role in etiology via its ability to transform B cells, and presence of clonal EBV genome within HL cells. Also, free circulating EBV genome fragments have been detected in pretreatment blood samples from HL patients [1]. EBV associated HL has been defined as cases with clonal EBV infection, EBV genome and gene products in the Reed Sternberg (HRS) cells [2]. Degree of such association varies with the type of technique utilized for detection of EBV, sub type of HL, geographical area, socio-economic class of the patients and various other clinico-pathological factors [3–5]. Though a few studies from India have been conducted to evaluate the prevalence and implications of EBV positivity in HL, the results remain equivocal. We evaluated the prevalence of EBV in classic HL in North Indian patients by immunohistochemistry (IHC) and real time polymerase chain reaction (PCR) in formalin fixed lymph node biopsy tissue. The association of EBV expression with demographic, clinical, histomorphological parameters and treatment response was also done.
Materials and Methods
Prospective enrolment of newly diagnosed classic HL cases diagnosed on morphological evaluation and IHC expression of CD15 and CD30 was done over 1 year(January 2016 to December 2016).The institute ethics committee approved the study. Written informed consent was taken from the patient or patient’s guardian. We excluded patients who had received chemotherapy or radiotherapy before tissue collection and also those with inadequate tissue samples.
We expected to recruit at least 64 patients for the study, this was based upon expected prevalence of 40% EBV (reported prevalence 20–70%) positivity amongst HL patients. The sample size was calculated using the Cochran formula [n = (z2pq/d2)]. The sample size was calculated for 95% confidence interval and an error allowance of 12%. The upper limit of sample size was kept open ended.
Clinical parameters including age, gender, clinical stage, bulky disease, B- symptoms, baseline computerized tomography scan findings, extra nodal infiltration, bone marrow infiltration, response after first chemotherapy, and relapse were recorded. All the patients had viral markers evaluated (HIV, HBsAg, HCV). Treatment response was evaluated according to the Indian Pediatric Oncology Group (InPOG) protocol. All patients were treated by the ABVD chemotherapy and radiotherapy if necessary [6]. The Hematoxylin and eosin stained section was evaluated to classify cases as mixed cellularity (MC) and nodular sclerosis (NS). The NS cases were further evaluated for various histomorphological features like capsule thickness, effacement of nodal architecture, nodule formation, residual follicles, granuloma, necrosis, RS cell morphology, sclerosis, and inflammatory cells infiltrate [7]. IHC for latent membrane protein LMP1 (Anti-EBV, LMP1- Dako, Product code- IS753, Mouse monoclonal antibody, Clone: CS.1–4) was done as per the standard protocol [8]. Any RS cell showing cytoplasmic positivity for LMP1 was considered positive [9]. The IHC was repeated if the staining intensity was smudgy. DNA was extracted from processed clinical samples using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) following the manufacturer’s protocol. A panel of oligonucleotide primers and dual labeled hydrolysis probes (Taqman) were synthesized by integrated DNA technology (IDT) with HPLC purification. EBV virus probe was tagged with HEX as reporter and quenched with BHQ-2. Probe tagged with Cy5 (648–668 nm) was a reporter and IAbRQSp (Iowa Black® RQSp) was a quencher at 3’ end. PCR result was analyzed on real-time PCR detection system (LightCycler480 II, Roche). Each sample DNA was tested for two reaction by primer/probe sets for EBV and human β-actin. The β-actin primer and probe set targets human β-actin gene which serves as an internal positive control for human nucleic acid. No template control (NTC) and positive controls (PC) for all primer/probe sets were included in each run. Known EBV virus DNA was used as positive control.Human specimen control (HSC) was used as a secondary control to validate the nucleic acid extraction procedure and reagent integrity [10, 11].
PCR result was analyzed as EBV not detected or detected. Cases with either/both (IHC/PCR) positive were considered as positive and both negative were considered as negative.
Statistical analysis was done using SPSS 21 (IBM corp). The association of various factors with EBV positivity was evaluated using Chi-square test. p values less than 0.05 were considered to be significant.
Results
The study included 88 IHC proven cases of HL. The patient characteristics are shown in Table 1. The age of the patients ranged from 3 years to 68 years. Median age was 8.5 years. Majority (69.3%) were pediatric patients with age less than 15 years. All the patients were immunocompetent. 84.1% were males. EBV was positive by either IHC or PCR in 79 patients (89.7%). EBV LMP1 was expressed by IHC in 70 cases (79.5%). Of the positive cases 23 had < 10% positive RS cells. Sixty-four cases (72.7%) were positive by PCR analysis. Positive detection by IHC or PCR alone was seen in only 11 and 9 cases respectively. Based upon the morphological characteristics 79.5% were nodular sclerosis and the remaining cases were of mixed cellularity.
Table 1.
Patient characteristics
| Characteristic | NS (70, 74.5%) | MC (18, 11.45%) |
|---|---|---|
| n (%) | n (%) | |
| Gender | ||
| Male | 59 (84.3) | 15 (83.3) |
| Female | 11 (15.7) | 3 (16.7) |
| Age (years) | ||
| < 15 | 48 (68.6) | 11 (61.1) |
| 15–50 | 19 (27.1) | 7 (38.9) |
| > 50 | 3 (4.3) | – |
| Stage | ||
| I and II | 19 (27.1) | 3 (16.7) |
| III and IV | 51 (72.9) | 15 (83.3) |
| B symptoms present | 57 (81.4) | 13 (72.22) |
| Bulky disease | 39 (55.7) | 9 (0.5) |
| Extranodal infiltration | 23 (32.8) | 14 (77.8) |
| Bone marrow infiltration | 2 (2.9) | 1 (5.5) |
| Relapse | 3 (4.3) | 1 (5.5) |
| Response after Ist cycle | ||
| CR + VGPR | 49 (0.7) | 15 (83.3) |
| PR | 19 (27.1) | 3 (16.7) |
| NR | 2 (2.9) | – |
NS: nodular sclerosis, MC mixed cellularity, CR complete remission, VGPR very good partial remission, PR partial remission, NR no remission
All cases of MC (n = 18) were positive for EBV (IHC 66.7%, PCR 83.3%). EBV was positive in 61 cases (87.1%) of NS (IHC 82.8%, PCR 70%) (Fig. 1). The proportion of EBV positive cases was higher in MC as compared to NS (100% and 87.1%), but this was not significant statistically. The association of various clinical parameters with EBV positivity was evaluated for NS cases (Fig. 2). There was statistically significant association between LMP1 detection and higher clinical stage (p value 0.04). The EBV LMP1 positive group also had a higher proportion of cases with extra nodal disease (95.7% vs. 4.3%), bone marrow infiltration (100% vs. nil), disease relapse (100% vs. nil), but the association was not significant statistically. Moreover, there was no statistically significant difference seen for age, gender, bulky disease, and treatment response.
Fig. 1.
Real time PCR plots for EBV. a EBV positive samples red line and EBV negative samples green line. b Internal control- β-actin gene (human housekeeping gene amplification), red line positive samples, green line no amplification for water as no template control (NTC) (color figure online)
Fig. 2.
Distribution of various clinical and treatment related factors between the nodular sclerosis sub-type with further characterization into EBV positive (blue bar) and negative sub-group (red bar). ^Denotes extranodal infiltration in spleen and liver.# CR complete remission, VGPR very good partial remission, PR partial remission, NR no remission. **Statistically significant p value 0.04. *Higher proportion of cases in EBV positive subgroup, not statistically significant (color figure online)
The association of various histomorphological parameters (Fig. 3a, b) with EBV positivity was separately evaluated for NS cases. The various parameters under the category of lymph node architectural alterations, inflammatory cell response and RS cell characteristics did not show statistically significant difference between the EBV positive and negative group. Though EBV positivity was seen in 100% cases of granuloma formation, and atypical lymphocytes the association was not statistically significant. A high proportion of histiocyte clusters (92.9%), large area of necrosis (91.7%), less prominent neutrophil response (92.6%) and less prominent eosinophil response (92%) were associated with EBV positivity, but even this difference was not statistically significant. (Figure 4)
Fig. 3.
a, b Distribution of various histomorphological factors between the EBV positive and negative group in patients with nodular sclerosis subtype of Hodgkin Lymphoma. *Higher proportion of cases in EBV positive subgroup, not statistically significant
Fig. 4.
Lymph node nodular sclerosis subtype of Hodgkin lymphoma: a Immunohistochemistry for LMP1 (200 ×), cytoplasmic staining. b–d Hematoxylin and eosin (400 ×) inflammatory changes, b Granuloma, c Eosinophils (arrow) with Reed Sternberg cells, d Neutrophils (arrow head)
Discussion
Eighty-eight cases of classic HL were evaluated for EBV status via LMP1 IHC expression and PCR analysis on formalin fixed tissue. Overall 90% of cases were positive for EBV, this is coherent with previous reports from India [12–15]. Our study had a higher proportion of NS cases with EBV positivity as compared to previous Indian (17–68%) and western reports [13–16]. NS cases from other studies conducted in paediatric and adult HL cases have demonstrated a similar high proportion (86-97%) of EBV positive cases [4, 13]. These results may be attributable to regional variations in patient population in India. Our patient population (both children and adult) did not show significant difference for EBV status with respect to age. This is in contrast to the epidemiological categories in HL by Jarrett et al. [12] proposing higher EBV association in children and elderly as compared to young adults. Majority of our cases were males, however the EBV association did not differ significantly with gender contrary to the previous report by Dinand [12]. Our results emphasize the association of EBV with North Indian HL independent of age, gender, and histological subtype.
The EBV positive cases presented with higher disease stage. This is in ambiguity to previous report of EBV expression in cases with earlier disease stage [4]. EBV LMP1 leads to constitutive activation of NF KB signaling in RS cells in classic HL. Additionally, the RS cells in EBV positive HL are able to locally suppress the cytotoxic T cell response by secretion of IL10 [17]. This immune evasion mechanism may contribute to higher disease stage in EBV positive cases.
In our study the detection rate of EBV by IHC for LMP1 (79.5%) was found to be higher than the PCR (72.7%) in contrast to previous reports [5]. Reason for low positivity by PCR might be because of false negative result, due to poor DNA retrieval from paraffin block or inability to detect viral DNA due to low copy number. Another possibility may be due to transformation of Hodgkin disease progenitor cells by “hit and run mechanism” of EBV [3]. The higher positivity by IHC might be due to false interpretation of the result because of cross reactivity and background staining [18]. In our study a case was considered positive if bright cytoplasmic positivity was seen in RS cell.
In NS cases several histomorphological parameters were evaluated to identify any morphological association with EBV status. The various parameters did not show statistically significant differences. The inflammatory cellular milieu in HL is an end result of the cytokine and chemokine production by RS cell. EBV modulates this response. A higher proportion of cases with necrosis, granuloma formation, histiocyte clusters, and presence of atypical lymphocytes were positive for EBV, however the difference was not significant. Also, the EBV positive case had a lower proportion of neutrophil and eosinophil infiltrate.
A high proportion of EBV positivity indicates pathogenic role in Hodgkin’s Lymphoma. The IHC for LMP1 showed positivity in a higher proportion of cases as compared to PCR analysis on formalin fixed tissue. There was significant statistical association between EBV positivity and higher clinical stage. No significant correlation of EBV status was seen with other demographic, clinical, histomorphological parameters and treatment response. An evaluation of the effect of EBV status in immunomodulation of tumour microenvironment may help in identifying the variable treatment response in EBV positive HL patients.
The study also had a few limitations, in situ hybridization was not utilized for EBV detection, and moreover, long term follow up and survival data of these patients will add further value to the study.
Conclusion
In our study population a high proportion of HL cases showed positivity for EBV indicating a pathogenic role. The positivity was independent of age, gender and histological subtype. Further evaluation of EBV positivity in modulation of tumor immunity may provide insights into variable treatment outcome in EBV positive cases.
Acknowledgements
We would like to acknowledge Mr. Shantanu Prakash for technical help in molecular detection of EBV in the formalin fixed tissue blocks from HL cases.
Funding
None.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have conflict of interest.
Ethical Approval
Approved by the institutional ethics committee.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Gandhi MK, Lambley E, Burrows J, Dua U, EllioT S, Shaw PJ, Prince HM, Wolf M, Clarke K, Underhill C, Mills T, Mollee P, Gill D, Marlon P, Seymour JF, Khanna R. Plasma Epstein–Barr virus (EBV) DNA is a biomarker for EBV-positive Hodgkin’s lymphoma. Clin Cancer Res. 2006;12:460–464. doi: 10.1158/1078-0432.CCR-05-2008. [DOI] [PubMed] [Google Scholar]
- 2.Jarrett RF. Risk factors for Hodgkin’s Lymphoma by EBV status and significance of detection of EBV genomes in serum of patients with EBV- associated Hodgkin’s Lymphoma. Leuk Lymphoma. 2003;44(Suppl 3):S27–S32. doi: 10.1080/10428190310001623801. [DOI] [PubMed] [Google Scholar]
- 3.Flavell KJ, Murray PG. Hodgkin’s disease and the Epstein–Barr virus. Mol Pathol. 2000;53(5):262–269. doi: 10.1136/mp.53.5.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Dinanad V, Dawar R, Arya S, Unni R, Mohanty B, Singh R. Hodgkin’s lymphoma in Indian children: prevalence and significance of Epstein–Barr virus detection in Hodgkin’s and Reed-Sternberg cells. Eur J Cancer. 2007;43(1):161–168. doi: 10.1016/j.ejca.2006.08.036. [DOI] [PubMed] [Google Scholar]
- 5.Qi ZL, Han XQ, Hu J, Wang GH, Gao JW, Wang X, Liang DY. Comparison of three methods for the detection of Epstein-Barr virus in Hodgkin’s lymphoma in paraffin-embedded tissues. Mol Med Rep. 2013;7:89–92. doi: 10.3892/mmr.2012.1163. [DOI] [PubMed] [Google Scholar]
- 6.Eichenauer DA, Engert A, André M, Federico M, Illidge T, Hutchings M, Ladetto M. Hodgkin’s lymphoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2014 doi: 10.1093/annonc/mdu181. [DOI] [PubMed] [Google Scholar]
- 7.Wasielewski SV, Franklin J, Fischer R, Hubner K, Hansmann ML, Diehl V, Georgii A, Wasielewski RV. Nodular sclerosing Hodgkin’s disease: new grading predicts prognosis in intermediate and advanced stages. Blood. 2002;05:1548. doi: 10.1182/blood-2002-05-1548. [DOI] [PubMed] [Google Scholar]
- 8.Rhodes A. Fixation of tissues. Bancroft’s theory and practice of histological techniques. 7. Amsterdam: Elsevier; 2013. pp. 69–94. [Google Scholar]
- 9.Herling M, Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP, Kliche KO, Nadali G, Viviani S, Bonfante V, Giardini R, Chilosi M, Kittas C. Expression of Epstein–Barr virus latent membrane protein-1 in Hodgkin and Reed–Sternberg cells of classical Hodgkin’s lymphoma: associations with presenting features, serum interleukin 10 levels, and clinical outcome. Clin Cancer Res. 2003;9(6):2114–2120. [PubMed] [Google Scholar]
- 10.Niesters HG, van Esser J, Fries E, Wolthers KC, Cornelissen J, Osterhaus AD. Development of a real-time quantitative assay for detection of Epstein–Barr virus. J Clin Microbiol. 2000;38:712–715. doi: 10.1128/JCM.38.2.712-715.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Prakash S, Jain A, Jain B. Development of novel triplex single-step real-time PCR assay for detection of Hepatitis Virus B and C simultaneously. Virology. 2016;31(492):101–107. doi: 10.1016/j.virol.2016.01.029. [DOI] [PubMed] [Google Scholar]
- 12.Dinand V, Sachdeva A, Datta S, Bhalla S, Kalara M, Wattal C, Radhakrishnan N. Plasma Epstein–Barr Virus(EBV) DNA as a biomarker for EBV-associated Hodgkin Lymphoma. Indian Paediatr. 2015;52:681–685. doi: 10.1007/s13312-015-0696-9. [DOI] [PubMed] [Google Scholar]
- 13.Naresh KN, Johnson J, Srinivas V, Soman CS, Saikia T, Advani SH, Badwe RA, Dinshaw KA, Muckaden M, Magrath I, Bhatia K. Epstein-Barr virus association in classical Hodgkin’s disease provides survival advantage to patients and correlates with higher expression of proliferation markers in Reed-Sternberg cells. Ann Oncol. 2001;11(1):91–96. doi: 10.1023/A:1008337100424. [DOI] [PubMed] [Google Scholar]
- 14.Karnik S, Srinivasan B, Nair S. Hodgkin’s Lymphoma: immunohistochemical features and its association with EBV LMP-1. Experience from a South Indian hospital. Pathology. 2003;35:207–211. doi: 10.1080/0031302031000123164. [DOI] [PubMed] [Google Scholar]
- 15.Gandhi MK, Tellam JT, Khanna R. Epstein–Barr virus-associated Hodgkin’s lymphoma. Br J Haematol. 2004;125(3):267–281. doi: 10.1111/j.1365-2141.2004.04902.x. [DOI] [PubMed] [Google Scholar]
- 16.Lingyun G, Xin W. Epstein–Barr virus-associated lymphoproliferative disorders: experimental and clinical developments. Int J Clin Exp Med. 2015;8(9):14656–14671. [PMC free article] [PubMed] [Google Scholar]
- 17.Skinnider BF, Mak TW. The role of cytokines in classical Hodgkin lymphoma. Blood. 2002;99(12):4283–4297. doi: 10.1182/blood-2002-01-0099. [DOI] [PubMed] [Google Scholar]
- 18.Ramos-Vara JA. Principles and methods of immunohistochemistry. Methods Mol Biol. 2011;691:83–96. doi: 10.1007/978-1-60761-849-2_5. [DOI] [PubMed] [Google Scholar]