Epstein-Barr virus (EBV) is a ubiquitous virus which belongs to the ? Herpesvirus subfamily.
It infects B cells, T cells and NK cells and in B cells establishes a lifelong infection known as latent infection and expresses virus cancer genes. In the primary infection, EBV in the saliva infects naive B cells present in the oropharynx.1 It then initiates a growth transforming
infection, causing transformation of naïve B cells into proliferating blasts and then establishes a latent infection in memory B cells. After convalescence, the virus persists latently in these memory B cells in an episomal form. These infected memory B cells can differentiate into plasma cells which further undergo lytic infection to produce more viruses. Newly infected naive B cells are controlled by Cytotoxic T lymphocytes, unless immunity is suppressed.
EBV positivity of 30% – 50% is found in patients in North America, Brazil, Taiwan, United Arab Emirates, and Western Europe, whereas it is seen in nearly 100% of children belonging to developing countries.2
EBV causes both benign and malignant diseases.3 It has been detected in a large subset of Hodgkin disease cases all over the world, especially in countries with poor socioeconomic conditions and among younger age.4
EBV attributed malignancies globally revealed that the highest mortality was in East Asia (47%) reflecting the fact that this region, which includes China, Democratic People’s Republic of Korea and Taiwan, has the highest prevalence of both gastric and nasopharyngeal carcinoma in the world.5
The following malignancies are associated with EBV3:
Burkitt lymphoma – 100% in endemic type and 30% in sporadic type.
Hodgkin disease(HD) also known as Hodgkin lymphoma – 60-80% in mixed cellularity type and 20-40% in nodular sclerosis type.
Post-transplant lymphoproliferative disorder – ;90%.
HIV associated lymphomas- 40%.
Nasopharyngeal carcinoma – 100% in high endemic areas and 80% in low endemic areas.
Oral cancers- 82.5%.
In India, burden of EBV positivity is very high and more and more malignancies are found to be associated with it however data is still emerging in different diseases. EBV association with Hodgkin Disease is being studied in India, wherein few studies described in literature showed EBV positivity in HD ranging from 31-82% in all ages, and 28-98% amongst the paediatric population.6-10
EBV association with HD has a bearing on Overall Survival (OS) / Disease Free Survival (DFS) of patients and thus warrants more stringent chemotherapy, therefore EBV profiling is essential as the management and clinical profile in EBV seropositive cases changes and targeted therapy is to be introduced accordingly.
REVIEW OF LITERATURE
Hodgkin disease(HD) was first described by Thomas Hodgkin in 1832.pileri Later, Carl Sternberg and Dorothy Reed independently described the so called “Diagnostic cells”, now known as Reed-Sternberg cells, in 1898 and 1902 respectively. Despite recent progress, it still continues to be one of the most fascinating hematopoietic malignancies, mainly because of its unique histopathological appearance with lymphoma cells in a reactive inflammatory background. It imposes technical challenge thus demanding rapid advances with respect to its pathogenesis. Furthermore, the phenotype of HD is unique among malignancies being at the crossroads of inflammation and malignancy.
HD is a malignant disease of the lymphoid tissue which originates from B lymphocyte(98%) and less common T lymphocyte (2%). It contributes to ;1% of all de novo neoplasms occurring worldwide annually. It is characterized by presence of Hodgkin/Reed-Sternberg (H/RS) cells and their variants in an inflammatory background composed mainly of reactive lymphocytes, plasma cells and eosinophils.(ZAHRA MOHZAHEB) A classic RS cell measures 20-60m in diameter with large rim of cytoplasm and atleast two nuclei with eosinophilic nucleoli covering ;50% nuclear area.pileri
The incidence of HD shows variations in geographic distribution, age group, social class and gender.ZM3 Western countries show a higher incidence of HD as compared to the Asian countries. In the developed nations, the onset of disease shows a bimodal distribution with a first peak in the third decade and a second peak after the age of fifty and men are affected by HD slightly more often than women among all subtypes (ZM)7. The annual age adjusted incidence rates in the UK and USA are 2.4 and 2.8 per 100,000 respectively (ZM)8. The developing nations however, show a predominance of HD in childhood with a decrease in incidence with rising age.(ZM) 7. Genetic factors seem to play a role in causation of HD.
Haim et al found that there is 3 to 9 fold increased risk of developing HD in family member of patients suffering from HD, with a relative risk of 3.1 among first degree relatives as compared with the controls.ZM44 There is a higher relative risk in males as compared to females and also in siblings of the cases than among parents and offsprings. A strong association is found between early birth order, high maternal education, low number of siblings and playmates, single nuclear family and occurrence of HD in younger patients in developed countries(ZM7. Role of an infectious agent is also suggested in agreement with the clinical characteristics of HD, e.g. fever, weight loss, night sweat and the laboratory findings of elevated erythrocyte sedimentation rate or interleukin 6 in the serum(ZM) 18.
HD is not an uncommon lymphoid tumor in India. 1,2 Data from referral cancer hospitals in India suggests difference in demographic profile of HD and its response to treatment as compared to the west. There are only few large-scale studies analyzing the immunoprofile of HD in India. (PATKAR IJC).
Hodgkin disease, has two main subtypes, classical Hodgkin disease (cHD) and nodular lymphocyte predominant Hodgkin disease(NLPHD). In India, NLPHD accounts for 11.97% cases of HD. This incidence is slightly higher as compared to western data wherein NLPHD accounts for 4-6% of cases. (PIJC)4,5 There is a difference in the clinical and pathological profile of Indian cHD as compared to the west. While in the west cHD still maintains its bimodal distribution with first peak from 15 to 30 years of age followed by a sharp fall and a second peak in the sixth decade, 6 the results from Indian population show sharply rising incidence from the age of five years peaking in the third decade. This is attributed to the high (18.89%) percentage of cases that occur before 15 years in India.(PIJC)## 23
EPSTEIN BARR VIRUS AND ITS ASSOCIATION WITH HD
Analysis of epidemiological data suggested the role of an infectious agent in the causation of Hodgkin disease. There has been rising evidence which support EBV’s association with HD.
There is a biological plausibility of EBV-mediated B cell transformation and presence of clonal EBV genomes within HD tumor cells, implying that infection has occurred before malignant transformation.
There is an association of symptomatic primary EBV infection with Infectious mononucleosis(IM) which later gives rise to HD. In the largest study involving 38,000 cases of IM it was seen that risk of developing HD after IM was increased by 2.5 times. In the same study it was also noted that since IM occurred mostly in adolescents, the risk of HD was 3.5 times higher in young adults.MJHID95,96 Another study suggests that HD tended to occur 2.9 years after IM.ZM22,23
Presence of distinctive EBV antibody titer profiles and viral loads both before and after HD diagnosis.
Difference in the demographic, clinical, and epidemiologic characteristics of EBV+ and EBV- HD.
Thereby strongly suggesting that these virally defined variants of HD are distinct entities and that their pathogenesis should be considered separately. Even after extensive research, no other infectious agent has been so far identified6-8.MJHID
THE ROLE OF EBV IN TRANSFORMING B CELLS TO H/RS CELLS
HD is divided into Nodular lymphocyte predominant HD(NLP-HD), and classical HD (cHD) which is further subclassified into mixed cellularity, nodular sclerosis, lymphocyte-rich and lymphocyte depleted subtypes17. Most cases of cHD and NLP-HD have been found to carry clonal somatic mutation in Ig V gene. Interestingly, H/RS cells constitute only 1% of the tumor mass and are shown to have strong NF-B overexpression18-21. H/RS cells of HD are further found to have lost the expression of typical B cell lineage genes and instead they show a strong expression of signaling molecules and transcription factors of other cell types22,23. In 40% of HD cases infection with EBV has been found. The virus remains in the malignant cells during the whole course of disease which is proven by the presence of clonal viral genomes in the tumor cells 24,25.MJHID
Epstein-Barr virus (EBV) is a ubiquitous virus belonging to the ? herpesvirus subfamily.KIMURA1)The primary target for EBV is B cell though it can also infect T or natural killer(NK) cells. It causes benign lymphoproliferative disease like infectious mononucleosis aswell as various kinds of lymphoid malignancies.KIMURA2-4) EBV infects B cells and establishes a life-long latent infection. The primary infection is limited to the naïve B cells in the oropharynx which are infected by cell free EBV in the saliva.7 The virus attaches to its primary receptor cell surface protein CD21 via the viral envelope glycoprotein gp350/220.8)
For penetration into the cell it requires viral glycoprotein complex gH-gL-gp42 and co-receptor HLA class II (Fig. 1).9)
Fig. 1 Attachment of EBV to B cells through viral glycoproteins and cellular receptors. The schematic diagram of Epstein-Barr virus (EBV) and cell surface of B-cell is shown. EBV infects B cells through the attachments of gp350/220 to CD21 and gH/gL/gp42 to HLA classII.
It then initiates a growth-transforming infection by causing naïve B cells to transform into proliferating blasts. If the host immunity is good then the proliferation of these EBV transformed cells is regulated by EBV-specific cytotoxic T lymphocytes (CTL)and NK cells but the response is insufficient to ensure complete eradication and the virus remain latent throughout life at low or intermittent levels of virion production.10) The age of the patient alongwith the genetic makeup appear to have significant contribution on the clinical manifestation of the disease. If the primary infection occurs early in the childhood, as seen in developing countries, it is usually asymptomatic. However, if the primary infection occurs during adolescence as is typical of developed nations, it progresses to infectious mononucleosis which usually resolves with the appearance of EBV specific immunity in the host. The virus though still persists latently at low levels in an episomal form in the memory B cells.10) (Fig. 2).
Fig. 2 EBV infection to lymphocytes. Epstein-Barr virus (EBV)-infected naïve B cells transform and proliferate as activated blasts, but are finally controlled by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. After convalescence, EBV persists as a latent infection with episomal DNA in memory B cells.
During the latency period various EBV nuclear antigens (EBNAs), latent membrane proteins (LMPs) and RNA are expressed which are responsible for immortalization and growth altering effects of infected B cells. Depending upon the viral antigens expression, four types of latency patterns are recognized, type 0-III, each of which is typical of the diseases associated with EBV.
TABLE1: LATENCY PATTERNS IN EBV INFECTION
LATENCY TYPE SEEN IN GENES EXPRESSED
TYPE 0 EBV infected memory
B cells EBER &/ or BART
TYPE 1 Burkitt lymphoma EBER,BART,EBNA1
(MOST COMMON) HD, gastric & nasopharyngeal carcinomas EBER, BART, EBNA1,
TYPE 3 Immunocompromised patients(HIV-AIDS/post transplant) EBER, BART, EBNA1,
LMP1, LMP2S, EBNA2,
EBNA 3S, EBNA LP
These virus-infected cells persist at a low level for the entire lifetime.7) Occasionally, these EBV-infected memory B cells can differentiate into plasma cells and undergo lytic infection producing viruses. Newly infected naïve B cells are kept under control by CTL unless host immunity is suppressed. In immunocompromised hosts, these transformed cells convert to proliferating blasts resulting in symptomatic disease, such as post-transplant lymphoproliferative disorders.2)
EBV can infect the epithelial cells of waldeyer’s ring and shed viruses during the primary infection. It does this via CD21 independent mechanism by attaching to the epithelial cells through gH.13 There has been increasing evidence which now suggests that EBV also infects T and NK cells during primary infection. Rarely, in the acute infectious mononucleosis patients, EBV-positive T and NK cells can also be seen.
MOLECULAR GENETICS OF HD(FLETCHER P-1369)
The histiogenesis of Reed-Sternberg cells has remained controversial. Histiocytes, interdigitating dendritic cells, follicular dendritic cells(FDC), myeloid cell, and lymphocyte have all been implicated as cell of origin.fletcher 47) It is now widely accepted that RS cells in cHD are of B cell lineage as proven by the expression of B cell specific activator protein PAX5, CD20, presence of IG gene rearrangement, and a pattern of genome expression compatible with B cells.47,48,183-185 RS cells show global downregulation of B cell gene expression program , which is unique among B cell neoplasms.186 They show features of germinal centre or post germinal centre B lymphocytes but are “crippled” either due to crippling mutations of the IG genes which is seen in around 25% cases or downregulation and disruption of the B cell transcription factors, such as OCT2, BOB.1 and PU.1.47,108,185,187-192
There is also presence of abberant somatic hypermutation targeting genes expressed in germinal centre cells(PIM1, PAX5, RhoH/TTF,MYC).133
Numerical chromosomal abnormalities are commonly seen in cHD, but no specific nonrandom chromosomal abnormalities are noted.185,230
The rearrangement of the locus encoding the MHC type II transcriptional activator CIITA leading to the transcription of variety of gene fusions occurs in upto 15%of cases, resulting in the downregulation of HLA class II expression and upregulation of molecules which suppress an antitumor immune response, such as PDL1 and PDL2.231
Gene expression profiling studies have shown that Reed-Sternberg cells exhibit decreased mRNA levels in nearly all established B-lineage–specific genes thereby affecting multiple components of signaling pathways active in B cells, including B-cell receptor signaling.233 Molecular groups with different clinical outcome are identified. The good prognosis group overexpresses genes involved in apoptotic induction and cell signaling, while the poor prognosis group is characterized by upregulation of genes involved in fibroblast activation, angiogenesis, extracellular matrix remodeling, cell proliferation, and downregulation of tumor suppressor genes.234 The malignant cells in cHD are thought to originate from preapoptotic germinal centre (GC) B cells since in 25% of cHD cases the H/RS cells carry B cell receptor (BCR)-destructive mutations3. Normally GC B cells harbouring BCR-destructive mutations or mutations that decrease the affinity towards the antigen during affinity maturation in the GC undergo cell death by apoptosis48,49. While, in cHD these cells are able to survive due to a transformation event which is likely the infection with EBV, as in vitro infection of GC B cells with EBV is shown to prevent these BCR-deficient GC B cells from undergoing apoptosis and hence transforming into long-lived cell lines50-52by expressing a BCR-mimic LMP2A which is also expressed in EBV+ H/RS cells53. Eventually, in long-term cultures LMP2A replaces the BCR-signal and BCR+ cells become dependent on the expression of LMP2A54. The rescue of BCR deficient GC B cells by EBV is also supported by the fact that the BCR-deficient HD cases are all EBV+, while only 40% of HD cases show an association with EBV17. EBV LMP2A confers early lymphomagenesis, where the signaling molecules of LMP2A and BCR are still expressed. NF-B, a transcription factor appears to play an important role in rescuing H/RS precursor cells. It does so by activating the expression of the anti-apoptotic DISC-inhibitor c-FLIP thereby inducing the expression of proinflammatory cytokines55-57. In EBV+ HD, latency II gene expression pattern is noted, including the expression of LMP1, LMP2A and EBNA1. LMP1 being a mimicker of a constitutively active CD40 receptor, acts as a strong activator of NF-B, and is presumably responsible for the constitutive NF-B expression in EBV+ HD cases23,58-60.mjhid
INTERACTION OF EBV+ HRS CELLS WITH THE MICRO-ENVIRONMENT AND THE IMMUNE SYSTEM
The clinical and pathologic features of cHD show an abnormal immune response which is thought to be the result of elaboration of many different types of cytokines by the malignant Reed-Sternberg (RS) cells or surrounding tissues81.Maximum cases of cHD cases express tumor necrosis factor receptor (TNFR) family members and their ligands, alongwith an unbalanced production of Th2 cytokines and chemokines. The production of cytokines is of importance in the immunopathogenesis of HD, as these factors can act both as autocrine growth factors or as factors initiating and sustaining the reactive infiltrate. Also, cytokines produced by cells of the microenvironment may contribute to stimulate HRS cell survival and proliferation. The presence of EBV may alter the expression of cytokines and chemokines. EBV favors a Th1 reaction in the HD microenvironment. This fact is supported by the expression of IL-12 and chemokines (IP-10, Mig, MIP-1), which causes Th1-cell differentiation, at higher levels in EBV + HD cases than in EBV – cases82. Accordingly, CD8+ T cells are more abundant in the reactive infiltrate of EBV+ cases. However, cell mediated immune response in EBV+ cases seems to be ineffective, as there occurs local suppression of cytotoxic T cells specifically targeting EBV antigens. This suppression of immune response may be attributed to the presence of IL-10, a potent anti-inflammatory cytokine which is frequently produced by RS cells in EBV+ cHD cases. LMP1 can cause cellular IL-10 expression in EBV+ cells83. According to a study by Herling et al analyzing 577 HD patients, IL-10 levels were reported to be higher in EBV+ cases84. IL-6 is another cytokine which has been reported to be expressed at increased levels in EBV+ tumors85.However, the associations between cytokine levels in peripheral blood and EBV tumor status have not always produced significant correlations86,87. A recent study demonstrated an increased expression of CCL20, a chemokine attracting Treg cells, is also found to be increased in the microenvironment of EBV+ versus EBV- RS cells88.Similarly another cytokine which has been reported to be expressed at increased levels in EBV+ tumors is autotaxin89. Autotaxin is an autocrine motility factor with lysophospholipase-D activity and generates lysophosphatidic acid (LPA)which enhances the growth and survival of HD cells. Specific down-regulation of autotaxin and in turn decreased LPA levels lead to reduced cell growth and viability. The development of molecular profiling techniques has made it possible to establish more comprehensive gene expression patterns of EBV+HD tissues. EBV+ tumors are characterized by a robust gene signature involving innate immunity and antiviral responses90. The molecular profiling has confirmed that EBV favors a Th1 reaction with simultaneous increased expression of IFN, CXCL9,CXCL10, and CXCL11/ITAC and elicits an antiviral response with overexpression of genes such as IVNS1ABP (NS1BP), PLSCR1, and OAS.
ROLE OF EBV IN VARIOUS SUBTYPES OF HODGKIN DISEASE
The role of Epstein Barr virus (EBV) in the onset of Hodgkin’s lymphoma was discussed in the past, however the research on this subject was difficult to perform due to a small number
of neoplastic cells characteristic for this type of tumor. Following the development of
sensitive DNA methods, association of EBV with this neoplasm was confirmed, along with the monoclonal presence of EBV genome2,3. The association of EBV with specific types of
Hodgkin disease varies. In developing countries EBV positivity is seen in almost 100% cases of cHD though there are differences in rural and urban regions. Mixed cellularity HD shows the highest prevalence of ~75% along with lymphocyte depleted cHD, nodular sclerosis type shows an intermediate EBV positivity of 10-25% cases. NLPHD shows the least positivity of 5%. WHO 429
METHODS OF DETECTION OF EBV
To diagnose a case of HD, for clinical management and also for research purposes, laboratory tests are required which can accurately detect EBV. The tests are also important to distinguish and arrive at definitive diagnosis in cases where overlapping histomorphological features are present, as seen in HD, anaplastic large cell lymphoma, or reactive lymphoid hyperplasia.7 The prognostic significance of the tests lies in the fact that in EBV associated cases, patient’s tumor burden can be monitored by employing molecular assays for viral load and treated using EBV targeted oncologic therapies.8-13guidelines margaret The various modalities available for detection of EBV in affected tissues include spot hybridization, in situ hybridization (ISH), and polymerase chain reaction(PCR).2-3 Various proteins elaborated by EBV can also be detected using immunohistochemistry like EBV nuclear antigen 1(EBNA 1) and latent membrane proteins (LMP1,LMP2a,LMP2b).zong li qui
EBV-encoded RNA (EBER1 and EBER2) is expressed abundantly, whereas EBER transcripts being non polyadenylated remain untranslated. EBER in situ hybridization remains the best test for detecting and localizing latent EBV in tissue samples.20-25 In latently infected cells more than one million copies per cell of EBER transcripts can be found.26 EBER in situ hybridization is widely used to identify cases of EBV associated Hodgkin disease and is considered as one of the best test however, the technique and its interpretation has various drawbacks. For example, false positive EBER interpretations can occur due to latent infection of background lymphocytes, nonspecific staining, or cross-reactivity with mucin, yeast, or plant materials.20 Similarly, false-negative results can be seen as a consequence of RNA degradation.27 To avoid this problem, a control stain for a ubiquitous cellular transcript can be used to ensure that RNA is preserved and available for hybridization in the cells under study. Out of various such transcripts studied, U6 cellular transcript is of particular interest as a control as it is similar to EBER in terms of size, abundance, and intranuclear distribution.28 An interesting observation is made of the fact that tumors which were shown to be EBV positive by other methods or at previous times, were reported to be false negative for EBER even though RNA was still well preserved in these cases.29-31 The possible explanation given for this is the hit and run hypothesis, according to which EBV might once have been present in a tumor, but the EBV genome subsequently was lost in part or completely.31,32 To support this hypothesis, it is important to define the criterias for interpretation of the analytic tests for EBV and to describe an approach for handling focal EBER expression among RS/H cells of the same tumor. Till date, such guidelines are lacking. EBV-related Hodgkin lymphoma can also be detected by immunohistochemical(IHC) methods employing viral LMP1. LMP1 is expressed in the cytoplasm and surface membrane of RS/H cells but is rarely expressed in latently infected background lymphocytes of Hodgkin lymphoma.33 It is also elaborated in the benign lymphocytes of infectious mononucleosis and in a small subset of lymphocytes in healthy, remotely infected viral carriers.34,35 IHC for LMP1 is more economic and rapid than EBER hybridization and more easily incorporated into routine clinical laboratories. It is performed using commercial antibodies on fixed, paraffin-embedded tissues.36 Results are to be interpreted with caution as false-positive staining is reported in poorly fixed tissues, in the cells of nervous system, and also in some uninfected hematopoietic elements, including eosinophils and plasma cells.20,36,37 False-negative immunostain results are obtained more frequently in the decalcified tissues.
Guidelines for Interpreting Epstein-Barr Virus–Encoded RNA (EBER) In Situ Hybridization and Latent Membrane Protein 1 (LMP1) Immunohistochemical Stains in Hodgkin Lymphoma
• The morphologist must be competent enough in distinguishing Reed-Sternberg/Hodgkin (RS/H) cells from non tumor cells. The cytologic features and distribution of RS/H cells should be matched with H;E-stained sections before interpreting EBER or LMP1 results.
• To call a case as positive, the EBER or LMP1 signal must be unequivocally localized to RS/H cells. The fraction of RS/H cells expressing EBER and LMP1 is different among cases, with most cases having a high fraction of positive tumor cells. For the purpose of identifying all Epstein-Barr virus–related Hodgkin cases, just one unequivocal RS/H cell expressing EBER or LMP1 should be considered sufficient to call a case positive. Equivocal cases frequently are resolved when EBER, LMP1, and H;E stains are assessed in parallel.
• The EBER signal is localized to the nucleus, sometimes sparing or rimming the nucleus. A negative EBER result can be interpreted as negative only if RNA is shown to be preserved
and available for hybridization in tumor cells. A varying fraction of background
lymphocytes express EBER (usually 0%-1%), and these must be differentiated from RS/H cells.
• The LMP1 signal is localized to the cytoplasm and surface membrane and it has a granular character. False-positive cytoplasmic staining can be seen in eosinophils and a fraction of
plasma cells. “Edge artifact” and other “background staining” may also contribute to false-positive signal in benign and malignant cells alike.
• As is the case with any histochemical stain, quality control measures should be used to ensure adequacy of staining, preservation of cytomorphologic features, and interpretive competency.
ASSOCIATION OF EBV WITH HD: INDIAN SCENARIO
AUTHOR SITE n(AGE) METHOD POSITIVITY
Radha et al.2 1997 Chennai 45(All ages) IHC 28%
Naresh et al.32000 Mumbai 110(All ages) IHC+ISH 78%
Karnik et al.4 2003 Vellore 100(All ages) IHC 82%
Rajalakshmi et al5,2006 Bangalore 40(All ages) IHC 55%
Veronique et al6, 2006 Delhi 145(Pediatrics) IHC+ISH 97%
Roshani et al7, 2017 Delhi 39(All ages) ISH 51%
Only a limited number of studies are available reflecting the Indian scenario of HD and EBV association. Majority of these studies employed immunohistochemistry for LMP1 to detect EBV infection, a few performed in situ hybridization for EBER. EBV positivity in Indian HD shows a wide range from 28-97%.
TABLE 2: EBV POSITIVITY IN INDIAN HD
A study from Chennai by Radha et al (1997) included 45 cases of HD in which EBV association was studied using LMP1 antigen expression in RS/H cells by IHC. They found EBV positivity in 31% cases. It was seen in 21.7% of mixed cellularity, 50% of nodular sclerosis and 100% of lymphocyte depletion subtypes, whereas lymphocyte predominance HD cases were negative for LMP1.
Another study from Mumbai by Naresh et al (2000) studied association of EBV with HD using LMP1 IHC and EBER-ISH in 110 cases. They included 52 cases of mixed cellularity, 41 cases of nodular sclerosis, 16 cases of lymphocyte rich HD and 1 case of lymphocyte depleted subtype.
The ages ranged from 4-61years with 50 (45%) patients being 14 years and 60(55%) patients being ;14years. 86 were male and 24 were female patients.
In their study cohort, 25(23%) cases belonged to stage I, 31(28%) to stage II, 38(35%) to stage III and 16(15%) to stage IV. In these cases EBER1 positivity was found in 86 out of 110 patients (78%) while LMP1 expression was seen in 56(51%) cases.
At Vellore, Karnik et al(2003) included 100 cases of HD and used LMP1 antigen to isolate EBV. They found LMP1 positivity in 82% of HD cases and most often in Nodular sclerosis subtype (86%). EBV LMP1 was found positive in 82% all HD and in 96% childhood cases.
In North India only two studies are available till date.
In a study from Delhi, Dinand et al (2006) exclusively involved with pediatric population. They included 145 cases of HD (less than 15 years) out of which 105 were Mixed cellularity, 33 Nodular sclerosis , 01 case each of Lymphocyte depleted, Lymphocyte rich HD and 02 cases of NLPHD. Three cases could not be subtyped.
They found EBV LMP1 expression in 131 out of 145 cases (90.3%). EBER 1;2 was carried out in 135 cases out of which 126(93.3%) showed positivity. With combined IHC and ISH, they detected EBV in 140 out of 145 cases (96.6%).
The latest study from Delhi by Gala et al (2017) employed ISH to detect EBV in 39 cases of HD of which 35 belonged to cHD and rest were NLPHD. EBER positivity was seen in 20 out of 39 (51%) cases of HD.
So far work on EBV association in HD in North Indian population has been limited. The present study includes untreated cases of HD of all age groups and intends to bring out the association by correlating with IHC for LMP1 antigen.
TABLE 3: STAGING OF HD: ANN ARBOR CLASSIFICATION
I Involvement of a single lymphatic site (i.e., nodal region, Waldeyer’s ring, thymus, or spleen) (I); or
localized involvement of a single extralymphatic organ or site in the absence of any lymph node involvement (IE).
II Involvement of two or more lymph node regions on the same side of the diaphragm (II); or
localized involvement of a single extralymphatic organ or site in association with regional lymph node involvement with or without involvement of other lymph node regions on the same side of the diaphragm (IIE).
III Involvement of lymph node regions on both sides of the diaphragm (III), which also may be accompanied by extralymphatic extension in association with adjacent lymph node involvement (IIIE) or by involvement of the spleen (IIIS) or both (IIIE,S).
IV Diffuse or disseminated involvement of one or more extralymphatic organs, with or without associated lymph node involvement; or
isolated extralymphatic organ involvement in the absence of adjacent regional lymph node involvement, but in conjunction with disease in distant site(s).
Stage IV includes any involvement of the liver or bone marrow, lungs (other than by direct extension from another site), or cerebrospinal fluid
DESIGNATIONS APPLICABLE TO ANY STAGE
A No symptoms.
B Fever (temperature >38ºC), drenching night sweats, unexplained loss of >10% of body weight within the preceding 6 months.
E Involvement of a single extranodal site that is contiguous or proximal to the known nodal site.
S Splenic involvement.
AIMS AND OBJECTIVES
PRIMARY OBJECTIVE: To study the expression of Epstein Barr Virus in Hodgkin Disease in North Indian population.
SECONDARY OBJECTIVE: To detect Epstein Barr Virus positivity in various subtypes of Hodgkin Disease and also in various demographic variables.
MATERIALS AND METHODS
The present study was conducted in Departments of Pathology, Surgery, Medicine, Paediatrics, and Otorhinolaryngology, Maulana Azad Medical College and associated hospitals, over a period of 1 and a half years from Feb 2017- Aug 2018
The study group included 26 Hodgkin disease cases for which lymph node excision biopsies were conducted in the Department of Surgery and Otorhinolaryngology, Maulana Azad Medical College and associated hospitals, New Delhi. An informed consent was obtained from all patients.
All newly diagnosed and untreated cases of Hodgkin disease.
All other hematolymphoid malignancies.
Detailed clinical history which includes history of unexplained weight loss, unexplained fever, chest pain, cough, shortness of breath, pruritis, pain at nodal site, bone pain, past history of prolonged illness and family history were taken. All relevant history of the swelling with respect to mode of onset, duration, progression, association with trauma or pain, whether accompanied by similar swelling elsewhere in the body or in the past were noted. History of secondary changes like softening, ulceration, fungation or inflammatory changes was also inquired.
General physical examination and a local examination was done in terms of size, shape, color, surface, number, consistency, overlying skin, local temperature and tenderness.
The tissue received as lymph node excision biopsy was grossed and routinely processed.
Formalin fixed paraffin embedded blocks were made after routine processing in automated tissue processor.
Four micron thick sections were cut from the paraffin block.
The slides were stained with hematoxylin and eosin and their morphology studied.
Tumor was characterized taking into account the histomorphology, lymph node architecture, presence of RS/H cells and accompanying inflammatory milieu of cells.
Immunohistochemistry(IHC) was put for LMP1, CD 15, CD30.
The buffers used were PBS with a pH of 7.0 for staining and citrate buffer with a pH of 6.0 for antigen retrieval.
The various IHC markers used were:
LMP1 (Dako, RTU))
CD15 (Skytek, RTU))
CD45 (Skytek, RTU)
CD3 (Novocastra, RTU)
CD20 (Skytek, RTU)
Immunopositivity was determined under light microscopy at 40X.
CD15 and CD30 were considered positive when cells showed membranous and cytoplasmic dot- like reactivity.
LMP1 was considered positive when even one unequivocal RS/H cell showed cytoplasmic and surface membrane reactivity with granular character and golgi zone accentuation.
HEMATOXYLIN AND EOSIN STAINING:
The sections were dewaxed in xylene and taken through graded alcohol and then brought to water.
The sections were stained with Harris Hematoxylin for 8 minutes.
They were then washed under running water until bluing occurred.
They were differentiated in 1% acid alcohol for 5 seconds and were washed under tap water again until the sections were blue.
They were then stained with Eosin Y for 2 minutes and then washed with distilled water.
They were dehydrated in graded alcohol solutions, passed through xylene which acted as a clearing agent.
Then finally the slides were mounted with DPX.
Cytoplasm: varying shades of pink.
IMMUNOHISTOCHEMISTRY STAINING TECHNIQUE:
Expression of all the immunomarkers was determined on four to five micron thick paraffin sections on poly L lysine coated slides. All steps were carried out in moist and humid container and care was taken to ensure that the sections remained moist throughout the procedure.
The staining area on the section slides was marked after comparing with parallel H&E stained section.
The sections were deparaffinised by putting them on a hot plate and by dipping them in xylene.
They were then hydrated with graded ethanol followed by being brought to water.
The sections were placed in 3% H2O2 in methanol (H2O2 block) for 30 minutes.
The slides were then put in a coplin jar filled with 10mM citrate buffer (pH 6) and heated in microwave for 10 cycles of 3 minute duration each accompanied by cooling in between 2 cycles which allowed unmasking of the epitope.
Three washes of PBS buffer were given without cooling at the end of 10th cycle.
The tissue was incubated with primary antibody overnight at 4 degree C.
Three washes with PBS buffer were given.
Secondary antibody (biotinylated goat antipolyvalent antibody) was added followed by incubation for 30 minutes.
Three washes with PBS buffer were given.
Tertiary antibody (peroxidase labelled streptavidin- peroxidase complex) was added followed by incubation for another 30 minutes.
Three washes of PBS were given.
Freshly prepared DAB was applied on the slides and the reaction monitored under the microscope.
The slides were immersed in distilled water as soon as a crisp brown cytoplasmic staining was seen in case of CD15 & CD30 and cytoplasmic-membranous staining was seen in LMP1.
The slides were counterstained with hematoxylin.
They were dehydrated in graded alcohol solutions.
They were then passed through xylene which acted as a clearing agent.
Then finally the slides were mounted with DPX.
Cytoplasmic & membranous dot like positivity with brown colour.
OBSERVATIONS AND RESULTS
The present study was conducted in Departments of Pathology, Surgery and Medicine, Maulana Azad Medical College and associated hospitals,
New Delhi. A total of 26 cases of Hodgkin disease diagnosed on lymph node excision biopsy were included in the present study. All the 26 cases in the present study were newly diagnosed cases, who had not received any therapy.
The age of the patient ranged from 5 to 55 years with a median age of 18 years. Age range for Mixed cellularity cases was 5 to 46 years with median age of 17 years. In the patients with Nodular sclerosis age range was 7 to 16 years with median age of 11.5 years. In Lymphocyte rich age of the patient was 14 years. In LDHD age ranged from 20 to 55 years with median age of 34years. (Figure 3).
TABLE 4: AGE AND SEX DISTRIBUTION OF THE STUDY POPULATION
MALE 6 8 1 4 0 1 20
FEMALE 1 4 0 0 1 0 6
M:F 6:1 2:1 – – – – 3.3:1
TOTAL 7 12 1 4 1 1 26
Out of 26 cases, 20 cases occurred in males and 6 in females with a male: female ratio of 3.3:1. (Figure 4).
CLINICAL PROFILE OF THE PATIENTS
The most common presenting complaint seen in patients was of unexplained fever & weight loss 50% (13/26 cases), followed by pain at nodal site 30.8%(8/26cases), cough & pruritis 11.5%(3/26 cases), shortness of breath 7.7%(2/26) & bone pain 3.8%(1/26cases) (Table 5).
TABLE 5. CLINICAL PROFILE OF THE PATIENTS
Clinical parameters No. of Cases
Unexplained fever 13 50
Unexplained weight loss 13 50
Nodal pain 8 30.8
Cough 3 11.5
Pruritis 3 11.5
Shortness of breath 2 7.7
Bone pain 1 3.8
On general physical examination pallor was found in 16 cases (64%), icterus was found in 02 cases (8%), clubbing was found in 01 case (4%). All cases had palpable lymphadenopathy.
In 02 cases (8%) hepatosplenomegaly was also noted. Bulky disease (size 7cm) was seen in 05 cases(19.2%)(Table 6).
TABLE 6. EXAMINATION FINDINGS
Finding Number of cases
Palpable lymphadenopathy 26 100
Pallor 16 64
Icterus 3 8
Clubbing 1 4
Hepatosplenomegaly 2 8
Bulky disease(7cm) 5 19.2
DURATION OF LYMPHADENOPATHY
Ten cases had lymphadenopathy for less than 6 months duration. In thirteen cases lymphadenopathy was present for 6-12 months. Three cases had lymphadenopathy for more than 1 year. Thus maximum number of patients(>50%) had lymphadenopathy for less than 1 year duration. No statistically significant correlation was found between duration of lymphadenopathy and age, clinical stage or diagnosis.(Figure 5.)
Clinical Staging was noted as per Ann Arbor classification for Hodgkin disease. Majority of the cases belonged to stage I- 17cases (65.4%), followed by stage II- 04cases (15.4%),stage III- 04cases (15.4%) ,stage IV- 1case (3.8%)
TABLE 7. ANN ARBOR CLASSIFICATION FOR HODGKIN DISEASE
STAGE NUMBER OF CASES
EARLY I 17 65.4
II 4 15.4
ADVANCED III 4 15.4
IV 1 3.8
Out of 26 cases of cHD, 20 were classified as Mixed cellularity, 02 Nodular sclerosis, 01 lymphocyte rich, and 03 lymphocyte depleted. (Figure 6)
TABLE 8:AGE WISE DISTRIBUTION OF VARIOUS SUBTYPES
Mixed cellularity 6 9 1 3 1 0
Nodular sclerosis 1 1 0 0 0 0
Lymphocyte rich 0 1 0 0 0 0
Lymphocyte depleted 0 1 0 1 0 1
Total(26) 07 12 01 04 01 01
TABLE 9: SEX DISTRIBUTION OF VARIOUS SUBTYPES
Mixed cellularity 14 06
Nodular sclerosis 02 0
Lymphocyte rich 01 0
Lymphocyte depleted 03 0
Total 20 06
TABLE 10: CORRELATION OF EBV STATUS WITH STAGE IN VARIOUS SUBTYPES OF HD
SUBTYPE MC, N=20
n(%) NS, N=2
n(%) LR, N=1
n(%) LD, N=3
EBV STATUS + – + – + – + – + –
STAGE I N=17 9(34.6) 4(15.4) 1(3.8) 0 1(3.8) 0 2(7.7) 0 13
N=4 1(3.8) 2(7.7) 1(3.8) 0 0 0 0 0 2
N=4 4(15.4) 0 0 0 0 0 0 0 4
N=1 0 0 0 0 0 0 0 1(3.8) 0
TOTAL 14(70) 6(30) 2(100) 0 1(100) 0 2(67) 1(33) 19 (73.1) 7
Type of specimen:
All the cases were diagnosed on histopathological examination of lymph node excision biopsy.
HD is a malignant disease of lymphoid tissue contributing <1% of all de novo neoplasms occurring worldwide every year. EBV has been postulated as a causative agent in the disease process. Upto 100% of HD in developing countries can be attributed to EBV though differences exist between rural and urban areas. LMP1 is a surrogate biomarker of EBV infection. LMPs alongwith other nuclear antigens and RNA are responsible for growth alteration and immortalization of infected B cells.
Thus the aim of the present study was to study the expression of LMP1 in various subtypes of HD.
This study comprised of 26 newly diagnosed untreated cases of HD presenting to the Surgery, Medicine departments of Maulana Azad Medical College and associated hospitals, Delhi. All the cases were diagnosed on lymph node excision biopsy.
In the present study the age of patients ranged from 5-55years with a median age of 18years however, we found a lower median age of presentation as compared to other Indian studies by Jain et al(32years), Goswami et al (34years) and Patkar et al(35years).
Majority of the cases occurred in males(76.92%),while females constituted 23.08% with M:F ratio of 3.3:1.
Mixed cellularity was the commonest subtype and occurred more commonly in males than females, cases of nodular sclerosis, lymphocyte rich and lymphocyte depleted too were male predominant. These findings are similar to the various studies which also show predominance in males except nodular sclerosis variant in which equal incidence is found in males and femaleswho 184,2759,3654,3695.
These can be explained by the social status of patients, since majority of the patients in the present study belonged to lower middle & lower economical strata and thus younger age group(<15years) wherein there is increased exposure to infectious agents early in childhood due to less hygenic environmental conditions, multiple playmates and siblings, overcrowded dwellings thus leading to increased risk of development of HD.zahramozaheb 19.
HD in western population shows a bimodal distribution with 1st peak in second to third decade and 2nd peak in the sixth decadepijc 6. No such distribution was seen in the present study or other Indian studies.ref
In the present study, B symptoms were recorded in 50% cases. Study by Goswami BK et al and Chandi et al reported frequency of B symptoms to be 79.17% and 73% respectively.Goswami,chandi7 However Dinand et al found B symptoms in 61.5% patients which is comparable to that seen in the present study.
Nodal pain was present in 30.8% patients, followed by cough, pruritis, shortness of breath and bone pains. However data from other studies correlating these parameters were not available.
In the current study, palpable lymphadenopathy was found to be consistently present in all patients, most common site being cervical nodes. Similar results were seen in study by Jain et al (92%). This may suggest a higher burden of disease at the time of diagnosis. Pallor was noted in 64% cases in the present study. Comparable results were reported by Dinand et al(61.9%) and Goswami BK et al(70.83%). >50% patients (88.5%)had lymphadenopathy for <1year, however no statistically significant correlation was found between duration of lymphadenopathy and age, clinical stage or diagnosis.
Bulky disease was seen in 19.2% cases which is lesser compared to other studies. Jain et al found bulky disease in 42% patients while Diehl et al found even higher incidence of bulky disease(58-68%)Jain, 16 However there was no significant correlation of bulky disease with the stage.
In the present study, mixed cellularity HD comprised 77%(20/26 cases) of the total cases. This was followed by lymphocyte depleted comprising of 11.5%(3/26 cases), nodular sclerosis 7.7%( 2/26 cases) and lymphocyte rich constituting 3.8%(1/26 cases).Goswami BK et al studied a total of 48 cases from North Eastern part of India and found mixed cellularity as the commonest subtype(45.83%) followed by nodular sclerosis (33.33%), lymphocyte depleted (4.17%) and lymphocyte rich (2.08%).ijhbtJain et al in their study from Western part of India studied a total of 125 patients and also found mixed cellularity as the most common subtype comprising 57%, followed by nodular sclerosis, lymphocyte predominant and NOS types in 32%, 2%, and 9%.jpgmDinand et al studied 145 cases from Northern India and found mixed cellularity HD in 72.4% cases, nodular sclerosis in 22.8% cases, NLPHD in 1.4% cases , lymphocyte rich and lymphocyte depleted classic HD in 0.7% cases.
While mixed cellularity remains the predominant subtype in Indian subcontinent, data from western countries have shown nodular sclerosis as the predominant subtype.Herling et al studied 577 cases of HD and found nodular sclerosis in 80.07% cases, followed by mixed cellularity in 19.41% cases and lymphocyte depleted in 0.52% cases.ccrStudy by Jarett et al included 461 cases of cHD and also found nodular sclerosis as the predominant subtype in 69.41%, mixed cellularity in 22.78%,lymphocyte rich in 3.47% and lymphocyte depleted in 0.65% cases. blood
Thus the findings of the present study are similar to the rest of Indian studies which have shown mixed cellularity as the predominant subtype of HD particularly one by Dinand et al, the difference from western data is quite striking where NS is the predominant subtype.
In the current study, 80.8% patients belonged to early stage, of which stage I were 65.4% and stage II were 15.4% whereas 19.2% patients belonged to advanced stage with 15.4% in stage III and 3.8% in stage IV.
IMMUNOHISTOCHEMICAL EXPRESSION OF EBV IN HD
EBV association with HD shows geographical variation. Available data suggests an association of EBV to be 70-100% in developing countries and 30-50% in developed countries.naresh5,6,10,21-28
In the present study 73.1%cases were found to be positive for EBV by LMP1 antigen detection. Similar results were obtained by Naresh et al. They studied 110 cases of cHD and found EBV positivity in 78% cases by EBER-in situ hybridization while with LMP1 a positivity of 51%. Karnik et al in their study involving 100 cases of HD found LMP1 positivity in 82% cases. Another study by Gala et al found EBV positivity of 51% however they employed EBER-ish as a method to detect infection. All three above studies were conducted in adult HD cases. In the paediatric age group Dinand et al have found EBV positivity in as high as 90.3% cases. India being a developing country therefore proves to be carrying a very high association of EBV with HD.
Data from western countries has shown a lesser association with EBV. Study by Jarrett et including 461 HD cases found EBV positivity in only 33.41% cases.
Glaser et al while studying diverse racial/ethnic group of 1032 patients found overall EBV positivity of 23% in whites, 28% in blacks, 46% in Hispanics and 40% in Asian/Pacific Islander patients. Study by Herling et al reported EBV positivity by LMP1 in 21% cases of HD. Similarly Cickusic et al also found EBV positivity in only 38.3 % of the patients studied.
CORRELATION OF EBV POSITIVITY WITH VARIOUS SUBTYPES OF HD
Among the various subtypes of HD in the current study, strongest association of EBV is observed in nodular sclerosis and lymphocyte rich HD, with positivity reaching upto 100%.
Mixed cellularity HD:
In the present study, 14 out of 20 (70%) mixed cellularity cases were positive for EBV LMP1. Naresh et al found EBV positivity in 86% mixed cellularity cases, whereas Karnik et al have reported 79% positive cases. Even higher positivity of EBV in mixed cellularity was seen in the study by Dinand et al (97.1%).
Among the western studies, one by Herling et al found EBV positive mixed cellularity HD in 39% patients only. Cickusic et al have reported EBV association in 25.6% HD patients whereas Jarrett et al reported it in 22.8% cases. Glaser et al found EBV positive mixed cellularity HD in 59.3% whites,42.1% blacks, 74.5% Hispanics and 80% Asian/Pacific islanders. From the above results it can be seen that EBV is more frequently associated with mixed cellularity in the developing countries than the developed.
Nodular sclerosis HD:
In the current study, nodular sclerosis was found to be EBV associated in 100% cases. Naresh et al found EBV association in 67% cases while Karnik et al found it in 86% cases. Dinand et al reported EBV in 97% nodular sclerosis HD cases.
Study by Herling et al reported 17% positivity of EBV in NS subtype, while Jarrett et al and Cickusic et al have reported 69.4% and 66.7% respectively.
Lymphocyte rich HD:
In the present study we came across 100% positivity in lymphocyte rich subtype aswell. Dinand et al and Naresh et al also found a high positivity of 100% and 81% in this subtype respectively.
In the western scenario, EBV association was found to be low. Study by Jarrett et al have reported a positivity of only 3.5%.
Lymphocyte depleted HD:
EBV association with LDHD in the present study was found to be 67%. Karnik et al reported it to be 75% whereas Dinand et al found 100% positivity in this subtype.
Jarrett et al in their study found only 0.65% EBV positivity in LDHD whereas Cickusic et al and Herling et al have found it to be 5.1% and 67% respectively.
CORRELATION OF EBV WITH STAGE OF DISEASE
Stage of the disease is an independent prognostic factor in HD.
EBV positivity was found maximally in patients with stage III (100%) of disease, followed by stage I (76.5%), stage II(50%).A higher overall EBV positivity was observed in advanced stage disease. We could not find Indian studies correlating EBV status and stage of disease.
In the study by Jarrett et al, EBV association in stage I,II,III and IV was found to be 30.3%, 26.2%, 23.4% and 20% respectively while Herling et al reported EBV positivity as 40%, 13%, 28% and 27% in the respective stages.
Majority of the patients in the present study belonged to early stage disease (stage I&II) which showed a lesser EBV positivity(71.4%) as compared to the advanced stage(stage III & IV) which showed a positivity of 80%. The present study highlights that HD in Indian context is predominantly a disease of young males, with mixed cellularity as the commonest subtype, highly associated with EBV in advanced stage.
The present study included 26 cases of HD. All 26 cases in the study were newly diagnosed and had not received any form of treatment.
They were all clinically diagnosed, staged according to the Ann arbor classification and diagnosis was confirmed on lymph node excision biopsy.
Patients were of younger age with age range of 5-55years with a median age of 18 years.
A loss of bimodal pattern of distribution was observed.
Majority of the cases occurred in males(76.92%) with a M:F ratio of 3.3:1.
Palpable lymphadenopathy was found in 100% patients, followed by pallor in 64% cases, B symptoms in 50% cases and nodal pain in 30.8%.
Bulky disease was noted in 19.2% cases.
More than half of the patients had lymphadenopathy for less than an year(88.5%).
Most of the patients presented with stage I(65.4%), followed by stage II&III (15.4%) and only 3.8% with stage IV suggesting a lower stage of presentation among Indian patients than western patients.
Mixed cellularity HD comprised 77%( 20cases), followed by lymphocyte depleted subtype 11.5%(3cases), nodular sclerosis 7.7%(2cases) and lymphocyte rich 3.8%(1case).
The median age of presentation was 17years in mixed cellularity, 11.5 years in nodular sclerosis, 14years in lymphocyte rich and 34 years in lymphocyte depleted subtype.
Male predominance was seen in mixed cellularity, lymphocyte rich and lymphocyte depleted while nodular sclerosis had equal incidence among males and females.
EBV by LMP1 was detected in total 19 out of 26 cases(73.1%).
Maximum association of EBV was seen to be in nodular sclerosis(2 out of 2 cases) and lymphocyte rich HD(1 out of 1 case) subtype constituting 100%. Mixed cellularity showed an association with EBV in 70% cases(14 out of 20).
EBV was significantly positive in patients with advanced stage disease(80%).
Mixed cellularity cases showed more EBV positivity in advanced stage disease(100%), while other subtypes showed a higher EBV association with early stage disease.
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