1. Molecular detection of Epstein- Barr virus (EBV) in patients with Non-Hodgkin Lymphoma attending in Haematology Units of Tertiary care Hospitals in Yangon Dr. Moh Moh Htun Director (Research) DMR Moh Moh Htun1, Tun Lwin Nyein2, Sein Win2, Aye Aye Gyi2, Mya Ohnmar1, Theingi Thet Paing Htun1, Hnin Nu Htwe1, Zar Zar Aung1 and Khine Moe Aung1

2. Introduction
Global cancer rates could further increase by 50% to 15 million new cases in the year 2020, according to the World Cancer Report from the World Health Organization in 2003 (WHO-2003)
Non-communicable diseases (NCDs) are the leading causes of death in the world
Nearly 80% of NCD deaths occur in low- and middle-income countries
It comprises of mainly cardiovascular diseases, cancers, diabetes and chronic lung diseases

3. Prevalence of Non Hodgkin Lymphoma (NHL) in the World
Is a common haematological malignancy in the world. It is increasing in incidence globally
Incidence of NHL is 5/ 100,000 populations and mortality rate is 2.5/ 100,000 populations in the world
NHL is 10th most common cancer and 2.7 % of all cancers in the world (2012)
NHL occurs in any age (children, teens and young adults)
EB viral- DNA is detected in 40 % of Non-Hodgkin lymphoma patients (Haruko T & Brenner MK, 2017)

4. Prevalence of NHL in Myanmar
Incidence of Lymphoma is rising gradually & 7th most common cancer in YGH (2014)
Non Hodgkin Lymphoma (NHL)-> 607 cases out of total cases of malignant cases (3.9%)in Yangon General Hospital according to Yangon Cancer Registry within seven years.
108 NHL cases out of 3586 total cancer cases (3%) in 2016
It was the sixth commonest cancer in that registry for both genders but the female are more common than male (1:1.4)

5. Classification of NHL
Working Formulation National Cancer Institute‘s (IWF) Low, Intermediate & High grade
Revised European –American Classification of Lymphoid Neoplasms (REAL) B& T cell origin
World Health Organization (WHO) Classification 2001, Precursor T/B /NK cells and Mature T/B /NK cells

6. The common types of NHL B- cell type T- cell type
Diffuse large B-cell lymphoma (DLBCL) Most common type (>80%)
Precursor T-lymphoblastic lymphoma
Follicular Lymphoma
Peripheral-T cell Lymphoma
CLL/Small lymphocytic Lymphoma (SLL)
Mantle cell Lymphoma
Extranodal T/Nkcell lymphoma
Adult T cell lymphoma
Marginal zone Lymphoma
Burkitt like Lymphoma
Lymphoplasmacytic & CNS Lymphoma
Anaplastic large cell Lymphoma
Aggressive NK cell lymphoma

7. The common types of NHL (cont)
B-cell Lymphomas are commonly occurred around the world
T-cell lymphomas are proportionally more common in Asia than in Western countries
The Incidence of T-cell lymphomas are 28.1% in China, 16.2% in India and 32-38% in Japan
Diffuse Large B Cell Lymphoma (DLBCL) is the most common type of NHL approximately one third of total cases of NHL in the world

8. Common NHL types in Asia
Extra nodal NHL %,
T-cell Lymphoma (PTCL) (unspecified) 22.4%
Natural Killer T-cell Lymphoma (NKTCL) 22.4%
Angioimmunoblastic T cell Lymphoma (AITL) 17.9%
ATLL - 25%
They have association with EBV and HTLV-1 infections in Asian areas

9. Major etiologic factors related to NHL
Genetic predisposition-cMYC or BCL2 mutation
Translocation- t(14,18)(q32;q21)
Viral and Bacterial infections (HIV, HTLV, EBV, HCV & H.pylori)
Exposure to chemicals- weed & insect killers
Environment – Ionizing & UV Radiation, dyes

10. The common clinical symptoms of NHL
Painless enlarged lymph nodes, abdominal pain, chest pain, cough and trouble breathing, fatigue, fever , weight loss and night sweating

11. Epstein Barr Virus (Oncogenic virus ) (Robaina TF, 2008)
is a member of the Herpesviridae family and Gammaherpesvirinae subfamily
It can mainly infect in lymphocytes and epithelial cells
Infectious mononucleosis and oral hairy leukoplakia (OHL) are caused by EBV
There is two genotypes -> EBV-1 and EBV-2 distinguished by divergent gene
sequences.
(Robaina TF, 2008)

12. Aims of the research
The study was carried out to detect the major subtypes of EBV in histologically confirmed Non- Hodgkin Lymphoma (NHL) patients who attended in Haematology Units of Tertiary care Hospitals in Yangon

13. Objectives of the research
To describe the clinical, social demographic data and histological types of NHL
To detect EBV subtypes in serum of patients with histologically confirmed Non-Hodgkin Lymphoma
To correlate the histological types and common subtypes of EBV in these cases.

14. Study design & populations
A cross sectional descriptive study involved 83 cases (47 cases 57% were male and 36 cases 43% were female) of Non Hodgkin Lymphoma (NHL) attending in Haematological Units, Yangon General Hospital and North Okkalapa General and Teaching Hospital, Yangon during August 2017 to September 2018

15. Study design & populations (cont)
After approval by the Ethics Review Committee of Department of Medical Research (2017) , informed consent was obtained from the subjects who participated in this study
All clinical data including fever, site of lymph node enlargement, cycles of chemo therapy and laboratory data (histology report) were collected by using proforma)

16. Materials and methods
The three milliliter (ml) of whole blood samples from all subjects were collected under aseptic condition.
All serum samples were kept in a -20ºC freezer for DNA extraction after separation of serum by centrifugation at 3000 rpm.
Detection of EBV DNA was done by polymerase chain reaction (PCR) with two sets of specific primers for EB NA-1 and EBNA-2.

17. Materials and methods (cont)
EBNA-1
Forward Primer - TGAATACCACCAAGAGGTG
((262 bp)262 )
Backward Primer- AGTTCCTTCGTCGGTAGTC
 EBNA-2
Forward Primer- TGGAAACCCGTCACTCTC
(300bp)
Backward Primer - TAATGGCATAGGTGGAATG

18. Detection of common genotypes of EBV
DNA from each sample will be carried out by using Qiagen DNA extraction kit and amplified by using EBV-1 and EBV-2 primer sets.
DNA substrate will amplify by using two sets of primers, dNTPs, magnesium sulphate, Taq polymerase and 10 X Buffer under 35 cycle of thermocycler according to specific temparture for denaturation, annealing and extension.

19. Results
In this study, 83 cases of histologically confirmed NHL were classified according to Revised European- American Lymphoma (REAL) clinical grouping of currently recognized NHL.
(1) Indolent chronic lymphomas
(2) Aggressive lymphoma
(3) Highly aggressive (acute lymphoma/ leukemia).

20. Age and sex distributions of NonHodgkin Lymphoma (NHL)
Age group (Year)
Male
Female
Total cases %
<20 7 1 8
9.6
21-40 12 2 14
16.9
41-60 18 36
43.3
61-80 10 24
29.0
>80 -
1.2
Total 47 83
100

21. Age and sex relation of NHL

22. Demographic data Age Mean age in years 50.15±17 Sex Male Female
47 (56.6%)
36 (43.4%)

23. Clinical data Background information Number of patients n (%)
Clinical symptoms
Fever
Yes No
Enlarged lymph nodes
Metastasis
Other symptoms (weight loss, cough & deafness)
HIV infection
Reactive
Non-reactive
24 (28.9%)
59 (71.1%)
30 (36.1%)
53 (63.9%)
22 (26.5%)
61 (73.5%)
0 (0%)
83 (100%)

24. Chemotherapy
Yes No
35 (42%)
48 (58%)
Clinical Units
YGH
NOGH
43 (51.8%)
40 (48.2%)
Residence area
Yangon
Bago
Ayeyawaddy
Magway
Mandalay
Sagine
Other regions
38 (46%)
12 (14.5%)
10 (12%)
9 (11%)
4 (5%)
6 (6.5%)

25. Proportion of EBV DNA in different histological types and grades of NHL
Total cases
Total %
EBV-DNA
Positive
EBV –DNA
Negative
Grade(I) Indolent chronic Lymphoma
Chronic indolent lymphoma
-Small lymphocytic (10)
-Follicular (14)
-Extranodal (10) 34 41
7 (20.6%)
27 (79.4%)
Grade(II) Aggressive Lymphoma
-DLBCL (34)
-Anaplastic (14) 48
57.8
8 (16.7%)
40 (83.3%)
Grade(III) Highly aggressive Lymphoma/leukemia
- Precursor B lymphoblastic lymphoma (1) 1
1.2
1 (100%)
0 (0%) 83
100 16 67

26. Detection of EBV-1 Type by PCR
EBV-DNA band at 262 bp P

27. Detection of EBV in NHL patients
Out of 83 NHL patients, (16) patients were Epstein –Barr virus (EBV) DNA positive (19.3%) and 67 patients (80.7%) were EBV- DNA negative by amplification of polymerase chain reaction (PCR) with specific primer set of EBNA-1 gene at 262 bp.
EBVNA- 2 was not detected in all NHL cases by PCR method by using specific EBVNA-2 primer. EBV-NA type 1 positive was found in 7/34 cases of Grade (I) NHL, 8/83 cases of Grade (II) NHL and 1/83 cases of Grade (III), respectively.

28. Discussion
The EB virus is one of the Herpesviridae families that maintain a life -long persistent infection in over 90% of adult healthy population with low copy number in memory B cells.
EBV Nuclear Antigen1 (EBV- NA1) is the only viral protein that expressed in all forms of latent stage in EBV related cancers.
EBV genotype 1 is most prevalent in all types of lymphoma in Pakistan study
The most common form of lymphoproliferative disorders related to EBV are B cell lymphomas; Hodgkin lymphoma (HL), NHL including Burkitt lymphoma and DLBCL.

29. Discussion
In this study, the most of the NHL patients participated in this research study were living in Yangon region (46%) and other regions such as Bago, Ayeyarwaddy and Magway.
All NHL cases are non-reactive for Human Immunodeficiency virus (HIV) infection and less clinical symptoms such as fever, cough, lymph node enlargement and metastatic signs and symptoms.
Male and female population in this study (1.3:1) but female are higher prevalence in common age group of 50- >60 years

30. Discussion
The most common histological type of NHL was Aggressive Lymphoma (Grade II) especially in Diffuse Large B cell lymphoma (DLBCL) 34/83 cases (41%).
The lowest histology type of NHL was one case/83 of Precursor B lymphoblastic lymphoma in Grade (III) Highly aggressive Lymphoma/leukemia.
DLBCL is the most common type of Aggressive NHL in adults and up to 40% of prevalence rate in the world.

31. Discussion EBV type 1 DNA positive (16) cases-
7/16 cases (20.6%) of Grade (I) Indolent chronic Lymphoma (NHL),
8/16 cases (16.7%) in Aggressive Grade (II) NHL
1/1 (100%) in Highly aggressive grade of NHL.

32. Discussion
EBV serotype 1 is more prevalent in Western countries and Asian countries including Pakistan.
All EBV positive samples of NHL are EBV serotype 1 and it will be carried out to genetic sequence for continuation of study.
There were not significant correlation between histological types and grades of NHL and EBV positive infection in this study. (p= 0.109)
EBV –DNA was detected in low grade and intermediate grade of NHL in this study.

33. Conclusion & Dissemination of message
EBV DNA may be one of indicators for early diagnosis of lymphoma and prognostic markers for monitoring of treatment response by radiation and chemotherapy.
The research findings related to EBV in lymphoproliferative disorders may be provided the application of EBV targeted therapy in miRNA (micro RNA) pathway especially for NHL.

34. Recommendation
Currently in the world, urgent to develop a safe and effective EB vaccine to prevent many diseases liked nasopharyngeal carcinoma, Burkitt's lymphoma, Hodgkin's lymphoma, breast cancer and stomach cancer
Molecular epigenetics research of EBV in common cancers of our country, is required for prevention and control of NCD including cancer

35. References
World Health Organization (WHO, 2003): The major findings of the World Cancer Report. Available at
Accessed date on 22nd Oct 2009
World Health Organization (WHO, 2010)Global status report on communicable diseases Available at Accessed date on 2nd August 2017 
Zahra M (2012) Epidemiology of Lymphoid Malignancy in Asia; Epidemiology Insights: edited by Dr. Maria De Lourdes Ribeiro de Souza Da Cunha. Pp , IBSN Available at Accessed date on 23rd February 2017
Soe Aung (2014) Cancer registry data, Yangon General Hospital. Detailed data for all patients both OPD and In –patients, Department of Radiotherapy and Medical Oncology; p

36. References
Hjalgrim H, Friborg J and Melbye M (2007) The epidemiology of EBV and its association with malignant disease. Available at NBK47424/ Accessed on 8th March 2017
Lingyun G and Xin W. (2007) Epstein- Barr Virus associated Lymphoproliferative disorders: experimental and and clinical development; Human Herpes virus: Biology, Therapy and Immunoprophylaxis , edited y Arvin A, Cambridge University press; 2007.
Haruko T and Brenner M K. (2017) Immunotherapy against cancer-related viruses. Springer Nature (Cell Research) (2017) 27:59-73.Available at Accessed on 20th June 2017
Cai Q, Chen K and Young K H (2015) Epstei –Barr virus –positive T/NK =cell lymphoproliferative disorders; Experimental & Molecular Medicine (2015) ,e133;doi: /emm Available at http;// html. Accessed on 8th march 2017

37. Acknowledgement
The researchers like to express our sincere thanks Director General and Board of Directors, ERC of Department of Medical Research for encouragement and allowing us to conduct the study. We would also thanks to medical doctors and nurses of Haematology Units of Yangon General Hospital (YGH) and North Okkalapa General and Teaching Hospital (NOGTH), Yangon. We extend our deepest thanks to participants who took part in this study.

38. Thank You !