|Year : 2011 | Volume
| Issue : 4 | Page : 720-724
|The association of Epstein-Barr Virus infection with multiple myeloma
Mohammad Hadi Sadeghian1, Hossein Ayatollahi1, Mohammad Reza Keramati2, Bahram Memar3, Saeed Amel Jamedar4, Maryam Mohammadnia Avval1, Maryam Sheikhi1, Gohar Shaghayegh1
1 Department of Hematology and Blood Bank, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2 Neonatal Research Center, Imam Reza Hospital, MUMS, Mashhad, Iran
3 Department of Pathology, Mashhad University of Medical Sciences, Mashhad, Iran
4 Department of Microbiology, Mashhad University of Medical Sciences, Mashhad, Iran
Click here for correspondence address and email
|Date of Web Publication||6-Jan-2012|
| Abstract|| |
Background: Multiple myeloma is a conventional term for clonal proliferation of plasma cells. It is suggested that viruses may have a significant role in the pathogenesis of multiple myeloma. Aims: The aim of this study was to evaluate whether there is an association between multiple myeloma and Epstein-Barr Virus (EBV) by the polymerase chain reaction (PCR) method. Materials and Methods: This case-control study was performed on 60 paraffin-embedded bone marrow biopsies (30 multiple myeloma and 30 normal bone marrow specimens) in the molecular pathology section of our hospital. The patients and control groups were matched according to gender and age. Several sections were cut from each paraffin blocks, and then the deoxyribonucleic acid (DNA) was extracted by the non-heating extraction method. In the next step, PCR was carried out for detection of EBV genome and finally its products were analyzed by electrophoresis. Results: DNA of EBV was detected in 10 patients of the case group (5 males and 5 females) and 3 subjects (2 males and 1 female) of the control group. The Pearson chi-square test showed significant difference between case and control group (P=0.03) for detection of the EBV genome. In myeloma patients, the mean white blood cell count was 9.05 + 4.02 and 5.20 + 2.02 Χ 10 9 /L in EBV positive and negative groups, respectively and a significant difference count was seen for the WBC count. Recommendations: Our results show an association between multiple myeloma and EBV infection.
Keywords: Epstein-Barr Virus , multiple myeloma, polymerase chain reaction
|How to cite this article:|
Sadeghian MH, Ayatollahi H, Keramati MR, Memar B, Jamedar SA, Avval MM, Sheikhi M, Shaghayegh G. The association of Epstein-Barr Virus infection with multiple myeloma. Indian J Pathol Microbiol 2011;54:720-4
|How to cite this URL:|
Sadeghian MH, Ayatollahi H, Keramati MR, Memar B, Jamedar SA, Avval MM, Sheikhi M, Shaghayegh G. The association of Epstein-Barr Virus infection with multiple myeloma. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Apr 24];54:720-4. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/720/91504
| Introduction|| |
Multiple myeloma (MM) is a malignant disorder of plasma cells, primarily occurring in the bone marrow. This neoplastic lesion is diagnosed based on pathology, radiologic, and clinical findings, including anemia, hypercalcemia, renal insufficiency, high serum immunoglobulin, and radiologic evidence of lytic bone lesions. The term of "plasma cell myeloma" is entered in the 2008 WHO (World Health Organization) classification of malignant B-cell lymphoma instead of MM.  Near to 1% of all neoplasm and 10% of hematologic malignancies in the United States are Multiple myeloma.  MM also accounts for about 20% of the deaths caused by hematologic neoplasms. 
There are some data suggesting the possible potential role of several infectious agents in the pathogenesis of MM. Montella et al.  implicated the association of hepatitis C virus infection with MM and Elira Dokekias et al.  reported plasma cell myeloma in human immunodeficiency virus (HIV)-infected patients. Although some authors have found a relationship between human herpes virus 8 (HHV-8), a member of the herpes virus family and MM ,, but this finding has not been confirmed by others. ,,
Epstein-Barr Virus (EBV) is another member of the herpesvirus family and causes infectious mononucleosis. It is well documented that this virus is associated with a number of malignancies such as nasopharyngeal carcinoma, a subset of Hodgkin lymphoma, the African (endemic) form of Burkitt lymphoma, B-cell lymphomas in immunosuppressed individuals and rare forms of T-cell lymphomas.  The role of EBV in pathogenesis of some form of lymphoprolifrative disorders is so important that even the name of this virus has been entered in classification of lymphoma and some EBV-related neoplasms (e.g., EBV+ diffuse large B-cell lymphoma of the elderly, pediatric follicular lymphoma, and EBV+ T-cell lymphoproliferative diseases of childhood) are highlighted in the new (2008) WHO classification.  There are few reports about detection of EBV genome in MM patients ,,, but this finding has not been confirmed by others. ,,
The aim of this study was to investigate whether there are differences between two groups of patients with MM and the control group for the presence of EBV deoxyribonucleic acid (DNA) in bone marrow tissue by the polymerase chain reaction (PCR) method.
| Material and Methods|| |
This case-control study financially was supported and ethically approved by the research vice chancellor of Mashhad University of Medical Sciences.
It was performed on 60 formalin-fixed paraffin embedded (FFPE) bone marrow biopsies in molecular pathology laboratory, Ghaem teaching Hospital, Mashhad, Iran.
Our materials included 30 FFPE of MM patients who had positive clinical and radiological finding of MM and also had more than 30% plasma cells in the bone marrow (patient group), and 30 FFPE of normal subjects had not increase plasma cells (control group). Control specimens were obtained from patients with lymphoma that had normal bone marrow morphology and they were matched with patients group for age and sex. The bone marrow biopsy in patients with lymphoma had been performed for determination of staging. Archived slides of two groups were reviewed by two pathologists for confirmation of diagnosis and tissue adequacy for extracting of DNA. We also reviewed medical history and previous laboratory reports of patients. We did not find any history of immunodeficiency diseases, transplantation, or immunosuppressive therapy in our patients.
Five to seven 20-μm-thick sections were cut from each FFPE specimens under sterile conditions, and then the DNA was extracted by using proteinase K and the non-heating DNA extraction method  DNA concentration was determined by using the Thermo Scientific NanoDrop 2000 Spectrophotometer and specimens with low DNA content (<20 ng/μL) were excluded from the study. PCR for EBV genome detection was performed by using the PCR Kits (DNA Technology JSC, PCR Kit, Mosscow, Russia).
Epstein-Barr Virus Detection
The appropriate numbers of paraffin sealed tubes were marked as the positive and negative controls. Then PCR Master Mix was prepared for numbers of reactions. 10 μl of PCR Master Mix and 0.5 μl unit of Taq polymerase (Hot Start Taq DNA Polymerase) were added into each paraffin-sealed tube and were mixed. 5 μl (100 ng) of DNA samples were added (except for positive and negative controls) and were spun at 1000 rpm for 3-5 s. The tubes were placed into the Applied Biosystems (ABI) Veriti Thermal Cycler and PCR was performed on a PCR Cycler with the program of 180 s at 94°C for the first step and then 45 cycles was run as follows: 50 s at 94°C, 50 s at 64°C, and 50 s at 72°C.
Amplified PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light by gel documentation instrument. PCR Kits with internal control (IC: 540 bp) would have positive results if we had: 1-DNA band corresponds to the band of the positive control (185-bp) or, 2-there are two DNA bands, one of which corresponds to the DNA band of the positive control and the second band corresponds to the DNA of the internal DNA band [Figure 1].
|Figure 1: Electrophoresis of amplified PCR products for EBV DNA Line 6: size marker, Line 1,3,4,9, and 10: patients with negative results of EBV, Line 2,5,7, and 8: patients with positive results of EBV, Line 12: negative control, Line 2,5,7, and 8: positive control|
Click here to view
Results of PCR in two groups of patients and normal were analyzed with SPSS (Statistical software for social analysis-version 11.5) by a specialist of statistics. Case and control groups were compared with the Pearson chi-square test and t-test for categorical variables (e.g. PCR results) and continuous variable (e.g. age) respectively. A P-value under 0.05 (P < 0.05) was considered significant.
| Results|| |
The age range in the control group was 40-80 years with 63.4 ± 10.70 years for mean ± standard deviation (SD). In the patients group (case group), it was from 40 to 80 years with 63.6 ± 9. 60 years for mean ± SD, and the t test showed no significant differences between the case and control group (P = 0.95). In case group, 14 patients (46.7%) and 16 patients (53.3%) were male and female respectively and in the control group 16 subjects (46.7%) were males and 14 normal individuals (46.7%) were females. No significant difference was found between case and control groups for gender (P = 0.61) by the Pearson chi-square test.
Positive PCR results for DNA detection of EBV were seen in 10 (33.3%) samples of the patient group and 3 (10.0%) samples of the 30 normal bone marrow tissues. The Pearson chi-square test showed a significant difference (P=0.03) for detection of EBV DNA between samples of MM and control groups [Table 1].
|Table 1: Summary results for two groups of case (EBV positive) and control (EBV negative)|
Click here to view
The mean age in MM patients with positive and negative genome of EBV was 58.20 ± 10.69 and 66.30 ± 7.96 respectively and t-test showed a significant difference for age distribution (P=0.03). In patients group, EBV was detected in 5 males and 5 females and we did not find a significant difference between two groups of EBV positive and negative for sex distribution (P=0.79). In myeloma patients, the mean white blood cell (WBC) count was 9.05 ± 4.02 and 5.20 ± 2.02 × 10 9 /L in EBV positive and negative groups, respectively. This difference was statistically significant; however, we did not find any significant differences in other laboratory results including serum calcium, erythrocyte sedimentation rate (ESR), complete blood count (CBC) index, between case and control groups [Table 2].
|Table 2: Comparison laboratory result in Epstein-Barr Virus positive patients with MM and normal subjects|
Click here to view
| Discussion|| |
MM is a relatively common disorder that described at first by an English physician in 1844.  The reported incidence in UK is 7.8 per 100 000 per year in adults  and the American Cancer Society estimated that more than 20,000 new cases of MM have been diagnosed during 2009 in the United State.  In Iran, hematopoietic malignancies are 4.36% and 5.52% of all cancers in females and males respectively and near to 10% of hematopoietic malignancies are plasma cell neoplasms. , Unevenly geographic distribution of MM has been documented and this disease is more frequent in some regions of Europe and North America.  This different distribution may be due to genetic and/or environmental factors (e.g. endemic infection).
The EBV was discovered by Tony Epstein and his student, Yvonne Barr in 1964, when they worked on samples derived from one type of endemic disease of African children.  It is well known, this B-lymphotropic virus that also called human herpesvirus 4 (HHV-4) can be associated with a variety of human tumors.  Patients with primary and secondary immunodeficiencies are more susceptible to develop EBV-related lymphoma than immunocompetent individual. , Concomitant EBV infection and MM are seen in immunocompromised patients. Ancy΄n et al.  and Cheng et al.  demonstrated EBV infection in patients with MM after renal transplantation and Voelkerding et al.  reported a plasma cell tumor with the presence of EBV DNA in tumor tissue but not in nontumor tissue in a patient with a history of acquired immune deficiency syndrome.
Our study showed an association between EBV and MM patients without any obvious history of immunodeficiency or transplantation. Our finding was concordant with the Min et al.  study. In their study, EBV-DNA was evaluated by the quantitative PCR method and positive results, with a significant difference (P=0.01), were observed in 56% of MM patients and 6.2% of the control groups, respectively.
Frequency of EBV in patients with MM is different. Hermouet et al.  detected the EBV genome in 36.4% of MM samples by real-time quantitative PCR in Portugal and Chang et al.  reported immunoreactivity for EBV in 7% of patients with plasmacytoma and MM by the immunohistochemistry method in Taiwan.
In contrast to our results, some other investigators have not observed any association between EBV and MM. Csire et al.  evaluated the presence of EBV in patients with MM using both serologic and nucleic acid amplification techniques and compared their results with the control group. They detected this virus in 36 (52%) of 69 MM patients and 23 (52%) of 44 normal subjects as a control group by the PCR method and their results did not show any significant difference between them.  Similarly, a study by Schönrich et al.  revealed no significant difference between control and MM patients for EBV antibodies (90%/91% EBV IgG1). In addition, Vega et al.  found a significant difference in EBV DNA between plasmablastic lymphoma and plasma cell myeloma so that EBV was positive in all patients with plasmablastic lymphoma but not in any cases of plasma cell myelomas.
In EBV-infected subjects, several mechanisms are involved in tumor genesis. The immune system involvement seems to be an important step in EBV-related oncogenesis. EBV infects humans' cells including B lymphocytes by using the complement receptor 2 (CR2). The infection of B lymphocytes is latent. Latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) are two important EBV genes that act as an oncogene, especially in immunosuppressed patients. LMP-1 prevents apoptosis by activating BCL2, and induces the expression of angiogenesis factors (e.g. VEGF, FGF-2, and COX2).  Furthermore, LMP-1 stimulates cell proliferation through induction of cytokines such as interleukin-6 (IL-6).  It is well known that IL-6 is a plasma cell growth factor and a powerful cytokine for proliferation of the myeloma cells.  Additionally, it may be presumed that the increase of IL-6 during EBV infection may trigger plasma cell proliferation and myeloma cells. 
Geographic distribution is an important factor that must be considered when making decisions about association of EBV and plasma cell myelomas. Our study showed that 10.0% normal subjects were positive for the EBV genome in bone marrow tissue by the PCR method. Although we did not find any document about the prevalence of EBV antibodies in the general population of Northeast Iran, but the frequency of this infection among children was reported around 81.3% by using the ELISA (VCA-IgG) method in southeast Iran.  In a similar study, Katebi et al.  reported high frequency of EBV (>90%) in patients with Hodgkin lymphoma by the immunohistochemistry method in Northeast Iran.
Our study was done on 30 patients with MM. This sample size may be too small for final decision; therefore, future study with a larger number of case groups is helpful to decrease the probable sampling error. Furthermore, complementary studies by using in situ hybridization or immunohistochemistry methods are needed to detect direct evidence of EBV in bone marrow tissue.
| Conclusion|| |
We find a statistically significant relationship between multiple myeloma and detection of EBV DNA in bone marrow tissues by the PCR method but because of the high prevalence of EBV infection in the control group, a similar study with greater number of patients can be helpful for the final decision.
| Acknowledgement|| |
This study was the result of a MD thesis and financially supported by the research vice chancellor of Mashhad University of Medical Sciences. The authors would like to thank Dr. Mohammad Khajeh Daluei for his invaluable recommendations and data analysis.
| References|| |
|1.||Bain BJ, Clark DM, Wilkins BS. Bone Marrow Pathology. 4 th ed. Hoboken, New Jersey, USA: Wiley-Blackwell; 2010. p. 421. |
|2.||Rhee FV, Anaissie E, Angtuaco E, Bartel T, Epstein J Nair B. Myeloma. In: Kaushansky K, Lichtman MA, Kipps TJ, Seligsohn U, Prchal PT, editors. Williams Hematology. 8th ed. New York: McGraw-Hill; 2010: p.1627-35. |
|3.||Chen W, Huang Q, Zuppan CW, Rowsell EH, Cao JD, Weiss LM, et al. Complete absence of KSHV/HHV-8 in posttransplant lymphoproliferative disorders: An immunohistochemical and molecular study of 52 cases. Am J Clin Pathol 2009;131:632-9. |
|4.||Montella M, Crispo A, Russo F, Ronga D, Tridente V, Tamburini M. Hepatitis C virus infection and new association with extrahepatic disease: Multiple myeloma. Haematologica 2000;85:883-4. |
|5.||Elira Dokekias A, Purhuence MF, Malanda F, Moyikoua A, Moutschen M. Multiple myeloma and HIV/AIDS infection. Three case reports. Tunis Med 2004;82:55-9. |
|6.||Rettig MB, Ma HJ, Vescio RA, Põld M, Schiller G, Belson D, et al. Kaposi's sarcoma-associated herpesvirus infection of bone marrow dendritic cells from multiple myeloma patients. Science 1997;276:1851-4. |
|7.||Ismail SI, Mahmoud IS, Salman MA, Sughayer MA, Mahafzah AM. Frequent detection of Human Herpes Virus-8 in bone marrow of Jordanian patients of multiple myeloma. Cancer Epidemiol 2011;35:471-4. |
|8.||Beksac M, Ma M, Akyerli C, Der Danielian M, Zhang L, Liu J, et al. Frequent demonstration of human herpesvirus 8 (HHV-8) in bone marrow biopsy samples from Turkish patients with multiple myeloma (MM). Leukemia 2001;15:1268-73. |
|9.||Sadeghian MH, Katebi M, Ayatollahi H, Keramati MR. Immuno-histochemical study association between human herpesvirus 8 and multiple myeloma. Int J Hematol 2008;88:283-6. |
|10.||Duprez R, Lacoste V, Hermouet S, Troussard X, Valensi F, Merle-Beral H, et al. Plasma-cell leukemia and human herpesvirus 8 infection. Leukemia 2004;18:1903-4. |
|11.||Kumar V, Abbas AK, Fausto N, Aster J. Robbins and Cotran pathologic basis of disease. 8 th ed. Pennsylvania: Elsevier Saunders; 2010. p. 355-6. |
|12.||Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4 th ed. Lyon, France: IARC Press; 2008. |
|13.||Ancý´n I, Sarra´ J, Peris J, Romagosa V, Domingo-Claros A, Gran˜ena A. Demonstration of Epstein-Barr Virus in a case of multiple myeloma after renal transplantation. Haematologica 2000;85:773-4. |
|14.||Tcheng WY, Said J, Hall T, Al-Akash S, Malogolowkin M, Feig SA. Post-transplant multiple myeloma in a pediatric renal transplant patient. Pediatr Blood Cancer 2006;47:218-23. |
|15.||Voelkerding KV, Sandhaus LM, Kim HC, Wilson J, Chittenden T, Levine AJ, et al. Plasma cell malignancy in the acquired immune deficiency syndrome. Association with Epstein-Barr Virus. Am J Clin Pathol 1989;92:222-8. |
|16.||Min W, Yongshun T, Shuhua Y. Detection for EB virus DNA of acute leukemia and multiple myeloma. Harbin Med J 2010;5:5-6. |
|17.||Csire M, Mikala G, Peto M, Ja´nosi J, Juha´sz A, Tordai A, et al. Detection of four lymphotropic herpesviruses in Hungarian patients with multiple myeloma and lymphoma. FEMS Immunol Med Microbiol 2007;49:62-7. |
|18.||Schönrich G, Raftery M, Schnitzler P, Rohr U, Goldschmidt H. Absence of a correlation between Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) and multiple myeloma. Blood 1998;92:3474-5. |
|19.||Shi SR, Cote RJ, Wu L, Liu C, Datar R, Shi Y, et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: Heating under the influence of pH. J Histochem Cytochem 2002;50:1005-11. |
|20.||Dispenzieri A, Lacy MQ, Greipp PR. Multiple Myeloma. In: Greer JP, Foerster J, Lukens JN. Wintrobe's Clinical Hematology. 12th ed. Philadelphia: Lippincott Williams and Wilkins; 2009. p. 2372-3. |
|21.||Detailed Guide: Multiple Myeloma What Are the Key Statistics About Multiple Myeloma?. American Cancer Society. Available from: http://bit.ly/dMwLDk. [Last accessed on 2009 May 28]. |
|22.||Mousavi SM, Gouya MM, Ramazani R, Davanlou M, Hajsadeghi N, Seddighi Z. Cancer incidence and mortality in Iran. Ann Oncol 2009;20:556-63. |
|23.||Cancer control office in center for disease control. Iranian annual cancer registrian report 2003. March 2005(1383). p. 115. |
|24.||Becker N. Epidemiology of multiple myeloma. Recent Results Cancer Res 2011;183:25-35. |
|25.||Cohen JI, Bollard CM, Khanna R, Pittaluga S. Current understanding of the role of Epstein-Barr Virus in lymphomagenesis and therapeutic approaches to EBV-associated lymphomas. Leuk Lymphoma. 2008;49 Suppl 1:27-34. |
|26.||Hermouet S, Sutton CA, Rose TM, Greenblatt RJ, Corre I, Garand R, et al. Qualitative and quantitative analysis of human herpesviruses in chronic and acute B cell lymphocytic leukemia and in multiple myeloma. Leukemia 2003;17:185-95. |
|27.||Chang ST, Liao YL, Lu CL, Chuang SS, Li CY. Plasmablastic cytomorphologic features in plasma cell neoplasms in immunocompetent patients are significantly associated with EBV. Am J Clin Pathol 2007;128:339. |
|28.||Vega F, Chang CC, Medeiros LJ, Udden MM, Cho-Vega JH, Lau CC, et al. Plasmablastic lymphomas and plasmablastic plasma cell myelomas have nearly identical immunophenotypic profiles. Mod Pathol 2005;18:806-15. |
|29.||Chen H, Hutt-Fletcher L, Cao L, Hayward SD. A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr Virus. J Virol 2003;77:4139-48. |
|30.||Kovacs E. Interleukin-6 leads to interleukin-10 production in several human multiple myeloma cell lines. Does interleukin-10 enhance the proliferation of these cells? Leuk Res 2010;34:912-6. |
|31.||Soleimani GH, Saneei Moghadam E, Gorgani F. Frequency of Epstein-Barr Virus (EBV) anti-antibody titer in 0-14 year old children referred to Aliasghar clinic in Zahedan. J Birjand Univ Med Sci 2008;14:40-4. |
|32.||Katebi M, Sharifi N, Tarhini M, Otrock ZK, Bazarbachi A, Kchour G. Frequency of Epstein-Barr Virus expression in various histological subtypes of Hodgkin's lymphoma. Histopathology 2008;52:775-7. |
Department of Hematology and Blood Bank, Endoscopic & Minimally Invasive Surgery Research Center, Ghaem Medical Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad
Source of Support: The research vice chancellor of Mashhad University of Medical Sciences, Conflict of Interest: None
[Table 1], [Table 2]
| Article Access Statistics|
| Viewed||5091 |
| Printed||164 |
| Emailed||2 |
| PDF Downloaded||225 |
| Comments ||[Add] |