This journal club discusses multiple myeloma and EBV. The document summarizes characteristics of multiple myeloma, its diagnostic workup, criteria for diagnosis, and role of infections like EBV, HCV and HIV in pathogenesis. It then summarizes a case-control study that found higher rates of EBV DNA in bone marrow of multiple myeloma patients compared to controls, as detected by PCR. It concludes there may be an association between EBV and multiple myeloma but larger studies are needed.

1. Journal club

3. Multiple myeloma
• Malignant disorder of plasma cells
• Primarily occurring in the bone marrow.
• 20% of the deaths by hematologic neoplasms.
• Diagnosis requires
Pathologic
Radiologic
Clinical findings

4. Initial Diagnostic Workup
• History and physical
• CBC
• BUN/creatinine, electrolytes
• Lactate dehydrogenase (LDH)
• Calcium/albumin
• Beta-2 microglobulin
• Serum free light chain (FLC) assay
• Serum quantitative immunoglobulins,
serum protein electrophoresis (SPEP),
serum immunofixation electrophoresis
(SIFE)
• 24-hr urine for total protein, urine
protein electrophoresis (UPEP), urine
immunofixation electrophoresis (UIFE)
• Skeletal survey
• Unilateral bone marrow aspirate +
biopsy, including bone marrow
immunohistochemistry and/or bone
marrow flow cytometry
• Cytogenetics
• Fluorescence in situ hybridization (FISH)
[del 13; del 17p13; t(4;14); t(11;14);
t(14;16); 1q21 amplification]

5. In some circumstances
• MRI
• CT scan (avoid contrast)
• PET/CT scan
• Tissue biopsy to diagnose a solitary osseous or extraosseous plasmacytoma
• Bone densitometry
• Plasma cell labeling index
• Staining of marrow and fat pad for amyloid
• Serum viscosity
• HLA typing

6. Multiple myeloma
• Clonal bone marrow plasma cells >10% / biopsy proven bony or
extramedullary plasmacytoma
+
• Any one of the following
• Evidence of end organ damage – CRAB
• Clonal BM plasma cells 60% or more
• Involved: uninvolved serum free light chain ratio >/= 100
• >1 focal lesions on MRI (size 5mm minimum)

7. Multiple myeloma defining event
CRAB
• C: Calcium elevation (> 11 mg/dL or > 1 mg/dL higher than ULN)
• R: Renal insufficiency (creatinine clearance < 40 mL/min or serum
creatinine > 2 mg/dL)
• A: Anemia (Hb < 10 g/dL or 2 g/dL < normal)
• B: Bone disease (≥ 1 lytic lesions on skeletal radiography, CT, or PET-
CT).

8. Role of infections
• There are studies suggesting the possible potential role of several
infectious agents in the pathogenesis of MM.
• Hepatitis C virus infection
• Human immunodeficiency virus (HIV)-infection
• Human herpes virus 8
• Epstein-Barr Virus (EBV)
Kumar V, Abbas AK, Fausto N, Aster J. Robbins and Cotran pathologic basis of disease. 8th ed.
Pennsylvania: Elsevier Saunders; 2010. p. 355-6.

9. EBV
• Member of herpesvirus family
• Infectious mononucleosis
• Associated with a number of malignancies such as
nasopharyngeal carcinoma,
Hodgkin lymphoma – Classical
Burkitt lymphoma
diffuse large B-cell lymphomas (immunosuppressed individuals)
T-cell lymphoma ( gamma –delta)
cutaneous T- cell lymphoproliferative disease
Angioimmunoblastic T cell lymphoma

10. AIM OF THE STUDY:
Investigate differences between two groups of patients for presence of
EBV DNA in bone marrow biopsy by PCR
MM group
Control group

11. MATERIAL AND METHODS:
• Case-control study
• 60 formalin-fixed paraffin embedded (FFPE) bone marrow biopsies.

12. SAMPLE – GENDER DISTRIBUTION
CASE GROUP CONTROL GROUP
MALE 14 16
FEMALE 16 14
TOTAL 30 30

13. Patient group
MM patients
positive clinical and
radiological finding
more than 30% plasma
cells in the bone marrow
30 FFPE marrow biopsies
Control group
Patients diagnosed with
lymphoma
Biopsy done for
determination of staging
Normal bone marrow
morphology without
increase in plasma cells
30 FFPE marrow biopsy
SUBJECTS

14. EXCLUSION CRITERIA
• History of immunodeficiency diseases
• transplantation
• immunosuppressive therapy

15. PROCEDURE
Sample selection
(control and patient
group)
60 FFPE sections of
bone marrow biopsy
DNA extraction by
non-heating
method
DNA concentration
determined by
spectrophotometer
PCR for EBV genome
detection
Electrophoresed on
2% agarose gel
Photographed by gel
documentation
instrument in UV
light
Statistical analysis

16. 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
(185bp)
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.

17. TEST RESULTS
• Positive PCR results for DNA detection of EBV –
• 10 (33.3%) samples of the patient group
• 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.

18. Patients group - EBV was detected in 5 males and 5 females.
No significant difference between two groups of EBV positive and negative for
sex distribution.
33.3% 10%

19. Other findings in the study
• In myeloma patients, the mean white blood cell (WBC) count
(variation) was 9.05 +/- 4.02 and 5.20 +/- 2.02 × 109 /L in EBV positive
and negative groups, respectively.
• This difference was statistically significant
• But no significant differences in other laboratory results –
serum calcium, erythrocyte sedimentation rate (ESR), complete
blood count (CBC), between case and control groups.

20. Reason for the difference in WBC count – not elaborated further.
Limitation

21. In the present study
• Association between EBV and MM patients without any obvious
history of immunodeficiency or transplantation.

22. Limitations of the study
• Smaller sample size
• Future study with a larger number of case groups is helpful (to
decrease the probable sampling error)
• High prevalence of EBV infection in the control group.
• Complementary studies - in situ hybridization or
immunohistochemistry methods to detect direct evidence of EBV in
tissue.

23. CONCLUSION
• A statistically significant relationship between multiple myeloma and
detection of EBV DNA in bone marrow tissues by the PCR method is
found.
• Similar study with greater number of patients can be helpful for the
final decision.

24. DISCUSSION
• Iran 6 to7.8 per 1,00,000
• Worldwide 4.3 to 5.8% per 1,00,000
• India is 1.9 to 3 per 1,00,000 (6,000 to 10,000 per year)
• Unevenly geographic distribution with higher frequency in Europe
and North America.
• Due to genetic and/or environmental factors (e.g. endemic infection).

25. Plasma cell disorders
• Smoldering Multiple myeloma
• Multiple myeloma
• Non IgM MGUS
• IgM MGUS
• Solitary plasmacytoma
• Solitary plasmacytoma with minimal bone marrow involvement
• Osteosclerotic myeloma

26. SMOLDERING MULTIPLE MYLEOMA
2 criterias should be met:
Serum monoclonal protein (IgG or IgA) ≥3 g/dL
or
Urinary monoclonal protein ≥500 mg/24 h
and/or
Clonal BM plasma cells 10% - 60%
• No myeloma defining events or amyloidosis (no CRAB)

27. MGUS – non IgM
All 3 criterias must be met
• Serum monoclonal protein (IgG or IgA or IgM) <3 g/dL
AND
• Clonal BM plasma cells <10%
AND
• No myeloma defining events (CRAB)

28. MGUS – IgM
All 3 criterias must be met
• Serum monoclonal protein (IgG or IgA or IgM) <3 g/dL
AND
• Bone marrow lymphoplasmacytic infiltrations <10%
AND
• No myeloma defining events (CRAB)

29. Solitary plasmacytoma
All 4 criterias must be met:
• Biopsy proven solitary lesion of bone / soft tissue with clonal
prolifteration of plasma cells.
• Normal bone marrow with no evidence of clonal plasma cells
• Normal skeletal survey and MRI/CT (Except for the primary leison)
• Absence of end organ damage.

30. Solitary plasmacytoma with minimal bone
marrow involvement
All 4 criterias must be met:
• Biopsy proven solitary lesion of bone / soft tissue with clonal
prolifteration of plasma cells.
• <10% clonal bone marrow plasma cells
• Normal skeletal survey and MRI/CT (Except for the primary leison)
• Absence of end organ damage.

31. Osteosclerotic myeloma
• Polyneuropathy
• Organomegaly
• Endocrinopathy
• Monoclonal gammopathy
• Skin changes

32. EBV
• Also called human herpesvirus 4 (HHV-4)
• Nature: B-lymphotropic virus

33. PATHOGENESIS
EBV Saliva
B-cells and
oropharyngeal
epithelial cells
Spreads to
underlying
lymphoid tissue
Infects mature
B-cells
(Reservoir)
EBV envelope
glycoprotein +
CR-2
binds to B cells
LYTIC OR
LATENT

34. Latent period
Virus persists as an extrachromosomal episome.
• EBNA-1
EBV dna binds to host cell during mitosis
• LMP-1
Promotes B-cell activation (mimicking CD-40) and proliferation
(activates BCL-2 and prevents apoptosis)
• EBNA-2
Activates cyclin D and promotes cell activation and replication
• IL-10
Suppresses anti-viral T-cell response

35. Role of EBV in MM pathogenesis
LMP-1 (Latent membrane protein)
• Activation of BCL-2 gene
Prevents apoptosis
• Induces expression of
angiogenesis factors (VEGF)
• IL-6 for cell proliferation
EBNA-2 (EB nuclear antigen-2)
• Cyclin D activation
Promotes cell activation
and replication

36. EBV + MM
Cases have been documented in
• Immunocompromised patients
• Post renal transplant status
UNIMPENDED B-
CELL ACTIVATION
POLYCLONAL
MONOCLONAL B-
CELL LYMPHOMA

37. CONCLUSION
• A statistically significant relationship between multiple myeloma and
detection of EBV DNA in bone marrow tissues by the PCR method is
found.
• Similar study with greater number of patients can be helpful for the
final decision.

39. CASE REPORT

40. Vyas Y, Salkar A, Bothale AK. Coexisting prostate adenocarcinoma with multiple myeloma: A rare case report.
Indian J Pathol Microbiol 2018;61:434-6.

41. • An 83-year-old male
• Chief complaint - lower back pain with incontinence of urine.
• On investigation –
Hemoglobin - 8.6 g%,
Total leukocyte count - 11,200/mm3
Platelet count - 73 × 109 /l
P/S - normocytic normochromic anemia with thrombocytopenia
Erythrocyte sedimentation rate - normal
Urine examination was normal

42. • Serum: Alkaline phosphatase - 446 IU/l (35–105)
LDH = 637 IU/l (225–450)
Prostate-specific antigen (PSA) =140 ng/l.
• Transrectal ultrasound - a hypoechoic prostatic mass with irregular
margins.
• Ultrasound-guided biopsy –
Adenocarcinoma prostate with Gleason score of 5+4

43. Most of the malignant cells
are arranged in cords.
Few ill formed glands seen.

44. MRI of the bones - Osteolytic lesions in the skull and vertebrae.

45. • Bone marrow aspiration and biopsy - 65% plasma cells.
• No evidence of secondary deposits of malignant epithelial cells

46. • IHC – CD 138 positive in marrow biopsy
• Immunoelectrophoresis – IgG lambda monoclonal gammopathy with
M-band positive.
• The final diagnosis –
Multiple myeloma with prostate adenocarcinoma .

47. Discussion
• The incidence of simultaneous occurrence of prostate
adenocarcinoma and hematolymphoid malignancy has been reported
as 1.2%.

48. Theories
Myeloma cells and stromal
cells
Immunosupression
IGF-1, IL-6, SDF-1, VEGF
Released into circulation
Proliferation of prostate cancer
cells

49. Theories
Myeloma cells
Vascular Endothelium derived Growth Factor (VEGF)
Mediator of angiogenesis
Overexpression of VEGF present in majority of prostate
cancers
Poor prognosis

50. Other factors
• Repeated antigenic stimulation of reticuloendothelial cells
• Genetic susceptibility for plasma cell dyscrasias in patients with
positive family history
• Epstein–Barr virus infection
• Lack of suppression of B-cells by T-cells (development of
gammopathy).

51. Summary
• Further genetic studies are required to know the association between
these two diseases.

53. PCR

57. 1 2 3 4 5 6 7 8 9 10 1211
control (IC:
ositive results
d corresponds
sitive control
e two DNA
orresponds to
positive control
corresponds to
al DNA band