2. Assessment of monoclonality in large B cell non-Hodgkins lymphoma
Lotfi N. M.Sc
Rastin M, Ph.D
Memar B, MD
Mahmoudi M. MD, Ph.D
Immunology Research Center, BuAli Research Institute, Mashhad University of Medical Sciences

3. Introduction

4. Subdivision of large B-cell lymphomas by morphology
Diffuse large B-cell lymphoma (DLBCL)
40% of adult Non-Hodgkin lymphomas
- Morphologically, genetically and clinically
heterogeneous group of large B cells tumors
Subdivision of large B-cell lymphomas by morphology
Centroblastic lymphoma
Immunoblastic lymphomas

5. Composed of large cells Resemble centroblasts or immunoblasts
Often with a mixture of the two
Clinical significance is yet debated
Centroblastic type (80% of the cases) is composed of :
- cells resembling germinal center centroblasts
- with one to three peripheral nucleoli
- a narrow rim of basophilic cytoplasm
-mixture of both immunoblasts and centroblasts

6. In crucial cases: Morphological criteria do not help distinguish
Diagnosis of NHL still represents a challenge
In reactive and malignant lymphoproliferations
Molecular Diagnostic methods are useful tools
Clonality assays are helpful

7. Demonstration of monoclonality in gene level
In B cell lymphomas is of great importance
Distinguish lymphomas from reactive lymphoid lesions
When morphological and immunophenotypic features are
difficult to interpret

8. Southern blot analysis of Ig genes:
Is the established method for monoclonality demonstration
However it is:
Slow
Complex
Requires fresh samples
Expensive

9. Detection of B cell monoclonoclonality
PCR
Polymerase chain reaction overcome limitations
- Offers :
Sensitive
Rapid
Simple
Flexible
Detection of B cell monoclonoclonality

10. PCR approach based on : -Amplification of VDJ region of rearranged Ig heavy chain gene in lymphoid tissues - Followed by size analysis of products On an electrophoretic gel.

11. DH VH JH CH 5´ 3´ DH-N-JH VH-N-DH-N-JH CDR3 5´ FR1 FR2 FR3 N D N FR4bp

12. Methods & Materials

13. Receiving sample blocks from Pathology archive
Reading OD and
PCR with beta actin
primer
Receiving sample blocks from Pathology archive
Electrophoresis and staining
Method
DNA Extraction
PCR with Specific primers 13

14. Samples obtained from:
archives of :
Imam Reza
Ghaem
Omid Hospitals
private laboratories in Mashhad
Samples include 44 DLBCL specimens
20 samples from tonsil and appendix as controls
These cases were reviewed by a pathologist for confirmation.

15. 5 µm sections were cut from paraffin blocks
Samples were deparraffinized
DNA extracted using proteinase K digestion method

16. Amplification of house keeping gene
ß -Actin gene amplified To confirm extracted
DNA quality

17. PCR reaction

18. Electrophoresis and staining
To analyze the PCR products :
15µl of each PCR product was electrophoresed on
8% PAGE
Stained with AgNo3

19. Results

20. MW
50 bp
FR3 N D JH
‍JH

21. FR3 N D
‍JH
50 bp MW

22. 100 bp
(c) MW
125bp

23. Results: - all samples had adequate DNA quality
- using primers amplifyng FRIII-JH region
- clonal IgH gene rearrangements
- demonstrated in 75%(33/44) of the cases.
Monoclonality
Positive Results
Number
Samples
%75 33 44
DLBCL %0 20
Control

24. Disccusion and conclusion

25. 1993 Fc- Linng et al 2002 Yon- Chin- Tai et al Adan-Begg et al 2003
Results
Year
Previous studies
56%
1993
Fc- Linng et al
54.3%
2002
Yon- Chin- Tai et al
47%
Adan-Begg et al
55%
2003
Timeap. Gurbitty et al

26. - Previoue Studies on Diffuse large B cell lymphomas - Reported 47%-55% with the FR3/JH - we succeed to increase the diagnosis to 75% - using new design of primers (degenarate primers)

27. PCR method is: - Sensitive & Reliable - for detecting B cell clonality Can be used as an accessory diagnostic tool to conventional assessment of lymphoprolyferative diseases.

28. Thanks for Your Attention

29. Thanks for Your Attention

30. 100 bp

31. M M