Assessment of monoclonality in large B cell non-Hodgkins

lymphoma

Lotfi N. M.ScRastin M, Ph.DMemar B, MD

Mahmoudi M. MD, Ph.D

Immunology Research Center, BuAli Research Institute, Mashhad University of

Medical Sciences

Centroblastic lymphoma

Immunoblastic lymphomas

Subdivision of large B-cell lymphomas by morphology

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

-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

- 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

Southern blot analysis of Ig genes:

Is the established method for monoclonality demonstration

However it is:

Slow

Complex

Requires fresh samples

Expensive

Polymerase chain reaction overcome limitations

- Offers :

Sensitive

Rapid

Simple

Flexible

Detection of B cell monoclonoclonality

PCR

VH-N-DH-N-JH

FR1 CDR1 FR2 CDR

2 FR3 N D N FR4

CDR3

3´5´

5´ 3´

CHJHDHVH

DH-N-JH

100-150 bp

Methods & Materials

DNA ExtractionPCR with Specific primers

Electrophoresis and staining

Method

Receiving sample blocks from Pathology

archive

Reading OD and PCR with beta actin

primer

Samples obtained from:

-archives of : Imam RezaGhaem 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.

5 µm sections were cut from paraffin blocks

Samples were deparraffinized

DNA extracted using proteinase K digestion method

ß -Actin gene amplified To confirm extracted DNA quality

PCR reaction

To analyze the PCR products :

15µl of each PCR product was electrophoresed on

8% PAGE

Stained with AgNo3

50 bp

FR3 N D N JH

FR3 N D N JH

FR3 N D N JH

MW 1 2 3 4 5 6

FR3 N

FR3 N

FR3 N

D N JH

D N JH

D JHN

50 bp

1 2 3 4 MW

100 bp

1 2 (c) 3 4 5 6 MW

125bp

Samples

Number

Positive Results

Monoclonality

DLBCL

4433%75

Control

200%0

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.

Disccusion and conclusion

Previous studies YearResults

Fc- Linng et al199356%

Yon- Chin- Tai et al

200254.3%

Adan-Begg et al200247%

Timeap. Gurbitty et al

200355%

Thanks for Your Attention

Thanks for Your Attention

100 bp

1 2 3 4 M 5 6 7 8 9 10 1 2 3 4 M 5 6