02 BOOK OF ABSTRACTS .indd

Molecular analysis of lymphoproliferative disorders using
standard PCR
Ira Kokovi} 1 , Rastko Golouh 2 , Janez Jan~ar 2 , Andreja Zidar 2 ,
Srdjan Novakovi} 1
1
Department of Molecular Diagnostics, Institute of Oncology, Zalo{ka 2, SI-1000 Ljubljana,
Slovenia; 2 Department of Pathology, Institute of Oncology, Zalo{ka 2, SI-1000 Ljubljana, Slovenia
Background: Demonstration of clonality and detection of specific genetic abnormalities
enable distinguishing between neoplastic lesions and reactive processes and, thus,
have important value in the diagnosis of lymphoid neoplasms. A clonal population of
lymphoid cells can be detected by PCR amplification of the rearranged immunoglobulin
heavy chain (IgH) gene in B-cell lymphomas and T-cell receptor γ chain (TcRγ) gene
in T-cell lymphomas. Similarly, the chromosome translocation t(14;18)(q32;q21) in
follicular lymphoma can be detected by PCR amplification of rearranged bcl-2/IgH
region. We have introduced PCR-based assays for clonality analysis, as well as for the
detection of t(14;18)(q32;q21) as adjunct tests in lymphoma diagnostics.
Methods: One hundred and sixty-eight biopsies of various lymphoproliferative
disorders collected at the Department of Pathology, Institute of Oncology over the
period 1997-2005, which could not be diagnosed using classical morphological and
immunophenotypic criteria, were analysed using established molecular techniques. All
tissue samples were fixed in phosphate buffered formalin and embedded in paraffin
(FFPE tissue). DNA was isolated according to the standard method. Rearranged IgH
and TcRγ genes were amplified using primers predicted from conserved sequences
in variable (V) and joining (J) gene segments. PCR products were analysed by
electrophoresis in 10% polyacrylamide gels, stained with ethidium bromide and
photographed under UV light.
Results: Using molecular methods we determined the clonality of 146/167 analysed
cases (87.4%). Fifty-four cases (32.3%) were monoclonal and 92 cases (55.1%) were
polyclonal. We could not determine the clonality of the remaining 21 cases (12.6%):
3 cases were scored as oligoclonal, 12 as »monoclonal in a polyclonal background«
and 6 cases as negative. Considering both, classical histopathological and molecular
findings we determined the type of lymphoid neoplasm in 94.6% of analysed cases.
There were 31.5% of B-cell neoplasms, 21.4% of T-cell neoplasms, 4.8% of Hodgkin’s
disease cases and 36.9% reactive lymphoid proliferations. We could not define the
type of lymphoid proliferation in the remaining 5.4% of cases. Cases suspected for
follicular lymphoma (26) have been analysed for the presence of t(14;18)(q32;q21).
Eight cases (30.8%) were positive, 15 cases (57.7%) were negative and 3 cases (11.5%)
were inconclusive.
Conclusions: Introduced PCR assays are simple, fast (the results can be obtained
within 2-3 days) and, what is most important in surgical pathology, can be applied on
small amounts of FFPE tissue. In the era of gene expression profiling, the detection
of clonality and specific genetic abnormalities by standard PCR methods still provides
important diagnostic and clinical information.
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