Abstract
In crucial cases the diagnosis of non-Hodgkin's lymphoma (NHL) still represents a challenge to the pathologist since morphological criteria do not always help to distinguish between reactive and malignant lymphoproliferations. Clonality assays are a useful supplement since monoclonal cell proliferation is strong evidence for malignancy. The polymerase chain reaction (PCR) can be utilized to establish the clonal origin of B-or T-cell lymphocyte populations by amplification of rearranged immunoglobulin and T-cell receptor (TCR) genes. In the present study DNA was isolated from a variety of neoplastic and nonneoplastic formalin-fixed, paraffin-embedded lymph nodes (n=62), cutaneous tissue (n=9), samples of miscellaneous origin (n=11), and, reported here for the first time, decalcified bone marrow samples (n=35). These samples were submitted to PCR-based assays directed against the immunoglobulin heavy-chain (IgH), immunoglobulin κ light-chain (IgLκ), and TCRγ chain genes. The impact of various decalcifying agents on the ability to amplify DNA was investigated by PCR-based amplification of a single copy gene. Buffered and nonbuffered EDTA was found not to impede amplification of DNA fragments up to 300 bp in length. In lymph node and cutaneous specimens monoclonality was detected in 83% of B-NHL cases using a seminested PCR approach for the amplification of IgH, whereas the same approach gave rise to monoclonal bands in 80% of bone marrow samples. The subsequent amplification of IgLκ helped to raise the sensitivity of detection to 94%. Monoclonality was detected in seven of nine T-cell NHLs by amplification of TCRγ. Most of the false-negative results were related to DNA extracted from centroblastic-centrocytic lymphoma and lymphoplasmocytic immunocytoma (37% negative each). PCR-based rearrangement analysis of immunoglobulin and TCR chain genes should be used in diagnostic pathology for cases which are histopathologically and immunohistochemically questionable. The application of clonality assays to bone marrow samples previously decalcified with EDTA provides a new tool for the detection of minimal residual disease.
Similar content being viewed by others
Abbreviations
- BALT :
-
bronchus-associated lymphoid tissue
- dNTP :
-
deoxynucleoside triphosphate
- Ig :
-
immunoglobulin
- IgH :
-
immunoglobulin heavy chain
- IgL :
-
immunoglobulin light chain
- MALT :
-
mucosa-associated lymphoid tissue
- NHL :
-
non-Hodgkin's lymphoma
- PCR :
-
polymerase chain reaction
- TCR :
-
T-cell receptor
References
Arnold A, Cossman J, Bakhshi A, Jaffe ES, Waldman TA, Kormeyer S (1983) Immunoglobulin-gene rearrangements as unique clonal markers in human lymphoid neoplasms. N Engl J Med 309:1593–1599
Brisco MJ, Tan LW, Orsborn AM, Morley AA (1990) Developement of a high sensitive assay, based on the polymerase chain reaction, for rare B-lymphocyte clones in a polyclonal population. B J Haematol 75:163–167
Cleary MI, Chao J, Warnke R, Sklar J (1984) Immunoglobulin gene rearrangement as a diagnostic criterion of B-cell lymphoma. Proc Natl Acad Sci USA 81:593–597
Crescenzi M, Seto M, Herzig GP, Weiss PD, Griffith RC, Krosmeyer SJ (1991) Thermostable DNA polymerase chain amplification of t(14; 18) chromosome breakpoints and detection of minimal residual disease. Proc Natl Sci USA 85:4469–4473
Griesser H (1993) Applied molecular genetics in the diagnosis of malignant non-Hodgkin's lymphoma. Diagn Mol Pathol 2:177–191
Gulley ML, Dent GA, Ross DW (1992) Classification and staging of lymphoma by molecular genetics. Cancer [Suppl] 69:1600–1606
Ichihara Y, Matsuoka H, Kurosawa Y (1988) Organisation of human immunoglobulin heavy chain diversity gene loci. EMBO J 7:4141–4150
Inghirami G, Szabolcs MJ, Yee HT, Corradini P, Casarman E, Knowles DM (1993) Detection of immunoglobulin gene rearrangement of B cell non Hodgkin's lymphomas and leukemias in fresh, unfixed and formalin-fixed, paraffin-embeeded tissue by polymerase chain reaction. Lab Invest 68:746–757
Küppers R, Zhao M, Rajewski K, Hansmann ML (1993) Detection of clonal B cell populations in paraffin-embedded tissues by polymerase chain reaction. Am J Pathol 143:230–239
Lennert K, Feller AC (1990) Histopathologie der Non-Hodgkin-Lymphome. Springer, Berlin Heidelberg New York, pp17–21
Ling FC, Clarke CE, Lillicrap D (1992) Positive immunoglobulin gene rearrangement study be the polymerase chain reaction in a colonic adenocarcinoma. Am J Clin Pathol 98:116–119
Lohmann D, Pütz B, Reich U, Böhm J, Präuer H, Höfler H (1993) Mutational spectrum of p53 gene in human small lung cancer and relationship to clinicopathological data. Am J Pathol 142:907–915
Negrin RS, Kiem HP, Schmidt-Wolf GH, Blume KG, Cleary ML (1991) Use of the polymerase chain reaction to monitor the effectiveness of ex vivo tumor cell purging. Blood 77:645–660
Pan LX, Diss TC, Peng HZ, Isaacson PG (1994) Clonality analysis of defined B-cell populations in archival tissue using microdissection and the polymerase chain reaction. Histopathol 24:323–327
Quertermous T, Murre C, Dialynas D, Duby AD, Strominger JL, Waldman TA, Seidman JG (1986) Human T-cell g chain genes: organisation, diversity, and rearrangement. Science 231:252–255
Schaefer HE (1988) Histologie und Histochemie am Paraffinschnitt. Verh Dtsch Ges Pathol 67:6–7
Seidman JG, Leder P (1978) The arrangement and rearrangement of antibody genes. Nature 302:575–581
Sklar J, Longtine J (1992) The clinical significance of antigen receptor gene rearrangements in lymphoid neoplasia. Cancer [Suppl] 70:1710–1718
Slack DN, McCarthy KP, Wiedemann LM, Sloane JP (1993) Evaluation of sensitivity, specificity, and reproducibility of an optimized method for detecting clonal rearrangements of immunoglobulin and T-cell receptor genes in formalin-fixed, paraffin-embedded sections. Diagn Mol Pathol 2:223–232
Stansfeld Diebold, Kapanci Y, Kelenyi G, Lennert K, Mioduszewska O, Noel H, Rilke F, Sundstrom C, Van Unnik JAM, Wright DH (1988) Updated Kiel classification for lymphomas Lancet I: 292–293, 603
Sukpanichnant S, Vnenecak-Jones CL, McCurley TL (1993) Detection of clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction in scrapings from archival hematoxylin and eosin-stained histologic sections: implications for molecular genetic studies of focal pathologic lesions. Diagn Mol Pathol 2:168–176
Trainor KJ, Brisco MJ, Story CJ, Morley AA (1990) Monoclonality in B-lymphoproliferative disorders detected at the DNA level. Blood 75:2220–2222
Trainor KJ, Brisco MJ, Wan JH, Neoh S, Grist S, Morley AA (1991) Gene rearrangement in B- and T-lymphoproliferative disease detected by the polymerase chain reaction. Blood 78:192–196
Wan JH, Trainor KJ, Brisco MJ, Morley AA (1990) Monoclonality in B cell lymphoma detected in paraffin wax embedded sections using the polymerase chain reaction. J Clin Pathol 43:888–890
Yunis JJ, Frizzera G, Oken MM, McKenna J, Theologides A, Arnesen M (1987) Multiple recurrent genomic defects in follicular lymphoma. N Engl J Med 316:79–84
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Weirich, G., Funk, A., Hoepner, I. et al. PCR-based assays for the detection of monoclonality in non-Hodgkin's lymphoma: application to formalin-fixed, paraffin-embedded tissue and decalcified bone marrow samples. J Mol Med 73, 235–241 (1995). https://doi.org/10.1007/BF00189923
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00189923