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.
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 kappa light-chain (IgL kappa), and TCR gamma 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 IgL kappa helped to raise the sensitivity of detection to 94%. Monoclonality was detected in seven of nine T-cell NHLs by amplification of TCR gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
Weirich
G
G
Institute of Pathology, School of Medicine, Technical University of Munich, Germany.
Funk
A
A
Hoepner
I
I
Heider
U
U
Noll
S
S
Pütz
B
B
Fellbaum
C
C
Höfler
H
H
D001483:Base Sequence; D001853:Bone Marrow / Q000473:pathology; D002999:Clone Cells; D005557:Formaldehyde; D015321:Gene Rearrangement; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008198:Lymph Nodes / Q000473:pathology; D008228:Lymphoma, Non-Hodgkin / Q000145:classification / Q000175:diagnosis* / Q000235:genetics; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D012867:Skin / Q000473:pathology; D016707:Tissue Fixation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007145:Immunoglobulin kappa-Chains; D016692:Receptors, Antigen, T-Cell, gamma-delta; D016159:Tumor Suppressor Protein p53; D005557:Formaldehyde