Detection of immunoglobulin gene rearrangement of B cell non-Hodgkin's lymphomas and leukemias in fresh, unfixed and formalin-fixed, paraffin-embedded tissue by polymerase chain reaction.
BACKGROUND
The majority of B cell nonHodgkin's lymphomas (NHLs) are composed of a genotypically identical cell population characterized by a unique immunoglobulin (Ig) VDJ gene rearrangement which is customarily documented by Southern blot hybridization analysis of fresh tissue. Sometimes, however, this approach cannot be used because of an insufficient quantity of tissue or the unavailability of fresh tissue. Therefore, alternative strategies should be designed in order to overcome these limitations.


METHODS
One possible alternative is the identification of Ig VDJ products of normal and neoplastic B cells by polymerase chain reaction (PCR) using mixed oligonucleotide primers recognizing the framework III region or Ig variable heavy chain leader sequences and universal Ig heavy chain joining region (JH) oligonucleotide primers. To determine whether the respective DNA samples are suitable for PCR amplification, control and unrelated genes should also be investigated (exon 5 of the p53 gene). In this study, genomic DNA was extracted from a well characterized panel of 139 human B cell lymphoid leukemias and NHLs derived from fresh (84) and/or paraffin-embedded (55) tissue, 19 normal peripheral lymphoid tissues, 9 Epstein-Barr virus infected lymphoblastoid cell lines and, as negative controls, 11 T cell LLs. Clonal Ig gene rearrangement products were assessed for the presence of a distinct PCR fragment after framework III-JH PCR amplification and electrophoretic separation and by DNA sequencing of the cloned PCR-Ig fragments.


RESULTS
Eighty-eight of the 139 (63%) B-NHLs consisting of 53/84 (63%) fresh, unfixed and 35/55 (64%) formalin-fixed, paraffin-embedded samples, exhibited distinct PCR bands. Using this approach we were able to identify a single clonal B cell population mixed with 1,000 nonB cells or 5 polyclonal B cells. There was no difference in the detection of monoclonality among different B-NHL categories. PCR fragments were not identified in any of 27 normal lymphoid tissues or 11 T-lymphoid leukemias. To detect a larger number of Ig gene rearrangement products, genomic DNA of 12 B-NHL/lymphoblastoid cell lines were investigated using VH-specific leader and JH oligonucleotides by PCR. A single PCR product was obtained in 9 of 12 (75%) cases and their clonality was documented by DNA sequencing of the cloned PCR fragments. The clonality of 11 of the 12 (92%) cases could be demonstrated using both PCR approaches.


CONCLUSIONS
Our results suggest that the monoclonality of human neoplastic B cells can be efficiently evaluated by PCR equally well from fresh, unfixed and formalin-fixed, paraffin-embedded tissues. This technique should prove to be a powerful tool in clinical diagnosis and research as well as in the retrospective analysis of archival pathologic specimens.
Inghirami
G
G
Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, New York.
Szabolcs
M J
MJ
Yee
H T
HT
Corradini
P
P
Cesarman
E
E
Knowles
D M
DM
D001483:Base Sequence; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics*; D005557:Formaldehyde; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015448:Leukemia, B-Cell / Q000235:genetics*; D016393:Lymphoma, B-Cell / Q000235:genetics*; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D004273:DNA, Neoplasm; D007135:Immunoglobulin Variable Region; D005557:Formaldehyde