Sensitive detection of clonal antigen receptor gene rearrangements for the
diagnosis and monitoring of lymphoid neoplasms by a polymerase chain
reaction-mediated ribonuclease protection assay
H Veelken, B Tycko and J Sklar
Department of Pathology, Brigham and Women's Hospital, Harvard Medical
School, Boston, MA 02115.
This report describes a novel assay involving the polymerase chain reaction
(PCR) and RNase protection for the rapid and sensitive detection of
malignant lymphoid cells by nucleotide sequences within their individual
rearranged gamma T-cell receptor (TCRG) genes. In this assay, clonal
rearrangements are amplified from the DNA of diagnostic tumor specimens
using a consensus V segment primer and a consensus J segment primer to
which the promoter for T7 RNA polymerase has been appended. The PCR product
from this amplification is transcribed into a radiolabeled RNA probe. Test
RNA transcribed from the opposite DNA strand is synthesized by similar
methods from TCRG genes of a subsequent biopsy specimen. The test RNA is
hybridized with the probe, and mismatched nucleotide sequences in the RNA
hybrids are digested by RNase A. Detection of fully protected probe by
means of polyacrylamide gel electrophoresis and autoradiography indicates
the presence of malignant cells in the test specimen. Dilution experiments
with DNA of cell lines from acute lymphoblastic leukemias (ALLs) show that
detection of one tumor cell among 10(5) normal bone marrow cells is usually
possible. Residual disease was also successfully detected in several cases
of ALL during clinical remission, including detection in one case at the
10(-5) level. The procedure described here may provide a simplified and
rapid method for the sensitive diagnosis and monitoring of lymphoid
malignancies. This procedure should be applicable to most antigen receptor
genes, and unlike most comparable methods, requires neither analysis of
nucleotide sequence nor synthesis of tumor-specific oligonucleotide probes
or primers.
Volume 78,
Issue 5,
pp. 1318-1326,
09/01/1991
Copyright © 1991 by The American Society of Hematology
