PCR-heteroduplex analysis of T-cell receptor gamma gene rearrangement in paraffin-embedded skin biopsies.
We developed a rapid, simple, and sensitive method for the detection of T-cell receptor-gamma (TCRgamma) gene rearrangements in paraffin-embedded skin biopsies. Available techniques often require either fresh tissue, several primer pairs, nested amplifications, or specialized electrophoresis steps such as denaturing gradient gel electrophoresis. Our method is based on heteroduplex analysis of polymerase chain reaction (PCR) products of the TCRgamma in a nondenaturing modified polyacrylamide gel using a single pair of primers and is adapted for paraffin-embedded tissue. When tested against Southern blot analysis, the PCR results correlated in 8 of 9 cases. Six mature cutaneous B-cell lymphomas and 29 inflammatory skin disorders all resulted in a polyclonal amplification pattern. When analyzing 3-mm or 4-mm punch biopsies of 51 cases of cutaneous T-cell lymphoma, 37 (72.5%) showed a clonal rearrangement with this technique. For 7 cases of patch stage mycosis fungoides, frozen tissue and formalin-fixed and paraffin-embedded tissue was available, and in 5 of 7 cases (71%), the results in frozen and paraffin-embedded tissue were concordant. One case showed a clonal pattern in frozen tissue but not in paraffin-embedded tissue, and one case was polyclonal in frozen tissue but monoclonal in paraffin-embedded tissue. Using serial dilutions of DNA from a T-cell ALL in a polyclonal background (tonsil), we established a sensitivity of 0.5%. Heteroduplex PCR of the TCRgamma is a rapid, sensitive, and inexpensive screening procedure as well as a useful adjunct to histologic analysis and immunophenotyping of cutaneous T-cell proliferations.
Kohler
S
S
Department of Pathology, Stanford University Medical Center, California 94305, USA.
Jones
C D
CD
Warnke
R A
RA
Zehnder
J L
JL
D001707:Biopsy, Needle; D015139:Blotting, Southern; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor / Q000235:genetics*; D019675:Genes, T-Cell Receptor gamma / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods; D006801:Humans; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000473:pathology; D016410:Lymphoma, T-Cell, Cutaneous / Q000235:genetics* / Q000473:pathology; D009182:Mycosis Fungoides / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology
D017931:DNA Primers; D004273:DNA, Neoplasm; D016692:Receptors, Antigen, T-Cell, gamma-delta