Clonality analysis of defined cell populations in paraffin-embedded tissuesections by RT-PCR amplification of X-linked G6PD gene

This paper establishes a method of clonality analysis using the reverse tra nscription-polymerase chain reaction (RT-PCR) to amplify X-linked G6PD tran scripts on defined cell populations microdissected from archival, paraffin- embedded tissue sections. Four known monoclonal low-grade B-cell lymphomas from females who were heterozygous (informative) at the 1131 exonic polymor phic locus of the G6PD gene were used to validate the method. Lymphoma and reactive lesions in each case were separated by microdissection, In order t o preserve the intact RNA species in the lesion, sections were digested on the slides before microdissection, A one-step RT-PCR was performed with a s ingle pair of primers, one of which contained a mismatched base adjacent to the polymorphic site, to generate a PvuI cutting site. Successful amplific ation and allele identification by PvuI digestion were achieved from all RN A samples studied. Three of four samples from non-neoplastic reactive lesio ns showed two bands with equal intensity, representing transcription of the two alleles of the G6PD gene, while the corresponding tumour samples demon strated a biased intensity in one allele, indicating monoclonality, To asse ss the method further, the clonal nature of in situ and invasive breast can cers was examined, along with adjacent normal breast tissue and hyperplasti c lesions from three informative females from our archives. Apart from the clusters of normal terminal duct-lobular units, all lesions were monoclonal , This result is in agreement with data derived from other X-linked gene st udies and loss of heterozygosity (LOH) analyses of pre-invasive breast dise ase. The results suggest that the clonality analysis method presented here is simple and reliable, and is therefore potentially applicable in a wide r ange of pathological conditions. Copyright (C) 2000 John Wiley & Sons, Ltd.