CLONAL X-INACTIVATION ANALYSIS OF HUMAN TUMORS USING THE HUMAN ANDROGEN RECEPTOR GENE (HUMARA) POLYMORPHISM - A NONRADIOACTIVE AND SEMIQUANTITATIVE STRATEGY APPLICABLE TO FRESH AND ARCHIVAL TISSUE

Assessment of clonality of cellular proliferations is important in exp erimental and clinical cancer research. X-chromosome inactivation stud ies are widely used to assess clonality, but most assays require relat ively large amounts of high molecular weight DNA. Two PCR-based strate gies, the phosphoglycerate kinase (PGK) and the human androgen recepto r (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and im mediate quantitation. This assay was validated by comparison with X-in activation patterns obtained by Southern analysis with the probes M27 beta and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter no dules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA e xtracted from formalin-fixed paraffin-embedded tissue (FPT) was analys ed with the two PCR-based methods and compared with X-inactivation pat terns determined by Southern analysis of high molecular weight (HMW) D NA. This modified HUMARA assay is reliable in most patients; as with o ther clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (u nder 15 years). Comparison of results obtained with HMW and FPT DNA yi elded consistent results for the HUMARA assay whereas the PGK PCR assa y was much less reliable. The HUMARA assay thus permits studies of sel ected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh a nd archival specimens. (C) 1997 Academic Press Limited.