Analysis of clonality by amplification of short tandem repeats

The clonal composition of cancers of the female reproductive tract was evaluated by analysis of patterns of X-chromosome inactivation. Using DNA extracted from frozen tissues or paraffin-embedded archival specimens as template, polymerase chain reaction (PCR) was performed to generate amplified DNA fragments of exon 1 of the X-linked androgen receptor gene, which contains a highly polymorphic trinucleotide repeat. Predigestion of tumor DNA with methylation-sensitive restriction endo-nuclease Hha I or Hpa II permitted selective PCR amplification from the methylated (uncleaved) allele. Of a total of 54 tumors analyzed, 50 cases showed heterozygosity (939r) and were therefore informative for clonal analysis. Monoclonal composition of the tumors was suggested in a total of 49 of 50 cases, including 12 adenocarcinomas of the uterine endometrium, 13 squamous cell carcinomas of the uterine cervix, 6 adenocarcinomas of the uterine en-docervix, and 18 epithelial tumors of the ovary. However, polyclonal composition was observed in one mucinous carcinoma of the ovary, in which we previously showed that both GGT—GAT and GGT—>GTT mutations are present in >209f of total K-rax copies in the tissue. Our studies demonstrate the utility of PCR amplification of highly polymorphic repetitive sequences for analysis of patterns of X-chromosome inactivation. This approach is practical for the analysis of clonal cell composition in a high proportion of both formalin-fixed and frozen archival tissues.