Improved Breast Cancer Detection Using a Novel In Situ Method to Visualize Clonality

摘要

We report the successful development of a sensitive clonality assay. The assay is based on a novel method we have developed termed 'biolithography' which applies photolithography, a process known in the semiconductor industry, to biologic material. Biolithography uses diazoquinone photoresist and targeted light irradiation to isolate DNA from selected cell populations under direct microscopic visualization with 100% specificity and sensitivity at the limit of PCR. Using biolithography, we have detected clonality from as few as 20 neoplastic cells. We have also discovered abnormal demethylation of the X chromosome at the human androgen receptor and PGK genes in several cases of breast carcinoma. Microsatellite analysis of markers closely linked to genes involved in X chromosome inactivation has identified loss of heterozygosity which correlates with demethylation. Consistent with the stepwise model of tumor progression, demethylation of both X chromosomes may reflect abnormal activation of X chromosomes. These studies demonstrate the utility of biolithography to discern subtle molecular changes in solid tumors which may exist in only microscopic regions. Efforts have also been made to increase the throughput of biolithography for potential translation to the clinical laboratory. Investigations of photoresists with ultra-high photosensitivity and laser driven systems are encouraging, and suggest the possibility of high-throughput systems. Biolithography may thus provide new opportunities for molecular diagnostics of solid tumors.