Technical Advance Whole Genome Amplification of Plasma-Circulating DNA Enables Expanded Screening for Allelic Imbalance in Plasma (2006)

Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments ( 5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified doublestranded DNA was blunted with T4 DNA polymerase, selfligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by 1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens. (J Mol Diagn 2006, 8:22–30; DOI: 10.2353/jmoldx.2006.050074) Solid malignant tumors release a significant amount of genomic DNA into the systemic circulation through cellular necrosis and apoptosis, and this circulating DNA can be exploited as an accessible tumor-specific biomarker. Tumor-released DNA can be detected as a result of specific genetic changes, including mutations, methylation, translocation, presence of viral genes, or allelic imbalance (AI), a marker for potential deletion of tumor suppressor genes. For example in breast cancer, tumor-released DNA in plasma provides a useful biomarker for breast cancer diagnosis and for monitoring relapse and metastasis. AI in particular is present in the majority of primary breast tumors and is also encountered in the plasma of breast cancer patients. In the great majority of cases, AI in plasma corresponds to alterations in the primary tumor of the respective patient. Plasma-circulating tumor DNA at diagnosis of breast cancer is a predictor of disease-free survival and correlates with clinicopathological features and disease stage. Although genetic alterations such as AI in plasmacirculating DNA have been shown to have clinical utility as biomarkers for early cancer detection or therapy monitoring, there are technical difficulties limiting their widespread application to cancer screening. Because of the genetic heterogeneity of tumors, no single biomarker is present in 100% of primary tumors. Further, plasmacirculating DNA of tumor origin often contains only a fraction of the primary tumor alterations that may reflect the existence of different clones within the same tumor. In addition, plasma-circulating DNA may contain normal DNA, which complicates detection of tumorspecific genetic changes. To increase sensitivity and