High Sensitivity Detection of Tumor Gene Mutations

It is widely accepted that cancer develops as a results of mutations in cell growth pathway related genes particularly those mutations that occur in growth factor receptor pathways such as the cell surface Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) pathways and in tumor suppressor genes such as p53, APC and PTEN. Detection of these mutations is critical not only for early detection of cancerous cells but also for decisions on what therapy should be applied to treat the patient. For example, mutations in the intracellular downstream signaling proteins rat sarcoma viral oncogenes KRAS, NRAS and HRAS can produce resistance to a particular therapeutic agent. In this context of ‘Precision Medicine’ tumor gene mutations have classically been measured in the DNA isolated from formalinfixed paraffin embedded (FFPE) tumor biopsy tissue. However, surgical tumor biopsies are not only invasive and risky, but are extremely difficult for inaccessible and fragile organs such as the lungs. A recent study confirmed that this standard prognostic procedure is woefully inadequate [1]. A localized tumor biopsy could miss mutations in a distal region of the tumor that might radically change a person’s chances for survival. And although biopsies can provide data about specific mutations that might make a tumor vulnerable to targeted therapies, that information is static and bound to become inaccurate as the cancer evolves. Minimally invasive procedures such as taking blood is simple in comparison, urine sampling is even simpler. Several groups have reported that the mutational landscape of a patient’s tumor can be measured simply by monitoring the mutational status of the circulating cell-free tumor DNA (ctDNA) in the patient’s blood. However, this requires a highly sensitive technique and to date most large oncology research centers have resorted to BEAMing PCR2 to achieve this. This is partly because tumor DNA is much harder to detect in the circulation due to the large excess of wildtype DNA. There is typically less of it in the blood. In people with very advanced cancers, tumors might be the source of most of the circulating DNA in the blood, but more commonly, ctDNA makes up barely 1% of the total and possibly as little as 0.01%. Several groups in recent years have reported using ctDNA to study patients who were being treated with EGFR inhibitors. Looking for example for known KRAS mutations that confer resistance; or for mutations that prevent drugs from binding to their target [3-5].