Extracellular Vesicle-Based Noninvasive Detection of PIK3CA Compound Mutation for Breast Cancer

There are multiple subtypes of breast cancer. Twelve percent to fifteen percent of women with advanced hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer harbor two mutations in a single gene called PIK3CA. However, the 5-year relative survival of metastatic HR+/HER2- breast cancer is only 30.4%. Since cancer genomes have different copies of this gene on different chromosomes, it is important to know if these mutations are on the same chromosome. If two mutations lie in the same PIK3CA copy (called compound PIK3CA mutations or double PIK3CA mutation in cis) -- on the same chromosome -- then they will make a protein that has two simultaneous alterations. If they lie on different copies -- on different chromosomes (called double PIK3CA mutations in trans) -- they will make two different proteins, each with only one alteration. It was recently discovered that patients that have two PIK3CA mutations in cis (cis-PIK3CA mutations) that make two alterations in the same protein are very responsive to a drug called a PI3K inhibitor. Women with double-PIK3CA-cis-mutant tumors who took alpelisib (PIQRAY) lived longer lives without their disease progressing. However, there is no clinical diagnostic technology available to detect these mutations in this double mutation configuration. Liquid biopsies are an important potential method to create a diagnostic for these cis-PIK3CA mutations. Liquid biopsies allow for non-invasive genetic testing when tissue biopsies are scarce or unreachable. This means that patients can get diagnosed with a simple blood draw instead of a surgical procedure. The targeted therapy PI3K inhibitor for cis-PIK3CA mutations finding clarifies the need for a liquid biopsy method to detect these cis-PIK3CA mutations. However, this is a large technical challenge because the mutations are located far apart on DNA or RNA, and the difficulty of the diagnosis increases as the distance on the DNA or RNA increases.

This long distance is uniquely suited to one liquid biopsy approach: Extracellular vesicles (EVs). EVs 'bud' off cancer cells and find their way into the blood stream. These tiny bits of cancer cells harbor cancer genomic DNA and RNA at sizes that are big enough in which to find these cis-PIK3CA mutations. But, these cancer EVs are diluted by the EVs that can bud off normal cells into the blood. This requires us to invent a new ultra-sensitive approach to measure these distant cis-PIK3CA mutations in EV DNA or RNA. The overarching challenge of this proposal is to enable rapid liquid biopsies of breast tumors from plasma using the unique Lipid NanoProbe (LNP) method of extracellular vesicle isolation AND measure cis-PIK3CA mutations despite the contamination by normal EVs. We anticipate those breakthrough technologies will enable significant impact in basic research and clinical translational advancement. Firstly, this liquid biopsy diagnostic technology will help millions of women with breast cancer and their physicians decide if alpelisib is right for them. Finally, measuring two mutations on the same piece of DNA at long distances, in the presence of lots of contaminating DNA, is very challenging. Our techniques will be applicable to other genes and other cancers. Thus, creating this diagnostic technology for breast cancer will directly help patients get the right treatment, and the technology will find broad use in other cancer and basic research.