Michaela J. Higgins

PURPOSE: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. EXPERIMENTAL DESIGN: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. RESULTS: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. CONCLUSIONS: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients.

In recent years the description of well-defined molecular subtypes of breast cancer, together with the identification of the driving genetic alterations and signaling pathways, has led to the clinical development of a number of successful molecular targeted agents. This is best exemplified in the subset of HER2-amplified breast cancers, in which an increasing number of active agents are changing the natural history of this aggressive disease. Other targets are under exploration, and the clinical development of these agents will require a change from the current large, randomized trials in unselected patient populations to smaller trials in groups with a molecularly defined tumor type. In addition, combinatorial approaches that act on the secondary mutations and/or compensatory pathways in resistant tumors may markedly improve on the effects of targeted agents used alone.

PURPOSE: We sought to evaluate the feasibility of detecting PIK3CA mutations in circulating tumor DNA (ctDNA) from plasma of patients with metastatic breast cancer using a novel technique called BEAMing. EXPERIMENTAL DESIGN: In a retrospective analysis, 49 tumor and temporally matched plasma samples from patients with breast cancer were screened for PIK3CA mutations by BEAMing. We then prospectively screened the ctDNA of 60 patients with metastatic breast cancer for PIK3CA mutations by BEAMing and compared the findings with results obtained by screening corresponding archival tumor tissue DNA using both sequencing and BEAMing. RESULTS: The overall frequency of PIK3CA mutations by BEAMing was similar in both patient cohorts (29% and 28.3%, respectively). In the retrospective cohort, the concordance of PIK3CA mutation status by BEAMing between formalin-fixed, paraffin-embedded (FFPE) samples and ctDNA from temporally matched plasma was 100% (34 of 34). In the prospective cohort, the concordance rate among 51 evaluable cases was 72.5% between BEAMing of ctDNA and sequencing of archival tumor tissue DNA. When the same archival tissue DNA was screened by both sequencing and BEAMing for PIK3CA mutations (n = 41 tissue samples), there was 100% concordance in the obtained results. CONCLUSIONS: Analysis of plasma-derived ctDNA for the detection of PIK3CA mutations in patients with metastatic breast cancer is feasible. Our results suggest that PIK3CA mutational status can change upon disease recurrence, emphasizing the importance of reassessing PIK3CA status on contemporary (not archival) biospecimens. These results have implications for the development of predictive biomarkers of response to targeted therapies.

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to "knock in" PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3beta phosphorylation. Paradoxically, the GSK3beta inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3beta target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3beta is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.