No abstract

Purpose Detecting circulating plasma tumor DNA (ptDNA) in early stage cancer patients 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 post-surgery blood samples were collected from early stage breast cancer patients (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. Pre-surgery plasma samples (n=29) were then analyzed for PIK3CA mutations using ddPCR. Of the fifteen PIK3CA mutations detected in tumors by ddPCR, fourteen of the corresponding mutations were detected in pre-surgical ptDNA, while no mutations were found in plasma from patients with PIK3CA wild type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation positive ptDNA pre-surgery had ddPCR analysis of post-surgery plasma, with five patients having detectable ptDNA post-surgery. 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 early stage breast cancer patients. 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.

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 GSK3␤ phosphorylation. Paradoxically, the GSK3␤ 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 GSK3␤ 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 GSK3␤ 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.GSK3␤ ͉ lithium ͉ mTOR ͉ phosphatidylinositol 3-kinase ͉ cancer