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.

Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

Purpose Mutations in the estrogen receptor-alpha (ER) gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Due to limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from metastatic breast cancer patients. Experimental Design We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild type ESR1 identified by next generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital polymerase chain reaction (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS and ptDNA ESR1 mutations were analyzed by ddPCR. Results In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in six of twelve patients (50%). Conclusions We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion.

Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as "negative" by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them. A great success in the treatment of breast cancer has come from the identification of human epidermal growth factor receptor 2 (HER2)/Neu (ERBB2) amplification/overexpression as a targetable driver in ∼20% of breast cancers (1). HER2 is a member of the ErbB family of transmembrane receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ErbB1), HER3 (ErbB3), and HER4 (ErbB4) (2). Activation of ErbB signaling causes receptor tyrosine autophosphorylation and induces interactions with cytoplasmic signal transduction partners that promote a wide variety of cellular processes including proliferation, motility, and escape from apoptosis. In addition to their key role in normal cellular growth and maintenance, the dysregulation of ErbB receptors has been extensively implicated in the development of numerous cancers (1).Whereas overexpression or amplification of HER2 has been well described to deregulate ErbB signaling, cancer genome sequencing studies have demonstrated that somatic point mutations in the HER2 gene occur in a number of cancers, including 2-4% of breast cancers (3-6). Importantly, these mutations are most often found in patients as single copies without amplification/overexpression of HER2 (HER2-"negative" breast cancers), though HER2 protein expression is often still present. Overexpression studies have implicated a number of these mutations as activating and oncogenic (4, 7-9). Additionally, one mutation, L755S, has been described to be associated with lapatinib resistance when overexpressed (4, 10).Past studies comparing overexpression of a mutant cDNA to single nucleotide knockin of mutant oncogenes have shown dramatic differences ...