Purpose: We aim to develop a digital PCR-based method for the quantitative detection of the two common epidermal growth factor receptor (EGFR) mutations (in-frame deletion at exon 19 and L858R at exon 21) in the plasma and tumor tissues of patients suffering from non-small cell lung cancers. These two mutations account for >85% of clinically important EGFR mutations associated with responsiveness to tyrosine kinase inhibitors. Experimental Design: DNA samples were analyzed using a microfluidics system that simultaneously performed 9,180 PCRs at nanoliter scale. A single-mutant DNA molecule in a clinical specimen could be detected and the quantities of mutant and wild-type sequences were precisely determined. Results: Exon 19 deletion and L858R mutation were detectable in 6 (17%) and 9 (26%) of 35 pretreatment plasma samples, respectively. When compared with the sequencing results of the tumor samples, the sensitivity and specificity of plasma EGFR mutation analysis were 92% and 100%, respectively. The plasma concentration of the mutant sequences correlated well with the clinical response. Decreased concentration was observed in all patients with partial or complete clinical remission, whereas persistence of mutation was observed in a patient with cancer progression. In one patient, tyrosine kinase inhibitor was stopped after an initial response and the tumor-associated EGFR mutation reemerged 4 weeks after stopping treatment. Conclusion: The sensitive detection and accurate quantification of low abundance EGFR mutations in tumor tissues and plasma by microfluidics digital PCR would be useful for predicting treatment response, monitoring disease progression and early detection of treatment failure associated with acquired drug resistance.

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Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorageindependent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer.

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