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Total amount of cfDNA extracted from each patient sample

Available therapies and clinical trials

Allelic fractions for all variants detected in tDNA and ctDNA* in patients for whom both plasma and tissue samples were obtained (n=50)

Summary of driver and resistance mutations in EGFR and ALK detected in ctDNA, tDNA, and both ctDNA and tDNA

Tissue next-generation sequencing gene coverage. A: Illumina TruSeq panel (47 genes), and B: Penn Precision Panel (20 genes)

Analysis of discordant EGFR driver and resistance mutations detected in tDNA or ctDNA

All mutations detected in patient ctDNA (n=102)

Circulating tumor DNA next-generation sequencing gene coverage

Purpose: The expanding number of targeted therapeutics for non–small cell lung cancer (NSCLC) necessitates real-time tumor genotyping, yet tissue biopsies are difficult to perform serially and often yield inadequate DNA for next-generation sequencing (NGS). We evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS.Experimental Design: A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of up to 70 genes and matched with tissue samples, when possible.Results: We detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible.Conclusions: Therapeutically targetable driver and resistance mutations can be detected by ctDNA NGS, even when tissue is unavailable, thus allowing more accurate diagnosis, improved patient management, and serial sampling to monitor disease progression and clonal evolution. Clin Cancer Res; 22(23); 5772–82. ©2016 AACR.

Previous evaluation of HER2 overexpression in salivary gland cancers indicated an incidence varying between 7 and 56%, with no clear difference among three histologically different subtypes. As part of a Phase II trial of trastuzumab for treatment of incurable salivary gland cancer, we screened 137 tumors for HER2 expression.Unstained sections of paraffin-embedded tumor samples were stained with p185/HER2 receptor antibody. Tumors with moderate (2+) to strong (3+) complete membrane staining in at least 10% of the tumor cells were scored as positive for overexpression.The overall frequency of overexpression for HER2 was 17% (23 of 137), whereas it was only 8% in the three most common histological subtypes screened. Overexpression was distinctly rare in the most common subtype screened, adenoid cystic carcinoma (4%, 3 of 70). Overexpression was very common in salivary duct cancers; 10 (83%) of 12 were positive for HER2. This observation is consistent with the typical high-grade histological features and aggressive behavior of this subtype as well as with its histogenetic similarity to breast cancer. Analysis based on histogenesis (intercalated duct versus excretory duct) indicated a higher frequency of overexpression in the latter (55%) than in the former (7%).Our overall results suggest that trastuzumab will not have a major role in treatment of salivary gland cancers of intercalated duct origin. Further systematic evaluation of trastuzumab in subtypes of excretory duct origin could be supported.