Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on sensitivity for detecting genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for non-invasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 cancer patients using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90–150 bp, and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90–150 bp improved detection of tumor DNA, with more than 2-fold median enrichment in >95% of cases, and more than 4-fold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with AUC>0.99 compared to AUC<0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC>0.91, compared to AUC<0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cell-free DNA for clinical applications, earlier diagnosis and study of tumor biology.

Purpose: We evaluated the clinical benefit of tumor molecular profiling to select treatment in the phase I setting. Experimental Design: Patients with advanced solid cancers and exhausted treatment options referred to a phase I unit were included in a prospective, single-center, singlearm open-label study (NCT02290522). Tumor biopsies were obtained for comprehensive genomic analysis including whole-exome sequencing and RNA sequencing. When possible, patients were treated with regimen matched to the genomic profile. Primary endpoint was progression-free survival (PFS). Results: From May 2013 to January 2017, a total of 591 patients were enrolled, with 500 patients undergoing biopsy. Genomic profiles were obtained in 460 patients and a potential actionable target was identified in 352 (70%) of 500 biopsied patients. A total of 101 patients (20%) received matched treatment based on either gene mutations or RNA expression levels of targets available in early clinical trials or off-label treatment. Objective response according to RECIST1.1 was observed in 15 of 101 patients (0% complete response, 15% partial response), with a median PFS of 12 weeks (95% confidence interval, 9.9-14.4). Conclusions: Our study supports the feasibility of genomic profiling to select patients in the phase I setting and suggests that genomic matching can be beneficial for a minor subset of patients with no other treatment options. Randomized studies may validate this assumption. See related commentary by Ratain, p. 1136

The detection of HPV DNA in MGG-stained FNA smears by PCR is a valid method that could be implemented in routine clinical practice. Cancer Cytopathol 2016;124:820-7. © 2016 American Cancer Society.