Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.

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.

IMPORTANCETherapies targeting the programmed cell death 1 (PD-1) receptor or its ligand (PD-L1), such as the humanized monoclonal antibody durvalumab, have shown durable clinical responses in several tumor types. However, concerns about the safety and feasibility of PD-1/PD-L1 blockade in HIV-1-infected individuals have led to the exclusion of these patients from clinical trials on cancer immunotherapies. OBJECTIVE To evaluate the feasibility and safety of durvalumab treatment in patients with advanced cancer and virologically controlled HIV-1 infection. DESIGN, SETTING, AND PARTICIPANTSThe DURVAST study was a nonrandomized, open-label, phase 2 clinical trial in patients with any solid tumor type in which anti-PD-1 or anti-PD-L1 antibodies have approved indications or for which there are data of antitumoral activity with no other available curative therapy. All patients had basal undetectable plasma viremia while undergoing combination antiretroviral therapy.INTERVENTIONS Treatment consisted of intravenous infusion of durvalumab (1500 mg every 4 weeks) until disease progression or unacceptable toxic effects. MAIN OUTCOMES AND MEASURESAdverse events were graded with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Tumor response was evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1.RESULTS A total of 20 HIV-1-infected patients with advanced cancer were enrolled; 16 (80%) were male, the median (range) age was 54 (30-73) years, and 12 (60%) had progressed with previous cancer treatment lines. A median (range) of 4 (1-16) cycles of durvalumab were administered. Drug-related adverse events were observed in 50% of patients, and all were grade 1 and 2 (mainly diarrhea, asthenia, and arthromyalgia). Four of 16 response-evaluable patients (25%) had a partial response. Five patients (31%) had stable disease, including 4 with durable stable disease (disease control rate of 50%). CD4 + and CD8 + T-cell counts and plasma HIV-1 viremia remained stable throughout the study.CONCLUSIONS AND RELEVANCE Durvalumab treatment was feasible and safe in HIV-1-infected patients with cancer receiving combination antiretroviral therapy. HIV-1-infected patients on suppressive antiretroviral therapy with advanced cancer should have access to cancer immunotherapy treatments.

Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centred at ~50 bp. We show that these 'ultrashort' cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.

Our study provides a clinically applicable assay that links PD1 mRNA abundance, activated CD8 T-cells and anti-PD1 efficacy.