Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Precision Radiotherapy: Reduction in Radiation for Oropharyngeal Cancer in the 30 ROC Trial.

BACKGROUND: Patients with human papillomavirus-related oropharyngeal cancers have excellent outcomes but experience clinically significant toxicities when treated with standard chemoradiotherapy (70 Gy). We hypothesized that functional imaging could identify patients who could be safely deescalated to 30 Gy of radiotherapy. METHODS: In 19 patients, pre- and intratreatment dynamic fluorine-18-labeled fluoromisonidazole positron emission tomography (PET) was used to assess tumor hypoxia. Patients without hypoxia at baseline or intratreatment received 30 Gy; patients with persistent hypoxia received 70 Gy. Neck dissection was performed at 4 months in deescalated patients to assess pathologic response. Magnetic resonance imaging (weekly), circulating plasma cell-free DNA, RNA-sequencing, and whole-genome sequencing (WGS) were performed to identify potential molecular determinants of response. Samples from an independent prospective study were obtained to reproduce molecular findings. All statistical tests were 2-sided. RESULTS: Fifteen of 19 patients had no hypoxia on baseline PET or resolution on intratreatment PET and were deescalated to 30 Gy. Of these 15 patients, 11 had a pathologic complete response. Two-year locoregional control and overall survival were 94.4% (95% confidence interval = 84.4% to 100%) and 94.7% (95% confidence interval = 85.2% to 100%), respectively. No acute grade 3 radiation-related toxicities were observed. Microenvironmental features on serial imaging correlated better with pathologic response than tumor burden metrics or circulating plasma cell-free DNA. A WGS-based DNA repair defect was associated with response (P = .02) and was reproduced in an independent cohort (P = .03). CONCLUSIONS: Deescalation of radiotherapy to 30 Gy on the basis of intratreatment hypoxia imaging was feasible, safe, and associated with minimal toxicity. A DNA repair defect identified by WGS was predictive of response. Intratherapy personalization of chemoradiotherapy may facilitate marked deescalation of radiotherapy.

Detection of Early Human Papillomavirus-Associated Cancers by Liquid Biopsy.

PURPOSE: A circulating tumor DNA (ctDNA) test to detect plasma Epstein-Barr viral DNA can be used to screen for early nasopharyngeal cancers; however, the reported sensitivity of viral ctDNA tests to detect human papillomavirus (HPV)-associated cancers is modest. We assessed the utility of droplet digital polymerase chain reaction (ddPCR) to detect early-stage HPV-associated cancers using sequential HPV16 and HPV33 assays that account for HPV subtype distribution and subtype sequence variants. PATIENTS AND METHODS: We collected plasma specimens from 97 HPV-positive patients with oropharyngeal squamous cell carcinoma and eight patients with HPV-positive anal squamous cell carcinoma, each with locoregionally confined disease. Negative controls included samples from seven patients with HPV-negative head and neck cancers and 20 individuals without cancer. RESULTS: Of 97 patients with nonmetastatic, locoregionally confined oropharyngeal squamous cell carcinoma, 90 patients had detectable HPV16 ctDNA and three patients had HPV33 ctDNA, indicating an overall sensitivity of 95.6%. Seven of eight patients with early anal cancer were HPV16 ctDNA positive. No HPV ctDNA was detected in 27 negative controls, indicating 100% specificity. HPV16 ctDNA was detected in 19 of 19 patients with low-volume disease, defined as patients with a single, asymptomatic positive lymph node (N1) or an isolated T1-2 asymptomatic primary tumor. HPV16 ctDNA levels directly corresponded to tumor responses to chemoradiation and surgery. CONCLUSION: With an updated understanding of HPV subtypes and sequence variation, HPV ctDNA by ddPCR is highly sensitive and specific, identifying HPV16 and HPV33 subtypes in a similar distribution as reported in major genomic profiling studies. The detection of small tumors indicates that HPV16 and HPV33 ctDNA ddPCR could be readily used in early detection screening trials and in disease response monitoring, analogous to Epstein-Barr virus DNA.