Serum, Cell-Free, HPV-Human DNA Junction Detection and HPV Typing for Predicting and Monitoring Cervical Cancer Recurrence

Reviewer #1 (Public review):

Van Arsdale and colleagues evaluated whether human-HPV DNA junctions could be detected in serum, cell-free DNA from 16 patients with cervical cancer by hybrid capture and Illumina sequencing. Junctions were identified in seven patients, and these junctions were concordant with junctions identified in tumor DNA except for one patient, suggesting that, in most cases, the cfDNA is originating from a clone of the primary tumor. Junction detection at 6 months was found to be statistically significant prognostic for recurrence. The study further validates that type-specific E7 DNA, which is essential for tumorigenesis, was detectable by PCR for most patient sera, but had no association with recurrence. Furthermore, the study provides additional evidence that tumors harboring non-alpha-9 clade HPVs had shorter recurrence-free survival and overall worse outcome from the study's patients, as well as reanalysis of TCGA data. However, these findings need to be more extensively discussed in the context of previous publications. One identified limitation of this approach is the detection of non-tumor HPVs, but this was only seen in one patient. The major shortcoming of this study is the limited number of patients that were evaluated, but for a retrospective study, this is a reasonable number of patients evaluated, and the findings are appropriately not overstated. The design, execution, and detailed analysis of the sequencing data are a major strength. This study provides important foundational evidence for further evaluating the clinical utility of HPV DNA detection from cfDNA and specifically assessing for integration junctions.

Reviewer #2 (Public review):

Summary:

The authors set out to identify cell-free HPV breakpoint junctions and assess their utility in identifying cervical cancer recurrence as a surrogate, tumor-specific assay. They added unrelated findings about a potential relationship between various viral types and cancer recurrence frequencies, concluding that clade alpha 9 types recurred at a lower rate than did non-alpha 9 viral types.

Strengths:

The authors analyzed 16 cervical cancer samples and matched serum samples collected initially or upon clinical treatments. An association between virus types and cancer recurrence frequencies is a novel finding that will likely induce further insights into HPV pathogenic mechanisms.

Weaknesses:

The main claims of this manuscript are only partially supported by the data as presented, because the sequencing data are not quantified and were not analyzed in a statistically adequate way. First, only one or at most two breakpoints are presented per tumor (Table 1). This finding is discrepant from many extensive, published genomics studies of HPV-positive cancers, in which many unique breakpoints are found frequently in individual cancers, ranging from 1 or 2 up to more than 100. Second, no information is provided about likely correlations between genomic DNA copy number at rearranged loci and breakpoint-identifying sequencing read counts. Third, no direct comparison is presented between supporting read counts from cancer samples and read counts from circulating cell-free DNA samples. Fourth, many of the initial cancer samples harbored no insertional breakpoints, so no correlation with breakpoints in the serum samples would be possible. Fifth, no mention was made about tumor heterogeneity, where a given breakpoint may not be present in every cell of the tumor. Previous literature about the general topic of using cell-free DNA breakpoints as a surrogate for cancer cells is not cited adequately. Findings about potential correlations between various viral types and variable recurrence rates are not well-supported by the authors' own data, because of the limited sample numbers studied. This section of the paper is relatively unrelated to the main thrust, which is about breakpoint detection.