Infection with oncogenic strains of human papillomavirus
(HPV),
such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis.
As an adjunct to traditional invasive tissue sampling methods, the
use of modern thermostable enzyme chemistries can aid in the development
of innovative assay workflows to extract and detect circulating HPV
DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully
generated a model system to replicate fragmented cHPV-DNA in human
plasma. Using this model system, we designed a novel thermostable
enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening
and robustly evaluated its analytical assay performance. Our findings
demonstrated that the use of thermostable enzymes provided faster
cHPV-DNA extraction and amplification, leading to an overall three-fold
improvement in overall assay time as compared to the current standard
assay workflow and achieving clinically relevant levels of analytical
specificity, sensitivity, and precision for accurate cHPV-DNA detection
with excellent 100% sensitivity and specificity in contrived human
plasma specimens. In summary, we have devised a rapid laboratory workflow
to facilitate the emerging use of liquid biopsies for minimally invasive,
rapid, and scalable HPV DNA testing. With facile assay modifications,
our thermostable enzyme-based cHPV-DNA assay can be utilized for the
detection of other clinically high-risk HPV genotypes.
e15034 Background: Identification of somatic variants in cancer by high-throughput sequencing has become common clinical practice largely because many of these variants may be predictive biomarkers for targeted therapies. However, there can be high sample QC failure rates for some assays (sometimes up to 40%), preventing the return of results that may affect patient treatment decisions. Pillar Biosciences has incorporated their patented SLIMamp technology into commercially available cancer NGS testing kits with the claim that these kits can successfully interrogate challenging formalin-fixed paraffin-embedded tissue (FFPET) samples with low tumor purity, poor DNA quality, and/or low input DNA, resulting in a high sample QC pass rate. The aim of this study was to substantiate that claim using Pillar’s amplicon-based oncoReveal Solid Tumor Panel. Methods: We acquired 48 tumor samples that had failed one or more pre-analytical QC sample parameters for whole exome sequencing (WES) from ATGC’s ISO15189 accredited diagnostic genomics laboratory. XING Genomic Services performed an exploratory data analysis using our pre-analytical QC assays to characterise the samples and then sequenced the samples in our ISO15189 accredited laboratory using the validated oncoReveal Solid Tumor Panel. Results: We were able to achieve high sequencing coverage (>3000X) for all 48 samples and explored the determinants of sample “success”. We were able to generate clinical reports for 45 samples (94%), of which 38 (79%) contained clinically actionable or significant variants that would not have otherwise been identified. Ten samples had a higher number of total variant calls with over-representation of C>T transitions representing stochastically-amplified, formalin-induced artefacts in samples with very low input template DNA. Of these, 7 cases had reportable variants and 3 were deemed unreportable. We demonstrated that DNA integrity is the major determinant of success even in samples with low input DNA or low tumor purity and were able to further refine pre-analytical and post-analytical QC metrics to better identify samples with poor quality DNA that can be sequenced reliably. Conclusions: In this study, we showed that the Pillar Biosciences oncoReveal Solid Tumor Panel, which uses SLIMam technology, was able to generate reliable, interpretable results for 94% of samples that failed pre-analytical QC for WES, substantiating Pillar’s claim.
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