Feasibility of a Novel Non-Invasive Swab Technique for Serial Whole-Exome Sequencing of Cervical Tumors During Chemoradiation Therapy

BACKGROUND: Clinically relevant genetic predictors of radiation response for cervical cancer are understudied due to the morbidity of repeat invasive biopsies required to obtain genetic material. Thus, we aimed to demonstrate the feasibility of a novel noninvasive cervical swab technique to (1) collect tumor DNA with adequate throughput to (2) perform whole-exome sequencing (WES) at serial time points over the course of chemoradiation therapy (CRT). METHODS: Cervical cancer tumor samples from patients undergoing chemoradiation were collected at baseline, at week 1, week 3, and at the completion of CRT (week 5) using a noninvasive swab-based biopsy technique. Swab samples were analyzed with whole-exome sequencing (WES) with mutation calling using a custom pipeline optimized for shallow whole-exome sequencing with low tumor purity (TP). Tumor mutation changes over the course of treatment were profiled. RESULTS: 216 samples were collected and successfully sequenced for 70 patients (94% of total number of tumor samples collected). A total of 33 patients had a complete set of samples at all four time points. The mean mapping rate was 98% for all samples, and the mean target coverage was 180. Estimated TP was greater than 5% for all samples. Overall mutation frequency decreased during CRT but mapping rate and mean target coverage remained at \u3e98% and \u3e180 reads at week 5. CONCLUSION: This study demonstrates the feasibility and application of a noninvasive swab-based technique for WES analysis which may be applied to investigate dynamic tumor mutational changes during treatment to identify novel genes which confer radiation resistance

MicroRNA-363 targets myosin 1B to reduce cellular migration in head and neck cancer.

BackgroundSquamous cell carcinoma of the head and neck (SCCHN) remains a prevalent and devastating disease. Recently, there has been an increase in SCCHN cases that are associated with high-risk human papillomavirus (HPV) infection. The clinical characteristics of HPV-positive and HPV-negative SCCHN are known to be different but their molecular features are only recently beginning to emerge. MicroRNAs (miRNAs, miRs) are small, non-coding RNAs that are likely to play significant roles in cancer initiation and progression where they may act as oncogenes or tumor suppressors. Previous studies in our laboratory showed that miR-363 is overexpressed in HPV-positive compared to HPV-negative SCCHN cell lines, and the HPV type 16-E6 oncoprotein upregulates miR-363 in SCCHN cell lines. However, the functional role of miR-363 in SCCHN in the context of HPV infection remains to be elucidated.MethodsWe analyzed miR-363 levels in SCCHN tumors with known HPV-status from The Cancer Genome Atlas (TCGA) and an independent cohort from our institution. Cell migration studies were conducted following the overexpression of miR-363 in HPV-negative cell lines. Bioinformatic tools and a luciferase reporter assay were utilized to confirm that miR-363 targets the 3'-UTR of myosin 1B (MYO1B). MYO1B mRNA and protein expression levels were evaluated following miR-363 overexpression in HPV-negative SCCHN cell lines. Small interfering RNA (siRNA) knockdown of MYO1B was performed to assess the phenotypic implication of reduced MYO1B expression in SCCHN cell lines.ResultsMiR-363 was found to be overexpressed in HPV-16-positive compared to the HPV-negative SCCHN tumors. Luciferase reporter assays performed in HPV-negative JHU028 cells confirmed that miR-363 targets one of its two potential binding sites in the 3'UTR of MYO1B. MYO1B mRNA and protein levels were reduced upon miR-363 overexpression in four HPV-negative SCCHN cell lines. Increased miR-363 expression or siRNA knockdown of MYO1B expression reduced Transwell migration of SCCHN cell lines, indicating that the miR-363-induced migration attenuation of SCCHN cells may act through MYO1B downregulation.ConclusionsThese findings demonstrate that the overexpression of miR-363 reduces cellular migration in head and neck cancer and reveal the biological relationship between miR-363, myosin 1b, and HPV-positive SCCHN

Top 50 gene alterations over time for 33 patients with all four-time points.

Heat map displaying the top 50 genes ranked by occurrence for 33 patients (132 samples) grouped by timepoint collection during chemoradiation.</p

Patient characteristics.

Patient characteristics.</p

Gene list with alteration type and frequency for all samples.

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Computational pipeline for whole exome sequencing data.

Workflow depicting preprocessing, variant calling and data analysis tools and parameters implemented to analyze WES data acquired from tumor DNA collected by cervical swab.</p

Top 50 gene alterations over time for 70 patients with paired normals.

Heat map displaying the top 50 genes ranked by occurrence for 70 patients (216 samples) grouped by timepoint collection during chemoradiation.</p

Overall gene alterations from swab acquired tumor samples (patients 2–30) is similar to landscape of TCGA cervical squamous cell carcinoma dataset.

(A) Ninety-four percent (1339/1430) of altered genes in baseline samples (defined as substitutions, insertions or deletions in gene) were also identified in the TCGA dataset, suggesting accurate identification of mutated genes related to cervical cancer. (B) The distribution of the top 30 most altered genes in study samples 2–30 and in TCGA(C) was also similar. (TIF)</p

CONSORT 2010 checklist of information to include when reporting a pilot or feasibility trial*.

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