22: Q&A

Anal squamous cell cancer (ASCC), an HPV-associated malignancy, is one of few cancers in the US that is increasing in incidence. Pathogenesis of ASCC is well described with progression from normal mucosa (NM) through stages of anal intraepithelial neoplasia (AIN), and is thought to be driven by the HPV E6 and E7 viral oncogenes. As most AIN3 do not progress to ASCC, biomarkers to identify high-risk AIN3 requiring intervention would be valuable. Using next generation RNA sequencing (RNA-Seq), we sought to identify patterns of differential gene expression across the spectrum of ASCC progression with the goal of identifying potential biomarkers for selective management of AIN3.

 


Methods:
Forty formalin-fixed paraffin embedded samples [14 ASCC, 17 AIN3, 9 normal mucosa (NM)] were obtained. Histologic diagnoses were confirmed, tissues microdissected, and RNA was extracted and purified. RNA was sequenced using the Illumina TruSeq RNA Exome platform.  Multi-step bioinformatics methodology was used to 1) generate principal component analysis (PCA) models 2) model differential expression of genes between normal, AIN3 and tumor samples via Partial Least Squares (PLS) and volcano plots and 3) identify pathways most commonly affected by differential expression across ASCC development.



Results:
As expected, PCA demonstrated clear 2-dimensional separation between NM and ASCC specimens. Gene expression in AIN3 trended toward that of NM samples. Significant differential expression was noted between NM, AIN3, and ASCC by volcano plot analysis. Of 1126 selected genes, 118 probes were significant for differential expression between AIN3 and ASCC with 50 discrete genes clustering AIN3 to ASCC.



Conclusions:
We have demonstrated clear differential gene expression between NM, AIN3, and ASCC. Furthermore, from a large panel of probes, we have identified a small subset of genes with discrete shared expression between some AIN3 and ASCC. Our findings lay the framework for further characterization to elucidate shared differentially expressed genes that may serve as biomarkers to identify those AIN3 lesions most likely to progress to ASCC, and thus may benefit from selective ablation.