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RNAscope®, has emerged as an important in-situ hybridisation method to validate mRNA expression within single cells whilst preserving tissue morphology in histological samples. The aim of this research was to compare the utility of various open-source and commercial image analysis methods, to quantify mRNA transcripts identified by RNAscope within formalin fixed paraffin embedded (FFPE) histological samples and cell monolayer preparations. Examination of MLH1 expression from 10 histological FFPE colorectal cancer specimens using four image analysis tools (Colour Deconvolution, SpotStudio, WEKA and the LEICA RNA-ISH algorithm) showed the WEKA tool as having the greatest level of agreement with manual quantification. Comparing image analysis methods to qRT-PCR for quantifying MLH1, GFI1 and TNFRSF11A expression within two colorectal cell lines results suggest that these image analysis methods perform at a similar level to qRT-PCR. Furthermore, we describe the strengths and limitations for each image analysis method when used in combination with RNAscope assays. Our study concludes that there are several freely available and commercial image analysis tools that enable reliable RNA in situ expression analysis, however operators need to consider factors, such as expected expression levels of target genes, software usability and functionality.

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Genome-wide expression studies using microarrays and RNAseq have increased our understanding of colorectal cancer development. Translating potential gene biomarkers from these studies for clinical utility has typically relied on PCR-based technology and immunohistochemistry. Results from these techniques are limited by tumour sample heterogeneity and the lack of correlation between mRNA transcript abundance and corresponding protein levels. The aim of this research was to investigate the clinical utility of the RNA in situ hybridisation technique, RNAscope®, for measuring intra-tumoural gene expression of potential prognostic markers in a colorectal cancer cohort. Two candidate gene markers (GFI1 and TNFRSF11A) assessed in this study were identified from a previous study led by the The Cancer Genome Atlas (TCGA) Network, and analysis was performed on 112 consecutively collected, archival FFPE colorectal cancer tumour samples. Consistent with the TCGA Network study, we found reduced GFI1 expression was associated with high-grade and left-sided tumours, and reduced TNFRSF11A expression was associated with metastasis and high nodal involvement. RNAscope® combined with image analysis also enabled quantification of GFI1 and TNFRSF11A mRNA expression levels at the single cell level, allowing cell-type determination. These data showed that reduced mRNA transcript abundance measured in patients with poorer prognosis occurred in carcinoma cells, and not lymphocytes, stromal cells or normal epithelial cells. To our knowledge, this is the first study to assess the intra-tumoural expression patterns of GFI1 and TNFRSF11A and to validate their microarray expression profiles using RNAscope. We also demonstrate the utility of RNAscope® technology to show that expression differences are derived from carcinoma cells rather than from cells located in the tumour microenvironment.

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BACKGROUND: Anal intra-epithelial neoplasia (AIN) is believed to be a precursor to squamous cell carcinoma of the anus. The risk of developing anal cancer in patients with AIN, although known to occur, has been thought to be relatively low. This study reviews our experience with AIN, reviewing the incidence and risk factors for development of invasive malignancy and the outcome of present management strategies. METHODS: This study examined a cohort of 72 patients identified from a prospective database with AIN from a single institution between January 1996 and December 2004. A single pathologist examined all pathological specimens. RESULTS: There were 72 patients (52 women) and the median age was 49 years (range, 18-81 years). We identified progression of AIN to invasive malignancy in eight patients despite undergoing surveillance. Regression following treatment or biopsy was seen in 25 patients. Four patients required stomas for incontinence following treatment. CONCLUSION: This study has shown a high rate of progression to invasive malignancy (11%) with AIN despite surveillance. The patients at risk of developing squamous cell carcinoma were the immunocompromised and those with genital intra-epithelial field change. Treatment of AIN has significant complications and despite treatment, invasive cancers do occur. Decisions made for treatment of AIN can affect treatment choices if invasive malignancy develops.

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