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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Respiratory Research 1/2017

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

Zeitschrift:
Respiratory Research > Ausgabe 1/2017
Autoren:
Andre Koper, Leo A. H. Zeef, Leena Joseph, Keith Kerr, John Gosney, Mark A. Lindsay, Richard Booton
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12931-016-0496-3) contains supplementary material, which is available to authorized users.

Abstract

Background

Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown.

Method

To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA.

Results

Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs.

Conclusions

Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.
Zusatzmaterial
Additional file 1: Figure S1. Laser microdissection of FFPE PSCC and ISCC. (DOC 2651 kb)
12931_2016_496_MOESM1_ESM.doc
Additional file 2: Table S1. RT-PCR primer. Table S2. Patient and sample information. Table S3. Differential RNA expression in ISCC versus paired controls (normal histology). Table S4. Differential RNA expression in PSCC versus paired controls. Table S5. Differential RNA expression in PSCC and ISCC. Table S6. Differential RNA expression in high-grade PSCC versus paired controls. Table S7. Differential RNA expression in low-grade PSCC versus paired controls. Table S8. Differential RNA expression in high-grade PSCC and low-grade PSCC. Table S9. Ingenuity Pathway Analysis. Table S10. IPA results for genes associated with Clusters 1-12. Table S11. RNA expression profiles across PSCC and ISCC. (DOC 5260 kb)
12931_2016_496_MOESM2_ESM.doc
Literatur
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