Background: Clinical tumour specimens are routinely preserved as formalin-fixed paraffin embedded (FFPE) tissues for histopathological analysis. These specimens are richly annotated with clinicopathological and prognostic information. However, molecular analysis of early stage FFPE cancer specimens is far more challenging than analysis using fresh tissue, or late staged FFPE specimens which tend to be larger. We have investigated integrative omics analysis from Stage I and IIIA colorectal cancer FFPE specimens by proteomic mass spectrometry, Nanostring gene expression analysis, DNA sequencing and CyToF immune cell profiling.
Methods: For proteomics 5x5 µm sections were macro-dissected on slide, processed using trypsin and SP3 purification. An EncyclopeDIA narrow window gas-phase fractionated chromatogram library empirically corrected with PROSIT was produced. Each sample was acquired via overlapping wide window DIA. All data were acquired on a QExactive HFX mass spectrometer over 140min using a 50cm x 75µm column. For Nanostring and DNA sequencing, mRNA and DNA was extracted from freshly cut 10x5 µm sections and quality checked by Bioanalyzer. Samples were run on Cancer Progression kit profiling 770 genes. We explored whole exome sequencing and targeted panel sequencing. For imaging mass cytometry a Hyperion CyToF system was used with 40 CD antigen markers on each 7 µm section.
Results: The extensive peptide chromatographic library enabled quantitative measurement of ~5300 proteins across all 12 samples. Statistical analysis identified proteins with roles in metastasis and cell migration when we dichotomised specimens based on lymph node positivity. Nanostring gene expression analysis identified factors absent from the proteomics analysis and revealed the importance of AP-1 TF activation in promoting metastasis. WES produced poor data due to fragmented DNA. Imaging mass cytometry was successfully implemented from archival sections and is under review.
Conclusion: Learnings for integrative omics analysis of minimally invasive FFPE CRC tumours has been established to underpin larger cohort studies.