# Advanced Omics and Radiobiological Tissue Archives: The Future in the Past
## 4 Omics Analysis on FFPE Samples in Radiobiological Archives
### Genomics and Transcriptomics  
Genomic approaches are widely applied in profiling clinical cancer samples and biomarker discovery of diagnosis and prognosis. Comparison of the mutational burden of a tumour to normal tissue provides insight into the tumour development due to environmental exposures by the finding of specific mutational signatures of genotoxins [94], but also allows to decipher the nature and timing of mutational processes and the contribution of even rare germline variants increasing cancer risk [95]. FFPE tissues, with all known difficulties, offer attractive candidates for a comprehensive investigation of the cancer genome. High-throughput transcriptomics, including microarrays and next-generation sequencing (NGS), is an emerging technology that enables the study of genomes, epigenomes, and transcriptomes using limited sample material, often used in cancer samples [3,96]. The potential of archival samples for high-throughput transcriptome profiling has been well identified and discussed [97]. Efforts are given to optimize microarray and next-generation sequencing technologies as well as associated bioinformatics tools to develop a compatible platform to low input extracted nucleic acids from FFPE tissues [61,98]. RNA extracted from FFPE samples is often not well compatible with standard RNA-sequencing methods, where poly(A) selection or ribosomal RNA (rRNAA) depletion-based methods are the gold standard. Both approaches suffer from a higher degree of RNA contamination and a lower number of alignable reads [96,99]. Several approaches have been developed to address the problem of degraded RNA in the sequencing analysis. [PERSON] et al. used the nCounter platform from Nanostring Technologies, which allows digital readout of up to 800 mRNA targets even when the RNA is degraded [100]. An exome capture approach for RNA-seq from FFPE samples was also developed. The platform maximizes RNA-seq libraries by including most reads in exons regardless of the RNA quality [101]. [PERSON] et al. developed a so-called FFPEcap-seq method suitable for sequencing capped 5-ends of FFPE RNA. To generate sequencing libraries, the platform combines enzymatic enrichment of 5-capped RNAs with template switching [96]. A recent comparison ranked platforms on their performance on FFPE samples with different storage times [97]. Thereby 3\({}^{\prime}\)-sequencing approaches from Lexogen and Qiagen were identified as highly reliable and cost-effective for old FFPE samples. Despite considerable progress in the application of whole-genome sequencing on FFPE samples, there are only a few studies available that analysed RNA and DNA isolated from FFPE materials from patients who received radiotherapy. These studies were mainly designed to identify the tumour signature or biomarkers of radioresistance of cancer. To investigate the mechanism underlying the poor outcome of radiochemotherapy with and without cetuximab in cervical cancer (CC) patients, [PERSON] et al. screened the hotspot mutations by target sequencing on FFPE samples from patients who were treated by chemotherapy combined with standard pelvic radiation therapy. The authors showed that alterations in the PIK3 CA pathway negatively affected the complete response to radiochemotherapy. The analysis showed that CC patients with PIK3 CA mutation had trends to poorer disease-free survival (DFS) at 2 years [102]. [PERSON] et al. analysed the mutation signatures of radioresistancet tumours by exon sequencing of 409 cancer-related genes in FFPE samples of uterine cervical cancer (UCC) patients who survived multiple rounds of radiotherapy [103]. The analysis indicated activating mutations in PIK3 CA and KRAS, and putative inactivating mutations in SMAD4. The authors further validated the association between this mutation signature and radioresistance by cell-based experiments [103]. The same team later analysed the genetic profile of FFPE tumours collected from UCC patients with local recurrence after carbon ion radiotherapy using the same analytical platform [104].