Comprehensive profiling (genomic, proteomic and metabolomic) using tissue samples is a key area of investigation for translational research and personalized medicine. Indeed, patterns of expression may be poised to replace single diagnostic biomarkers, particularly in the case of genomic profiling for cancer research.
Unfortunately, preanalytical variables (e.g., tissue manipulation, warm or cold ischemia, storage conditions) influence expression levels in tissue samples, ultimately impacting study reproducibility and interpretation downstream. For this reason, markers for tissue sample quality are imperative.
Veneroni et al. (2016) recently evaluated under vacuum storage (UVS) as a fiscally sound, green alternative for specimen storage between surgical excision and sample processing, focusing on features pertinent to –omics studies.1 Briefly, UVS entails sealing tissue samples in plastic bags held at 4°C after surgical harvesting and prior to sample processing and storage as an alternative to formalin fixation.
To accomplish this, the research team collected paired tumor and normal matched samples from 16 patients (eight breast cancer, five colon cancer, three lung cancer) as well as tumor tissue from five patients with mesenchymal tumors. They dissected these samples within 20 minutes of excision and divided them into three to five aliquots. They snap-froze one of these aliquots in liquid nitrogen (T0) and subjected the others to UVS, holding for various time points (1 hour [T1], 24 hours [T2], 48 hours [T3] and 72 hours [T4]), prior to snap-freezing and storage at −80°C.
The researchers performed histomorphology, peptidome and Western blot analyses on all samples (163 specimens total). Fewer specimens were suitable for gene expression (84 specimens) and metabolic profiling (132 specimens). A total of 66 samples underwent all evaluated techniques.
Histomorphology with standard staining and immunohistochemical staining for tissue-specific antigens evidenced well-preserved structural integrity, high protein stability (Mib-1) and anti-vimentin antibodies for all time points. Principal variance component analysis indicated minimal impact for gene expression or proteomic data and slightly greater impact for some metabolites.
The team noted no significant change in RNA quality up to 48 hours (and likely longer). They did observe 18 genes with significantly altered expression over time; however, these alterations generally occurred in normal tissue samples, not tumor samples. Notably, the team also reported phosphoprotein conservation (mTOR and STAT3) for 24 hours to 48 hours. For mass spectrometry, UVS preserved diagnostic peptide features.
In terms of metabolite profiling, the team observed recurrent alterations for each tissue type. In particular, choline, alanine and valine showed increased signal intensity, while creatine and phosphocreatine displayed lower variations. They note that in comparison with macromolecules like RNA and proteins, small metabolites are likely more sensitive to preanalytical factors (e.g., warm ischemia, tissue handling). They suggest that choline may prove to be a useful marker for monitoring tissue quality for metabolomics applications.
Overall, Veneroni et al. call for further investigations of UVS. They suggest its feasibility for tissue specimens destined for histological, transcriptomic and proteomic studies (up to 48 hours UVS, likely longer) but indicate that its applicability for metabolomics may be more limited.
1. Veneroni, S., et al. (2016) “Applicability of under vacuum fresh tissue sealing and cooling to omics analysis of tumor tissues,” Biopreservation and Biobanking [Epub ahead of print], doi: 10.1089/bio.2015.0093.