Colorectal cancer is the third most common cancer, affecting 1.8 million new patients and causing 862,000 deaths every year (1). Studies show that eating processed or red meat and drinking alcohol may increase the risk for colorectal cancer (2). Treatment can include surgery, radiation, chemotherapy, targeted therapy or immunotherapy.
With the advances in screening technologies over the past decade, many cancer patients now benefit from personalized medicine. Genomic, gene expression and proteomic studies can help clinicians choose the best treatment options for cancer patients, including colorectal cancer patients. Measuring cancer DNA changes can also help doctors monitor disease progression in patients. These ‘omics studies are also an integral part of biomedical research looking for novel cancer therapeutics and diagnostics.
For personalized medicine studies to work, researchers and diagnostic labs need access to high quality biological samples. Many studies have shown that preanalytical variables, such as storage temperature, can drastically affect sample quality (3). Enzymatic damage to nucleic acids and proteins occurs in samples kept at room temperature, or even on ice, for extended periods of time. Similarly, repeated freeze-thaw cycles can cause RNA, DNA and protein to degrade in biological samples (4,5).
Sample degradation adversely affects experimental results. For example, even small levels of RNA degradation can cause significant variation in gene expression (6).
How Biobanking Affects Colorectal Cancer Samples
A recent study looked at how storage temperature and freeze-thaw cycles affect samples of colorectal cancer and adjacent normal colorectal tissue (7). The samples came from the Tissue Bank of the Sixth Affiliated Hospital at Sun Yat-sen University. This is the largest colorectal tissue bank in Southern China, with more than 110,000 samples collected from 6,230 patients since 2007.
The study assessed how RNA, DNA and protein quality changed in 144 paired samples of normal and colorectal cancer tissue stored at room temperature or on ice for up to 48 hours. RNA, DNA and protein in these samples appeared to be stable for up to 48 hours on ice.
However, at room temperature, RNA started to degrade after 8 hours in colorectal cancer tissues and lost quality after 24 hours in normal tissues. Likewise, DNA in colorectal cancer tissues started to degrade after 24 hours at room temperature. Expression levels of almost one third of the proteins analyzed also changed significantly if samples were stored at room temperature.
While room temperature storage negatively affected RNA, DNA and protein. Freeze-thaw cycles seemed to only affect protein quality in this study. Researchers allowed samples to thaw on ice then refroze them at -80°C. RNA and DNA integrity remained the same after 9 freeze-thaw cycles. However, more than half of the analyzed proteins (157/298 proteins, including 37/65 phosphorylated proteins) showed altered expression after 7 freeze-thaw cycles.
This study shows that RNA and DNA in colorectal cancer tissue may degrade more quickly than nucleic acids in normal tissue. Data from the study also suggest that proteins in colorectal cancer tissue could be more sensitive to freeze-thaw cycles than RNA and DNA. This is a somewhat surprising finding as previously published studies have found the opposite. For example, samples from gastrointestinal cancers and adjacent normal tissue can be susceptible to RNA degradation after one freeze-thaw cycle (5). However, these samples were snap frozen in liquid nitrogen and not collected in RNAlater which stabilizes RNA.
Another surprising finding from this study is that RNA, DNA and protein all seemed to be stable when stored on ice for up to 48 hours. Again this may be because the samples were stored in RNAlater.
These results should be interpreted with caution because even if global RNA levels appear stable, gene expression can still be negatively affected by variabilities in sample handling (6). Therefore best practice guidelines still recommend storing samples in aliquots to minimize freeze-thaw cycles.
- Cancer Factsheet. World Health Organization. (Online) Accessed 17 October, 2019 at https://www.who.int/news-room/fact-sheets/detail/cancer
- Colorectal cancer statistics. World Cancer Research Fund (Online) Accessed 17 October, 2019 at https://www.wcrf.org/dietandcancer/cancer-trends/colorectal-cancer-statistics
- NCI Best Practices for Biospecimen Resources. National Cancer Institute. 2016 (Online) Accessed June 17, 2019
- Ji et al. The Impact of Repeated Freeze-Thaw Cycles on the Quality of Biomolecules in Four Different Tissues. Biopreserv and Biobank. 2017
- Hu et al. Influence of Freeze-Thaw Cycles on RNA Integrity of Gastrointestinal Cancer and Matched Adjacent Tissues. Biopreserv and Biobank. 2017
- Botling J, Edlund K, Segersten U. et al. Impact of thawing on RNA integrity and gene expression analysis in fresh frozen tissue. Diagn Mol Pathol. 2009
- Fan et al. Impact of Cold Ischemic Time and Freeze-Thaw Cycles on RNA, DNA and Protein Quality in Colorectal Cancer Tissues Biobanking. J. Cancer. 2019