References 1. Holyoake A., et al. Development of a multiplex RNA urine test for the detection and stratification of transitional cell carcinoma of the bladder. Clin Cancer Res (2008) 14 (3): 742–749. DOI: 10.1158/1078-0432.CCR-07-1672 2. Dawson S.J., et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. (2013) 368 (13): 1199–209. DOI: 10.1056/NEJMoa1213261 3. Khandelwal A., et al. Long non-Coding RNA: A New Paradigm for Lung Cancer. Mol Carcinog (2015) 54: 1235–51. DOI: 10.1002/mc.22362 4. Bulbul A., Leal A. & Husain H. Applications of cell-free circulating tumor DNA detection in EGFR mutant lung cancer. J Thorac Dis. (2020 May) 12 (5): 2877–2882. DOI: 10.21037/ jtd.2020.01.66 5. https://diagnostics.roche.com/global/en/products/ params/cobas-egfr-mutation-test-v2.html 6. Gutschner T. and Diederichs S. The hallmarks of cancer: a long non-coding RNA point of view. RNA Biol. (2012) 9(6): 703–719. DOI: 10.4161/ rna.20481 7. Li Z.X., et al. MALAT1: a potential biomarker in cancer. Cancer Manag Res. (2018) 10: 6757–6768. DOI: 10.2147/CMAR.S169406 8. Wang W.W., et al. Combination of long noncoding RNA MALAT1 and carcinoembryonic antigen for the diagnosis of malignant pleural effusion caused by lung cancer. OncoTargets and Therapy. (2018) 11: 2333—2344. DOI: 10.2147/OTT.S157551 9. Oshi M., et al. Urine as a source of liquid biopsy for cancer. Cancers (2021) 13 (11): 2652. DOI: 10.3390/cancers13112652 10. Ferro M., et al. Personalized medicine liquid biopsy biomarkers in urine: A route towards molecular diagnosis and personalized medicine of bladder cancer. J Personalized Medicine 11(237) (2021) 11: 237. DOI: 10.3390/jpm11030237 11. Sidana A., et al. Schema and cancer detection rates for transperineal prostate biopsy templates: a review. Therapeutic Advances in Urology. (2022) 14. DOI: 10.1177/17562872221105019 12.Durand X. Progensa™ PCA3 test for prostate cancer. Expert Rev Mol Diagn. (2011) 11 (2): 137-44. DOI: 10.1586/erm.10.122
PCA3, first identified in 1995, is a prostate specific, nonprotein coding RNA that is significantly over-expressed in prostate cancer, without any correlation to prostatic volume and/or other prostatic diseases (e.g. prostatitis). Several studies have demonstrated that PCA3 can be used as a prognostic marker of prostate cancer, especially in conjunction with other predictive markers, and quantitative PCA3 urine tests are already utilized in clinical practice 12 . In the following study, Air-Dryable ™ Direct RNA/DNA qPCR Urine was tested in a 10-fold serial dilution of RNA (10,000, 1,000, 100 and 10 copies) in the presence of 5% DNase-treated human urine and
known copy number concentrations of the long non-coding RNA target (PCA3), and run on a QuantStudio using 50°C for 10 minutes, 95°C for 3 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds. The results demonstrate that Air-Dryable™ Direct RNA/DNA qPCR Urine is able to detect PSA 3 from urine with high sensitivity in human urine. Minimal residual disease (MRD) In addition to early cancer screening, there is currently a concerted focus on post- treatment disease monitoring to look for minimal residual disease (MRD) where cancer cells persist in the body despite treatment. These cells are below the resolution and detection levels of imaging studies and are considered a principal cause of cancer recurrence. The concept of liquid biopsy for minimal/measurable residual disease is relatively well-developed in Leukemia, where increasingly sensitive sampling techniques have enabled clinicians to detect MRD at lower and lower levels. In addition, solid tumor surveillance, which has historically been challenging compared to hematologic malignancies due to the limited accessibility of tumor cells, is now also possible with the increase in sensitivity and specificity of liquid biopsy assays. Conclusion Liquid biopsies are revolutionizing the field of clinical oncology, offering ease in tumor sampling, continuous monitoring by repeated sampling, screening for therapeutic resistance and the opportunity to develop personalized therapeutic regimens. Though the technology is still evolving, its non-invasive nature promises to open up new eras in clinical oncology. Previous limitations around qPCR sensitivity and reproducibility are starting to be overcome and solutions are now available that address the challenges associated with liquid biopsy testing. Using crude samples to increase sensitivity and reduce assay complexity and cost is one answer that enables easier integration in the clinic and possibly even point of care, which could create profound improvement in clinical outcomes and the medical expenditure related to cancer care.
Figure 5. Detection of prostate cancer antigen 3 (PCA3) for prostate cancer down to 10 copies of RNA in samples containing 5% DNase-treated human urine
Figure 5: Prostate cancer antigen 3 (PCA3), a long non-coding RNA also known as DD3, which is located on the long arm of chromosome 9, had higher expression levels in prostate cancer tissues than in adjacent non-cancerous tissues, and showed no detectable levels in normal tissue and other prostate-related diseases. This specific characteristic of PCA3 makes it an ideal biomarker for prostate cancerization, as it is not confused with patient age, prostate inflammation, prostate volume or trauma as other biomarkers can be. Air-dried Air-Dryable ™ Direct RNA/DNA qPCR Urine was used in a 10-fold serial dilution of RNA (10,000, 1,000, 100 and 10 copies respectively), in the presence of 5% urine. The results illustrate that Air-Dryable ™ Direct RNA/DNA qPCR Urine was tested in a 10-fold serial dilution of RNA (10,000, 1,000, 100 and 10 copies) in the presence of 5% DNase-treated-human urine and known copy number concentrations of the long non-coding RNA target (PCA3), and run on a QuantStudio using 50°C for 10 minutes, 95°C for 3 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds. The results demonstrate that Air-Dryable ™ Direct RNA/DNA qPCR Urine is able to detect PSA 3 from urine with high sensitivity in human urine.
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