Meridian is a primary manufacturer of specialized high-quality molecular reagents and offers solutions to a wide range of industries to diagnose and treat diseases, discover new therapeutics or develop tests for environmental, food and cosmetic safety.
Advances in qPCR-based liquid biopsy for cancer detection & its applications
Cancer is a leading cause of death globally, killing nearly 10 million people in 2020, with breast cancer, lung cancer and colon cancer representing the three most common types 1 . Diagnostic strategies focused on early detection and regular screening has been shown to significantly increase survival rates. A new innovation in the cancer diagnostic market that is transforming cancer testing is called liquid biopsy. It enables cancer screening from non-invasive clinical samples, such as blood, urine, saliva and stool which is significantly less intrusive than surgical biopsy. In addition, it removes any risk of surgical haemorrhage, hematoma, tissue damage or the potential for seeding tumor cells into surrounding tissues, and it enables a safe, easily repeatable practice for cancer diagnosis, prognosis and treatment.
Introduction Cancer is a complex and dynamic disease, making it difficult to diagnose and predict how an individual’s cancer will progress and respond to treatment. While each tumor is unique, biomarkers help to provide very specific indicators of tumor behaviour and disease progression. Traditionally, tumor tissue was the only reliable specimen that could be used for confirming diagnosis, monitoring disease progression or assessing the response to treatment. However, our understanding of cancer has evolved and new, more sensitive technologies are emerging. The most advanced novel technology to date is liquid biopsy, a non-invasive technique that relies on the detection and isolation of tumor-specific molecules such as proteins, miRNAs, exosomes, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) from specimens such as blood, urine, saliva and stool. The first liquid biopsy test based on CTC enumeration was approved by the U.S. Food and Drug Administration (FDA) in 2013 and three years later in 2016, a second test was approved. It was the first PCR-based ctDNA test designed to detect epidermal growth factor receptor (EGFR) gene mutations from the blood of patients with non–small cell lung cancer (NSCLC). PCR-based detection of ctDNA in liquid biopsy is considered an attractive approach to molecular cancer screening as ctDNA is
highly specific, very sensitive, and simple to isolate and analyze using qPCR techniques. ctDNA are small DNA fragments that are shed by a tumor into the bloodstream and the level of ctDNA represents the relative tumor burden. Studies have shown that for breast cancer, a ctDNA test can detect disease progression five months before radiographic evidence 2 . In addition, ctDNA has demonstrated a greater breast cancer detection rate and stronger correlation with changes in tumor burden, and provides
field of biomarkers, RNA exhibits several advantages over DNA - the expression pattern of several RNA molecules, in particular long non-coding RNAs (lncRNAs), is highly tissue- and disease-state specific, and RNA expression is more dynamic than DNA, fluctuating according to the internal needs of the cells. Studies have shown that lncRNAs in particular, play a critical role in cancer progression and can be used as a diagnostic and prognostic marker, capable of identifying stage-specific tumors 3 .
the earliest gauge of response to treatment, compared to the traditional protein cancer biomarker CA 15-3. Additional advantages of ctDNA over CA15-3 include the molecular capability to analyze the cancer genome and metastatic heterogeneity, all non-invasively. Overall, ctDNA analysis by liquid
Several cancers exhibit specific genetic mutations that effect how they respond to treatment, either increasing the cancer’s sensitivity to the drug, or more frequently seen, increasing the cancer’s resistance to the drug.
biopsy is relatively lower in cost compared to traditional biopsy methods and could potentially be much more readily accessible by applying it at the point-of care in routine clinical practice on a point-of-care instrument. Other, newer cancer molecular biomarkers that are not in approved tests, but have gained significant interest in recent years, are non-coding RNAs (ncRNAs). In the
The potential of lncRNAs as biomarkers continues to be explored, especially in the challenging areas of minimal residual disease (MRD), auxiliary staging, real-time monitoring of drug resistance, and risk of metastatic relapse and prognosis.
www.meridianbioscience.com/lifescience
Applications for liquid biopsy cancer testing Genotyping patients – screening for first line therapy for non-small-cell lung cancer (NSCLC). The need to genotype cancer patients prior to treatment is fundamentally important for selecting the appropriate therapy and drugs. Several cancers exhibit specific genetic mutations that effect how they respond to treatment, either increasing the cancer’s sensitivity to the drug, or more frequently seen, increasing the cancer’s resistance to the drug. In lung cancer, one of the most frequently occurring mutations is the exon 19 deletion mutation in EGFR, and it is associated with sensitivity to EGFR tyrosine kinase inhibitors, the first-line treatment for non-small-cell lung cancer (NSCLC). Several EGFR mutation detection assays are already EU- and FDA-approved and used in the management of lung cancer patients for initial genotyping, monitoring drug-resistant clones, and evaluating the response to treatment 4 . However, these assays require DNA extraction prior to qPCR analysis, which not only increases the complexity and cost of the assay, but the potential for contamination and carryover inhibitors which can impact the accuracy of the assay. In order to optimize the workflow, manufacturers of EGFR mutation detection assays have developed specific DNA sample preparation kits as a separate test component 5 . Ideally, the need for DNA extraction and purification could be avoided altogether and qPCR detection of EGFR mutations could be carried out directly on the sample. In the following study, Meridian’s Air-Dryable ™ Direct DNA qPCR Blood mix was examined for its ability to detect single copies of the EGFR exon-19 mutation (Fig. 1) . Known copy numbers of synthetic EGFR Exon 19 Deletion and an EGFR Exon 19 wildtype (negative control), was used in the presence of plasma extract with a standard reaction protocol (95°C for 3 minutes, followed by 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds) using wet or lyophilized (see MDX092 User Guide) Air-Dryable ™ Direct DNA qPCR Blood mix. For EGFR mutation analysis, plasma is often the preferred sample type as hemolysis can occur in whole blood samples and cause interference with many types of laboratory tests. However, plasma samples must be stabilized with anticoagulants to prevent clotting and these additives can cause inhibition in a PCR reaction. Meridian’s
Figure 1. Extraction-free qPCR reduces complexity and increases sensitivity
Figure 1: Known copy numbers of synthetic EGFR Exon 19 Deletion and an EGFR Exon 19 wildtype (negative control), was used in the presence of plasma extract with a standard reaction protocol (95°C for 3 minutes, followed by 50 cycles of 95°C for 10 seconds and 60°C for 25 seconds) using wet or lyophilized (see MDX092 User Guide) Air-Dryable ™ Direct DNA qPCR Blood mix. The advantage of drying down the Air-Dryable ™ Direct DNA qPCR Blood is that more sample can be added to the reaction. As illustrated in (A) when the amount of eluted sample is increased from 35% (orange) to 100% (brown), the sensitivity increases by almost 10-fold. This increase in sensitivity enables the detection of single copies of the E19del-EGFR to be detectable (B, light blue) within a 20 µ L reaction (gDNA standards, EGFR Exon 19 wildtype (Negative Control – brown)).
Air-Dryable ™ Direct DNA qPCR Blood (MDX092) mix was tested for its inhibitor- tolerance to the anticoagulant heparin in a qPCR study detecting an Exon19 mutation. Specifically, a ratio of 0.1% of E19del-EGFR to 99.9% wild type DNA was spiked into a sample containing 20% heparin-plasma. Air-Dryable ™ Direct DNA qPCR Blood was tested alongside KAPA Probe Force and TaqMan ™ Universal Master Mix for its ability to amplify the mutation. Across both concentrations of mutant DNA (10 copies and 1,000 copies), Air-Dryable ™ Direct DNA
qPCR Blood was the only mix to produce an amplification curve, demonstrating its ability to perform in the presence of up to 20% heparin. In contrast, the other mixes were unable to generate a result. Using specimen-specific mixes such as Air- Dryable ™ Direct DNA qPCR Blood confers several advantages over universal mixes, most notably, a greater ability to perform in the presence of inhibitors, and an increased sensitivity enabling extraction-free qPCR analysis.
Figure 2. The effect of 20% heparin-plasma on qPCR sensitivity
Figure 2: Detection of epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. Here a deletion in Exon 19 known to be sensitive to current chemotherapy was analysed, by spiking a known amount containing 0.1% of E19del-EGFR from 99.9% wild type DNA into 20% heparin-plasma. We compared the sensitivity of Air-Dryable ™ Direct DNA qPCR Blood (MDX092) (red) to detect this mutant against A) KAPA Probe Force (blue) and B) TaqMan ™ Universal Master Mix (green). The results illustrate the greater tolerance and so better sensitivity of MDX092 to plasma and heparin anti-coagulant, allowing down to 10 copies of the mutation to be detected.
Figure 3. Relative detection sensitivity of MALAT1 -- a potential lncRNA biomarker for several different types of cancer
include cyclin dependent kinase 1 (CDC2), Insulin Like Growth Factor Binding Protein 5 (IGFBP5) and Midkine (MDK). Bladder Cancer Markers In the following study, Meridian’s Air- Dryable ™ Direct RNA/DNA qPCR Urine (MDX151) was compared to UltraPlex ™ 1-Step ToughMix for its ability to amplify bladder cancer makers CDC2, IGFBP5 and MDK in samples containing 10% urine (Figure 4). Specifically, reactions were set up with 10% DNase-treated-human urine and 200 pg of CDC2, IGFBP5 or MDK RNA and run either with air-dried Air-Dryable ™ Direct RNA/DNA qPCR Urine or wet UltraPlex ™ 1-Step ToughMix on a QuantStudio using 50°C for 10 minutes, 95°C for 2 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 20 seconds. Across all three markers, Air-Dryable ™ Direct RNA/DNA qPCR Urine showed a lower ct value demonstrating that it is able to detect cancer related RNA markers from urine faster and with higher sensitivity compared to other mixes. Prostate Cancer Markers Currently, the gold-standard screening assay for prostate cancer is the PSA (prostate specific antigen) test, and elevated levels that suggest the presence of cancer are further examined by transrectal or trans- perineal biopsy. Although this strategy has proven to be a very effective diagnostic algorithm, cancer detection rates from tissue biopsy are low, varying between 36% - 54% as not all elevated PSA antigen results are caused by cancer 11 . In addition, repeat biopsies are often required and there is a risk of severe infection and hospitalization with each procedure.
QuantStudio using 50°C for 10 minutes, 95°C for 3 minutes and 40 cycles of 95°C for 10 seconds and 60 °C for 25 seconds (Fig. 3) . Direct detection of molecular biomarkers from urine Advances in liquid biopsy have largely centred around blood specimens, however other body fluids such as stool, saliva and urine can also be used. In particular, urine has demonstrated great promise in urological cancers and non-urological cancers such as prostate cancer 9 and offers significant benefits over blood as it is completely non-invasive, can be collected in large volumes, and is a rich source for ctDNA. Several liquid biopsy assays based on qPCR methods for bladder cancer detection and monitoring are already FDA-approved and have helped to lower the economic burden of bladder cancer patient surveillance and to increase patient compliance 10 . Most of the available tests examine a panel of mRNA biomarkers whose overexpression has been linked stages 1-4 of bladder cancer, and Figure 3: Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is one of the few well-studied long non-coding RNAs (lncRNAs), which presents high expression and enhanced activity in numerous tumor tissues and cells, playing a critical role in tumor invasion and metastasis. In this study, the sensitivity of Air-Dryable ™ Direct RNA/DNA qPCR Blood (MDX121) (red) to detect MALAT1 was compared against QuantaBio UltraPlex ™ 1-Step ToughMix in the presence of 5% DNase-treated-serum. The results illustrate the greater sensitivity of MDX121 with serum, suggesting its greater suitability lncRNA biomarker screening.
Direct detection of lncRNA biomarkers from blood - Detecting early-stage cancers using lncRNA biomarkers LncRNAs are defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. Research has shown that lncRNA, exert distinct biological functions through diverse molecular mechanisms 6 . Several studies have validated the use of exosomal lncRNAs as minimally invasive diagnostic and prognostic markers in several types of cancers. MALTA1(metastasis associated in lung adenocarcinoma transcript 1) is a 8.8kb long non-coding RNA (lncRNA) that has attracted significant attention in the past couple of years as a potential biomarker for cancer diagnosis as well as prognosis. MALAT1 is widely expressed in normal tissues and is aberrant expressed in cancers, suggesting that it plays a role in cancer pathophysiology and disease progression 7 . Studies examining diagnostic testing for lung-cancer associated malignant pleural effusion (LC-MPE), have found that combinations of MALAT1 and carcinoembryonic antigen (CEA), a tumor marker used in the detection of several cancers including lung cancer, provided higher sensitivity and accuracy in predicting LC-MPE than CEA alone 8 . In the following study, Meridian’s Air-Dryable Direct DNA qPCR Blood was tested in the presence of 5% DNase-treated-serum (to remove genomic DNA for RNA detection) for its sensitivity to detect MALAT1. DNase I was added to serum and incubated for 2 h at 37°C followed by heat inactivation at 95°C for 10 minutes, and reactions set up with 5% of this DNase-treated-serum and known copy number concentrations of the long non- coding RNA target (MALAT1), and run with either Air-Dryable Direct DNA qPCR Blood or UltraPlex ™ 1-Step ToughMix on a
Figure 4. Detection of tumor-related mRNA markers (CDC2 kinase, IGFBP5 and MDK) in samples containing 10% urine
A) CDC2
B) IGFBP5
C) MDK
Figure 4: Meridian’s Air-Dryable ™ Direct RNA/DNA qPCR Urine (red) was compared to UltraPlex ™ 1-Step ToughMix (grey)for its ability to amplify bladder cancer makers CDC2, IGFBP5 and MDK in samples containing 10% urine. Specifically, reactions were set up with 10% DNase-treated-human urine and 200 pg of CDC2, IGFBP5 or MDK RNA and run either with air-dried Air-Dryable ™ Direct RNA/ DNA qPCR Urine or wet UltraPlex ™ 1-Step ToughMix on a QuantStudio using 50°C for 10 minutes, 95°C for 2 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 20 seconds. Across all three markers, Air-Dryable ™ Direct RNA/DNA qPCR Urine showed a lower ct value demonstrating that it is able to detect cancer related RNA markers from urine faster and with higher sensitivity compared to other mixes.
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.
Ordering information: USA 5171 Wilfong Road Memphis, Tennessee 38134 Phone: +1 901-382-8716 Fax: +1 901-333-8223
Email: info@meridianlifescience.com Orders: orders@meridianlifescience.com www.meridianbioscience.com/lifescience
Connect with us:
ISO 13485 Certified
05/23
Page 1 Page 2 Page 3 Page 4Powered by FlippingBook