Circulating cell-free tumor DNA (cfDNA) is released by solid tumors into the blood stream. There is a lot of interest among the medical and biobanking communities in using cfDNA as a relatively non-invasive way of predicting cancer prognosis and monitoring disease progression. Cell-free tumor DNA can be isolated from blood samples and quantified in biobanks or laboratories. However, this is challenging because cfDNA is only present in blood samples at very low concentrations. Biobanks must overcome this limitation and use standardized methods to accurately quantify cell-free tumor DNA, if this cfDNA is to be used to monitor a patient’s response to treatment.
A Simple, Affordable Cell-Free Tumour DNA Assay for Biobanks
The industry is working to develop a simple, affordable, standardized assay to accurately measure total cell-free tumor DNA from each sample before doing further analysis. As part of this effort, researchers from the University of Modena and Reggio Emilia in Italy recently analyzed whether Qubit fluorimetry or NanoDrop spectrophotometry can accurately quantify cfDNA levels in patients with malignant melanoma or prostate cancer and in healthy controls (1).
NanoDrop uses UV spectrophotometry to measure DNA quantity and quality. NanoDrop can measure single stranded DNA (ssDNA), double stranded DNA (dsDNA) and RNA. However, it cannot tell the difference between ssDNA and dsDNA within a single sample. In contrast, Qubit measures fluorescence from dyes that bind to either single stranded or double stranded DNA. Two separate kits must be used to make these measurements but this method can reliably quantify ssDNA versus dsDNA.
In their study published in Clinica Chimica Acta, the authors compared cell-free tumor DNA quantification by Qubit, NanoDrop and real time PCR. For the real time PCR assay, the researchers used Taqman probes to measure levels of the Amyloid beta Precursor Protein (APP) gene. Real time PCR is considered the gold standard for DNA quantification in biobanks. However, this method is more expensive and time consuming than using the Qubit or NanoDrop methods.
The authors found significant differences in the amount of cell-free tumor DNA detected by each assay. Real time PCR detected significantly lower levels of cfDNA in each sample than NanoDrop or Qubit. The authors tested Qubit with both the single strand and double strand DNA assay kits. The Qubit single strand assay kit detected twice as much cell-free tumor DNA than any other method in almost all samples, indicating that this test has a higher sensitivity for single stranded DNA, particularly low concentration ssDNA. However, all methods showed large standard deviations in each patient population.
Cost of Cell-Free Tumor DNA Quantification in Biobanks
The authors also conducted a cost and time analysis of each cell-free tumour DNA quantification method. They found that the NanoDrop method was the fastest and the cheapest, although it is not clear whether they factored in the cost of purchasing the NanoDrop machine which retails for over four times the price of the Qubit. The Qubit method was also significantly cheaper and faster than real time PCR.
Conclusions
The authors concluded that the Qubit ssDNA kit gives the best cell-free tumour DNA quantification, whereas NanoDrop gives an estimate of cfDNA purity as well as quantity. Therefore, they suggest using these two methods for initial cfDNA quantification and quality assessment before doing any further patient-specific analysis by next-generation sequencing or real time PCR.
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