The existence of extracellular cell free DNA (cfDNA) was detected in blood as early as 1948. In 1977 it was documented for the first time that levels of cell free tumor DNA (cftDNA) were significantly higher in cancer patients than in healthy persons. In the last decade, human whole genome sequencing has revealed the relationship between somatic mutations in oncogenes, tumor suppression genes, and cancer development. This led to the rediscovery of cftDNA as a valuable resource for diagnosis, prognosis, treatment decisions, and follow-up monitoring of cancer patients. Next generation sequencing has led to a number of interesting findings that could pave the way for doctors to use less invasive techniques for cancer detection, offering actionable diagnostic information at an early stage of cancer development.
In February of 2014, a group from Johns Hopkins presented a comprehensive report in Science Translational Medicine which moved the application of ctDNA analysis as a cancer diagnostic to a new level (1). This study analyzed ctDNA from 640 patients with 18 cancer types at four clinical stages, and was able to present several interesting conclusions:
- ctDNA could be detected in more than 50% of localized tumors and more than 75% of advanced tumors. In most tumor types, levels of ctDNA correlated with the stage of cancer.
- Detection of cancer based on ctDNA analysis is more sensitive than analysis of circulating tumor cells (CTC). ctDNA and associated mutations were always detected at higher levels in blood of cancer patients, and could even be detected when CTC were absent. This observation suggests that ctDNA does not originate from CTC.
- ctDNA and localized tumors had the same mutations and/or rearrangements, confirming that most ctDNA is shed to the bloodstream by tumor cells.
- It is possible to predict the resistance to EGFR blockage treatment of colon cancer by acquisition of new mutations in KRAS, BRAF, NRAS and EGFR, which could help tailor personal treatment during disease progression.
- 47% of patients with stage I disease had detectable ctDNA in plasma.
The most promising finding from this research was that mutations found in ctDNA could be used to detect and treat colon cancer during stage I growth. However, many problems have to be solved before a ctDNA test can be used for routine clinical diagnosis. Currently, it is difficult to determine the origin and location of a tumor from analysis of ctDNA in the blood of an asymptomatic person, because the majority of known mutations occur in many different types of tumors. Moreover, as few as 50 million malignant cells can produce detectable amounts of cftDNA in blood (2), which is far below the detection limit of current imaging methods. Hopefully, the results presented will encourage more prospective studies using analysis of cftDNA by next generation sequencing to produce highly informative data, which will help narrow the suspected origin of mutated ctDNA and allow intervention prior to the progression of disease.
In conclusion, this report validates ctDNA analysis for tumor diagnosis as a promising, non-invasive method for screening, diagnosis, treatment decisions and monitoring of human cancer. The spread of cftDNA analysis promises to generate further information for broad implementation into clinical applications.
Bioo Scientific developed the NEXTflex™ Cell Free DNA-Seq Kit to facilitate the analysis of cfDNA for studies such as these. The NEXTflex Cell Free DNA-Seq Kit has been optimized for the construction of DNA-seq libraries from cell-free fetal or circulating tumor DNA. With a short, two hour DNA library prep protocol, this kit can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina® platforms. The NEXTflex™ 1-step End-Repair and Adenylation protocol simplifies workflow and shortens hands-on library construction time. In addition, the availability of up to 192 unique adapter barcodes facilitates high-throughput applications.
(1) Bettegowda C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014; 6: 224a24.
(2) Diaz LA Jr. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancer. Nature 2012; 486:537-540.