Cell Free DNA – the “floating remnants” that may lead to the hidden wrecks (tumors) under the boundless ocean

Figure 1: Is Cell-Free DNA our next hope to cancer detection?

Do you know that our marine environment is succumbing into severe pollution from corroding shipwrecks? With water making up to 71% of the earth surface, looking for shipwrecks that had sunk into deep waters is synonymous to searching for a needle in a haystack.

Does that actually resemble a kind of disease in our body too? Yes, that is what I am talking about; Cancer and Tumors. While all tumors are not necessarily cancerous, malignant tumors are essentially cancer. Regardless of tumors being benign or malignant, they are basically tissues that are composed of cells whose DNA are mutated, rendering them the abnormal capability to proliferate exponentially without regulation. These usually result in abnormal lesions or swelling of an organ, increasing the competition for “nutrients” with the healthy normal cells, thereby posing potential health hazards to the host. Similarly, removal of these tumor cells will halt the deteriorating health impacts and allow the host to regain its health. Unfortunately, searching for these tumors is not as simple as it seems to be.

It is a widely acknowledged fact that sooner a cancer is diagnosed and treated, better the prognosis. However, a clinically proven circulating biomarker to locate the specific location of the malignant cells remains a mystery. Sure, there are serum-based tumor-markers such as carcinoma antigen-125 (CA-125), carcinoembryonic antigen (CEA), and prostate-specific antigen (PSA) which are traditionally used to detect, diagnose, and manage some types of cancer. Although, the levels of these tumor markers are found to be significantly elevated in cancer patientsit does not confirm cancer diagnosis. Take PSA that is used to screen men for prostate cancer for example.  Though increased PSA level is associated with prostate cancer, there are non-prostate cancer patients with elevated PSA levels.  Therefore measurements of tumor markers are usually combined with other tests, such as biopsies, to diagnose cancer. 

Figure 2: Biopsy needle needed to draw a column of lung tissue to confirm lung cancer diagnosis

Needle-tissue biopsy is traditionally the gold standard for cancer diagnosis. However, this is a highly invasive expensive medical procedure that involves puncturing a needle into the body to obtain tissue samples for more in-depth examination of the cells. Hence, scientists have been diligently researching for the past 2 decades for alternatives that could by-pass needle-tissue biopsy and make cancer diagnosis more efficient and less invasive.

Taking a step back, if floating debris from shipwrecks could lead to the exact location of its host, is there something similar that can aid the physicians to detect the presence of tumors?

Figure 3: Can scientists uncover the mystery of these floating cell free DNA in relevance to Tumors?

Yes, that floating debris in relation with tumors is Cell-Free DNA. In fact, cell-free DNA is not a novel finding. Its existence was first discovered back in 1948 by Mandel and Matis, although debates on the clinical application of cell-free DNA in tumor diagnosis only began in1977.

As the name implies, “cell-free DNA” refers to DNA compounds not found in a cell. Traditionally, DNA is usually localized in protecting organelles such as nucleus and mitochondria. The exciting breakthrough begins when serum DNA levels are significantly elevated in cancer patients compared to healthy individuals, especially in patients with metastatic Tumors. In normal individuals, concentration of cell-free DNA varies from 0 – 100ng/ml of blood.

However, this extravagant surge in cell-free DNA concentration is not limited to cancer patients. Patients languishing in clinical conditions such as rheumatoid disease, trauma, myocardial infarction, fever and inflammatory diseases have similar phenomenon. Although their presence has been well documented in various clinical conditions, the origin and their liberation mechanism remains poorly understood.

Figure 4: Scientists believe that DNA is usually stored safely in the nucleus of cells.

Figure 5: Scientists suspects that when there is a tumor infected tissue adjacent to a blood vessel, there is a tendency for DNA to leak out of the abnormal tumor cells when they undergo necrosis or apoptosis. Their concentration is in relevance to the stage of the cancer.

Majority of the scientists have unanimously believed tumors are a major source of these DNA fragments that are released into the circulation when they undergo necrosis or apoptosis. In addition, a team of scientist observed that the quantity of circulating cell-free DNA correlated with the stage of cancer. With only 47% of the patients in stage 1 cancer having detectable cell-free DNA, the percentage of patients in progressing cancer stages II, III and IV was 55, 69 and 82 percent, respectively. Moreover, they also highlighted that the increasing concentration of cell-free DNA reflects the advancing stages of cancer (1). Therefore, this opens the possibility of determining how advanced a patient’s cancer is just by measuring the circulating level of cell-free DNA.

To further prove the correlation between circulating levels of cell-free DNA and the stages of cancer, Bettegowda and colleagues observed that patients with lower blood levels of cell-free DNA lived significantly longer than those with higher levels of cell-free DNA (2).

Since most studies up to this stage have illustrated that increased cell-free DNA is the hallmark to distinguish cancer patients from healthy individuals, this opens the possibility of using cell-free DNA as an early tumor marker. With the knowledge that tumors arise from genetic mutations, detecting these genetic mutations could be the means to discover an early diagnostic marker. Promisingly, there are a number of studies that have demonstrated tumor-specific genetic and epigenetic changes in the cell-free DNA of cancer patients such as N-ras mutations (acute myelogenous leukaemia (3), K-ras mutations (colorectal carcinoma) (4) and p53 mutations (breast cancer) (5) to name a few.

We now know that concentration of cell-free DNA in the circulation reflects the presence of tumors. The next challenge is to determine the location of the tumors for early cancer detection. There is a definite need to identify a specific sequence or modification on the cell fragments that can specifically identify the source. As a recent breakthrough in 2012, Agostini M and his colleagues found that ALU247can be a specific marker on the cell-free DNA in discriminating breast cancer patients from non-cancer subjects (6).

Since there is so much interest and promising results from the available studies, does that mean, cell free DNA is seeing the light at the end of the tunnel?

Unfortunately, studies regarding the application of cell-free DNA in routine clinical diagnostics are only at its budding stage. In order to succeed, there are some major details to be ironed out. The foremost limitation is that the majority of the research was done with small sample size (n < 100). For clinical application, there is a need to have a huge sample size to smooth out details such as gender differences, racial/national diversities and age group differences. Hence the conclusion derived from the basis of the power of statistical tests used in these studies remain questionable.

Second challenge is the establishment of reference intervals for the healthy population. This aspect requires an immense amount of resources such as extensive monitoring, documentation and meticulous consideration to all variable attributes from pre-analytical to analytical process.

Third issue is the variation observed in the various protocols and assays. Different studies have used different sample collecting tubes, different DNA isolation, quantitation and detection methods, while downstream processing procedures such as centrifugation speed have not been standardized as well too.

Therefore, although cell-free DNA is an exciting breakthrough in the field of clinical diagnostics for early cancer detection and monitoring, there are huge complicated gaps yet to fill. Only when the above limitations are overcome, the “shipwreck – tumors/cancer” will remain secluded, while scientists will continue to “dive” deeper to look for the elusive “debris – cell free DNA”.

Figure 6: Can cell-free DNA be one of the clinical biomarker in our stable of diagnostic assays for early cancer detection and prevention in future? 

References

1. Spindler KL, Pallisgaard N, Andersen RF, Brandslund I, Jakobsen A. Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer. PloS one. 2015;10(4):e0108247. Epub 2015/04/16.
2. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Science translational medicine. 2014;6(224):224ra24. Epub 2014/02/21.
3. Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. British journal of haematology. 1994;86(4):774-9. Epub 1994/04/01.
4. Kopreski MS, Benko FA, Kwee C, Leitzel KE, Eskander E, Lipton A, et al. Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer. British journal of cancer. 1997;76(10):1293-9. Epub 1997/01/01.
5. Silva JM, Dominguez G, Garcia JM, Gonzalez R, Villanueva MJ, Navarro F, et al. Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer research. 1999;59(13):3251-6. Epub 1999/07/09.
6. Agostini M, Enzo MV, Bedin C, Belardinelli V, Goldin E, Del Bianco P, et al. Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients. Cancer biomarkers : section A of Disease markers. 2012;11(2-3):89-98. Epub 2011/01/01.

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