Imagine if, instead of painful and invasive tissue biopsies of solid tumors, all that a physician needed was a 10 mL blood sample (about a tablespoon’s worth) to diagnose cancer, inform treatment decisions, or monitor the response of a tumor to treatment. Often, when a tumor is identified, doctors will surgically remove a piece of the tumor—that is, perform a biopsy—and subject the removed tumor tissue to an array of tests. These tests provide crucial information about the genetic makeup of the tumor and the stage (severity) of the disease that is used when making decisions about how to treat it.

Traditional biopsy methods are invasive procedures that often involve a surgeon’s scalpel and anesthesia, both of which carry inherent health risks. However, recent publications from Johns Hopkins [1] and Stanford School of Medicine [2, 3] show that the time of much simpler and less risky blood-based biopsies may be in our near future. Researchers have established non-invasive techniques that enable doctors to identify tumor masses that may be too small to show up using other diagnostic tests (like imaging), or to hone in on rare mutations known to be associated with particular disease progression patterns. Gone will be the necessity of being cut by a knife: instead, important diagnostic information could be obtained with a routine blood draw, which could even be included as part of an annual check-up.

Cancer cell mutations affect disease prognosis and inform treatment decisions

Cancerous cells carry multiple mutations that distinguish them from normal, healthy cells. Identification of these mutations has implications in the prognosis and treatment regimes that are prescribed. For instance, EGFR inhibitors are often given to block the division of tumor cells, but patients with colorectal carcinomas carrying mutations in the genes KRAS and BRAF are not given EGFR inhibitors because these drugs won’t prevent tumor cell division if KRAS and BRAF are mutated [4].  Traditional methods for finding cancer mutation signatures involve analysis of biopsy specimens; however, some tumors are difficult to access surgically (e.g. ovaries), the surrounding non-cancerous cells may contaminate the sample, and biopsy samples may degrade and become untestable over time [5].

As early as 1977, scientists noticed a heightened presence of cell-free DNA in the bloodstream of cancer patients compared to healthy individuals [4], which is thought to be the result of dying tumor cells leaking genetic material into the bloodstream. More recently, researchers wondered whether this “circulating tumor DNA” (ctDNA) could be used instead of a biopsy to obtain genetic material from tumors in order to find cancer mutation signatures.

A paper published recently in Science Translational Medicine [1] sought to analyze ctDNA present in the blood of patients for the presence of different cancer mutation signatures, which could inform treatment options.  It was the first time that researchers demonstrated the rarity of ctDNA in patients with brain tumors, as well as the variability in the amount of ctDNA present in blood across a panel of different cancers – abundant in ovarian, colon and breast cancers, less so in metastatic kidney, prostate, or thyroid cancers [1, 6]. Of particular interest for patients with colorectal cancer was the finding that the cancer-associated gene KRAS mutated over time in response to treatment, and that these changes were detectable with their ctDNA technology. This finding suggests that development of drug resistance in cancers could be monitored with analyses of ctDNA in serial blood draws during a patient’s treatment period. Finally, the researchers also reported that the concentration of ctDNA increased with disease progression, but even patients with stage I or II disease (early, and more readily treatable with surgery) had identifiable tumor DNA in their circulation, albeit at a lower frequency, a finding that holds promise for early disease detection [1].

 BEAM me up: MacGyver-ing detection of minute concentrations of ctDNA

One technical difficulty in using ctDNA to obtain information about tumors is that the mutated tumor sequences are present in very small quantities relative to the normal DNA fragments also found in the ctDNA released by cancer cells. To circumvent this obstacle, researchers generate more DNA fragments by, essentially, copy-and-pasting the DNA fragments (amplification) to increase their presence in a sample, a process that increases the number of fragments of both normal and mutated DNA. After this first step of amplification, a second round of amplification attaches many copies of the same DNA fragment to a single magnetic bead so that the signal of that particular sequence can be picked up from amidst all of the other sequences in the sample. Amplified sequences attached to magnetic beads can then be pulled out of the reaction mixture with a magnet: When held against the side of a tube, the beads adhere to that side, and the liquid can be removed, leaving just the DNA attached to the beads behind and allowing sequences to be sorted and quantified [7, 8]. By comparing the amount of mutant DNA to the amount of normal DNA, researchers can monitor the relative concentration of mutant DNA in the blood. This technique, called BEAMing (beads, emulsion, amplification, magnetics) was used in the study described above to detect and identify the ctDNA in patients’ blood samples (See Figure).

 

Figure 1 ~ Detecting mutant DNA in blood samples using magnetic beads

It’s all very exciting, but what’s the catch?

While it is exciting to imagine that a simple blood draw could tell doctors what they need to know to treat cancer more effectively, there are still many obstacles that must be overcome before we will see “liquid biopsies” joining the ranks of standard medical tests. Not the least of it is the psychological impact to patients with advanced disease who may find out earlier that their cancer has progressed: While earlier detection of a worsening disease could allow patients to abandon an ineffective treatment, it does not always mean a better overall outcome for the patient [1, 9]. From a technical standpoint, one disadvantage of BEAMing is that doctors already need to know exactly which mutations they’re looking for, whereas with other techniques involving sequencing, doctors can detect novel mutations that influence cancer progression.

Using ctDNA to learn about tumors holds great promise for patients and caregivers alike as researchers improve on the precision and reliability of such diagnostic techniques. Imagine identifying a tumor and having its development followed closely in response to a particular treatment regime, all with a simple blood draw.

Yi-Jang Lin is a first-year (soon to be second, gasp) graduate student in Biological and Biomedical Sciences.

References

[1] Bettegowda, C, M Sausen, RJ Leary, I Kinde, Y Wang, N Agrawal, BR Bartlett, H Wang, B Luber, RM Alani, ES Antonarakis, NS Azad, A Bardelli, H Brem, JL Cameron, CC Lee, LA Fecher, GL Gallia, P Gibbs, D Le, RB Giuntoli, M Goggins, MD Hogarty, M Holdhoff, SM Hong, Y Jia, HH Juhl, JJ Kim, G Siravegna, DA Laheru, C Lauricella, M Lim, EJ Lipson, SKN Marie, GJ Netto, KS Oliner, A Olivi, L Olsson, GJ Riggins, A Sartore-Bianchi, K Schmidt, IM Shih, SM Oba-Shinjo, S Siena, D Theodorescu, J Tie, TT Harkins, S Veronese, TL Wang, JD Weingart, CL Wolfgang, LD Wood, D Xing, RH Hruban, J Wu, PJ Allen, CM Schmidt, MA Choti, VE Velculescu, KW Kinzler, B Vogelstein, N Papadopoulos, and LA Diaz Jr. 2014. Detection of circulating tumor DNA in early- and late-stage human malignancies. Science Translational Medicine, 6(224): 1-12.

[2] Newman, AM, SV Bratman, J To, JF Wynne, NCW Eclov, LA Modlin, CL Liu, JW Neal, HA Wakelee, RE Merritt, JB Shrager, BW Loo Jr, AA Alizadeh, and M Diehn. 2014. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature Medicine, advanced online publication.

[3] Brooks, M. Ultrasensitive ‘liquid biopsy’ may detect many solid tumors. Medscape Medical News, April 7 2014. http://www.medscape.com/viewarticle/823188

[4] Sidransky, D. 2002. Emerging molecular markers of cancer. Nature Reviews Cancer, 2: 210-19.

[5] Diehl, F, and E Smergeliene. BEAMing for cancer. Genetic Engineering and Biotechnology News, 33(15): September 1 2013. http://www.genengnews.com/gen-articles/beaming-for-cancer/4972/

[6] Anderson, A. Cross-cancer analysis clarifies characteristics of circulating tumor DNA. GenomeWeb Daily News, February 19 2014. http://www.genomeweb.com/pcrsample-prep/cross-cancer-analysis-clarifies-characteristics-circulating-tumor-dna

[7] Dressman, D, H Yan, G Traverso, KW Kinzler, and B Vogelstein. 2003. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proceedings of the National Academy of Sciences, 100(15): 8817-22.

[8] Kinde, I, J Wu, N Papadopoulos, KW Kinzler, and B Vogelstein. 2011. Detection and quantification of rare mutations with massively parallel sequencing. Proceedings of the National Academy of Sciences, 108(23): 9530-35.

[9] Pollack, A. Sidestepping the biopsy with new tools to spot cancer. The New York Times, April 7 2014.