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Circulating Tumor DNA Methodologies

Introduction

The analysis of cell-free DNA or liquid biopsy is emerging as a noninvasive clinical tool to aid in achieving accurate diagnoses. It is also a valuable research tool to determine biomarkers of disease, identify drug targets, and ascertain the factors responsible for drug resistance. Circulating tumor DNA refers to cell-free DNA that are fragments of DNA released from tumor cells and appearing in the blood circulation.

Major challenges to cancer survival include tumor metastasis and resistance to chemotherapy. Tumor tissue is heterogeneous and exhibits a diversity of morphological features and can change over time. Effectively analyzing these tissues using traditional methods would require multiple biopsies. Besides being quite costly, this approach would be unendurable and harmful to patients.

Analysis of circulating tumor DNA in blood addresses a number of the limitations associated with tissue biopsy. Using this form of liquid biopsy is a rapid, noninvasive, and even cost-effective means to answer many questions such as, but not limited to, those regarding the risk of tumor recurrence, the likelihood of treatment success, and risks analysis for prevention.  Circulating tumor DNA detection methods have also uncovered information regarding possible mechanisms of chemotherapeutic resistance, as well as potential therapeutic targets for cancer treatment.

Clinical Utility of Cell-Free Tumor DNA Detection

The ability to determine the risk of recurrence and treatment resistance would provide opportunities to design treatment approaches to increase therapeutic success. Collecting tissue from metastatic deposits is not only invasive, but often provides results that have limited reliability1. Also, it is not uncommon for target tissues to be inaccessible. This is where the detection of cell-free tumor DNA enhances the diagnostic and surveillance efforts.

Lebofsky et al conducted studies using de novo detection of somatic mutations using plasma DNA and next-generation sequencing (NGS)2. The results were compared to those of traditional metastasis analysis. The researchers found that detection using circulating tumor DNA was as accurate as tissue biopsies. In some cases, it detected mutations that tissue biopsy failed or could not be applied. These findings show that the clinical use of circulating tumor DNA may be a viable alternative to tissue biopsy given the accuracy, low invasiveness, and reduced costs.

As stated earlier, tumor is a factor that limits cancer survival. Effective therapeutic approaches can be better implemented with the early prediction of tumor recurrence after surgery. However, there is not yet a routine efficient means to determine if a given patient may have residual disease after surgery or chemotherapy.

Tie et al used massively parallel sequencing-based methods to determine the predictive value of circulating tumor DNA detection3. In patients previously treated via surgery and/or chemotherapy for colon cancer, cell-free DNA was still detected, indicating the potential for recurrence. It was later (approximately 2.5 years after treatment) noted that the majority of those did have tumor recurrences. Therefore, it is possible to identify patients at risk of recurrence by detecting circulating tumor DNA.

Rothé et al used NGS to analyze tumor tissue and blood plasma samples from patients with breast cancer4. Tumor tissue and plasma results were concordant in 76% of patients. This is another indication of the potential of circulating tumor DNA as an alternative to tissue biopsy for tumor evaluation.

Cancer treatment evaluation

After surgical and other cancer treatment, it is important to monitor progress throughout and after providing adjuvant therapies. The information gleaned allows the detection of recurrence and the design of therapeutic strategies to enhance post-treatment survival. Zhou et al performed massive parallel sequencing assays to detect somatic mutations in tumor tissues and plasma samples of patients treated for colorectal cancer5. They found that the circulating tumor DNA levels correlated with tumor progression.

Sequence-based DNA monitoring methods have been used to quantify circulating tumor DNA before, during, and after treatment of patients with B-cell lymphomas. This method was more sensitive and had higher specificity compared to imaging techniques6. This is another cancer type where the application of circulating tumor methods shows much promise as an alternative to other traditional methods to monitor treatment outcomes.

Screening for mutations in patients at high risk for cancer development could inform regarding early disease states, and is valuable for taking preventive treatment measures. This could apply, for example, to individuals with a strong family history of cancer occurrence, smokers, and those with a genetic predisposition to a cancer type. Next-generation sequencing was used by Roschewski et al analyze circulating tumor DNA in patients with large-B-cell lymphoma7. Patients at risk of recurrence were identified before any clinical signs of disease were detectable.

Advantages of Circulating Tumor DNA Analysis vs. Tissue Biopsy

As previously noted, there are a number of advantages of circulating tumor DNA analysis when compared to traditional tissue biopsy. In the case of tumor biopsy, this approach is specific to localized tumor sites, is invasive, and tends to be expensive. The application of circulating tumor DNA detection is noninvasive, has a lower cost compared to tissue biopsy, and does not depend on the site of the solid tumor.

Tissue biopsy limitations that are overcome by performing circulating tumor DNA analysis (liquid biopsy) include the difficulties with tumor heterogeneity, and the need to apply the method to a limited anatomic location of the tumor. Complications also occur when only a limited amount of tissue is accessible. If multiple analyses are needed, repeated tissue biopsies would not be feasible.

Methods to Analyze Circulating Tumor DNA

Droplet-based digital PCR for detection of mutations in circulating tumor DNA from plasma of patients is also a means to detect mutations and tumor status. With this methodology, a plasma sample is diluted and separated into a large number of droplets. Essentially, thousands of amplification reactions take place from a single sample.

Next-generation sequencing methodology is a powerful means to determine and identify mutations. Large numbers of genes can be analyzed in a single run. It is a high-throughput technology that provides assay sensitivity and specificity8. Variant calling using liquid biopsy samples also provides high sensitivity, particularly with the application of NGS. The ability to detect mutations from a blood sample is a promising clinical application that can revolutionize cancer diagnosis and management.

Conclusions

The detection and analysis of tumor DNA in circulating blood provides a safer, sensitive, and cost-effective means to achieve diagnostic and surveillance efforts in cancer monitoring and treatment. Circulating tumor DNA methodologies primarily involve the use of NGS to identify biomarkers of tumor cells without subjecting patients to repeated invasive surgical procedures associated with tissue biopsies. There are many instances where it is not feasible to perform a traditional tissue biopsy. This is overcome with the ability to obtain a blood sample that can be analyzed using NGS, particularly given the sensitivity and accuracy of the methods. With the continuing advances in NGS technology, detection and analysis of circulating tumor DNA can rapidly become a viable alternative to traditional tumor analysis technologies.

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References

  1. Criscitiello, C., Andre, F., Thompson, A.M., De Laurentiis, M., Esposito, A., Gelao, L., Fumagalli, L., Locatelli, M., Minchella, I., Orsi, F., Goldhirsch, A., Curigliano, G., 2014. Biopsy confirmation of metastatic sites in breast cancer patients: clinical impact and future perspectives. Breast Cancer Res. 16, 205.
  1. Lebofsky R, Decraene C, Bernard V, Kamal M, Blin A, Leroy Q, Rio Frio T, Pierron G, Callens C, Bieche I, Saliou A, Madic J, Rouleau E, Bidard FC, Lantz O, Stern MH, Le Tourneau C, Pierga JY. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized medicine in a prospective trial across all tumor types. Mol Oncol. 2015 Apr;9(4):783-90.
  1. Tie J, Wang Y, Tomasetti C, Li L, Springer S, Kinde I, Silliman N, Tacey M, Wong HL, Christie M, Kosmider S, Skinner I, Wong R, Steel M, Tran B, Desai J, Jones I, Haydon A, Hayes T, Price TJ, Strausberg RL, Diaz LA Jr, Papadopoulos N, Kinzler KW, Vogelstein B, Gibbs P. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci Transl Med. 2016 Jul 6;8(346):346ra92.
  1. Rothé F, Laes JF, Lambrechts D, Smeets D, Vincent D, Maetens M, Fumagalli D, Michiels S, Drisis S, Moerman C, Detiffe JP, Larsimont D, Awada A, Piccart M, Sotiriou C, Ignatiadis M. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer. Ann Oncol. 2014 Oct;25(10):1959-65.
  1. Zhou J, Chang L, Guan Y, Yang L, Xia X, Cui L, Yi X, Lin G. Application of Circulating Tumor DNA as a Non-Invasive Tool for Monitoring the Progression of Colorectal Cancer. PLoS One. 2016 Jul 26;11(7):e0159708.
  1. Kwok M, Wu SP, Mo C, Summers T, Roschewski M. Circulating Tumor DNA to Monitor Therapy for Aggressive B-Cell Lymphomas. Curr Treat Options Oncol. 2016 Sep;17(9):47.
  1. Roschewski M, Dunleavy K, Pittaluga S, Moorhead M, Pepin F, Kong K, Shovlin M, Jaffe ES, Staudt LM, Lai C, Steinberg SM, Chen CC, Zheng J, Willis TD, Faham M, Wilson WH. Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B-cell lymphoma: a correlative biomarker study.
  1. Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA, Merritt RE, Shrager JB, Loo BW Jr, Alizadeh AA, Diehn M. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. 2014 May;20(5):548-54.

Written by Macrogen Corp.

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