circulating tumor DNA

Genetic alterations can determine the natural history of cancer and its treatment response.

With further advances in DNA sequencing technology, multiple novel genetic alterations will be discovered which could be exploited as prognostic, predictive and pharmacodynamic biomarkers in the development and use of cancer therapeutics.

As such, the importance in clinical practice of efficient and robust somatic mutation testing in solid tumours cannot be overemphasized in the current era of personalized medicine.

However, significant challenges remain regarding the testing of genetic biomarkers in clinical practice.

Reliance on archived formalin fixed, paraffin embedded tumour, obtained from diagnostic biopsies, for testing somatic genetic alterations could restrict the scientific community in asking relevant questions about a patient’s cancer biology.

Problems inherent with using formalin fixed, archival tissue are well recognized and difficult to resolve. It could be argued that to achieve rapid and efficient incorporation of genetic biomarkers into clinical practice, somatic mutation testing in cancer patients should be simpler, less invasive using a readily available clinical sample, whilst maintaining robustness and reproducibility.

In this regard, use of circulating free DNA (cfDNA) from plasma or serum as an alternative and/or additional source of DNA to test cancer specific genetic alterations is an attractive proposition.

In light of encouraging results from recent studies, this mini review will discuss the current role and future potential of somatic mutation testing from circulating or cell free DNA derived from the blood of patients with solid tumours.

Synopsis

 Cell-free DNA in blood (cfDNA) represents a promising biomarker for cancer diagnosis.
 Total cfDNA concentration showed a scarce discriminatory power between patients and controls.
 A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.
 Detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in the peripheral blood of patients with solid tumors has been widely studied for the early detection of metastatic spread.

According to cancer types

 Pancreatic cancer

• The clinical distinction between cancer and chronic pancreatitis is difficult in patients with pancreatic masses.
• LOH analysis in serum DNA may be a substantial help for diagnosing pancreatic masses.

 Prostate cancer (23269609)

• Cell-free circulating DNA in plasma and serum may serve as a biomarker for malignant tumor detection and follow up in patients with a variety of solid tumors including prostate cancer.
• In healthy patients, DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe.
• Cell-free circulating DNA levels were measured by a quantitative real-time PCR method with a set of primers targeted to amplify the consensus ALU apoptotic versus necrotic origin.
• Prostate cancer patients before and 3 months after diagnosis showed cell-free circulating DNA released at apoptotic and non-apoptotic cell death.
• Interestingly, all patients after 6 months demonstrated DNA released at non-apoptotic cell.
• The principal source of cell-free circulating DNA is of apoptotic and non-apoptotic cell death.
• However, during treatment, this feature could change.
• Therefore, the study of cell-free circulating DNA would be important to follow the evolution of the disease during the treatment.
• Genomic profiling of cell-free DNA in blood and bone marrow of prostate cancer patients. PMID: 20683729

 non-small cell lung cancer

• Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality.
• New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy.
• Cell free DNA (cfDNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC).

 melanoma (23209607)

• An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A) could improve the diagnostic performance in melanoma. (23209607)

 breast cancer (23143630)

• Cell-free DNA (cfDNA) in the plasma of patients with both malignant and benign breast lesions was analyzed to determine whether the findings may have diagnostic and prognostic implications and to analyze the association between the levels of cfDNA and prognostic parameters.
• The level of cfDNA can be easily quantified using plasma samples.
• Thus, level of plasma cfDNA might constitute an important noninvasive diagnostic and prognostic valuable tool in cancer breast patients’ management. (23143630)
• SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer. (23136251)
• There is a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor. (23136251)

 Esophageal cancer

• Non-Invasive Detection of Esophageal Cancer using Genetic Changes in Circulating Cell-Free DNA. Ardekani@avicenna.ac.ir PMID: 23407878

 Gastric cancer

• Assessment of SOX17 DNA methylation in cell free DNA from patients with operable gastric cancer. Association with prognostic variables and survival. PMID: 23403728

 Ovarian cancer

• LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival. Kuhlmann JD, Schwarzenbach H, Wimberger P, Poetsch M, Kimmig R, Kasimir-Bauer S. BMC Cancer. 2012 Jul 31;12:325. doi : 10.1186/1471-2407-12-325 PMID: 22849543 [Free]

Reviews

 Circulating tumor cells and circulating tumor DNA. Alix-Panabières C, Schwarzenbach H, Pantel K. Annu Rev Med. 2012;63:199-215. doi : 10.1146/annurev-med-062310-094219 PMID: 22053740

 Cell-free nucleic acids as biomarkers in cancer patients. Schwarzenbach H, Hoon DS, Pantel K. Nat Rev Cancer. 2011 Jun;11(6):426-37. doi : 10.1038/nrc3066 PMID: 21562580

 Screening for circulating nucleic acids and caspase activity in the peripheral blood as potential diagnostic tools in lung cancer. Roth C, Kasimir-Bauer S, Pantel K, Schwarzenbach H. Mol Oncol. 2011 Jun;5(3):281-91. doi : 10.1016/j.molonc.2011.02.002 PMID: 21398193

Open References

 The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV. Int J Mol Sci. 2013 Sep 13;14(9):18925-58. doi : 10.3390/ijms140918925 PMID: 24065096 [Free]

 Multiparametric analysis of cell-free DNA in melanoma patients. Salvianti F, Pinzani P, Verderio P, Ciniselli CM, Massi D, De Giorgi V, Grazzini M, Pazzagli M, Orlando C. PLoS One. 2012;7(11):e49843. PMID: 23209607 [Free]

 LOH at 6q and 10q in fractionated circulating DNA of ovarian cancer patients is predictive for tumor cell spread and overall survival. Kuhlmann JD, Schwarzenbach H, Wimberger P, Poetsch M, Kimmig R, Kasimir-Bauer S. BMC Cancer. 2012 Jul 31;12:325. doi : 10.1186/1471-2407-12-325 PMID: 22849543 [Free]

 A modified extraction method of circulating free DNA for epidermal growth factor receptor mutation analysis. Yuan H, Zhu ZZ, Lu Y, Liu F, Zhang W, Huang G, Zhu G, Jiang B. Yonsei Med J. 2012 Jan 1;53(1):132-7. PMID: 22187243 (Free)

 Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours. Aung KL, Board RE, Ellison G, Donald E, Ward T, Clack G, Ranson M, Hughes A, Newman W, Dive C. Hugo J. 2010 Dec;4(1-4):11-21. PMID: 22132062 (Free)

 Genomic analysis of circulating cell free DNA infers breast cancer dormancy. Shaw JA, Page K, Blighe K, Hava N, Guttery D, Ward B, Brown J, Ruangpratheep C, Stebbing J, Payne R, Palmieri C, Cleator S, Walker RA, Coombes RC. Genome Res. 2011 Oct 11. PMID: 21990379 (Free)

 Comparative evaluation of cell-free tumor DNA in blood and disseminated tumor cells in bone marrow of patients with primary breast cancer. Schwarzenbach H, Pantel K, Kemper B, Beeger C, Otterbach F, Kimmig R, Kasimir-Bauer S. Breast Cancer Res. 2009;11(5):R71. doi : 10.1186/bcr2404 PMID: 19772563 [Free]

 Cell-free tumor DNA in blood plasma as a marker for circulating tumor cells in prostate cancer. Schwarzenbach H, Alix-Panabières C, Müller I, Letang N, Vendrell JP, Rebillard X, Pantel K. Clin Cancer Res. 2009 Feb 1;15(3):1032-8. doi : 10.1158/1078-0432.CCR-08-1910 PMID: 19188176 [Free]

 Identification of loss of heterozygosity on circulating free DNA in peripheral blood of prostate cancer patients: potential and technical improvements. Müller I, Beeger C, Alix-Panabières C, Rebillard X, Pantel K, Schwarzenbach H. Clin Chem. 2008 Apr;54(4):688-96. doi : 10.1373/clinchem.2007.099333 PMID: 18281424 [Free]

 A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer. Schwarzenbach H, Müller V, Beeger C, Gottberg M, Stahmann N, Pantel K. Breast Cancer Res. 2007;9(5):R66. PMID: 17915011 [Free]

 Similar patterns of loss of heterozygosity in serum of adenocarcinoma of the distal oesophagus and the cardia in early diagnosis. Wachowiak R, Kaifi JT, Schurr PG, Merkert P, Yekebas E, Schwarzenbach H, Strate T, Sauter G, Izbicki JR. Anticancer Res. 2007 Jan-Feb;27(1A):477-81. PMID: 17352270 [Free]

References

 Detection of EGFR T790M mutation in plasma DNA from patients refractory to EGFR tyrosine kinase inhibitor. Sakai K, Horiike A, Irwin DL, Kudo K, Fujita Y, Tanimoto A, Sakatani T, Saito R, Kaburaki K, Yanagitani N, Ohyanagi F, Nishio M, Nishio K. Cancer Sci. 2013 May 31. doi : 10.1111/cas.12211 PMID: 23721103

 Characterization of cell-free circulating DNA in plasma in patients with prostate cancer. Delgado PO, Alves BC, de Sousa Gehrke F, Kuniyoshi RK, Wroclavski ML, Del Giglio A, Fonseca FL. Tumour Biol. 2012 Dec 27. PMID: 23269609

 The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer. Nygaard AD, Garm Spindler KL, Pallisgaard N, Andersen RF, Jakobsen A. Lung Cancer. 2012 Dec 10. PMID: 23238036

 Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Leary RJ, Sausen M, Kinde I, Papadopoulos N, Carpten JD, Craig D, O’Shaughnessy J, Kinzler KW, Parmigiani G, Vogelstein B, Diaz LA Jr, Velculescu VE. Sci Transl Med. 2012 Nov 28;4(162):162ra154. PMID: 23197571

 Free circulating tumor DNA as a diagnostic marker for breast cancer. Hashad D, Sorour A, Ghazal A, Talaat I. J Clin Lab Anal. 2012 Nov;26(6):467-72. PMID: 23143630

 SOX17 Promoter Methylation in Circulating Tumor Cells and Matched Cell-Free DNA Isolated from Plasma of Patients with Breast Cancer. Chimonidou M, Strati A, Malamos N, Georgoulias V, Lianidou ES. Clin Chem. 2012 Nov 7. PMID: 23136251

 Cell-free DNA in the circulation as a potential cancer biomarker. Kohler C, Barekati Z, Radpour R, Zhong XY. Anticancer Res. 2011 Aug;31(8):2623-8. PMID: 21778314

 Loss of heterozygosity at tumor suppressor genes detectable on fractionated circulating cell-free tumor DNA as indicator of breast cancer progression. Schwarzenbach H, Eichelser C, Kropidlowski J, Janni W, Rack B, Pantel K. Clin Cancer Res. 2012 Oct 15;18(20):5719-30. doi : 10.1158/1078-0432.CCR-12-0142 PMID: 23014523

 Circulating cell-free DNA: a promising marker of pathologic tumor response in rectal cancer patients receiving preoperative chemoradiotherapy. Agostini M, Pucciarelli S, Enzo MV, Del Bianco P, Briarava M, Bedin C, Maretto I, Friso ML, Lonardi S, Mescoli C, Toppan P, Urso E, Nitti D. Ann Surg Oncol. 2011 Sep;18(9):2461-8. PMID: 21416156

 Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients. Agostini M, Enzo MV, Bedin C, Belardinelli V, Goldin E, Del Bianco P, Maschietto E, D’Angelo E, Izzi L, Saccani A, Zavagno G, Nitti D. Cancer Biomark. 2012;11(2-3):89-98. PMID: 23011155

 Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing. Forshew, T., Murtaza, M., Parkinson, C., Gale, D., Tsui, D.W.Y., Kaper, F., Dawson, S.J., Piskorz, A.M., Jimenez-Linan, M., Bentley, D., Hadfield, J., May, A.P., Caldas, C., Brenton, J.D., Rosenfeld, N. Science Translational Medicine May 31, 2012.

 The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy. Payne RE, Hava NL, Page K, Blighe K, Ward B, Slade M, Brown J, Guttery DS, Zaidi SA, Stebbing J, Jacob J, Yagüe E, Shaw JA, Coombes RC. Br J Cancer. 2011 Dec 13. PMID: 22166803

 Evaluation of cell-free tumour DNA and RNA in patients with breast cancer and benign breast disease. Schwarzenbach H, Müller V, Milde-Langosch K, Steinbach B, Pantel K. Mol Biosyst. 2011 Oct;7(10):2848-54. doi : 10.1039/c1mb05197k PMID: 21785770

 Genomic profiling of cell-free DNA in blood and bone marrow of prostate cancer patients. Schwarzenbach H, Chun FK, Isbarn H, Huland H, Pantel K. J Cancer Res Clin Oncol. 2011 May;137(5):811-9. doi : 10.1007/s00432-010-0941-5 PMID: 20683729

 Quantification of free plasma DNA before and after chemotherapy in patients with advanced epithelial ovarian cancer. Capizzi E, Gabusi E, Grigioni AD, De Iaco P, Rosati M, Zamagni C, Fiorentino M. Diagn Mol Pathol. 2008 Mar;17(1):34-8. PMID: 18303408

 Microsatellite analysis in serum DNA as a diagnostic tool for distinction of patients with unknown pancreatic masses. Wachowiak R, Kaifi J, Schwarzenbach H, Yekebas E, Merkert P, Schurr P, Hansen B, Reichelt U, Strate T, Pantel K, Izbicki JR. Diagn Mol Pathol. 2007 Sep;16(3):174-8. PMID: 17721326

 Current status and future potential of somatic mutation testing from circulating free DNA in patients with solid tumours. Aung KL, Board RE, Ellison G, Donald E, Ward T, Clack G, Ranson M, Hughes A, Newman W, Dive C. Hugo J. 2010 Dec;4(1-4):11-21. PMID: 22132062

 Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer. Schwarzenbach H, Stoehlmacher J, Pantel K, Goekkurt E. Ann N Y Acad Sci. 2008 Aug;1137:190-6. doi : 10.1196/annals.1448.025 PMID: 18837946

 Microsatellite analysis of allelic imbalance in tumour and blood from patients with prostate cancer. Schwarzenbach H, Chun FK, Müller I, Seidel C, Urban K, Erbersdobler A, Huland H, Pantel K, Friedrich MG. BJU Int. 2008 Jul;102(2):253-8. doi : 10.1111/j.1464-410X.2008.07600.x PMID: 18336598

 Microsatellite analysis in serum DNA as a diagnostic tool for distinction of patients with unknown pancreatic masses. Wachowiak R, Kaifi J, Schwarzenbach H, Yekebas E, Merkert P, Schurr P, Hansen B, Reichelt U, Strate T, Pantel K, Izbicki JR. Diagn Mol Pathol. 2007 Sep;16(3):174-8. PMID: 17721326

 Detection of tumor-specific DNA in blood and bone marrow plasma from patients with prostate cancer. Schwarzenbach H, Chun FK, Lange I, Carpenter S, Gottberg M, Erbersdobler A, Friedrich MG, Huland H, Pantel K. Int J Cancer. 2007 Apr 1;120(7):1465-71. PMID: 17205532

 Comparison of genetic alterations detected in circulating microsatellite DNA in blood plasma samples of patients with prostate cancer and benign prostatic hyperplasia. Müller I, Urban K, Pantel K, Schwarzenbach H. Ann N Y Acad Sci. 2006 Sep;1075:222-9. PMID: 17108215

 Circulating tumour-associated plasma DNA represents an independent and informative predictor of prostate cancer. Chun FK, Müller I, Lange I, Friedrich MG, Erbersdobler A, Karakiewicz PI, Graefen M, Pantel K, Huland H, Schwarzenbach H. BJU Int. 2006 Sep;98(3):544-8. PMID: 16925751

 Detection and characterization of circulating microsatellite-DNA in blood of patients with breast cancer. Schwarzenbach H, Müller V, Stahmann N, Pantel K. Ann N Y Acad Sci. 2004 Jun;1022:25-32. PMID: 15251935