Methodological, biological and clinical aspects of circulating free DNA in metastatic colorectal cancer

Figures & data

Table 1. Studies which have included a comparison of total cell-free DNA levels in colorectal cancer and controls.

Reference	Patients	Normalcohort	Total cfDNA analyzed	Plasma mutations analyzed	Results
El-Gayar et al. [15] 2016	50	20 Healthy 10 Polyps	+	(+)	Higher in CRC AUC 0.90
Hao et al. [20] 2014	204	110 Healthy 61 Polyps	+	–	Higher in CRC AUC 0.89
Spindler et al. [58] 2014	86 Stage IV	93 Healthy 53 With comorbidity	+	+	Higher in CRC AUC 0.86
Spindler et al. [63] 2014	229 Stage IV	93 Healthy	+	+	Higher in CRC AUC 0.94
da Silva et al. [63] 2013	50	30 Healthy	+	–	Higher in CRC
Perkins et al. [64] 2013	25 Stage IV	20 Healthy	+	+	Higher in CRC
Qi et al. [65] 2012	31	92 Healthy 30 Polyps	+	–	Higher in CRC AUC 0.904
Mead et al. [66] 2011	24	35 Healthy 26 Polyps	+	–	Higher in CRC than benign GI disease and healthy controls AUC 0.76
Czeiger et al. [67] 2011	38	34 Healthy	+	–	Higher in CRC AUC 0.84
Danese et al. [68] 2010	118 (28 Stage IV)	26 Healthy 49 Polyps	+	–	Higher in CRC than benign GI disease and healthy controls AUC 0.93
Frattini et al. [69] 2008	70 (6 Dukes D)	20 Healthy	+	+	Higher in CRC
Boni et al. [70] 2007	67	67 Healthy	+	–	Higher in CRC
Flamini et al. [71] 2006	75 (12 Dukes D)	75 Healthy	+	–	Higher in CRC AUC 0.86
Diehl et al. [47] 2005	33 + 56	10 Healthy	+	–	Higher in Dukes C-D than healthy, not significantly higher in polyps or Dukes A
Shapiro et al. [72] 1983	29	88 Healthy	+	–	Higher in CRC than benign GI disease and healthy controls

AUC: area under the curve; cfDNA: cell-free DNA; CRC: colorectal cancer; GI: gastrointestinal.

Table 2. Studies investigating the correlation between circulating DNA and carcinoembryonic antigen.

Reference	cfDNA measure	Number of patients	Results of direct correlation between cfDNA and CEA	Comparison of cfDNA and CEA
El-Gayar et al. [15] 2016	DNA II and cfDNA (Alu repeats)	50	r = 0.45, p < 0.001	NA in relation to outcome Better diagnostic performance AUC 0.90 (DNA II) vs. 0.86 (CEA)
Tie et al. [82] 2015	ctDNA (various)	53	NA	Performed better than CEA Correlation to tumor burden: r = 0.05 (ctDNA) and 0.03 (CEA) Correlation to response: AUC 0.73 (ctDNA) and 0.52 (CEA)
Siravegna et al. [83] 2015	ctDNA (various)	10	NA	Case studies, no visual correlation
Spindler et al. [33] 2015	cfDNA (gCYG) ctDNA (KRAS)	134	r = 0.37, p > 0.05	Performed better than CEA ctDNA strong prognostic marker CEA failed in uni- and multivariate analysis
Bettegowda et al. [41] 2014	ctDNA (KRAS)	69	NA	CEA used as measure of tumor burden. ctDNA less likely to be detectable in low CEA patients. Positive correlation between ctDNA and CEA levels in those with detectable KRAS mutations. Association in univariate analysis 0.439 (95% CI 0.22–0.658), p < 0.001
Hao et al. [20] 2014	cfDNA and DNA II (Alu repeats)	204	NA	Performed better than CEA NA in relation to outcome Better diagnostic performance AUC 0.89 (DNA II) vs. 0.78 (CEA)
Diaz et al. [39] 2012	cfDNA ctDNA (KRAS)	29	NA Case studies, visual correlation ctDNA mirrors CEA, but no statistical analysis performed	Case studies, visual correlation ctDNA mirrors CEA in relation to tumor burden
Qi et al. [65] 2012	cfDNA (Alubased)	31	r = 0.232, p > 0.05	Better diagnostic performance (intestinal polyps vs. CRC) AUC 0.848 (cfDNA) vs. 0.0.68 (CEA)
Spindler et al. [32] 2012, 2014	cfDNA (gCYG) ctDNA (KRAS)	105	r = 0.29, p > 0.05	Perform better than CEA in uni- and multivariate analysis* (unpublished data)
Mead et al. [66] 2011	cfDNA (Line1) ctDNA (Alu repeat II)	24		Better diagnostic performance (intestinal polyps vs. CRC) AUC 0.756/0.772 (cfDNA/DNA II) vs. 0.0.0.596 (CEA)
Czeiger et al. [67] 2011	(cfDNA) fluorescent analysis	38	p = 0.18, p = 0.29	Diagnostic performance AUC 0.84 (cfDNA) NA for CEA Significantly higher cfDNA levels in patient who died from CRC than cured, performed better in combination with CEA
Danese et al. [68] 2010	cfDNA (GADPH)	118	r = 0.028, p = 0.78	Better diagnostic performance AUC 0.93 (cfDNA) vs. 0.68 (CEA)
Frattini et al. [69] 2008	cfDNA ctDNA (KRAS and p16 promotor hypermethylation Kit)	70	NA Some correlation by 2x2 cross tabulation	No direct comparison of performed in correlation to clinical outcome after surgery for CRC
Diehl et al. [81] 2008	ctDNA (various) (BEAMing)	18	r^2 = 0.20, p < 0.001	Performed better than CEA in relation to prediction of recurrence-free survival after surgery (p = 0.03)
Boni et al. [70] 2007	cfDNA (RNAse P gene)	67	NA	Better diagnostic performance (mean values compared in CRC and healthy)
Flamini et al. [71] 2006	cfDNA (GADPH)	75	rs = 0.16, p = 0.17	Better diagnostic performance AUC 0.86 (cfDNA) vs. 0.82 (CEA), optimal combined (AUC 0.92)
Ryan et al. [80] 2002	ctDNA (KRAS2)	78	NA	Better diagnostic performance (cross tabulation)
Shapiro et al. [72] 1983	cfDNA (radioimmunoassay)	63	r = 0.14	Better diagnostic performance (cross tabulation, χ^2-tests)

AUC: area under the curve; BEAMing: beads, emulsion, amplification, and magnetics; CEA: carcinoembryonic antigen; cfDNA: cell-free DNA; CRC: colorectal cancer; ctDNA: circulating tumor DNA; DNA II: DNA integrity index; GADPH: glyceraldehyde 3-phosphate dehydrogenase; KRAS: Kirsten ras; NA: not addressed; RNAse: P ribonuclease P gene.

Table 3 List of studies having investigated cfDNA and/or tumor-specific mutations in serial blood samples in metastatic colorectal cancer patients.

Reference	Patients with mCRC	Therapy	Markers	Methods	Results
Misale et al. [40] 2012	3.	EGFR inh.	KRAS	BEAMing	Emergence prior to progression
Diaz et al. [39] 2012	28.	EGFR inh.	KRAS	BEAMing	9/24 developed mutations at progression
Spindler et al. [94] 2014	105	EGFR inh.	KRAS, BRAF cfDNA	qPCR	Emergence of mutations prior to progression Decrease of cfDNA and plasma mutations in responders, no decrease in non-responders
Bettegowda et al. [41] 2014	24 (20 from ref. Diaz)	EGFR inh.	Multiple alterations	BEAMing	A total of 70 mutations emerged in the 24 patients
Mohan et al. [93] 2014	10	EGFR inh.	Multiple alterations	Plasma-seq	Gain of KRAS mutations in 4 patients Acquired KRAS copy number changes 4/10
Tie et al. [82] 2015	53	First-line chemotherapy	KRAS	Digital genomic assay Safe-SeqS	Spike Day 3 in 3 responders 10-fold drop correlated to good response
Wong et al. [60] 2015	35	Regorafinib	Multiple alterations	BEAMing	Decline of ctDNA in responders, and those with long PFS Emergence of new mutations?
Siravegna et al. [83] 2015	16 2 3	EGFR inh.	Multiple alterations	ddPCR NGS	Emergence of mutations prior to progression Decline after withdrawal Potential for re-challenge illustrated

BEAMing: beads, emulsion, amplification, and magnetics; BRAF: proto-oncogene B-Raf; cfDNA: cell-free DNA; ctDNA: circulating tumor DNA; EGFR inhibitor: epidermal growth factor receptor inhibitor; KRAS: Kirsten ras; qPCR: quantitative PCR.

El-Gayar D, El-Abd N, Hassan N, et al. Increased free circulation DNA integrity Index as a serum biomarker in patients with colorectal carcinoma. Asian Pac J Cancer Prev. 2016;17:939–944.

 PubMedGoogle Scholar

Hao TS, Shi W, Shen XJ, et al. Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer. Br J Cancer. 2014;111:1482–1489.

 PubMed Web of Science ®Google Scholar

Hansen TF, Andersen RF, Pallisgaard N, et al. A three weekly schedule of irinotecan and panitumumab as third-line treatment for patients with wild type KRAS metastatic colorectal cancer: Results from. A phase II trial. Colorec Cancer. 2014;3:135–145.

 Google Scholar

da Silva Filho BF, Gurgel AP, Neto MÁ, et al. Circulating cell-free DNA in serum as a biomarker of colorectal cancer. J Clin Pathol. 2013;66:775–778.

 PubMed Web of Science ®Google Scholar

Perkins G, Yap TA, et al. Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers. PLoS One. 2012;7:e47020.

 PubMed Web of Science ®Google Scholar

Qi J, Qian C, Shi W, et al. Alu-based cell-free DNA: a potential complementary biomarker for diagnosis of colorectal cancer. Clin Biochem. 2013;46:64–9.

 PubMed Web of Science ®Google Scholar

Mead R, Duku M, Bhandari P, et al. Circulating tumour markers can define patients with normal colons, benign polyps, and cancers. Br J Cancer. 2011;105:239–245.

 PubMed Web of Science ®Google Scholar

Czeiger D, Shaked G, Eini H, et al. Measurement of circulating cell-freeDNA levesl by a new simple flouroscent test in patients with primary colorectal cancer. Am J Clin Pathol. 2011;135:264–270.

 PubMed Web of Science ®Google Scholar

Danese E, Montagnana M, Minicozzi AM, et al. Real-time polymerase chain reaction quantification of free DNA in serum of patients with polyps and colorectal cancers. Clin Chem Lab Med. 2010;48:1665–8.

 PubMed Web of Science ®Google Scholar

Frattini M, Gallino G, Signoroni S, et al. Quantitative and qualitative characterization of plasma DNA identifies primary and recurrent colorectal cancer. Cancer Lett. 2008;263:170–81.

 PubMed Web of Science ®Google Scholar

Boni L, Cassinotti E, Canziani M, et al. Free circulating DNA as possible tumour marker in colorectal cancer. Surg Oncol. 2007;16 (Suppl 1):S29–S31.

 PubMed Web of Science ®Google Scholar

Flamini E, Mercatali L, Nanni O, et al. Free DNA and carcinoembryonic antigen serum levels: an important combination for diagnosis of colorectal cancer. Clin Cancer Res. 2006;12:6985–6988.

 PubMed Web of Science ®Google Scholar

Diehl F, Li M, Dressman D, et al. Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci USA. 2005;102:16368–16373.

 PubMed Web of Science ®Google Scholar

Shapiro B, Chakrabarty M, Cohn EM, et al. Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease. Cancer. 1983;51:2116–2120.

 PubMed Web of Science ®Google Scholar

Tie J, Kinde I, Wang Y, et al. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann Oncol. 2015;26:1715–1722.

 PubMed Web of Science ®Google Scholar

Siravegna G, Mussolin B, Buscarino M, et al. A.Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat Med. 2015;21:795–801.

 PubMed Web of Science ®Google Scholar

Spindler KL, Pallisgaard N, Appelt A, et al. Clinical utility of KRAS status in circulating plasma DNA compared to archival tumor tissue from patients with metastatic colorectal cancer treated with anti-EGFR therapy. Eur J Cancer. 2015;51:2678–2685.

 PubMed Web of Science ®Google Scholar

Bettegowda C, Sausen M, Leary RJ, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014;6:224ra24.

 PubMed Web of Science ®Google Scholar

Diaz LA, Jr, Williams RT, Wu J, et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 2012;486:537–540.

 PubMed Web of Science ®Google Scholar

Spindler KL, Pallisgaard N, Vogelius I, et al. Quantitative cell free DNA, KRAS and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clin Cancer Res. 2012;18:1177–1185.

 PubMed Web of Science ®Google Scholar

Diehl F, Schmidt K, Choti MA. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14:985–990.

 PubMed Web of Science ®Google Scholar

Ryan BM, Lefort F, McManus R, et al. A prospective study of circulating mutant KRAS2 in the serum of patients with colorectal neoplasia: strong prognostic indicator in postoperative follow up. Gut. 2003;52:101–108.

 PubMed Web of Science ®Google Scholar

Misale S, Yaeger R, Hobor S, et al. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature. 2012;486:532–536.

 PubMed Web of Science ®Google Scholar

Spindler KL, Pallisgaard N, Andersen RF, et al. Changes in mutational status during third-line treatment for metastatic colorectal cancer–results of consecutive measurement of cell free DNA, KRAS and BRAF in the plasma. Int J Cancer. 2014;135:2215–2222.

 PubMed Web of Science ®Google Scholar

Kidess E, Heirich K, Wiggin M, et al. Mutation profiling of tumor DNA from plasma and tumor tissue of colorectal cancer patients with a novel, high-sensitivity multiplexed mutation detection platform. Oncotarget. 2015;6:2549–2561.

 PubMed Web of Science ®Google Scholar

Wong AL, Lim JS, Sinha A, et al. Tumour pharmacodynamics and circulating cell free DNA in patients with refractory colorectal carcinoma treated with regorafenib. J Transl Med. 2015;13:57.

 PubMed Web of Science ®Google Scholar