Circulating free tumor DNA and colorectal cancer

Gastroentérologie Clinique et Biologique (2010) 34, 662—681

SEMINAR

Circulating free tumor DNA and colorectal cancer ADN libre circulant d’origine tumorale et cancer colorectal T. Lecomte a,∗,b,c, N. Ceze a,c, É. Dorval a,c, P. Laurent-Puig d a

Université Franc¸ois-Rabelais, parc Grandmont, 37200 Tours, France CNRS, UMR 6239 (GICC université Francois-Rabelais), 31, avenue Monge, 37200 Tours, France c Service d’hépatogastroentérologie et de cancérologie digestive, CHRU de Tours, 37044 Tours cedex 09, France d Inserm UMR-S775 « base moléculaire de la réponse aux xénobiotiques », université Paris-Descartes, France b

Available online 15 September 2010

Summary Cancer is characterized by multiple somatic genetic and epigenetic alterations that could be useful as molecular markers for detecting tumor DNA in different bodily fluids. In patients with various diseases as well as in healthy subjects, circulating plasma and serum carry small amounts of non-cell-bound DNA. In this free circulating DNA, tumor-associated molecular alterations can be detected in patients who have cancer. In many instances, the alterations identified are the same as those found in the primary tumor tissue, thereby suggesting tumor origin from a fraction of the circulating free DNA. In fact, various types of DNA alterations described in colorectal cancer have been detected in the circulating free DNA of patients with colorectal cancer. These alterations include KRAS2, APC and TP53 mutations, DNA hypermethylation, microsatellite instability (MSI) and loss of heterozygosity (LOH). Also, advances in polymerase chain reaction (PCR)-based technology now allow the detection and quantification of extremely small amounts of tumor-derived circulating free DNA in colorectal cancer patients. The present report summarizes the literature available so far on the mechanisms of circulating free DNA, and on the studies aimed at assessing the clinical and biological significance of tumor-derived circulating free DNA in colorectal cancer patients. Thus, tumor-derived circulating free DNA could serve as a marker for the diagnosis, prognosis and early detection of recurrence, thereby significantly improving the monitoring of colorectal cancer patients. © 2010 Elsevier Masson SAS. All rights reserved. Résumé Les altérations génétiques ou épigénétiques somatiques des cellules tumorales constituent un moyen d’identification moléculaire de la présence d’ADN tumoral dans un prélèvement biologique. De l’ADN libre est présent dans le sang et, chez les patients atteints de cancer, une fraction de cet ADN est d’origine tumorale. Chez des patients atteints de cancer, l’origine tumorale d’une fraction de l’ADN libre circulant a été démontrée au moyen de la détection d’altérations génétiques de l’ADN libre circulant plasmatique et/ou sérique identiques à celles mises en évidence au niveau de l’ADN extrait de la tumeur. Les altérations

∗

Corresponding author. E-mail address: lecomt [email protected] (T. Lecomte).

0399-8320/$ – see front matter © 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.gcb.2009.04.015

Circulating free tumor DNA and colorectal cancer

663

génétiques et épigénétiques les plus communes décrites dans le cancer colorectal ont été détectées au niveau de l’ADN libre circulant plasmatique et/ou sérique de patients atteints de cancer colorectal. Il s’agit principalement de mutations de l’oncogène KRAS2, de mutations des gènes suppresseurs de tumeurs APC et TP53, d’altérations de marqueurs microsatellites et d’anomalies de la méthylation de l’ADN tumoral. Grâce aux développements technologiques considérables réalisés dans le domaine de l’analyse de l’ADN, la recherche de nouveaux biomarqueurs du cancer colorectal basée sur la détection de l’ADN libre d’origine tumorale sérique ou plasmatique ou extrait d’autres prélèvements biologiques ouvre de nouvelles voies pour la mise au point de tests relativement simples et peu onéreux pour le dépistage, le diagnostic précoce de récidive, l’évaluation du pronostic et la prédiction de la réponse aux traitements. © 2010 Elsevier Masson SAS. Tous droits réservés.

Introduction A number of alterations occurring in cells and biological molecules during the course of neoplastic processes can be considered markers of cancer, and advances in genomics and proteomics now make it possible to identify such markers in plasma and serum, as well as in other bodily fluids such as urine, pancreatic juice, fecal matter and saliva. This makes these biological media highly useful for screening and making the diagnosis, and for determining the patient’s prognosis and therapeutic assessment. In patients with solid tumors, management decisions are made on the basis of clinical, histological and, more rarely, molecular factors, yet the overall picture—–given the heterogeneous nature of tumorigenesis—–remains imperfect, making it difficult to determine the most appropriate diagnostic or therapeutic strategy. However, one of the characteristic features of cancer cells is the presence of genetic anomalies (amplifications, deletions, point mutations, chromosomal translocations, microsatellite instability [MSI]) and/or epigenetic alterations (promoter gene hypermethylation, acetylation) that perturb the expression of the genes controlling critical cell processes, such as proliferation, differentiation, the cell cycle, apoptosis and angiogenesis [1]. Thus, an improved knowledge of the molecular biology of cancer can be expected to open the way for new perspectives not only in fundamental research (better understanding of carcinogenesis), but also in drug development (for example, the currently rapidly developing ‘target-therapy’ approach) and clinical management (use of biomarkers to establish the diagnosis or prognosis, or to predict response to treatment). Previous research has demonstrated that either plasma or serum can contain a small quantity of free (non-cell-bound) circulating DNA. Concentrations of this free circulating DNA, in the order of a few nanograms per milliliter (ng/mL), increase significantly in patients with cancer compared with healthy subjects [2]. Also, there is clear evidence that, in cancer patients, a fraction of this DNA is tumor-derived, as the genetic and/or epigenetic molecular alterations characteristic of the tumor are found in the free DNA circulating in the plasma or serum. In fact, molecular alterations characteristic of the majority of solid tumors have been detected in the serum or plasma of cancer patients [3]. However, most of the research into the detection of free tumor-derived DNA in

blood samples, and in other biological samples such as fecal matter, urine or pancreatic juice, is still in the preliminary stage. Nevertheless, this molecular approach to the detection of free circulating tumor-associated DNA in non-tumor biological samples introduces exciting new perspectives for the diagnosis and follow-up of patients with cancer.

Free cirulating DNA in plasma and serum Free circulating DNA was first reported in serum from patients with systemic lupus erythematosus, rheumathoid arthitis and glomerulonephritis [4—8]. Leon et al. [2] were the first to report that cancer patients presented with higher levels of such non-cell-bound circulating DNA in their blood than did patients with non-cancerous diseases. These findings were later confirmed in a series of patients with gastrointestinal cancer [9]. The mean concentration of free DNA circulating in serum was 118 ng/mL in patients with benign gastrointestinal disease versus 412 ng/mL in patients with malignant gastrointestinal disease. In a more recent study, the mean concentration of free circulating DNA measured in plasma, using a more sensitive method— –quantitative polymerase chain reaction (PCR) assay—–was 219 ng/mL (10—1200 ng/mL) in patients with cancer and 3.7 ng/mL in healthy subjects [10]. In the subgroup of patients with colorectal cancer (n = 7), the mean concentration of free circulating DNA was 194 ng/mL [25—980 ng/mL] In that study, analysis of the size of the free circulating DNA fragments suggested that this DNA could have arisen from both tumor cell lysis and apoptosis. The main studies investigating the quantification of free circulating DNA in plasma or serum in colorectal cancer are summarized in Table 1. Overall, these studies confirm the lack of any clear link between plasma or serum levels of free circulating DNA and tumor stage, and that high levels of free circulating DNA can be observed early in the disease process. However, high levels of free circulating DNA in plasma or serum are not specific to malignant disease, but have also been found in benign conditions (benign inflammatory disease, trauma) [11—16]. Comparisons between levels of free circulating DNA and the tumor markers commonly used in colon cancer (CEA), prostate cancer (PSA) and breast cancer (CA 15-3) have demonstrated them to be significantly correlated [17,18]. In colorectal cancer, the diagnostic value of serum levels of free circulating DNA appears to be greater

664 Table 1

T. Lecomte et al. Quantification of free circulating DNA in the plasma or serum of patients with colorectal cancer.

Number of patients

DNA in plasma (ng/mL)

66 healthy subjects

318

21 patients with colorectal cancer

609

88 healthy subjects 199 patients with gastrointestinal disorders (including 76 with colorectal cancer) 187 patients with benign intestinal disease 26 patients with metastatic (liver) colorectal cancer

DNA in serum (ng/mL)

14

Quantification method

Authors

Radioimmunological assay

Cox et al., 1976 [117]

Radioimmunological assay

Shapiro et al., 1983 [9]

Real-time quantitative PCR

Thijssen et al., 2002 [22]

412

118 10.6

47.6

58 patients with colorectal cancer (measurable concentrations in 25 patients) 11 patients with colorectal adenoma

105—709

Fluorometry

Lecomte et al., 2002 [56]

14

Real-time quantitative PCR

Diehl et al., 2005 [39]

22 patients with colorectal cancer Stage A or B (n = 16) Stage D (n = 6)

12 158 Spectrometry

Ito et al., 2003 [118]

Real-time quantitative PCR

Flamini et al., 2006 [19]

Fluorometry

Frattini et al., 2008 [18]

Real-time quantitative PCR

Boni et al., 2007 [119]

7 healthy subjects

320

46 patients with colorectal cancer Stage I Stage II Stage III Stage IV

560 720 609 769

75 healthy subjects

7.7

75 patients with colorectal cancer: Stage A (n = 18) Stage B (n = 19) Stage C (n = 26) Stage D (n = 12)

35.8 37.5 41.7 22.4 47.1

20 healthy subjects

5

70 patients with colorectal cancer

437

67 healthy subjects

0.85

67 patients with colorectal cancer

4771

than that of CEA [19]. Also, the level of free circulating DNA has been reported to be negatively correlated with the prognosis: patients with high levels have a poorer prognosis, especially in lung cancer [20,21], and in a series of 26 patients who underwent potentially curative resection of hepatic metastases from colorectal cancer, a high level of free circulating DNA preoperatively was predictive of recurrence [22]. Few data are available on the time course of free circulating DNA levels in cancer patients. One recent report suggested that increased levels found during the follow-up of patients who had undergone potentially curative surgery for colorectal cancer were associated with recurrence [18].

Also, in that study, which involved 70 patients, declining levels of free circulating DNA, from plasma samples drawn 4 and 10 months after surgery, were associated with no recurrence of disease. Free DNA circulating in serum or plasma is composed of double-stranded DNA fragments that vary in size from 0.5 to 21 kb (of human origin), as was demonstrated by Stroun et al. [23]. Two types of free circulating DNA have been described [10,24—27]. One comprises long fragments of DNA of different sizes generated by cell necrosis, while the other is made up of smaller fragments of DNA (185—200 pb) corresponding to nucleosomal DNA generated by apoptosis. This

Circulating free tumor DNA and colorectal cancer latter type of DNA is more specific of cell death observed in normal tissue [25,28]. The definitive proof that a fraction of this non-cell-bound circulating DNA is of tumor origin was provided in 1994 by Sorenson et al. [29], who detected mutations of the KRAS2 oncogene in the plasma of patients with pancreatic cancer. The KRAS2 oncogene mutations detected in free plasma-circulating DNA were identical to those detected in the primary pancreatic tumor found in these same patients [30—33]. Since that early work was done, somatic molecular alterations specific to tumor DNA have been detected in plasma or serum for the majority of solid tumors [3]. Similarly, most of the genetic and/or epigenetic somatic alterations described in solid tumors have been detected in the free plasma- or serum-circulating DNA in cancer patients [3]. The precise origin of this cell-free DNA fraction, arising from tumors and circulating in the bloodstream, is not known. Several hypotheses have been proposed but, so far, none has been backed by solid experimental evidence. Various theories have it that tumor DNA enters the bloodstream subsequent to tumor cell apoptosis or necrosis within the tumor mass, that it is actively transported across the tumor cell membrane, or that it arises from the lysis of circulating tumor cells or micrometastases [25,34—36]. In addition, few data are available on the plasma or serum clearance of free circulating DNA, although it has been suggested that it is eliminated rapidly from the plasma compartment [37,38]. Based on data for fetal DNA clearance from maternal blood, the half-life of free circulating tumor-derived DNA in blood appears to be in the order of 16 min [38,39]. Recent data favor the elimination of free circulating DNA via urine [40,41]. It is also likely that plasma clearance of free circulating DNA is the result of hepatic elimination and the action of plasma nucleases. It has even been hypothesized that free circulating tumor-derived DNA could have ‘genometastatic’ properties, implying functionality [42—46].

Molecular alterations of plasma or serum free circulating DNA in colorectal cancer When precisely characterized, genetic or epigenetic molecular alterations of cancer-cell DNA should offer a means of identifying the presence of cancer-cell DNA in biological samples. PCR has enabled the development of sensitive, specific and reproducible molecular-biology techniques that can demonstrate the presence of one altered gene copy in 10,000 to 100,000 normal copies. Such diagnostic techniques have been used for years to detect residual disease in bone-marrow samples from patients treated for malignant hematological disorders [47]. This type of approach has also been applied, mainly in pediatrics, to a few solid tumors such as Ewing’s sarcoma, as it can identify the chimeric transcript EWS/FLI-1 resulting from the t(11-22)(q24,q12) translocation [48]. Given the specific expression of such translocation in 90% of patients, reverse transcription PCR (RT-PCR) can successfully detect the EWS/FLI-1 fusion mRNA after reverse transcription to cDNA in bone marrow and circulating tumor cells in patients with Ewing’s sarcoma. These translocations are specific initiating events for a given tumor type and, thus, have major

665 diagnostic value that is also useful for monitoring disease progression. However, the fusion-gene tumor model that produces specific chimeric transcripts is an exception among the more commonly encountered solid tumors. For the majority of solid tumors, the acquired genomic anomalies are much more complex because of their genetic heterogeneity, the intricate mix of several carcinogenic models within a given tumor type and the accumulation of progressive genetic and/or epigenetic events. For these reasons, the biological anomalies with the most potential as markers for the most common solid tumors are molecular alterations of tumor DNA, some of which recur with a certain frequency in the same type of solid tumor. In colorectal cancer, for example, as well as other types of solid tumors, four main types of tumor DNA molecular alterations, found in different biological fluids, have been evaluated as disease markers: mutations of tumor and oncogene suppressor genes; alterations of microsatellite markers (loss of heterozygous status and MSI); hypermethylation of the gene promoters generally implicated in tumorigenic processes; and mutations of mitochondrial DNA. The molecular-biology techniques capable of detecting these molecular alterations are, a priori, highly specific because, first, the alteration involves only the DNA of tumor cells or cells undergoing malignant transformation and, second, all tumor cells would then theoretically express the alteration because of clonal proliferation. The sensitivity of a test based on this approach depends on the prevalence of the molecular alteration under consideration within the primary tumor and on the performance of the detection technique. This means that searching for more than one molecular alteration would be an easy way to increase sensitivity.

Oncogene and tumor suppressor gene mutations Among the genetic alterations frequently observed in colorectal cancer, mutations of the oncogene KRAS2, and of tumor suppressor genes TP53 and APC, have been detected in the serum or plasma of patients with colorectal cancer [39,49—60]. Mutations of the oncogene KRAS2 were the first genetic alterations to be identified in cell-free circulating DNA in patients with colorectal cancer. Most of the studies published on the topic are devoted to this marker, which has incited particular interest for several reasons:

• there is a high prevalence (approximately 50%) of oncogene KRAS2 mutations in colorectal cancer [61,62]; • oncogene KRAS2 mutations are among the earliest genetic alterations to occur during the process of colorectal carcinogenesis—–often as early as the adenoma stage [63]; • around 80% of patients with colorectal cancer exhibit a narrow spectrum of oncogene KRAS2 mutations, with changes located on either codon 12 or, less frequently, on codon 13 or, even more rarely, on codon 61 [64,65]; • highly specific and highly sensitive PCR techniques developed for detecting oncogene KRAS2 mutations have been facilitated by the fact that these alterations tend to concentrated in two regions (codons 12 and 13) and that the mutations observed are point mutations.

666

Table 2

Detection of oncogene KRAS2 mutations in the plasma or serum of patients with colorectal cancer (positive test rate). Marker

Blood sample

Patients included (n)

Detection rate among all included patients (% [n/n])

Mutation rate in all patients with CRC (% [n/n])

Detection rate in patients with mutated CRC (% [n/n])

Controls (n)

Rate of detection in controls (n/n)

Anker et al., 1997 [51] De Kok et al., 1997 [50] Kopreski et al., 1997 [49] Hibi et al., 1998 [53] Ryan et al., 2000 [120]

Mutations on codon 12 Mutations on codons 12/13 Mutations on codon 12 Mutations on codons 12/13 Mutations on codon 12

P

14

43 (6/14)

50 (7/14)

86 (6/7)

6

0

S

14

43 (6/14)

50 (7/14)

86 (6/7)

—

—

P or S

31

39 (12/31)

32 (6/19)

83 (5/6)

28

0/28

S

44

7 (3/44)

36 (16/44)

19 (3/16)

—

—

S

76 (including 12 adenomas)

42 (32/76)

86 (32/37)

5

0/5

Ward et al., 2000 [121] Salbe et al., 2000 [122] Kopreski et al., 2000 [123]

Mutations on codon 12 Mutations on codon 12 Mutations on codon 12

S

100

8 (8/100)

48.6 (37/76) (7/12 adenomas) 28 (28/100)

28.5 (8/28)

—

—

S

35

17 (6/35)

37 (13/35)

38.5 (5/13)

22

0/22

P

8 CRC, 62 adenomas

62.5 (5/8) 40 (25/62)

100 (5/5) 80 (20/25)

170

37/170

Mulcahy et al., 2000 [124] Lauschke et al., 2001 [125] Lecomte et al., 2002 [56] Ito et al., 2002 [126] Borchers et al., 2002 [127]

Mutations on codon 12 Mutations on codon 12

P

14

62.5 (5/8) 35.5 (22/62) 43 (6/14)

50 (7/14)

86 (6/7)

11

0/11

S

30

20 (6/30)

73 (22/30)

27 (6/22)

—

—

Mutations on codons 12/13 Mutations on codon 12 Mutations on codon 12

P

58

17 (10/58)

38 (22/58)

45 (10/22)

—

—

S

90

12 (11/90)

34 (31/90)

35 (11/31)

—

—

S

16 CRC, 6 adenomas, 3 Crohn’s, 4 UC

24 (7/29) (5/16 CRC, 2/4 UC)

—

—

20

0/20

T. Lecomte et al.

Authors

Authors

Marker

Blood sample

Patients included (n)

Detection rate among all included patients (% [n/n])

Mutation rate in all patients with CRC (% [n/n])

Detection rate in patients with mutated CRC (% [n/n])

Controls (n)

Rate of detection in controls (n/n)

Ryan et al., 2003 [111] Lilleberg et al., 2004 [57]

S

78a

41 (32/78)

53 (41/78)

76 (31/41)

20

0/20

P

20

35 (7/20)

35 (7/20)

100 (7/7)

—

—

Wang et al., 2004 [105] Wang et al., 2004 [128]

Mutations on codons 12/13 Mutations on codons 12/13/61 Mutations on codons 12/13 Mutations on codon 12

S

104

15 (16/104)

45 (47/104)

34 (16/47)

50

0/50

S

2 CRC, 10 adenomas

42 (5/12)

83 (5/6)

—

—

Hsieh et al., 2005 [106] Lindforss et al., 2005 [129]

Mutations on codons 12/13 Mutations on codons 12/13

S

118

39 (21/54)

50 (6/12) (1 CRC, 5 adenomas) 46 (54/118)

39 (21/54)

—

—

P

25

64 (16/25)

69 (9/13) preop, 53 (8/15) postop

—

—

Bazan et al., 2006 [58] Mora et al., 2006 [72] Frattini et al., 2008 [18]

Mutations on codons 12/13 Mutations on codon 12 Mutations on codons 12/13

P

66

41 (9/22) preop, 33 (8/24) postop 12 (8/66)

52 (29/66)

28 (8/29)

27

0/27

P

82 CRC, 3 adenomas 18

8 (7/82)

41 (34/82)

21 (7/34)

2

0

39 (7/18)

39 (7/18)

100 (7/7)

—

—

P

Circulating free tumor DNA and colorectal cancer

Table 2 (Continued)

CRC: colorectal cancer; P: plasma; S: serum; UC: ulcerative colitis; preop: preoperative; postop: postoperative; a Including nine high-grade dysplastic tubulovillous adenomas

667

668 Table 2 summarizes the current data for this genetic marker identified in the free circulating DNA of patients with colorectal cancer. Mutations of the KRAS2 oncogene can be detected in the plasma or serum of 25—30% of patients with colorectal cancer. Such a low rate of detection in the free circulating DNA in these patients is because this type of alteration only affects 40—50% of these tumors. On considering only tumors harboring KRAS2 mutations, the rate of detection of the same alteration in the free circulating DNA in plasma or serum as in tumor tissue is around 50%. When the search for this alteration is positive in a plasma or serum sample, the most common context is a patient with advanced, usually metastatic, cancer, thereby suggesting that the size of the tumor-derived free circulating DNA fraction is greater in more advanced disease. Nevertheless, when the data from available studies are pooled, their interpretation becomes rather more problematic due to the dissimilar methods used to collect, preserve and store samples (serum or plasma), and the variety of methods used to extract the circulating DNA and to detect the oncogene KRAS2 mutations. Moreover, in certain studies, the search for the genetic alteration was limited to patients known to have tumors expressing KRAS2 oncogene mutations. This suggests that, on the basis of the available data—–which are mainly from case-control studies and, therefore, include only a small number of patients and controls—–any evaluation of the sensitivity or specificity of this biomarker for colorectal cancer would lack validity. The largest study on this topic published so far was by Kopreski et al. [66], which evaluated the detection of oncogene KRAS2 mutations in the plasma of patients undergoing colonoscopy. In the study, the diagnosis of colorectal cancer was retained in eight patients and that of adenoma in 62, while colonoscopy was considered normal in 170 patients, with the oncogene KRAS2 mutation identified in the plasma of 62.5% (5/8), 35.5% (22/62) and 22% (37/170), respectively, of these patient subgroups. A search for the oncogene KRAS2 mutation was also performed using DNA extracted from tissue samples of the adenomas and cancers, as well as from biopsies of colonic mucosa taken from 65 patients whose colonoscopy was considered normal. However, agreement between detection of the oncogene KRAS2 mutation in plasma samples versus colorectal samples was not 100%, demonstrating a potential for false-positive results. When testing for the same mutations in DNA extracted from colonic samples (cancer, adenoma, normal mucosa), the rate of detection of the oncogene KRAS2 mutation in plasma was 83% (29/35), with the following distribution: cancer: 5/5; adenoma: 20/25; and normal mucosa: 4/5. In patients with normal colonoscopy for whom no colonic mucosa sample had been collected, the search for the KRAS2 oncogene mutation in plasma (n = 105) was positive in 28 cases. Of these 28 patients, 24 were considered to be at risk of colorectal cancer. This discrepancy can be attributed to other precancerous lesions of the colon, including very-early-stage aberrant crypts exhibiting the oncogene KRAS2 mutation, but for which colonoscopic sensitivity is not 100%. These data also offer an explanation for those reported cases of patients with colorectal cancer whose plasma samples were positive for the oncogene KRAS2 mutation despite the lack of a KRAS2 mutation in the tumor, or where the mutation identified in plasma was different from that iden-

T. Lecomte et al. tified in the tumor [33]. The possibility of false positives related to the restriction fragment length polymorphism (RFLP) PCR technique, used to detect KRAS2 oncogene mutations, has also been reported [33]. Another hypothesis to explain these discordant findings is clonal heterogeneity within the tumor, which is also suggested by the allelic loss in microsatellite markers of lung and colon cancers [67—72]. Nevertheless, given the low sensitivity of tests designed to detect oncogene KRAS2 mutations in the plasma or serum of patients with colorectal cancer, additional molecular alterations that are potentially useful as tumor DNA markers should be analyzed to improve the efficacy of the detection of tumor-derived free circulating DNA in serum or plasma. Two such tested examples of other genes frequently mutated in colorectal cancer are the tumor suppressor genes TP53 and APC. The gene coding for protein p53 is mutated in 60—70% of colorectal cancers [73,74]. Fearon and Vogelstein [75] demonstrated that mutations of the TP53 gene occur later in the process of colorectal tumorigenesis than do mutations of the oncogene KRAS2. However, mutations affecting the TP53 gene in colorectal cancer are highly diverse in their position on the gene and in their nature; they affect more than 200 codons, mainly localized at a central position on the gene. Also, they are not exclusively point mutations. Nevertheless, certain codons are more frequently involved. Mutations affecting codons 175, 245, 248, 249, 273 and 282 account for approximately 30% of all somatic mutations currently described in cancer [76]. Codons 175, 248 and 273 are the three leading ‘hot spots’ for mutations in all types of cancer, including colon cancer. Because of the wide spectrum of gene TP53 mutations in colorectal cancer, detection methods need to analyse a large region of the gene, using the most complex PCR techniques for detection of oncogene KRAS2 mutations. In the first such study reported, an allele-specific PCR technique (mismatch ligation assay), developed for each point mutation detected in the tumor DNA extract, was applied in the search for TP53 gene mutations in free circulating DNA in blood samples [53]. In most studies published so far, the search for TP53 gene mutations has focused on the region between exons 4 and 8, where the most commonly encountered TP53 mutations in colorectal cancer are located. Table 3 summarizes the available data so far on TP53 gene mutations in free circulating DNA in patients with colorectal cancer. Finding the TP53 gene mutation in the plasma or serum of patients with colorectal cancer is positive in about 15% of cases. In patients with tumors expressing a TP53 gene mutation, the same TP53 gene mutation is detected in the free circulating DNA in around 40% of cases. One study looked for mutations on codons 175 and 248 of this gene in the free circulating DNA of 240 patients undergoing colonoscopy [55]. Of these patients, 143 presented with gastrointestinal symptoms and 92 were asymptomatic with a high risk of colorectal cancer. Mutations of codon 175 or 248 on the TP53 gene were identified in the circulating DNA of two patients with adenoma and one with a hyperplastic polyp; yet, only one of these three lesions exhibited a mutation of codon 175 identical to that detected in the plasma. In the eight patients with colorectal cancer and the 61 others with adenoma, no mutations of codons 175 and/or 248 could be detected in plasma. As with the findings for mutations of the KRAS2 oncogene, this type of diagnostic test in a population

— 0/20 3/232b — — 0/50 — 0/27

CRC: colorectal cancer; P: plasma; S: serum; a Search for TP53 gene mutation in DNA extracted from tumor in 33 patients. b Including two patients (adenoma and hyperplastic polyp, respectively)

— 20 232 7 — 50 — 27 70 (7/10) 75 (3/4) 0/7 13.6 (3/22) 100 (10/10) 34 (13/38) 37 (16/43) 33 (7/21) (5 patients not tested) 33 (10/33)a 24 (4/17) 0/7 48 (22/46) 100 (10/10) 36.5 (38/104) 36.5 (43/118) 39 (26/66) 16 (7/44) 29 (5/17) 0/8 6 (3/46) 50 (10/20) 12.5 (13/104) 14 (16/118) 11 (7/66) Hibi et al., 1998 [53] Exons 5—8 Mayall et al., 1998 [52] Exons 5—8 Gocke et al., 2000 [55] Codons 175/248 Ito et al., 2003 [118] Exons 5—8 Lilleberg et al., 2004 [57] Exons 2—11 Wang et al., 2004 [105] Exons 4—8 Hsieh et al., 2005 [106] Exons 4—8 Bazan et al., 2006 [58] Exons 5—8

44 17 8 46 20 104 118 66 S P P S P S S P

Detection rate in patients with a mutated CRC (% [n/n]) Mutation rate in all patients with CRC (% [n/n]) Detection rate in all included patients (% [n/n]) Blood sample Patients included (n) Marker Authors

Table 3

Detection of suppressor gene TP53 mutations in the plasma or serum of patients with colorectal cancer (positive test rate).

Controls (n) Detection rate in controls (n/n)

Circulating free tumor DNA and colorectal cancer

669 of patients undergoing colonoscopy raises the possibility of false-positive results. Mutations of the APC gene are observed in 60—70% of sporadic colorectal cancer patients and also occur early in the adenoma—cancer sequence [77,78]. Around 80% of alterations are located between codons 1280 and 1500 of exon 15 [77,79]. As somatic mutations of the APC gene are concentrated on exon 15, the search for APC gene mutations in cell-free circulating DNA in patients with colorectal cancer has focused on this small region of the gene. Table 4 summarizes the current data on APC gene mutations in the free circulating DNA of such patients, in whom the search for the APC gene mutation is positive in plasma or serum in about 20%. On considering all patients with tumors exhibiting an APC gene mutation, the rate of detection in the free circulating DNA is the same as observed for the tumor itself—–around 45%. A quantitative approach has recently been developed for the detection of APC gene mutations in patients with colorectal cancer and adenoma [39]. The number of plasma copies of the APC gene and the fraction of mutated copies detected in the free circulating DNA in plasma correlated with the stage of disease: the numbers of APC gene mutation copies detected per milliliter of plasma (mean values) were 75,900, 3900, 4000, and 6300, for colorectal cancer stages IV, III, I, and adenoma, respectively; as percentages, the plasma copies carrying the mutated APC gene accounted for 11, 0.94, 0.04%, and 0.02%, of copies, respectively. Mutations of the oncogene BRAF have been reported in 5—10% of colorectal cancers and, in more than 90% of cases, the mutation was the activating mutation 1799 T → A (V600E; valine substitution at position 600 for glutamic acid) [80,81]. The rate of this mutation is especially high in sporadic cancers exhibiting the MSI phenotype, but low in those exhibiting the microsatellite stability (MSS) phenotype [81]. However, there has been only one report of the detection of the BRAF mutation in plasma from patients with colorectal cancer [57]. In the two patients with tumors expressing the BRAF oncogene mutation, the same BRAF mutation was detected in plasma. It is noteworthy that both of these tumors were free of any mutations of the KRAS2 oncogene, suggesting that these two types of alterations could be mututally exclusive and associated with two different tumorigenic pathways leading to colorectal cancer.

Microsatellite alterations Thus, the tumor genome can also be studied by characterizing micro satellite markers. Two types of molecular alterations are commonly observed in cancer and, particularly, colorectal cancer. One is chromosomal instability, which is generally seen as an allelic imbalance or LOH resulting in loss of chromosomal material, or as defective DNA repair leading to MSI [82]. The results of studies evaluating microsatellite alterations in cell-free circulating DNA in patients with colorectal cancer are summarized in Table 5. Markers of microsatellite alterations are observed in the plasma or serum of around 35% of patients with colorectal cancer and, in patients with tumors exhibiting at least one marker of microsatellite alteration, the rate of detection of the same marker is, on average, around 30% in the

670 Table 4

T. Lecomte et al. Detection of APC gene mutations in the plasma or serum of patients with colorectal cancer (positive test rate). Mutation rate in all patients with CRC (% [n/n])

Detection rate in patients with a mutated CRC (% [n/n])

Controls (n)

Detection rate in controls (n)

12.5 (1/8)

12.5 (1/8)

100 (1/1)

—

—

22 (3/11)

5

0

80 (20/25)

—

—

—

—

50

0

35 (18/52)

—

—

52 (17/33)

10

0

Marker

Blood sample

Gocke et al., 2000 [55] Ling et al., 2000 [54] Lauschke et al., 2001 [125] Lilleberg et al., 2004 [57] Wang et al., 2004 [105]

Mutations on exon 15 Mutations on exon 15 Mutations on exon 15

P

8

P

11

27 (3/11)

64 (7/11)

S

65

31 (20/65)

38 (25/65)

Mutations on exon 15

P

20

40 (8/20)

40 (8/20)

Mutations on exon 15, codons 1254—1631 Exon 15

S

104

13.5 (14/104)

44.2 (46/104)

30.4 (14/46)

S

118

15 (18/118)

44 (52/118)

P

33

52 (17/33)

Hsieh et al., 2005 [106] Diehl et al., 2005 [39]

Mutations on exon 15, codons 1209—1581

Patients included (n)

Detection rate in all included patients ([n/n])

Authors

100 (33/33)

100 (8/8)

CRC: colorectal cancer; P: plasma; S: serum.

free circulating DNA. However, the data currently available are inconsistent, with the rate of detection varying from 0 to 60% across studies. In addition, there have been reports of detection artifacts for MSI or LOH in poor-quality DNA, as well as false-positive LOH when the DNA concentration is low [83—86]. For all these reasons, this type of molecular alteration of tumor DNA does not appear to be a good marker for the detection of tumor DNA circulating in the bloodstream.

DNA epigenetic alterations Another way to determine the tumor origin of a fraction in the circulating DNA is to detect hypermethylation of promoter genes. Unlike point somatic DNA mutations associated with tumorigenic processes, which generally produce a wide spectrum of mutations for a given gene, DNA methylation is only observed in the CpG islands of a gene [87]. Methylation of the promoter of the tumor suppressor gene p161NK4a is an epigenetic molecular alteration of DNA that is the most widely studied for characterizing cell-free circulating DNA in serum or plasma in the search for tumor-derived DNA, particularly in colorectal cancer. The main reasons for choosing this type of molecular alteration are the high prevalence of these epigenetic molecular alterations of tumor-derived DNA in colorectal cancer, its early occurrence in the tumorigenic process and the development of methylation-specific PCR (MSP), a simple, robust method for detecting CpG-island hypermethylation of promoter genes [88—91]. MSP is sensitive and specific, and recent devel-

opments have allowed quantitative measurements of DNA methylation [88,92—94]. However, the risk with qualitative techniques is the detection of false-positive results, related to the fact that hypermethylation of the regulator sequences of certain genes is a physiological process present in normal cells, albeit in proportions that are considerably lower than that observed in tumors [95,96]. The discriminating element allowing distinction between cancer patients with this type of molecular alteration of tumor DNA and healthy subjects is the serum or plasma concentration of hypermethylated sequences of a gene used as a biomarker, as measured by quantitative PCR. The use of this quantitative method also enables a distinction between false and true positives through more accurate assessment of the degree of methylation of the tested DNA. Consequently, the rate of false positives using a test based on the characterization of this type of molecular alteration of tumor DNA in diverse biological fluids is decreased considerably with the quantitative method [97]. In colorectal cancer, numerous genes implicated in the cell cycle, cell adhesion and DNA repair—–such as APC, hMLH1, CDH1 (E-cadherin) and p161NKa—–present methylation anomalies that contribute to the initiation and progression of the colorectal carcinogenic process [90]. Hypermethylation of the promoter gene hMLH1 is the first tumor DNA methylation anomaly to be demonstrated in the sera of patients with the MSI phenotype of sporadic colorectal cancer [98]. Other, more recent, studies have demonstrated genes that have promoters which are often hypermethylated in colorectal cancer, and the results of studies looking for gene hypermethylation in the free circulating DNA in blood samples are summarized in Table 6.

Detection of markers of microsatellite alterations in the plasma or serum of patients with colorectal cancer (positive test rate).

Authors

Marker

Blood sample

Patients included (n)

Detection rate in all included patients (% [n/n]) SP

Hibi et al., 1998 [53]

8 markers

S

44

microsatellitesa Kolble et al., 1999 [130]

D1S243

27

D5S82 D8S264 D12S1689 D15S127 D15S129 D17S796 D17S1813 D18S70

Detection rate in patients with a mutated CRC (% [n/n])

Controls (n)

Detection rate in controls (n/n)

LOH: 70 (31/44) MSI: 34 (15/44) 15 (4/27)

LOH: 0/31

—

—

10

0/10

—

—

SM

LOH: 0/44 MSI: 0/44

S

Mutation rate in all patients with CRC (% [n/n])

4 (1/27) 22 (6/27) 18 (5/27) 15 (4/27) 18 (5/27) 4 (1/27) 15 (4/27) 30 (8/27) 11 (3/27) Total: 59 (16/27)

37 (10/27) 59 (16/27) 26 (7/27) 41 (11/27) 18 (5/27) 55 (15/27) 48 (13/27) 52 (14/27) Total: 96 (26/27)

LOH: 26 (7/27) MSI: 48 (13/27) Taback et al., 2006 [131]

SP/SM D4S175 D4S1586 D5S299 D8S133 D8S264 D15S127 TP53 D17S1832 D17S796 D18S58 D18S61

33b

NR 17 (4/23) 0 12 (3/25) 5 (1/19) 0 7 (2/29) 7 (2/28) 5 (1/22) 0 0 3 (1/32) Total: 27 (9/23)

MSI: 0/15 0 (0/4) 50 (5/10) 40 (4/10) 43 (3/7) 45.5 (5/11) 20 (1/5) 20 (3/15) 46 (6/13) 7 (1/14) Total: 54 (14/26) LOH: 35 (7/20) MSI: 93 (13/14) —

Circulating free tumor DNA and colorectal cancer

Table 5

17 (4/23) 5 (1/19) 12 (3/25) 11 (2/19) 5 (1/22) 0 4 (1/28) 9 (2/22) 7 (2/28) 4 (1/23) 0 Total: 33 (11/33)

49 (16/33)

671

58 (11/19)

Total: 70 (14/20)

58 (11/19)

Total: 70 (14/20) Total: 70 (14/20)

58 (11/19) TP53-D1 or TP53-PENTA

CRC: colorectal cancer; P: plasma; S: serum; SP: peripheral serum; SM: intraoperative mesenteric serum; LOH; loss of heterozygosity; MSI: microsatellite instability; NR: not reported. a D18S55, D18S58, D18S61, D18S69, CHRNB1, D17S786, D8S254, D8S133. b Including four adenomas.

35 (7/20) 35 (7/20) 35 (7/20) 20 P D5S346 Lilleberg et al., 2004 [57]

SP

Marker Authors

Table 5 (Continued)

Blood sample

Patients included (n)

Detection rate in all included patients (% [n/n])

SM

Detection rate in patients with a mutated CRC (% [n/n]) Mutation rate in all patients with CRC (% [n/n])

—

Controls (n)

—

T. Lecomte et al.

Detection rate in controls (n/n)

672

In patients with colorectal cancer, tests are positive for gene hypermethylation in plasma or serum samples in 25% of cases. On testing, all patients with tumors expressing at least one hypermethylated gene, the same gene hypermethylation as seen in the tumor is detected in free circulating DNA in 40% of patients.

Mutations of mitochondrial DNA Although short, the mitochondrial genome may have several advantages as a potential marker for colorectal cancer. First, there are several copies per mitochondria and several thousand mitochondria per cell. Second, the rate of mitochondrial DNA mutation is about 10 times higher than for nuclear DNA. Also, in addition to the fact that mitochondrial DNA mutation is frequent in colorectal cancer, mutated mitochondrial DNA is homoplasmic because of a replication advantage over normal mitochondrial DNA [99—103]. These observations suggest that mutated mitochondrial DNA may be a potentially useful molecular marker for colorectal cancer. One study investigated this potential marker in 77 patients with colorectal cancer and found a mutation rate of 9% (7/77) for tumor-associated mitochondrial DNA [104] and, of these seven patients, the same DNA mutation was identified in the cell-free circulating DNA in plasma from one patient.

Panels of DNA molecular alteration Given the relative heterogeneity of the tumor DNA molecular anomalies observed in sporadic colorectal cancers, tests designed to identify free circulating tumor DNA based on a panel of tumor DNA molecular alterations could theoretically detect the greatest number of sporadic tumors and increase test sensitivity. Tumor DNA molecular alterations are selected for the test panel according to their prevalence in colorectal cancer. Table 7 presents the results obtained with one such panel of alterations. Two studies reported by the same team showed that a panel including mutations of the KRAS2, TP53 and APC genes enabled the identification of at least one genetic alteration in tumor tissue from about 75% of patients with colorectal cancer [105,106]. Also, at least one mutation of the same genes (the same mutation as in the tumor) could be detected in the serum of 45% of these patients. On including all patients from the different studies investigating the use of a panel of alterations, the detection rate for mutations in the serum or plasma of patients with colorectal cancer was about 35%. This rate is, however, of limited value, as the altered molecular panels differed for the different studies, which included only small numbers of patients and used various molecular-biology detection techniques. Further research is needed to evaluate the feasibility and reproducibility of other molecular alteration panels. In addition, selection of a broader panel of molecular alterations—–including, for example, mutations of the KRAS2, TP53 and APC genes combined with hypermethylation of the p16INKa and hMLH1 genes—–would boost the percentage of colorectal tumors exhibiting at least one detectable tumor-DNA molecular alteration to a theoretical rate of above 85%.

Detection of gene methylation in the plasma or serum of patients with colorectal cancer (positive test rate).

Authors

Marker

Blood sample

Patients included (n)

Detection rate in all included patients(% [n/n])

Mutation rate in all patients with CRC(% [n/n])

Detection rate in patients with a mutated CRC (% [n/n])

Controls (n)

Detection rate in controls (% [n/n])

Grady et al., 2001 [98]

hMLH1

S

19

16 (3/19)

47 (9/19)a

33 (3/9)

—

—

Lecomte et al., 2002 [56]

P16INK4A

P

58

36 (21/58)

53 (31/58)

68 (21/31)

—

—

Zou et al., 2002 [132]

P16INK4A

Sb

52

27 (14/52)

38 (20/52)

70 (14/20)

44

0/44

Nakayama et al., 2002 [133]

P16INK4A

S

94

14 (13/94)

47 (44/94)

30 (13/44)

—

—

Nakayama et al., 2003 [134]

P16INK4A

S

11

63 (7/11)

73 (8/11)

88 (7/8)

—

—

Yamaguchi et al., 2003 [135]

DAPK

S

122

11.5 (14/122)

55 (67/122)

21 (14/67)

—

—

Leung et al., 2005 [109]

HLTF

S

49

32.7 (15/49

—

—

41

10 (4/41)

P

66

42.9 (19/49) 6.1 (3/49) Total: 57 (28/49) 4 (3/66)

21 (14/66)

21 (3/14)

—

—

SP/SM

16

SM

SP

—

—

—

—

6 (1/16) 0 0 0 0 Total: 6 (1/16)

142

19 (3/16) 13 (2/16) 6 (1/16) 0 0 Total: 38 (6/16) 24 (34/141)

—

—

20

—

64 (30/47)

90 (27/30)

101c

53.3 (57/101)

61 (11/18)

100 (11/11)

—

—

hMLH1 APC Bazan et al., 2006 [58]

P16INK4A

Taback et al., 2006 [131] APC MGMT RASSFIA P16INK4A RAR-ˇ2 Wallner et al., 2006 [110]

HLTF

S

HPP1 hMLH1 Ebert et al., 2006 [136]

ALX4

S

47

15 (21/141) 34 (48/141) Total: NR 57 (27/47)

Frattini et al., 2008 [18]

P16INK4A

P

18

61 (11/18)

673

CRC: colorectal cancer; P: plasma; S: serum; SP: peripheral serum; SM: intraoperative mesenteric serum; NR; not reported. a MSI + in nine cases. b Plasma for 10 controls. c Thirty-six patients with adenomas, 13 with hyperplastic polyps, 52 with normal colonoscopy

Circulating free tumor DNA and colorectal cancer

Table 6

674

Table 7

Detection by a panel of tumor DNA molecular alterations in the plasma or serum of patients with colorectal cancer (positive test rate). Marker

Blood sample

Patients included (n)

Detection rate in all included patients(% [n/n])

Mutation rate in all patients with CRC (% [n/n])

Detection rate in patients with a mutated CRC (% [n/n])

Hibi et al., 1998 [53]

KRAS2 (codons 12/13) TP53 (exons 5—8) LOH/MSI

S

44

Gocke et al., 2000 [55]

TP53 (Codons 175/248) APC (exon 15)

P

8

Lauschke et al., 2001 [125]

KRAS2 (codon 12) APC

S

30

Lecomte et al., 2002 [56]

KRAS2 (codons 12/13) P16INK4A

P

58

Wang et al., 2004 [105]

KRAS2 (codons 12/13) TP53 (exons 4—8) APC (exon 15)

S

104

Lilleberg et al., 2004 [57]

KRAS2 (codons 12/13/61) TP53 (exons 2—11) APC (exon 15) LOH/MSI BRAF

P

20

Hsieh et al., 2005 [106]

KRAS2 (codons 12/13) TP53 (exons 4—8) APC (exon 15)

S

118

Bazan et al., 2006 [58]

KRAS2 (codons 12/13) TP53 (exons 5—8) P16INK4A

P

66

Frattini et al., 2008 [18]

KRAS2 (codons 12/13) P16INK4A

P

18

7 (3/44) 16 (7/44) 0 (0/44) Total: 23 (10/44) 0/8 1/8 Total: 12.5 (1/8) 20 (6/30) 37 (11/30) Total: 53 (16/30) 17 (10/58) 36 (21/58) Total: 45 (26/58) 15 (16/104) 12.5 (13/104) 13.5 (14/104) Total: 34.5 (35/104) 35 (7/20) 50 (10/20) 40 (8/20) 70 (14/20) 10 (2/20) Total: 100 (20/20) 18 (21/118) 14 (16/118) 15 (18/118) Total: 35 (41/118) 12 (8/66) 7.4 (7/66) 4.5 (3/66) Total: 26 (17/66) 39 (7/18) 61 (11/18) Total: 72

36 (16/44) 33 (10/33) 80 (35/44) Total: NR 0/7 1/8 Total: 12.5 (1/8) 73 (22/30) 43 (13/30) Total: 83 (25/30) 38 (22/58) 53 (31/58) Total: 67 (39/58) 45 (47/104) 36.5 (38/104) 44.2 (46/104) Total: 75 (78/104) 35 (7/20) 50 (10/20) 40 (8/20) 70 (14/20) 10 2/20 Total: 100 (20/20) 46 (54/118) 36 (43/118) 44 (52/118) Total: 77 (91/118) 44 (29/66) 41 (27/66) 21 (14/66) Total: 76 (50/66) 39 (7/18) 61 (11/18) Total: 72

19 (3/16) 70 (7/10) 0 (0/44) Total: 10/25 0 1/1 Total: 1/1 27 (6/22) 85 (11/13) Total: 64 (16/25) 45 (10/22) 68 (21/31) Total: 70 (26/37) 34 (16/47) 34.2 (13/38) 30.4 (14/46) Total: 46 (36/78) 100 (7/7) 100 (10/10) 100 (8/8) 70 (14/20) 10 (2/20) Total: 100 (20/20) 39 (21/54) 37 (16/43) 35 (18/52) Total: 45 (41/91) 28 (8/29) 27 (7/26) 21 (3/14) Total: 34 (17/50) 100 (7/7) 100 (11/11) Total: 100

CRC: colorectal cancer; P: plasma; S: serum; LOH; loss of heterozygosity; MSI: microsatellite instability; NR; not reported.

T. Lecomte et al.

Authors

Circulating free tumor DNA and colorectal cancer Another approach would be to use more sensitive quantitative molecular-biology techniques to detect genetic alterations in the plasma or serum of patients with colorectal cancer. In a recent series of 18 patients with colorectal cancer and exhibiting at least one mutation of the KRAS2, TP53, APC and/or PI3K genes, use of a highly sensitive quantitative analysis of DNA alterations to search for plasma tumor DNA allowed the detection of free circulating tumorassociated DNA with the same genetic alterations as found in the tumor in all cases, as well as the quantification of the number of mutated tumor DNA copies circulating for each of the genetic alterations identified in the tumor [59].

Free tumor-derived circulating DNA in plasma or serum: a biomarker with prognostic value and/or predictive of treatment effect in colorectal cancer In light of the growing complexity of management strategies for colorectal cancer, it is essential to validate any new prognostic factors that could be used to identify subgroups of patients who may be expected to benefit from the various therapeutic strategies. For a given type of cancer at a given stage, the expected beneficial effect of a particular therapeutic strategy is never observed in all patients. The reason is undoubtedly related to the biological heterogeneity of most solid tumors. Therefore, it is logical to search for new prognostic factors among the determinants of such biological heterogeneity—–for example, among the molecular events characteristic of tumorigenesis. This is well illustrated by stage II and III colon cancer. Although the beneficial effects of adjuvant chemotherapy are well established for stage III colon cancer, it is observed in only 15% of patients given adjuvant chemotherapy, while recurrence is observed in 30% of those receiving adjuvant chemotherapy [107]. Moreover, adjuvant chemotherapy is useless for 75% of patients with stage II colon cancer treated by surgery alone, but is potentially of value in the other 25% who go on to develop recurrent disease. In terms of identifying new markers with prognostic value and which may be predictive of response to treatment, analysis of free circulating tumor-derived DNA is an avenue of research that may potentially provide at least a partial solution to the problem. Several stages of investigation are necessary to validate the prognostic or predictive value of a biological marker [108]. The first, analytical stage is to determine the sensitivity, specificity, and positive and negative predictive value of biomarkers in a small series of patients. The second stage is to validate the test in case-control studies and in trials including larger numbers of patients. The third stage needs to establish more precisely the link between the marker and the disease course, which requires longitudinal prospective studies with sufficient sample size as to allow robust statistical analyses. The final stage depends on the results of these studies, as they are then used to design multicenter interventional trials to establish marker-based diagnostic, therapeutic and surveillance strategies. Thus far, the data available on the use of tumor-derived free circulating DNA in blood samples as a marker of cancer are only preliminary. Studies investigating the use of molecular alterations char-

675 acteristic of colorectal cancer detected in free circulating DNA to determine their prognostic value and/or ability to predict the effect of treatment are summarized in Table 8. Most reported studies have included only a small number of patients in whom the marker was detected at diagnosis, and few studies have examined the evolution of the marker over the course of the disease. In colorectal cancer, detection of molecular alterations in the free circulating DNA in plasma or serum appears to be positively correlated with advanced disease, suggesting that this type of marker could have prognostic value [105,106,109,110]. However, in two large-scale studies, no such association was found [58,111]. In most studies, the detection of tumor DNA alterations in free circulating DNA involved blood samples drawn preoperatively, before surgery for colorectal cancer. Preoperative detection in free circulating DNA of the two most widely studied tumor-DNA molecular alterations (DNA methylation anomalies and point mutations of the KRAS2 oncogene) appears to be associated with a higher risk of recurrence [56,58,105,106], whereas only one study evaluated the prognostic value of detecting such a biomarker (KRAS2 oncogene mutation) during the surveillance period in patients with potentially curative surgery for colorectal cancer in parallel with the preoperative evaluation [1,111]. Detection of KRAS2 oncogene mutations in non-cell-bound circulating DNA in serum was positive during the follow-up in 27% of patients (16/60) with tumors exhibiting a KRAS2 oncogene mutation. Of these patients, 63% (16/60) developed a recurrence. The median time from the positive test to diagnosis of recurrence was 4 months (range 0—16). A concomitant increase in CEA was reported in three out of 10 of these patients. The relative risk of disease was significantly associated with detection of the KRAS2 oncogene mutation in free circulating DNA during follow-up. On multivariate analyses, detection of this genetic alteration in the free circulating DNA during the follow-up of 85 patients operated on for Dukes’ A, B or C tumor was an independent prognostic factor predictive of disease recurrence. In that study, the preoperative detection of a KRAS2 oncogene mutation in the free circulating DNA was not related to tumor stage and was also not an independent prognostic factor of recurrence. The value of this marker to predict the effect of chemotherapy has not been evaluated in colorectal cancer. The prognostic value of detecting tumor-derived DNA in the free circulating DNA fraction before potentially curative treatment could be related to the ‘genometastatic’ properties of free circulating tumor DNA [112]. It is worth noting that the association between detection of tumor molecular alterations in the blood and prognosis might be biased by the fact that certain tumor genetic alterations are linked to the disease prognosis; for example, in colorectal cancer, tumor DNA molecular alterations such as TP53 gene mutations, hypermethylation of the promoter of the p16INK4a gene, MSI, and LOH on chromosome 18q are all associated with the disease prognosis [113]. The corollary of the potential prognostic value and/or capacity to predict the effect of treatment with this type of marker is the application of such a biological parameter in the elaboration of new therapeutic strategies. Such an approach has become increasingly pertinent since the advent of therapies targeting the link between certain

676 Table 8 Tumor-derived free circulating DNA demonstrated by the presence of DNA molecular alterations in colorectal cancer: Prognostic studies and/or studies predictive of treatment effects. Disease stage

Patients (n)

Blood sample

Time of blood sample

Marker(s)

Patients with alterations in blood (% [n/n])

Value of marker

Authors

Stage I—IV

58

P

Preop

KRAS2 gene mutations

45 (26/58)

Prognosis (recurrence-free and overall survival)

Lecomte et al., 2002 [56]

p16INK4a gene methylation KRAS2 gene mutations

31/78 (preop)

Prognosis (recurrence)

Ryan et al., 2003 [111]

9/94 (postop) 35 (36/104)

Prognosis (recurrence)

Wang et al., 2004 [105]

57 (28/49)

Prognosis (trend for overall survival)

Leung et al., 2005 [109]

35 (41/118)

Prognosis (recurrence)

Hseih et al., 2005 [106]

9/25 (preop)

No link with survival (recurrence-free and overall survival)

Lindforss et al., 2005 [129]

Prognosis (recurrence)

Bazan et al., 2006 [58]

Prognosis (overall survival)

Wallner et al., 2006 [110]

Prognosis (recurrence)

Frattini et al., 2008 [18]

Potentially curative surgery

123

S

Preop (n = 78)

Stages II—IV

104

S

Postop (n = 94) Intraop

Stages I—IV

49

S

Before treatment

Stages I—IV

118

S

Preop

Stages I—IV

25

P

Preop/postop

Stages I—IV

66

P

Preop

Stages I—IV

141

S

Before treatment

Stages II/III/IV

18

P

Preop/postop

KRAS2, TP53 and APC gene mutations APC, hMLH1 and HLTF gene methylations (quantitative MSP) KRAS2, TP53 and APC gene mutations KRAS2 gene mutations

KRAS2 and TP53 gene mutations p16INK4a gene methylation HLTF, HPP1 and hMLH1 gene methylations

24 (34/141) 15 (21/141) 34 (48/141) 100 (7/7) 100 (11/11)

P: plasma; S: serum; preop: preoperative; postop: postoperative; intraop: intraoperative; MSP: methylation-specific polymerase chain reaction.

T. Lecomte et al.

KRAS2 gene mutations p16INK4a gene methylation

8/25 (postop) 76 (50/66)

Circulating free tumor DNA and colorectal cancer tumor molecular anomalies and response to treatment. In metastatic colorectal cancer, for example, a link was recently clearly demonstrated between the lack of a KRAS2 oncogene mutation in tumor DNA, usually extracted from a surgical specimen of the primary tumor, and the efficacy of anti-EGFR (epidermal growth factor) monoclonal antibodies (cetuximab or panitumumab) [114,115]. With the potential molecular heterogeneity of cancer not only at different sites of disease, but also over time, and considering the difficulty in obtaining a tumor sample at treatment onset, the detection of tumor genetic alterations in cell-free circulating DNA during the disease follow-up constitutes a highly relevant approach for choosing a treatment with efficacy, as determined by the specific genetic alterations of the tumor to be treated. Thus, the search for mutations of the KRAS2 oncogene in free circulating DNA may be of value for determining which patients might benefit the most from anti-EGFR treatment [116].

Conclusion The identification of new markers of colorectal cancer responds to the need for new sensitive and specific biological tests to enable earlier diagnosis, more accurate evaluation of the prognosis and earlier detection of recurrent disease, while also offering new directions towards the development of other diagnostic and/or therapeutic strategies to improve the prognosis. Cell-free circulating tumor-associated DNA, easily detectable in blood and possibly urine, is a potentially attractive candidate for this type of research. The feasibility of detecting tumor DNA circulating free in plasma and/or serum via identification of tumor DNA molecular alterations is clearly established, so such an approach could be developed as a source of new biomarkers for colorectal cancer. In parallel with the considerable technological developments in DNA analysis, such an approach, based on the detection of tumor-derived free circulating DNA in serum or plasma, or as found in extracts from other biological samples, offers new avenues for the development of relatively simple, low-cost tests for screening, early diagnosis of recurrence, evaluation of prognosis and prediction of response to treatment. It also opens up a new pathway to residual disease in patients with the most common solid tumors, as is already the case with many types of malignant hematological diseases. Nevertheless, the wide variety of methods used to detect this type of marker, as well as the lack of standardized tests, hinders their development. Given the present state of knowledge and considering the potential evolution in molecular-biology techniques, it is difficult to examine either the cost element for this type of molecular test or the practical modalities for using this type of test. However, what is needed at present is validation of this approach through new prospective studies that include large series of patients and apply standardized techniques before further examination of the adapted applications of these new markers in colorectal cancer are possible. Several multicenter prospective studies are currently ongoing, including the ALGECOL study (looking for genetic alterations in the serum of patients with stage II—III colorectal cancer and their impact on prognosis), which is now in the recruitment phase.

677

Conflict of interest statement None.

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