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REVIEWS Screening for colorectal cancer: established and emerging modalities Nikhil Pawa, Tan Arulampalam and John D. Norton Abstract | It has been estimated that >95% of cases of colorectal cancer (CRC) would benefit from curative surgery if diagnosis was made at an early or premalignant polyp stage of disease. Over the past 10 years, most developed nation states have implemented mass population screening programs, which are typically targeted at the older (at-risk) age group (>50–60 years old). Conventional screening largely relies on periodic patient-centric investigation, particularly involving colonoscopy and flexible sigmoidoscopy, or else on the fecal occult blood test. These methods are compromised by either low cost-effectiveness or limited diagnostic accuracy. Advances in the development of diagnostic molecular markers for CRC have yielded an expanding list of potential new screening modalities based on investigations of patient stool (for colonocyte DNA mutations, epigenetic changes or microRNA expression) or blood specimens (for plasma DNA mutations, epigenetic changes, heteroplasmic mitochondrial DNA mutations, leukocyte transcriptome profile, plasma microRNA expression or protein and autoantibody expression). In this Review, we present a critical evaluation of the performance data and relative merits of these various new potential methods. None of these molecular diagnostic methods have yet been evaluated beyond the proof-of-principle and pilot-scale study stage and it could be some years before they replace existing methods for population screening in CRC. Pawa, N. et al. Nat. Rev. Gastroenterol. Hepatol. advance online publication 1 November 2011; doi:10.1038/nrgastro.2011.205

Introduction Colorectal cancer (CRC) is one of the most prevalent forms of malignancy worldwide with approximately 1 million new cases diagnosed each year, accounting for 500,000 deaths.1 The pathogenesis of CRC involves the progressive accumulation of gene mutations over a time-scale of years as the disease advances.1 Most cases of early stage disease are clinically silent and, as a result, approximately 55% of patients in the UK present with lymph node or distant metastases.2 The stage of disease at diagnosis is the single most important predictor of prognosis and disease-free survival.3 The overall 5‑year age-standardized survival rate for CRC in Europe ranges from only 52% to 62%.2,4 It has been estimated that over 95% of cases of CRC would benefit from curative surgery if diagnosis was made at an early or premalignant polyp stage, and several large-scale studies have demonstrated a statistically signifi­cant reduction in mortality with implementa­ tion of mass CRC screening programs.5,6 It is perhaps relevant that during their lifetime, an estimated 40–50% of the population in developed nations will develop one or more polyps, although the majority of these polyps will remain benign.7 Broadly speaking, screening strategies are of two types: those that involve direct investigation of the patient, often with an invasive procedure, and those based on laboratory analysis of patient specimens (typically feces Competing interests The authors declare no competing interests.

or peripheral blood) to detect disease biomarkers. Both strategies target the higher-risk older-aged population (>50–60 years old). An ideal screening strategy needs to fulfill four important criteria: acceptable performance in terms of positive and negative predictive value for the target population; a reduction in mortality from the disease; acceptable levels of patient compliance; and low enough cost to be affordable within the context of the specific health-care system. No such ideal diag­ nostic screening procedure for CRC currently exists, and the main screening modalities that have so far been adopted (invasive diagnosis of the patient and the fecal occult blood test [FOBT]) represent a trade-off between the above four criteria. Several authors have recently reviewed the current state of practice and investigation in this field.5,6,8,9 The purpose of this Review is to summarize the different diagnostic screening strategies that have been or are being investigated (Table 1; Box 1), and to explore the potential of emerging technologies in the application of molecular markers for population screening for CRC.

Established screening modalities Direct investigation of the patient Colonoscopy Colonoscopy is regarded as the gold standard for the diagnosis of colorectal polyps and malignancy, and has gained increased popularity in the USA and Canada over the past 10–20 years; however, Poland is currently the only European country utilizing colonoscopy as the sole

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Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK (N. Pawa, J. D. Norton). ICENI Centre, Department of Surgery, Colchester Hospital University NHS Foundation Trust, Turner Road, Colchester CO4 5JL, UK (T. Arulampalam). Correspondence to: J. D. Norton jnorton@essex.ac.uk

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REVIEWS Key points ■■ Approximately 1 million new cases of colorectal cancer are diagnosed each year ■■ Population screening for detection of disease at an early stage has demonstrated a significant reduction in mortality ■■ The fecal occult blood test is the most widely adopted screening method, but is compromised by poor reliability and patient compliance ■■ Although flexible sigmoidoscopy is cost-effective, more definitive direct investigative procedures (such as colonoscopy) are prohibitively expensive for most national screening programs ■■ An expanding list of potential new screening methods based on the detection of DNA, RNA or protein molecular markers in patient stool or blood specimens is currently being evaluated ■■ The introduction of screening based on molecular markers is likely to be an incremental process; established screening modalities are likely to remain in widespread use for some years

screening method for individuals aged 55–66 years.10 Although its use is clearly associated with a decrease in CRC-related deaths, no direct evidence from random­ ized controlled trials has demonstrated the effectiveness of colonoscopy in reducing mortality from CRC, and therefore inferences are drawn from indirect evidence. In a prospective observational study of asymptom­ atic average-risk individuals, Kahi et al. 11 reported a 65% reduction in CRC mortality and 67% reduction in disease incidence with colonoscopy screening. Similarly, Rabenck and colleagues12 identified an

inverse association between mortality and increased rate of complete colonoscopy. A large population-based, case–­controlled study demonstrated a marked mortality reduction for left-sided lesions, but with very little reduction for right-sided tumors in patients who underwent colonoscopy.13 This observation concurs with similar findings from a cross-sectional study of the German population.14 Despite some confounding issues in these studies, such as variability in procedural performance at different centers, they do question the presumed superiority of colonoscopy as a screening tool, especially for tumors of the proximal colon. Under the auspices of the Northern-European Initiative on Colorectal Cancer (NordICC), a large multicenter randomized trial is currently underway that aims to establish the clinical impact of colonoscopy screening on CRC incidence and mortality, with the final results expected in 2026.15 Another area of concern with colonoscopy is that it is a very operator-dependent procedure; a ‘miss rate’ of between 6% and 12% has been identified for larger polyps and 5% for cancers.16 As an invasive procedure, it is also not without some attendant risk of complications, such as bowel perforation. The rate of major complications has been reported at 0.2%, a figure representing approximately 10 times that of any other cancer screening test.17 However, the main disadvantage of colonoscopy is its high cost, making it unaffordable as a population screening method for most of the global health-care sector.18

Table 1 | Current CRC screening guidelines Risk

European Union Guidelines 2010125

American Cancer Society Guidelines 2011126

Average risk

Screening age: 50–74 years Screening options: fecal occult blood test (2 yearly)

Screening age: 50 years (45 years for African Americans) Screening options: fecal occult blood test (yearly)* CT colonography (5 yearly)* Flexible sigmoidoscopy (5 yearly)* Double contrast barium enema (5 yearly)* Colonoscopy (10 yearly)

Increased risk (history of polyps or other risk factors) Low risk (1–2 adenomas [<10 mm])

Routine screening: fecal occult blood test after polyps removed

Colonoscopy 5–10 years after polyps removed

Intermediate risk (3–4 small adenomas or 1 ≥10 mm)

Colonoscopy 3 years after polyps removed. 1 negative colonoscopy, extend interval to 5 years; 2 consecutive normal exams, return to routine screening

Colonoscopy 3 years after polyps removed

High risk (5 or more adenomas or 1 ≥20 mm)

Colonoscopy within 1 year of removal of polyps, followed by 3 yearly surveillance

Colonoscopy within 3 years of removal of polyps. Consider genetic condition

History of cancer (resected)

pT1 lesions: surveillance as high-risk adenomas

Colonoscopy within 1 year of surgery

Family history (1st degree relative)

Not to be excluded from screening program (commence at 40 years‡)

Commence 5 yearly colonoscopy at age 40

IBD

Specific colonoscopy surveillance programs at shorter intervals

Colonoscopy every 1–2 years with biopsies for dysplasia

Familial adenomatous polyposis

Specific colonoscopy surveillance programs at shorter intervals with genetic testing clinic

Commence yearly flexible sigmoidoscopy at age 10. Consider colectomy if genetic test positive

Hereditary nonpolyposis CRC

Specific colonoscopy surveillance programs at shorter intervals with genetic testing clinic

Colonoscopy every 1–2 years from age 20. Genetic testing including 1st degree relatives

Hereditary risk

*Colonoscopy if positive. ‡Barcelona declaration. Abbreviation: CRC, colorectal cancer.

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REVIEWS Barium enema The double-contrast barium enema examination has a reported sensitivity of between 85% and 97% for CRCs and 73% for adenomas >7 mm when used as a screening tool.16 However, the bowel preparation involved in this labor-intensive procedure, together with its high cost, has limited its popularity.16 Additionally, a lack of welldesigned studies assessing its effectiveness as a screening tool has led to further skepticism, particularly with the introduction of CT colonography. Glick and colleagues19 generated a model of 16 different screening strategies to analyze the cost-effectiveness of the barium enema examination. The model illustrated the cost-effectiveness of the examination every 5 years, but only assuming the reported high sensitivity of early studies.16 However, in a population-based study, Toma and colleagues 20 reported the chance of missing a cancer at barium enema examina­tion to be one in five. CT colonography CT colonography has been recommended by the American College of Gastroenterology as a screening method at an interval of 5 years. Studies have shown sensitivities and specificities for the detection of lesions that are comparable to other screening methods. To date, the effectiveness of this screening tool in reducing CRC mortality and incidence has not been established, although meta-analyses have confirmed a high sensitivity and specificity for the identification of large polyps (≥10 mm), but not so high for smaller polyps.21,22 An additional disadvantage of CT colonography as a screening tool is the extracolonic findings described in up to 66% of cases, with 16% of individuals requiring further evaluation.23 Reports on the cost-­effectiveness of this method as a screening tool in comparison with colonoscopy have been encouraging, although the additional costs associated with investigation and reporting of extra-colonic findings must also be considered.24,25 On the other hand, CT colonography could offer some additional benefits with the opportunistic identification of aortic aneurysm and other specific intra-abdominal conditions. Flexible sigmoidoscopy Flexible sigmoidoscopy has the advantage that is requires less bowel preparation and usually does not require sedation for the patient, but has the disadvantage of only detecting carcinomas or adenomas in the distal colon. The Norwegian Colorectal Cancer Prevention group found no reduction in the incidence of CRC or mortality with flexible sigmoidoscopy screening after 7 years follow up.26 By contrast, a larger-scale study in the UK reported a 33% reduction in CRC incidence and a 43% reduction in mortality in a randomized population of 57,237 individuals undergoing sigmoidoscopy with a median follow-up time of 11 years.27 A similar reduction in both the incidence and mortality of CRC was reported by the Italian randomized controlled SCORE trial, in which individuals aged 55–64 years were randomly allocated to receive either flexible sigmoidoscopy

Box 1 | Challenges of CRC screening practices Direct investigation of the patient ■■ Colonoscopy is an operator-dependent procedure with variable miss rates and a low risk of perforation ■■ High costs associated with colonoscopy limit its use in a mass screening program ■■ The barium enema examination has a low sensitivity, particularly for small adenomas ■■ CT colonography can be associated with a high rate of extracolonic findings requiring further investigation

Investigation of patient specimens ■■ The fecal occult blood test (FOBT) is associated with a high false-positive rate, necessitating a strict diet before sampling ■■ Poor patient compliance rates are reported with the FOBT ■■ Immunochemical FOBT has better sensitivity and specificity although variable sample storage conditions can cause false-negative results ■■ The fecal DNA marker examination offers a more robust screening tool; however, cost-effectiveness remains an issue ■■ Established serum antigens CEA and CA19‑9 show low sensitivity for diagnosis, with CEA offering benefit in disease monitoring

(n = 17,136) or no intervention (n = 17,136).28 On the basis of the UK and Italian studies, a single flexible sigmoido­ scopy at age 55 would outperform the FOBT (see below) by a considerable margin. Furthermore, a cost-analysis model demonstrated flexible sigmoidoscopy to be cost saving, with or without the FOBT, whilst acknowledging the considerable investment involved when introducing a new screening option.29

Fecal hemoglobin Table 2 presents a snap-shot view of currently established diagnostic procedures utilizing molecular markers for CRC. As an inexpensive, noninvasive method, the FOBT is the most widely used screening method for CRC worldwide. In a number of independent studies, screening by FOBT has been shown to be associated with a statistically significant reduction in disease-related mortal­ity. For example, a meta-analysis of six trials using the Hemoccult® (Beckman Coulter, Inc., Brea, CA) test reported a 16% reduction in CRC mortality in the popula­tions offered screening.30 The traditional FOBT is based upon guaiac detection of peroxidise activity of the heme moiety in fecal specimens and is, therefore, susceptible to interference from bleeding of both upper and lower gastrointestinal origin. Similarly, ingestion of certain foods and drugs can yield false-positive results, necessitating a strict 3-day diet that eliminates meats, aspirin and other NSAIDs before sampling. The protocol consists of collecting two samples from three consecutive bowel movements. Positive results require a follow-up colonoscopy for definitive diagnosis. Patient compliance remains an important issue, with up-take rates of only 53.4–66.8% being reported. This problem is exacerbated by the annual or biannual testing required in many screening protocols. Nevertheless, this method is the most popular screening tool to date in Europe. As of 2007, 12 countries are basing their population screening

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REVIEWS Table 2 | Established screening methods for CRC using molecular markers Marker

Sensitivity (%)

Specificity (%)

Status

Fecal hemoglobin: guaiac fecal occult blood test127,128

15–30, 14.3

95, 79.2

In use

Immunohistochemistry129

94.1

87.5

In use

Serum carcinoembryonic antigen (CEA)

56

95

In use

Serum CA 19‑9131

26

98

In use

Fecal DNA markers (KRAS, APC, L‑DNA, p53)132–134

91, 74, 87.5

93, 97, 82

Clinical validation

Cytological assay: circulating tumor cells

32, 62, 94

ND, ND, 94

Clinical validation

130

50,51,135

Abbreviation: ND, not determined.

Loss or mutation of APC/β-catenin/ axin genes Normal epithelium

Mutation of KRAS/BRAF

Aberrant crypts foci

Early adenoma

Loss or mutation of DCC

Intermediate adenoma

Loss or mutation of p53

Late adenoma

Other alterations

Carcinoma

Metastatic disease

Genomic instability

Figure 1 | Temporal sequence of major genetic changes involved in the pathogenesis of colorectal cancer.

programs on the FOBT, the majority of which organize colonoscopy for positive results.31,32 The immunochemical FOBT relies on specific detection of human hemoglobin in stools, making it more selective for bleeding of colorectal origin. An increase in patient compliance is also expected as no dietary restrictions are required, and sample collection is easier. A more recent version of the immunochemical test determines quantitative levels of hemoglobin in stools. With a threshold of 100 ng hemoglobin per ml, this test has a sensitivity of 76.5% and a specificity of 95.3%, which is similar to colonscopic findings (for adenomas and cancers). 33 However, suboptimal conditions of sample storage, particularly increased temperature, can lead to a higher rate of false-negative results with the immunochemical FOBT.34 Results from a French CRC screening program demon­strated that immunochemical testing identified twice as many advanced cancers in comparison with the Hemoccult II® FOBT, albeit with a similar specifi­ city.35 Van Rossum et al.36 conducted a random population-based comparative study between the guaiac and immunochemical FOBT and reported a notably higher participation rate for the immunochemical test. The guaiac-based test also substantially underestimated the prevalence of advanced adenomas and cancers. A Spanish study also concluded that the immunochemical test is markedly superior to the guaiac test in detecting advanced adenomas and CRC.37 At present, the immunochemical FOBT remains the preferred option as a FOBT screening tool with its improved patient compliance, better performance and automated quantitative measurement; the test is also probably less costly compared with the traditional guaiac FOBT. 38,39 As discussed by Ward et al., 18 the various national screening programs using the FOBT do incur substantial hidden extra costs associated with large

numbers of follow-up colonoscopy investigations. To date, only one randomized controlled trial has assessed the efficacy of the FOBT as a screening method. With a follow-up time of 8 years, and using flexible sigmoido­ scopy as a follow-up for positive results, a 32% reduction in rectal cancer mortality was reported, but with no reduction in mortality for colon cancer nor incidence of CRC.40

Fecal DNA markers Cells shed from the colonic mucosa into the feces can be examined to detect the gene mutations or epi­ genetic markers that are the hallmarks of adenomas and carcinomas.41 In contrast to mucosal bleeding, the shedding of colonocytes is believed to be a continuous process; stool-derived DNA markers therefore facilitate a more robust screening strategy. A panel of DNA markers can be assessed for mutations that arise in the adenoma→carcinoma sequence, typically, APC, KRAS and p53 (Figure 1). This approach requires a larger sample of stool than the FOBT. Recommendations for a 5‑year screening interval have been made;41 however, at present, insufficient evidence exists to support this strategy. Imperiale et al.42 conducted a large populationbased study that compared fecal DNA testing with the FOBT. The study utilized a DNA marker panel consisting of 21 mutations and demonstrated a sensitivity of 52% for invasive cancers and 41% for invasive cancers plus adenomas. These results compare with a sensitivity of 13% (invasive cancers) and 14% (invasive cancers plus adenomas) obtained using the FOBT for the same popula­tion. Screening using fecal DNA markers thus demonstrated a higher sensitivity than the FOBT, although the two tests yielded similar specificities.42 Subsequent improvements to fecal DNA testing have led to three distinct changes in this technology: the addition of a DNA stabilization buffer; altering the

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REVIEWS DNA capture assay; and adding a promoter methylation marker for the vimentin gene.23 In a study that compared this second-generation fecal DNA assay with Hemoccult® and Hemoccult SENSA® FOBT, Ahlquist et al.43 reported a 46% detection rate of advanced adenoma and CRC lesions using the DNA test compared with 16% for Hemoccult® and 24% with Hemoccult SENSA ® . The inclusion of additional methylation-based gene markers in fecal DNA testing offers the prospect of further improvements in performance.44 Although fecal DNA testing is gaining wider accept­ ance as a screening tool, concerns still exist regarding its cost. Song et al.45 used a Markov model to compare cost-­effectiveness of various screening methods. The analysis found the FOBT and colonoscopy to be more cost­effective than fecal DNA testing.45 It has been suggested that by using the second-generation fecal DNA testing platform, fewer markers will be necessary, which could potentially reduce the overall cost.46 Further larger-scale studies involving the screening of average-risk popula­ tion groups are necessary to validate the cost-benefits of fecal DNA testing.

Cytological assays The presence of low levels of circulating tumor cells in the peripheral blood of patients with carcinomas of epithelial origin has been well documented. 47–49 In patients with CRC, the detection of such circulating tumor cells is usually associated with progressive or metastatic disease and in some centers is used routinely as a marker of progressive disease and to monitor therapeutic response.48,50–55 Cytological detection of occult peripheral blood tumor cells relies on antibodies to epithelial antigens (typically epithelial cell adhesion molecule [EpCAM] or cyto­keratin) that are expressed by cancers of epi­t helial origin but are absent from hematopoietic cells.47–49,56 However, the poor sensitivity of commercial cell counting instruments that are based on immuno­magnetic bead purifica­tion (such as CellSearch® [Johnson and Johnson, New Brunswick, NJ]) precludes their use in population-screening applications; circulating tumor cells are detected in only around 20–60% of patients with metastatic disease using these instruments.53 Combining immunomagnetic purification with immunocyto­chemistry for microscopic detection of gastrointestinal-specific cytokeratin‑20-­positive cells reportedly detects circulating tumor cells in 62% of patients with CRC.51 A higher sensitivity can be achieved for detecting low cell numbers of circulating occult tumor cells by direct immunofluorescence microscopy of peripheral blood cells using automated image capture and analysis systems.57–59 The development of an automated micro­ fluidic ‘lab-on‑a-chip’ device for capturing, quantitating and analyzing circulating tumor cells directly from a peripheral blood sample should afford an even higher sensitivity and diagnostic accuracy.60,61 Such an instrument would readily lend itself to use in a routine diagnostic laboratory. However, although cytological assays of circulating tumor cells are undeniably of value

in stratifying patients with progressive or metastatic disease and in monitoring therapeutic response,48,50–55 the current level of sensitivity attainable is too low for applications in popula­tion screening, particularly for early-stage disease.

Established serum antigen markers The high molecular weight glycoprotein, carcino­ embryonic antigen (CEA), is found in embryonic tissue and colorectal malignancies; it has a half-life of 2 weeks.62 High CEA levels are associated with cancer progression, and levels decrease after resection. However, high levels of CEA can also occur in other malignancies (such as breast, stomach and pancreatic cancer), not to mention IBD, liver disease and pancreatitis.2,63,64 Its role in screening is limited because levels of this antigen typically do not increase until the tumor has penetrated the serosa, and although levels rise with metastatic liver disease they rarely increase with peritoneal involvement. Fletcher et al.65 demonstrated that by using a cut-off value of 2.5 μg/l, screening of a normal population would result in a sensitivity of 30–40%. In a systematic review by Hundt and colleagues,66 19 studies were included reporting on the performance of CEA for early CRC detection. The overall sensitivity varied between 43% and 69%. A clear increase in the sensitivity was identified with tumor stage; 8% for Stage I up to 89% for Stage IV. A specificity of >90% was reported in 11 studies.66 On the basis of these results, the clinical value of CEA lies more in monitoring disease progression and as a prognostic marker after diagnosis rather than for screening. The CA19‑9 antigen is the second most investigated gastrointestinal tumor marker. Although it is the best available marker for the detection of pancreatic carcinoma, it remains less sensitive for the detection of CRC. A review of 13 studies evaluating its role in the early detection of CRC, using a common cut-off of 37 units/ml, reported overall sensitivities ranging from 18% to 65%, with specificities of >90% in most studies. Unfortunately, comparing sensitivity by tumor stage revealed 0% for Stage I and up to 67% for Stage IV, making it inappropriate as a population screening tool.66

Emerging screening modalities Protein markers in serum and plasma Over the past 10 years, a variety of proteomics-based strategies have been pursued in the quest for serum biomarkers of CRC (Table 3).67 With the limited applic­ ability of CEA and CA19‑9 antigens, a number of alternative candidate diagnostic protein markers have emerged from studies of the tumor tissue proteome. These include nuclear matrix proteins, CCSA‑2, CCSA‑3, CCSA‑4, matrix metalloproteinase 9, S100A8 and S100A9.68–71 Although the individual sensitivities and specificities of these markers in pilot studies fall short of acceptable performance for population screening, their relative ease of quantitation by immunoassay lends itself to develop­ment as part of an extended panel of serum protein biomarkers.

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REVIEWS Table 3 | Emerging molecular marker strategies for CRC screening Marker

Cases

Controls

Sensitivity (%)

Specificity (%)

CCSA‑3, CCSA‑468

28

107

91

82–91

CCSA‑2

27

40

89

84

MMP‑970

90

46

78

77

S100A8, S100A971

88

21

41–44

95

MAPKAPK3, ACVR2B

52

42

83

74

SELDI-TOF136

55

92

91

93

SELDI-TOF

55

92

89

92

SELDI-TOF85

37

31

65–90

65–90

SELDI-TOF138

62

31

95

91

139

SELDI-TOF

55

92

91

93

MALDI-TOF87

66

50

95

90

MALDI-TOF140

66

50

98

96

TMEF2, NGFR, SEPT9107

135

185

30–52

95

SEPT9

90

155

55–71

86–95

SEPT9141

183

126

72

90

APC, KRAS, p53142

104

50

13‑34

100

APC, MLH1, HLTF

49

41

6–57

90–100

5-gene panel: CDA, MGC20553, BANK1, BCNP1, MS4A1112

43

49

88

64

7-gene panel: ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1, IL2RB113

202

208

72

70

7-gene panel: ANXA3, CLEC4D, TNFAIP6, LMNB1, PRRG4, VNN1, IL2RB114

99

111

61

77

Fecal microRNA122

10

8

74

79

mRNA-L6144

187

45

79

100

MicroRNA

90

50

89

70

Serum peptide markers

69

76

137

DNA markers

108

143

RNA markers

145

The existence of circulating tumor-associated auto­ antigens in patients with cancer has been known for some time. However, only since 2010 have tumor-­associated serum autoantibodies been systematically evaluated as potential disease markers. 72,73 At least in part, this renewed interest has been facilitated by the availability of high-density protein microarray technologies, based on either commercial protein arrays or phage display expression libraries. Preliminary studies in lung, ovarian and pancreatic cancer illustrate how extended panels of arrayed antigens afford both high sensitivity and specificity in distinguishing patients with cancer from healthy individuals.74,75 In CRC, Badel et al.76 used a commercial protein microarray platform representing 8,000 proteins to identify 43 proteins that are differentially recognized by tumoral and reference sera. In 2009, a diagnostic ELISA based on a combination of two of these proteins (MAPKAPK3 and ACVR2B) yielded specificity and sensi­tivity values of 73.9% and 83.3%, respectively, in distinguishing patients with CRC from healthy individuals.76 More recently, the same laboratory identified two further markers from a phage display screen, MST1/STK4 and SULF1, that improve the diagnostic performance of the

assay.77 These studies highlight the potential of serological analysis of arrays displaying the complete human proteome (seromics) for the comprehensive identification of circulating autoantibodies that could be used to develop robust immunoassay screening methods for CRC. This concept is a fairly new area of investigation in the quest for serum markers that could be used as a multiplexed panel in ELISA or other solid phase assay platforms in population screening for CRC. Although considerable development work remains to be done, the approach is particularly attractive because it would ultimately utilize immunoassay-type methodology with its inherent low cost, robustness and established use. The most intensively investigated field in serum proteomics over the past 10 years has been in the application of surface-enhanced laser desorption–ionization time-of-flight mass spectrometry (SELDI-TOF MS) and matrix-assisted laser desorption–ionization timeof-flight mass spectrometry (MALDI-TOF MS). 78 In SELDI-TOF MS, serum or plasma samples are applied directly to protein chip surfaces that differentially retain proteins on the basis of their physicochemical properties. In MALDI-TOF MS, protein samples are first

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REVIEWS a

b

TOF mass spectrometer Laser pulse

MALDI plate MALDI plate

Positively charged ions

Serum protein samples spotted on plate with matrix

Sample

c

Peak mass values

Detector

High voltage

d

Acquired spectra

Dataset spreadsheet

Samples

45058.0

36082.6

27107.2

18131.8

9158.4

181.0 1200.05

3159.94

e

5119.84

7079.74

9039.63

Normalized expression values of matched peaks

10999.53

Predictive model (heat map) Normal sera CRC sera

Test sera

Normalized expression levels of discriminating peaks

Figure 2 | Schematic outline of serum or plasma protein expression profiling by MALDI-TOF mass spectrometry. a | Serum or plasma protein samples from matched healthy controls and from patients with a confirmed diagnosis of CRC are crystallized with a matrix compound as spots on the stainless steel MALDI plate. Test serum samples for screening can also be included. b | In the mass spectrometer, individual sample spots are pulsed with a high energy laser to ionize the complex mixture of proteins and peptides. Under the influence of an electric field, positively charged protein and peptide ions are accelerated down the ���flight tube’. The speed of flight, and hence time taken to impact the detector is determined by the charge-to-mass (m/z) ratio of the protein. For uniformly charged species, this time interval is proportional mass. c | Detector output time data from multiple laser pulses is converted to a Da mass scale (strictly speaking m/z) that together with recorded amplitude data is represented as a spectrum. d | The spectral graph data is exported as a simple digital text file so that matching peaks and their normalized ion intensities across all samples can be identified. e | Various algorithms can be used to identify ‘diagnostic’ marker peaks whose amplitude discriminates between CRC and normal serum. A diagnosis of test sera is then made by determining whether the amplitude of marker peaks most closely resembles normal or CRC serum. Abbreviations: CRC, colorectal cancer; MALDI-TOF, matrix-assisted laser desorption–ionization time-of-flight.

co-crystallized with an energy-absorbing matrix, usually with prior ‘off-line’ purification, before laser irradiation in the mass spectrometer. In both techniques, the laser desorbs the complex mixture of proteins, which are then detected by their mass-to-charge ratio in the time-offlight analyzer to generate a spectral profile ‘fingerprint’ representing numerous proteins and/or peptides in the sample (Figure 2). In numerous published studies, serum spectral profiles that distinguish between healthy indivi­ duals and patients with cancer have been reported.79–82 Similarly, in CRC, both SELDI-TOF MS and MALDITOF MS profiling has yielded impressive results in terms of sensitivity and specificity (Table 3).67,83,84 Engwegen

and colleagues85 conducted one of the first studies using SELDI-TOF MS to detect and validate biomarker proteins for CRC in serum. Two sets of serum samples were analyzed, the first containing samples from healthy controls or patients with stage IV cancer, and the second comprising samples from controls or patients with cancer of all stages. The CRC profiles were then compared with other cancer serum samples. The results identified five biomarker proteins able to reliably distinguish between healthy controls and patients with CRC. Sensitivities and specificities of between 65% and 90% were reported. However, a comparison with other cancer serum samples revealed that these biomarkers were not solely specific for

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REVIEWS CRC.85 This apparent lack of specificity for CRC has subsequently been confirmed in other studies.86 In another study, De Noo and colleagues87 used MALDI-TOF MS to compare 66 preoperative serum samples from patients who had CRC with 50 samples from healthy controls. 34 of 37 patients with early-stage disease, and all the patients with late-stage disease, were correctly classified as having cancer.87 So far, only one study has investigated the use of SELDI-TOF MS and MALDI-TOF MS profiling of proteins in urine from patients with CRC; Ward et al.88 reported a sensitivity of 78% and a specificity of 87% when comparing 67 patients with CRC and 72 non-cancer control patients. Smith and colleagues89 used SELDI-TOF MS to assess the ability of proteomics in predicting and monitoring the response of patients with locally advanced rectal cancer to neoadjuvant chemoradiotherapy. Sequential serum samples were taken from 20 patients; these samples identi­f ied a total of 14 protein peaks that collectively differ­entiated between good (9) and poor (11) responders with a sensitivity of 87.5% and a specificity of 80%. The study supports the analysis of dynamic tumor responses as an alternative predictive tool.89 A pilot study utilizing MALDI-TOF MS profiling of preoperative and post­ operative serum from patients with CRC suggests that this technique might also be useful in monitoring disease course in patients after surgery.90 As discussed by Petricoin et al.,91 the ‘tumor-specific’ proteins (as detected by MALDI-TOF MS and SELDITOF MS) do not, in most cases, represent genuine biomarkers. In the few cases in which the identities of discriminating protein peaks have been determined, they have been found to comprise degradation products of larger, usually abundant serum proteins. The origin of the tumor-­specific ‘peptidome/degradome’ in cancer sera seems to arise from the actions of specific proteases, most likely released from tumor-associated stromal cells. Although this finding does not invalidate their value as diagnostic markers, the reliability and reproducibility of SELDI-TOF MS and, to a lesser extent, MALDITOF MS platforms has, however, attracted consider­able criticism. As discussed elsewhere, these concerns arise from considera­tions of sample storage and processing, instrumenta­tion (particularly with older SELDI-TOF instruments) and in bio­informatics data analysis culminating in statistical ‘overfitting’ of predictive models.78,92,93 In addition, such studies in CRC have so far been limited to relatively small-scale patient cohorts, sometimes without appropriate matched controls. Although the technique is low-cost in terms of reagents and lends itself to high-throughput semi-­automated sample processing, it suffers the drawback of being inherently semi­quantitative at best. In addition, a high level of technical skill is required in quality control and data analysis and interpretation. Hence, most authorities would not envisage SELDI-TOF MS or MALDI-TOF MS instrumentation platforms, at least in their present form, being used in routine (population) screening of serum samples. Despite these reservations, the technique still represents a worthwhile approach for the discovery and

identification of serum markers for CRC that can subsequently be utilized in population screening using more robust immuno­sorbent assays. Under the auspices of the Human Plasma Proteome Project (HUPO), the efforts of numerous collaborating laboratories are being facilitated by advances in instrumentation of tandem mass spectrometers to elucidate the complete protein composition of human plasma. To date, around 10,000 proteins have been identified.94 Given that accurate quantitation of protein abundance is now routinely achievable using this type of mass spectro­ meter, it seems highly likely that this resource will have a major effect on the identification of diagnostic plasma protein markers for many cancer types.95 The resolution capabilities of advanced tandem mass spectrometers, when coupled with upstream purification, far exceeds that of conventional MALDI-TOF MS or SELDI-TOF MS, and is particularly suited to high throughput, multi­plexed detection and quantitation of discriminating protein and peptide biomarkers that are likely to be of low abundance.96 Translating such biomarker discovery into an inexpensive ‘lab-on‑a-chip’ test kit will be greatly facilitated by technologies for multiplexed analysis using integrated barcode chips, detecting proteins in the subfemtomolar concentration range by using single­molecule ELISA and magnetic nanosensor platforms with resolution down to attomolar concentrations and extensive linear dynamic range.97–99

DNA markers in serum and plasma As with circulating tumor cells, free plasma nucleic acid of tumor cell origin has been well documented to occur in patients with a number of cancer types, although its representation is very low compared with DNA and RNA of non-tumor origin.100,101 Nonetheless, it does represent a potential source of tumor biomarkers.102 With the advent of massively parallel sequencing combined with accurate quantitation by PCR amplification, Diehl et al.103 were able to precisely quantitate levels of plasma DNA of tumor origin in patients with CRC by using a panel of somatic mutations in genes that are commonly mutated in this disease (TP53, KRAS, APC and PIK3CA) (Figure 1). This method enabled the tumor dynamics in individual patients to be monitored after surgery or chemotherapy. Leary et al.104 used a different approach to identify tumor-specific translocations that were then used as markers for monitoring levels of circulating tumor-specific DNA in a small cohort of patients with CRC or breast cancer. Finally, He et al.105 showed that heteroplasmic mutations in mitochondrial DNA occur in CRC tumor cells and, by virtue of the high copy number of mitochondrial DNA (relative to nuclear DNA), can be readily detected and quantified in patient’s plasma. However, how useful these approaches would be for population screening in CRC is not clear; apart from the prohibitive cost, a priori knowledge of the target mutations in individual patient’s tumor cells is required. This methodology is likely to be most useful for ‘personalized’ monitoring of treatment response and for stratifying patients using markers such as KRAS mutations that

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REVIEWS are predictive of poor response to therapies targeting the epidermal growth factor receptor.106 As an alternative to using gene mutations as markers, several studies have exploited tumor-associated epi­ genetic methylation changes as markers to detect circulating plasma DNA of tumor origin. 107 A simple PCR-based assay of plasma to detect methylation of SEPT9 reportedly identifies patients with CRC with a sensitivity of 70% and a specificity of 90%.108,109 This performance compares favorably with both the FOBT and PCR screening of fecal DNA. The assay can also be automated and is now commercially available as the Epi proColon Early Detection Assay kit® (Epigenomics, Berlin, Germany).109 The addition of further markers to this blood screen test might further improve its perform­ ance in the future.110 However, its high cost does not compare favorably with an inexpensive annual FOBT and decadal colonoscopy.109

RNA markers in blood cells, plasma and feces The peripheral blood transcriptome is a potential source of discriminating diagnostic markers for a number of diseases. 111 By microarray gene expression profiling of leukocyte mRNA, Han et al.112 generated a five-gene discriminating marker panel (CDA, MGC20553, BANK1, BCNP1 and MS4A1) from genes that were differ­entially expressed between blood cells of patients with CRC and healthy individuals. The reported sensitivity was 88% with a specificity of 64% on an independent test set of normal and CRC samples.112 In a subsequent study, a seven-gene marker panel (ANXA3, CLEC4D, LMNB1, PRRG4, TNFAIP6, VNN1 and IL2RB) had a sensitivity of 82% and a specificity of 64%.113 Although the diagnostic performance in both studies was comparable, the absence of any overlap between the two diagnostic marker panels highlights the well-recognized ‘instability’ problem that afflicts classification based on gene markers obtained from differential expression profiling. The seven-gene marker panel has been validated in an independent popula­t ion study with a reported perform­a nce of 61% and 77% for sensitivity and specificity, respectively.114 MicroRNAs (miRNAs) have attracted considerable attention as diagnostic or prognostic markers when applied to tumor tissue from many cancers, including CRC,115,116 and circulating miRNAs are emerging as potential biomarkers for population screening.117,118 One study has shown that circulating plasma miRNA levels in patients with gastric cancers largely reflect those in matched tumor tissue; two miRNAs in particular (miR‑106b and miR‑106a/let-7a) discriminate between normal and gastric cancer plasma with high sensitivity and specificity (receiver-operating characteristic curve: 0.721 for miR‑106b and 0.879 for miR‑106a/let-7a).119 Studies in CRC have shown that circulating levels of miRNA‑29a, miRNA‑92a and miRNA‑17 display a statisti­cally significant increase in CRC plasma compared with controls.120,121 Quantitation of plasma miRNA markers thus offers a worthwhile avenue for future investigation as an approach for CRC screening. Finally,

miRNA seems to be remarkably well-preserved in exfoliated colonocytes from feces. The stools of patients with CRC reportedly display unique and identifiable patterns of miRNA expression. In a preliminary study, miRNA expression profiling gave an overall sensitivity and specificity of 74.1% and 79.0%, respectively.122

Conclusions Despite major investment and important scientific advancements over the past 15 years, very few new biomarkers for cancer diagnosis have so far been approved for clinical use.123,124 For CRC screening, established programs with proven, albeit less-than-ideal efficacy, such as the FOBT and patient-centric investigative procedures, are likely to remain in place for some years to come. Indeed, the potential of low-cost flexible sigmoidoscopy for statistically significant reduction in the incidence and mortality of CRC27,28 illustrates how evidence-based approaches in conventional screening programs can have a remarkable effect without the need for major investment in basic science and subsequent clinical validation, with its attendant less-than-certain outcome. Nonetheless, on the basis of the numerous precedents of the paradigm shift of conventional diagnostics towards molecular diagnostics in the health-care sector, it seems likely that population screening for CRC using molecular markers will eventually replace conventional methods. Herein lies a dilemma—of the plethora of different strategies that are currently being developed and evaluated, there is no obvious clear ‘winner’ at the present time; none of methods have been evaluated beyond proof-of-principle and pilot-scale study. This field is rapidly progressing, however, with major new advances being made every year. From the viewpoint of patient compliance, a blood-sampling-based test is clearly preferable to a stool test. Markers based on nucleic acid are usually more readily translatable into a test-kit platform but, together with cytological detection, might have theoretical limits of predictive accuracy. Nucleic acid detection is also quite costly, particularly when it involves a PCR procedure. Plasma protein markers, on the other hand, offer considerably more potential, at least in the longer term, for two main reasons. First, only a small fraction of the total human ‘plasmasome’ has so far been explored, and therefore an enormous untapped reservoir of potential disease markers is waiting to be discovered. These potential disease markers include not only the individual plasma proteins and peptides themselves but also a vast diversity of their post-translationally modified derivatives. Second, diagnostic immunoassay test kits for proteins have been in widespread use for many years in a variety of health-care settings. They are generally robust and inexpensive to mass-produce. In addition, the technology for more sensitive, second-generation protein assay platforms already exists. 97–99 It seems likely that the introduction of molecular marker screening methods will be an incremental process over the next few years. Moreover, national health-care programs are unlikely to divert resources away from conventional screening

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REVIEWS methods that have taken many years of planning and infrastructure investment to establish. It is therefore probable that the implementation of new technology for popula­tion screening in CRC will rely heavily on the activities of the commercial biopharmceutical sector rather than on publicly funded ventures, particularly during a period of global economic austerity. 1. 2.

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Screening for colorectal cancer : established and emerging modalities