As CRCs increase in size, they may bleed. Bleeding is often occult and may be intermittent. gFOBt identifies the presence of heme in stool and hence, may detect bleeding from a CRC. In this test, stool is placed on guaiac-impregnated paper; in the presence of heme, a peroxidase reaction occurs, causing the paper to turn blue. This is a qualitative assessment, that is, the test reader must decide whether or not the color change is adequate. Patients who are deemed gFOB-positive are subsequently referred for a diagnostic colonic investigation, usually colonoscopy.

To account for the potentially intermittent nature of CRC bleeding, stool sampling is usually required from several separate stool samples. The test is also not specific to human blood: any heme present in the stool sample can interact with the guaiac, including non-human heme in ingested red meat. Foods with naturally high levels of peroxidase (such as horseradish, broccoli, cauliflower, or cantaloupe) can also cause a positive reaction [21]. Dietary modifications may reduce the false positive rate, but this is at the expense of reduced participation due to the added complexity. Testing can be performed on rehydrated (more sensitive, less specific) or non-rehydrated samples (less sensitive, more specific). gFOBt is also limited by its poor sensitivity for detection of adenomas and CRC compared to other screening modalities [17], although its availability, lower cost than immunochemical tests, established use, and randomized controlled trial (RCT) evidence base demonstrating its effectiveness in reducing CRC mortality have led to it being the most frequently used fecal occult blood test in early CRC screening studies. In contrast, no RCTs have been conducted for fecal immunochemical testing (FIT) thus far [22].

A meta-analysis of three trials of biennial gFOBt including over 172,000 people demonstrated a relative reduction in CRC mortality by 15% [23]. Four large-scale trials have examined gFOBt as a screening tool for CRC, showing positivity rates of 0.8%–5.3% for non-rehydrated samples and 1.7%–14.3% for rehydrated samples (Table 2) [24-27]. The English BCSP currently uses non-rehydrated samples for the gFOBt and previously demonstrated a positivity rate of 2% [15,28]. A similar rate has been also shown in Scotland (2.1% positivity; age of screened individuals, 50–69 years) and France (2.8% positivity; age of screened individuals, 50–74 years) [29], and a higher positivity rate of 3.6% was observed in Finland (age of screened individuals, 60–69 years [30]; an earlier study in Finland with 60- to 64-year-old individuals reported a gFOBt positivity of 2.1%) [31]. However, the rate of CRC diagnosis after a positive gFOBt varies between 7% and 21% in England, Scotland, and France [28,29,32], but is only 3.6% in Finland [33]. A similar low CRC detection after a positive gFOBt was observed in Croatia (6.8% positivity; CRC incidence after positive gFOBt, 3.8%) [34], but uptake of this program was low, at less than 20%. These programs and their outcomes are summarized in Table 3. It should be noted that the definition of test positivity differs from program to program. In England, double sampling is required from 3 separate stools, and a positive test is defined as 5 or 6 of 6 windows being positive for heme. A repeat kit is requested for 1–4 positive windows; if any window is positive in the repeat test, the findings are deemed positive. If no windows are positive in the repeat kit, a third kit is performed; if any window is positive in that kit, the participant is deemed to have a positive result. If no windows are positive in the third kit, the participant is deemed to have had a negative test [35]. In both the French and Finnish programs, a test is deemed positive if any of the 6 windows are positive for heme [29,30]. In the three previously described trials of biennial gFOBt screening as well, the definition of positivity differed: the Nottingham study required 5 or 6 positive windows, or 1–4 positive windows followed by 1–6 positive windows in a repeat test [24], and both the Minnesota and Funen studies deemed 1–6 windows positive for heme as a positive test [25,26].

When compared against FS screening (with the expectation that positive gFOBt results would lead to colonoscopy as would FS that identified adenomas) in a Cochrane review, both gFOBt and FS screening were found to reduce CRC mortality when compared to no screening at all (FS: RR, 0.72 [95% confidence interval [CI], 0.65–0.79]; gFOBt: RR, 0.86 [95% CI, 0.80–0.92]), but no conclusion could be reached as to whether one method was better than the other because only low quality data were available [36]. However, as gFOBt has been shown to have a lower sensitivity for detection of advanced adenomas (11%–41% compared to 50%–81% for FS), it is probably less effective at reducing CRC incidence [37].

Five countries have an established, fully rolled-out gFOBtbased screening program. Uptake of screening in these countries is 68.8% in Finland (although the programme is being implemented via a randomized trial) [30], 55.4% in the UK [15], 53%–55.3% in Scotland [32], 34.4% in France [29], and 19.9% in Croatia [34]. Many factors play a part in the reasons for non-participation in screening, including socioeconomic status, ethnicity, age, and gender [38]. The nature of the test procedure itself has also been reported as a reason for non-participation in CRC screening [39,40]: this is one of the reasons that many gFOBtbased programs are moving to FIT-based screening.

FIT analyzes stool samples for the presence of blood using a mono- or polyclonal antibody to human hemoglobin. When hemoglobin is present, it forms a complex with the antibody, which can be quantified [41]. FIT has several advantages over gFOBt. Unlike gFOBt, it is not affected by false positive results from food, as it is specific for human hemoglobin. FIT requires fewer sampling episodes, often with only one sample being required [42]. It is also more specific for a lower GI source of bleeding than gFOBt is [22]. A meta-analysis of FIT accuracy demonstrated that its overall pooled sensitivity for CRC is 0.79 (95% CI, 0.69–0.86) and specificity is 0.94 (95% CI, 0.92–0.95) [43]. Further, the advanced adenoma yield over two rounds of screening did not differ irrespective of whether 1 or 2 samples were taken at each screening round, suggesting that single sample testing is adequate [44].

As it allows quantitative analysis of stool blood content, FIT has varied cut-off levels for detection, and the analysis process is automated. Review of the existing literature demonstrated a decreased positivity rate and increased specificity and positive predictive value for CRC and advanced adenomas with increasing cut-off levels [22]. Frequently used cut-off points are 20, 50, and 100 μg/g (100, 250, and 400 ng/mL respectively) [43]. This ability to vary the “positive test” cut-off allows screening programs to adjust the level to provide an acceptable threshold for further investigations without exceeding the capacity of the endoscopy service, which will deliver those investigations. In established gFOBt programs, a FIT cut-off to provide the same positivity rate as for gFOBt may be used: for example, in Scotland, a cut-off of 80 μg/g (400 ng/mL) provided approximately the same positivity level as gFOBt of 2.4% [42]. An alternative approach was used in a Dutch study where they aimed to detect the same amount of advanced adenoma using FIT as with gFOBt. They found a cut-off of 75 ng/mL lead to a similar rate of advanced adenomas as the gFOBt program [45]. Consideration has also been given to the use of a lower cutoff level, with a resulting higher positivity and consequently, higher CRC and advanced adenoma detection rates (ADRs). It was demonstrated that a lower cut-off could allow extension of the screening interval as positivity rates and CRC and advanced ADRs were similar to those in the standard screening scenario [46]. It has been recommended that standardization of FIT reporting is required if it is to become a routine part of CRC screening [47].

FIT is in use in a number of screening programs, many of which are still in the pilot or roll-out phases [17]. Few have yet reported outcomes of screening with FIT outside of a trial setting. There are no trials comparing the outcomes of screening with FIT versus no screening, but comparisons of FIT versus gFOBt have been reported. All available outcomes of FIT are summarized in Table 4 [42,48-50]. Patient compliance and satisfaction with FIT have been shown to be greater than with those with gFOBt. The participation rate was reported to be 12% higher for FIT than gFOBt [49], even when dietary restrictions for the gFOBt arm were lifted. This suggested the simpler method and requirement for only a single sampling episode rather than 3 episodes affected the likelihood of completing the screening process.

Stool DNA testing is a developing area of investigation. Stool samples are analyzed for DNA markers of neoplasia, since the cells of CRCs and advanced adenomas contain abnormal DNA markers (such as aberrantly methylated promoter regions, mutant K-ras, and β-actin) [51]. Early stool DNA tests detected only one marker, but newer tests are multi-target and have demonstrated higher sensitivity than FIT for advanced adenomas (42% vs. 23%) and CRC (92% vs. 72%) [51]. However, this improved sensitivity comes at the expense of higher test positivity (16% vs. 7%, for FIT) and will therefore place greater demands on endoscopy services [51].

Other stool tests such as those for RNA markers, protein markers, and microbiome-based biomarkers are also being investigated for use in CRC screening, but further work is required before these find common usage. Current limitations are related to high costs and practicalities as a large volume of stool is required [51].

FS screens for adenomas using a flexible endoscope inserted into the distal colon, with the aim of examining at least the rectum and sigmoid and, if possible, as far as the splenic flexure. There are variations in practice with regard to the bowel cleansing preparation required (enema preparation versus full oral bowel cleansing), depth of insertion desired, medications used (none, Entonox, or intravenous sedation), and the threshold for referral for a completion colonoscopy. There have been four RCTs of FS screening individuals in the age range 55–74 years conducted in Europe [52-54] and America [55]. These are summarized in Table 5 [50,52-57]. The European studies offered once-only FS, whereas the American study offered subsequent FS after 3–5 years. The primary goal of FS screening, particularly when used toward the lower end of the age range, is to detect and resect premalignant adenomatous polyps, preventing cancer development. Pooled analysis of these studies estimated a risk reduction of 18% for CRC incidence and 28% for deaths from CRC [58]. Further, a more recent pooled analysis of three of these four studies (two of the European studies and the American trial) evaluated the effect of age and gender on CRC incidence and mortality, demonstrating a reduction of 24% and 33%, respectively, in men regardless of age, but a smaller effect in women, and in women younger than 60 years rather than older than 60 [59].

Compared to gFOBt or FIT, FS has a higher detection rate of advanced neoplasia (i.e., advanced adenomas and CRC) [49] but has a lower specificity and sensitivity than colonoscopy for both advanced adenomas and CRC [37].

On the basis of the findings from the UK FS screening trial [52], the English BCSP recently added a one-off FS screening for all 55-year-olds. This trial now has 17 years of follow-up data and shows that a single FS results in reduction in CRC incidence and mortality of 26% and 30%, respectively, in intention-to-treat analyses [60]. Initial pilot sites commenced in 2013 with roll-out to all screening centers planned by 2018. An early pilot of the program demonstrated an ADR of 9.8% [61], and first-wave pilot sites have reported similar rates [62]. This rate initially appears lower than the 12.1% ADR reported in the UK trial, but two factors affect this figure: first, the population age being screened differs (55–60 years in the study versus 55 years in the screening program) and as already discussed, the ADR increases with age; second, the ADR in the screening program is calculated from histological analysis from the index procedure only, whereas in the trial, a composite ADR was calculated based on adenomas detected at both the screening FS and the subsequent colonoscopy.

A meta-analysis considering survival benefit (i.e., the longterm benefits over several years versus the immediate harms screening exposes participants to) suggested FS may be an appropriate screening test to offer to older adults (when compared to no screening), provided they have a life expectancy of more than 10 years, as the time taken to benefit for an absolute risk reduction of 1 in 1,000 was 9.4 years [63]. A Cochrane review of FS screening suggested similar effectiveness of gFOBt, but in the absence of any trial data comparing the two screening processes, it could not conclude that one method was better than the other [36].

FS is deemed to be a low-risk procedure (in the three RCTs of FS screening, there were two perforations in almost 54,000 procedures) [56,57,64], although subsequent colonoscopy based on initial FS findings carries some additional risk. To date, only England offers an organized national program of FS screening, with a pilot program running in Norway [17] and some areas of Italy offering FS screening [65]. Uptake in England was 44.1% in the first year of the program, with significant variation across the country [62]. Uptake was lower than in the gFOBt arm of the BCSP, where it approached 60% [66]. Posttest questionnaires were utilized during the pilot phase of the program, demonstrating overall high levels of satisfaction and low levels of embarrassment [67]. However, 43% of the participants reported moderate or severe pain during the procedure, a level underestimated by endoscopists [62] and suggests a need to evaluate comfort levels further within the program. It is not yet known how experience of this FS program at 55 years of age will affect uptake and involvement in the gFOBt arm of screening starting at 60 years of age, nor is it known what the effect of prior FS screening will be on the pathological yield of subsequent FOB/FIT screening.

Examination of the entire colon using a flexible colonoscope is the “gold standard” investigation for CRC. Screening with colonoscopy has the primary aim of detecting CRC and a secondary aim of detecting and removing adenomas. It allows direct assessment of the entire colonic mucosa, although visualization is rarely 100%. A post-colonoscopy CRC (PCCRC, a CRC diagnosed within 3 years of negative colonoscopy) suggests that a cancer or premalignant polyp may have been missed at colonoscopy. A nation-wide study in England demonstrated this occurred at a rate of 8.6% [68], and this should be considered when counseling participants. The PCCRC rate is an important indicator of the quality of colonoscopy, and it has been demonstrated that endoscopists with higher ADR have lower PCCRC rates [69,70]. As such, ADR is used as a key surrogate marker for colonoscopy quality. Colonoscopy should therefore be of the highest quality to maintain the benefits of screening, and serial audits of practice in the UK have shown improvements in the key performance indicators (KPIs) for diagnostic colonoscopy [9,71]. In England, BCSP colonoscopies are performed by screening-accredited colonoscopists who maintain high KPI standards. The BCSP and its data collection system have ensured improvements in KPIs and more robust quality assurance [72]. In other countries, specific screening accreditation is often not required, although the need for high-quality procedures is well recognized.

Colonoscopy is occasionally offered as the initial screening tool of choice in countries where screening is offered opportunistically. Only Poland has an organized screening program with colonoscopy as the primary screening test. This program started as an opportunistic screening program [73] and has randomized 55- to 64-year-old individuals to an immediate or postponed invitation to screening since 2012 [74]. In America, guidelines recommend colonoscopy screening commencing at age 50 years, with 10-yearly procedures [16]. This screening is opportunistic and is offered via the patients’ healthcare provider and is not part of an organized program. The American College of Gastroenterology guidelines [16] suggest a range of alternatives that can be offered depending on patient preference.

Although strong evidence backs the use of endoscopic screening, there is currently little proof that colonoscopy is superior to FS in terms of mortality reduction. Two large-scale RCTs are underway to explore the outcomes of screening colonoscopy [75,76], but no results are expected until the next decade. Cohort studies of patients undergoing colonoscopy and clearance of polyps demonstrate reduction of CRC incidence by up to 90% [77,78], but these are not asymptomatic populations.

In most countries, colonoscopy is used as the second step in the screening process after FS or fecal occult blood testing (with gFOBt or FIT) [17]. This is primarily because of cost and resource constraints and has been demonstrated to be a cost-effective measure [17,79].

One of the drawbacks of colonoscopy as a screening tool is the potential for complications. These include bleeding, bowel perforation, complications of sedation, and complications of bowel cleansing preparations. Overall, low rates of serious complications have been reported by the English BCSP (bleeding requiring transfusion, 0.04%; perforation, 0.06%) and the French screening program (overall rate of serious complications, 0.06%) [15,28,29,80].

Colonoscopy can be an uncomfortable procedure, and overall patient experience (taking into account not just discomfort but embarrassment, bloating, and other aspects) should be maintained at an acceptable level to ensure it is a viable screening option [67,72,81,82]. Work on comfort during screening has been undertaken in the English BCSP, and colonoscopy and FS procedures have been largely well tolerated, with no or minimal pain reported in 64.3% and 79% cases, respectively, and severe pain reported in only 1.3% and 1%, respectively [72,81]. However, it should be noted that these comfort scores were recorded by the nursing staff, and there appeared to be a discrepancy between these scores and those reported by patients; in qualitative studies, over 25% of patients reported experiencing more discomfort than expected with both screening modalities [67,83].

Examination using colon capsule endoscopy (CCE) allows minimally invasive assessment of the large bowel. Bowel cleansing is required, in similar fashion to colonoscopy, with European guidelines recommending the use of 4 liters of polypethylene glycol for preparation [84]. Two meta-analyses of CCE showed this procedure had a sensitivity of 71%–73% for detection of all polyps and 68%–69% for significant findings (defined as any polyp of 6 mm or larger or more than 3 polyps), with a specificity of 75%–89% and 82%–86%, respectively [85,86]. While these findings are comparable to those of other noninvasive measures, concerns have been raised about its accuracy, particularly when bowel preparation is considered; one study showed good or excellent preparation quality in only 52% of cases, with subsequent higher adenoma and cancer detection at colonoscopy [87]. It should of course be noted that individuals with lesions detected during CCE require colonoscopy for removal of the identified lesions, as CCE cannot be used to obtain histological samples. It has been shown to be well tolerated by patients [88], and in areas where CRC screening participation is poor, it has also been shown to improve uptake [89]. CCE is a potentially attractive modality for CRC screening but is currently limited by availability, expense, and the need for further procedures if lesions are detected, and further research is required before its use becomes commonplace [90].