Is Negative Fecal Immunochemical Test Reassuring? The Cumulative Risk of Colorectal Cancer after Multiple Negative Tests.

INTRODUCTION
The reported risk of colorectal cancer (CRC) is approximately 0.1% (1 in 1,000) for individuals aged ≥50 years, according to data from cancer registries and clinical trials.1,2 The fecal immunochemical test (FIT) is widely used for CRC screening. Clinicians typically refer patients for diagnostic colonoscopy following a positive FIT result, while a negative FIT result is generally interpreted as indicating a low risk of CRC. This perception is based on the high sensitivity of FIT, which ranges from 84% to 94% at a cutoff value of 10 to 20 μg hemoglobin (Hb)/g feces.3-9 Given that individuals with a negative FIT result, typically defined as <20 μg Hb/g feces, outnumber those with a positive result by about 20 to 1, and comprise approximately 95% of the average-risk population aged 50 and older,1 performing diagnostic colonoscopy on all those with negative results would be inefficient and a misuse of medical resources. Consequently, CRC risk in individuals with negative FIT results is often estimated using cancer registry data rather than direct diagnostic colonoscopy. However, CRC incidences reported by cancer registries may not be directly comparable to those identified via colonoscopy. A key limitation of registry data is reporting delay, which affects all cancer registry systems. Unlike immediate diagnosis via colonoscopy, CRC in individuals with a negative FIT result may go undetected until symptoms emerge, leading to delayed reporting. This delay can be 8 to 15 times longer, resulting in an apparent underestimation of CRC incidence in registry data compared to direct colonoscopy findings.2,10-14 Therefore, it is important to distinguish whether CRC cases are identified via cancer registries or direct colonoscopy.
To evaluate the CRC risk following negative results of FIT and to assess the impact of delayed reporting in cancer registry data, we conducted a multi-faceted analysis. First, we monitored CRC cases year after year following the first two rounds of FIT to compare the risk differences between positive and negative results.15 Second, based on the understanding that negative colonoscopy findings are generally considered reliable for up to 10 years,16,17 we applied a similar 10-year observation period to estimate CRC incidence rates following one or multiple consecutive negative FIT results. Finally, we examined CRC-specific and all-cause mortality among individuals with persistently negative FIT results to assess overall risk reduction.

MATERIALS AND METHODS

1. Study population
The cohort consisted of 141,982 individuals aged 50 years and older, with no known history of CRC, who participated in a health surveillance program conducted by the MJ Health Management Institution across Taiwan between 1994 and 2008.18 Each participant completed a health history questionnaire covering lifestyle and medical conditions. All laboratory tests were performed at a central laboratory. Informed consent was waived for this study, which was approved by the Institutional Review Board of China Medical University Hospital in Taiwan (Approval Code: N/A/CRREC-107-092 [CR-3]).

2. FIT test
From 1994 to 2008, an automated OC-Sensor test (Eiken Chemical Company, Tokyo, Japan) was used to measure the concentration of human Hb in fecal samples.1,19,20 A negative FIT result was defined as <20 μg Hb/g feces. The screening program operated continuously without synchronized “rounds” for all participants; however, the recommended screening interval in this retrospective study was every 2 years. The number of visits for each individual was counted as consecutive screening rounds, with approximately half of the participants returning for subsequent rounds (Supplementary Fig. 1).

3. Follow-up
We matched the identification document of each participant with the National Cancer Registry to identify incident CRC cases and with the National Death File to identify CRC deaths.21,22 A total of 1,700 incident CRC cases and 557 CRC-related deaths were identified among individuals aged ≥50 years through the end of 2008. The Taiwan National Cancer Registry, established in the 1980s, mandates reporting by all hospitals upon confirmation of a cancer diagnosis and maintains a coverage rate and data accuracy exceeding 98%.

4. Statistics
FIT sensitivity for CRC was defined as the proportion of CRC cases with a positive FIT result divided by the total number of CRC cases (both FIT-positive and FIT-negative). CRC cases observed after the first two rounds of FIT screening were reported annually for both positive and negative FIT groups. Age-adjusted incidence and mortality rates were standardized to the 2008 Taiwanese population. To reduce bias from the declining CRC incidence and mortality observed with repeatedly negative FIT results, often due to the “healthy returnee effect,” inverse probability weighting (IPW) was applied. Propensity scores were estimated using a logistic regression model that included age, sex, smoking, alcohol consumption, hypertension, and diabetes. This adjustment approximated a pseudo-population in which FIT results were independent of the observed covariates. Poisson regression was used to compare incidence and mortality rates between groups. A linear regression analysis was used to assess trends in incidence and mortality, with statistical significance defined as p<0.05. The number needed to screen23 to detect one CRC case over the study period was calculated as the reciprocal of the positive predictive value. The positive predictive value was defined as the number of true-positive CRC cases divided by the total number of positive FIT results (both true and false positives), without adjusting for follow-up time or censoring.1,19,20 All statistical analyses were performed using SAS software, version 9.2 (SAS Institute, Cary, NC, USA).

RESULTS

1. Baseline characteristics
Table 1 shows that individuals who consistently returned for additional FIT screenings exhibited the “healthy returnee effect,” characterized by decreasing rates of current smoking, hypertension, and diabetes over successive rounds. Compared to those with positive FIT results, individuals with negative FIT results showed relatively small differences in CRC risk across age groups (Supplementary Table 1) and no significant differences by sex (Supplementary Fig. 2).

2. CRC cases identified after the first two rounds of FIT
Table 2 presents CRC cases identified via the cancer registry after the first two rounds of FIT, tracked over a 2- to 10-year follow-up period. Although individuals with a positive FIT initially accounted for more CRC cases, this trend reversed over time, with an accelerated rise in CRC incidence beginning 2 years after a one-time negative FIT result. Nearly two-thirds of all CRC cases during the entire study period were detected in participants who had only a single negative FIT, surpassing the number of cases observed in those with a positive result. Due to the slow-growing nature of early-stage CRC in individuals with initially negative FITs, approximately 8 to 10 times more cases emerged by the end of the study than within the first 2 years. The proportion of CRC cases with negative FIT results increased from 30% (113/378) in the first 2 years to 58% (987/1,700) by the study's end after the first round, and from 31% (24/78) to 60% (261/432) in the second round (Fig. 1).

3. CRC risks after continuously negative FIT results
Table 3 shows that the proportion of “continuously negative” FIT results declined gradually from 94% to 91%, 88%, 86%, and 84% over the first five rounds, after excluding individuals with positive FITs in each round. Compared to the initial cohort (Fig. 2), CRC incidence was reduced by 37%, 47%, 67%, 77%, and 90% over the first five rounds, respectively. Corresponding reductions in CRC mortality were 33%, 58%, 80%, 91%, and 96%. Both CRC incidence and mortality showed consistent downward trends with an increasing number of negative FIT rounds (p for trend <0.05). After IPW adjustment, CRC incidence and mortality remained significantly lower with each additional round of negative FIT results during the first five rounds (p<0.05). However, cumulative incidence and mortality rates in later screening rounds may be underestimated, potentially leading to an overestimation of observed risk reduction if not properly adjusted. This decreasing trend in CRC risk with cumulative negative FIT results was also reflected in a reduction in all-cause mortality (p for trend <0.05) (Table 4).

4. CRC risk by FIT results in each round of FIT
Table 5 evaluates risk reduction within actual returning sub-cohorts in each round, thereby eliminating the influence of the healthy returnee effect (Supplementary Fig. 3). Regardless of prior FIT history, CRC incidence among participants with positive FIT results was 9 to 11 times higher than among those with negative FIT results in each round. Among participants with negative FITs, CRC risk was consistently lower in those without a prior history of positive FIT results compared to those without such exclusions.

5. Literature review
Supplementary Table 2 summarizes reported FIT sensitivity for CRC detection from the literature.9,10,15,24-26 These studies suggest that estimating FIT sensitivity using colonoscopy-confirmed cases for positive FITs and registry-based cases for negative FITs may overestimate the true sensitivity of FIT in detecting CRC.9,15,25Additionally, prior studies consistently report lower FIT sensitivity for detecting intramucosal or T1-stage CRC.9 Our findings are consistent with these observations, showing a decline in CRC incidence following multiple rounds of continuously negative FIT results, as detailed in Supplementary Table 3.27-33

DISCUSSION
FIT is recommended to triage patients over 40 with abdominal symptoms for suspected CRC, given the low risk associated with a negative result within the first 2 years, as reported in cancer registry data.3-6 However, annual monitoring of cancer incidence over a decade has revealed 5 to 10 times more CRC cases than those observed within the first 2 years following a negative FIT. This suggests that the long-term CRC risk following a negative FIT is substantial. Relying solely on a single negative FIT result to rule out CRC is misleading, as a significant residual risk remains. Our findings indicate that the risk of CRC becomes apparent after 2 years, supporting the need for biennial FIT screening to mitigate the long-term risk.
Most CRC cases are confirmed through diagnostic colonoscopy, and we assumed that the cancer registry provided comparable accuracy in identifying CRC cases following negative FITs. Early-stage CRC may develop slowly over 5 to 10 years before being clinically detected and registered. Given that the surveillance period after a negative colonoscopy is typically 10 years, a corresponding 10-year follow-up via five biennial FIT rounds with consistently negative results is necessary to match that level of risk reduction.16,17 Although more CRC cases are initially detected among individuals with positive FIT results, those with negative results may eventually surpass them in cumulative case numbers. This indicates that FIT can miss early-stage cancers.2,7-10,15 This is supported by higher cancer detection rates via direct colonoscopy compared to registry data,2,10-14 underscoring colonoscopy’s superior ability to identify cancers that FIT might overlook, particularly early-stage CRC. False-negative FITs may delay diagnosis, allowing early cancers to progress to more advanced stages. In addition to age, our prior research has identified several independent CRC risk factors, including a family history of CRC, Hb <10 g/dL, recent black stools or changes in bowel habits, and serum carcinoembryonic antigen ≥5 ng/mL.1 The probability of detecting CRC increases with the number of risk factors present. Therefore, individuals exhibiting any of these factors should be prioritized for diagnostic colonoscopy, even in the absence of a positive FIT.
The U.K. joint guideline considers a negative FIT result indicative of negligible CRC risk, recommending colonoscopy only for positive FITs.34 This stance is based on high FIT sensitivity derived from combining diagnostic colonoscopy results for positive FITs with cancer registry data for negative FITs. However, this combined approach may overestimate FIT’s sensitivity, as it blends different outcome sources and limits the observation window to 2 years.9,15,25 The reported high sensitivity assumes consistency across disparate data sources, which is further constrained by short follow-up period.
Our findings highlight the importance of continuing CRC screening after a negative FIT result, challenging the notion that a single negative result significantly lowers risk. It is crucial to document consecutively negative FIT outcomes, as each additional negative result reduces risk, conserves medical resources, and supports the need for ongoing screening. In comparison to positive FITs in each round, individuals with a one-time negative FIT carried only one out of 10 of the CRC risk. However, within 10 years, a single negative FIT still retained 63% of the initial risk. Risk decreased to 53% after the second negative FIT, 37% after the third, 23% after the fourth, and only 10% after the fifth. Completing five negative rounds over 10 years lowered CRC risk to levels comparable to those seen after a single negative colonoscopy.16,17 Continued screening remains essential, as risks re-emerge with any subsequent positive FIT (Tables 3 and 5).
The literature and our data indicate that combining colonoscopy outcomes for positive FITs with cancer registry outcomes for negative FITs overestimates FIT’s sensitivity (Supplementary Table 2).9,10,15,24-26 To our knowledge, we are the first to explore the mechanism behind this overestimation, which arises from mixing different outcome measurements.
Since roughly 95% of average-risk individuals aged ≥50 have negative FIT results,1 routine colonoscopy for all would be impractical. About 84% of our cohort maintained consecutively negative results across five rounds, supporting FIT as an efficient screening strategy that reduces the need for colonoscopies. We are the first to report stepwise CRC risk reduction over five rounds, reaching just 10% of the original risk without external intervention. Previous studies reported CRC risk reduction after 2 to 4 rounds of FIT,27-33 but did not fully address the contribution of excluding positive FIT cases to this effect.
The observed decrease in all-cause mortality, rather than an increase, suggests minimal competing risk, consistent with our prior study,19 which established a dose-response relationship between FIT levels and both CRC and all-cause mortality. Consequently, all-cause mortality logically declined among those with consistently negative FITs, after excluding higher-risk FIT-positive individuals.
A key strength of this study is its large sample size and extended 10-year follow-up. This timeframe was critical, as 803 incident CRC cases were identified after the first round and 219 after the second among individuals with a single negative FIT, highlighting the need for long-term monitoring.
This study has some important limitations. First, a major limitation of this study is the substantial loss to follow-up observed across screening rounds, with only approximately one-third of participants returning for a fifth round of FIT screening. This high attrition rate introduces a risk of bias, which may significantly affect the observed reductions in CRC incidence and mortality over time. Although FIT-positive individuals were excluded from these analyses, those who remained in the cohort were more likely to be younger, female, and have healthier baseline characteristics (e.g., lower smoking and comorbidity rates). The apparent decline in CRC risk may partially reflect the changing composition of the population rather than the true benefit of repeated screening. Based on extrapolations from Table 3, we assume those loss to follow-up had CRC risks equal to or modestly higher than retained participants and find that high attrition may result in underestimation of cumulative CRC incidence by 30% to 40% and mortality by 40% to 60% by Round 5 (Supplementary Table 4). This suggests the protective effect of repeated negative FITs may be overstated but it persisted.
Second, our study lacked data on colonoscopy follow-ups for those who tested positive for FIT. We primarily highlight the need for as many as five rounds (10 years) of continuously negative FITs to reduce the initial risk to approximately one out of 10, which is akin to the risk level observed after 10 years from a single negative colonoscopy finding.16,17 While colonoscopy may detect CRC slightly earlier, its impact on overall numbers was limited due to the smaller positive rate (3% to 5%) of FIT. The overall CRC incidence or mortality rationally can be diminished for the individuals with positive FITs if they undergo colonoscopy follow-up. Follow-up colonoscopy compliance was left to participant discretion, especially before Taiwan’s biennial FIT program started in 2004. This program began a decade after our study (1994 to 2008) and initially achieved only a 4.8% annual response rate with a 3.6% to 4.3% FIT positivity rate.35,36
Third, the absence of confirmatory colonoscopy in FIT-negative individuals remains a major limitation. Relying solely on delayed cancer registry data potentially underreported CRC incidence, particularly among interval cancers or advanced neoplasia not detectable by FIT. Given that the number of individuals with a negative FIT result, performing diagnostic colonoscopy on all individuals with negative FIT results would be inefficient and a misuse of medical resources. We demonstrated a reduction in cancer risk following negative FIT results, not solely based on registered incidence but also corroborated by consistent findings from National Death File-based mortality. Ultimately, relative survival should be considered the primary criterion for evaluating the effectiveness of a screening test such as the negative FIT.
Fourth, the “healthy returnee effect” may overestimate the risk reduction among individuals with consistently negative FIT results. Our sensitivity analysis of negative FIT results in each round shows that the CRC risk for individuals with continuously negative FITs was consistently lower than for those who did not exclude a history of positive FIT (Table 5). Furthermore, IPW is used to adjust for potential selection bias due to non-random dropout or healthier screening continuation across rounds. As a result, the cumulative incidence and mortality rates reported in later screening rounds may underestimate the true burden of CRC (Table 3). However, The IPW-adjusted CRC incidence and mortality were consistently reduced with each additional round of negative FITs during the first five rounds (p<0.05). We highlighted that both CRC risks and all-cause mortality rates are likely to decrease progressively for individuals maintaining continuously negative FIT results, even if the extent of risk reduction might be somewhat overestimated. This phenomenon could serve as an encouragement and motivation for the public to engage in further follow-up screenings with consistently negative FIT outcomes.
Fifth, the cancer risk identified in this study might be specific to this cohort and not universally applicable. Our cohort likely had higher health literacy and motivation than the general population aged ≥50 in Taiwan, particularly concerning diabetes, smoking, and alcohol use. This may have exaggerated the effectiveness of repeated negative FITs in reducing CRC risk (Supplementary Table 5),37,38 although surveillance bias may still contribute to the relatively higher CRC incidence observed in the MJ cohort. Nevertheless, the cancer risks, expressed as hazard ratios (HRs), were internally standardized, making them independent of cohort effects. Moreover, the large sample size, drawn from a broad population, and the extended follow-up period of 15 years likely reduced the variability typically seen in a single cohort.
Finally, we did not stratify CRC risk by age and sex across multiple FIT rounds. However, a twofold increase in HR per decade of age was observed primarily among individuals with positive FIT results. Given that the CRC risk associated with a positive FIT is approximately ten times higher than that for a negative FIT, the absolute risk differences by age in the negative FIT group were minimal. Additionally, the absolute differences in CRC risk by age or sex among individuals with negative FIT results were negligible when using a cutoff value of 20 μg Hb/g feces (Supplementary Fig. 2). We also employed a “passive follow-up” approach by linking participants' identification records with the National Death and Cancer Registry databases.21,22 Although some individuals traveled abroad, most eventually returned. The proportion of individuals with negative FIT results who moved abroad is likely similar to those who remained, as FIT results are not associated with self-perceived health status. While occasional deaths have occurred overseas, most of these cases were reported back to Taiwan in order to access financial or survival-related benefits.
We concluded that a one-time negative FIT cannot ensure the absence of CRC risk within the first 2 years, and the risk doubled every 2 years and reached 5 to 10 times higher within 10 years. A key limitation is the absence of confirmatory colonoscopy in FIT-negative individuals. Relying on cancer registry alone may miss interval cancers or advanced neoplasia that were not detected by FIT, potentially biasing the results toward lower observed risk. Combining diagnostic colonoscopy for positive FIT results with cancer registry data for negative FIT results may overestimate the sensitivity of FIT in detecting CRC. Biennial FIT screening should be maintained for individuals with repeatedly negative results, as the CRC incidence can decrease with additional rounds of negative FITs and the risks re-emerge once positive FIT occurs. High-risk individuals with one or more risk factors, such as a family history of CRC, Hb levels below 10 g/dL, recent occurrence of black-colored stools or changes in bowel habits, and a carcinoembryonic antigen level ≥5 ng/mL, may still benefit from colonoscopy despite negative FIT results. The association between continuously negative FITs and reduced all-cause mortality should also be recognized.