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. 2023 Feb 23;20(2):e1004185. doi: 10.1371/journal.pmed.1004185

Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study in Sweden

Jiangwei Sun 1,*, Fang Fang 2, Ola Olén 3,4,5, Mingyang Song 6,7,8, Jonas Halfvarson 9, Bjorn Roelstraete 1, Hamed Khalili 7,8,10, Jonas F Ludvigsson 1,11,12
Editor: Sanjay Basu13
PMCID: PMC9949679  PMID: 36821532

Abstract

Background

Although evidence suggests a persistently decreased risk of colorectal cancer for up to 10 years among individuals with a negative endoscopic biopsy result (i.e., normal mucosa), concerns have been raised about other long-term health outcomes among these individuals. In this study, we aimed to explore the long-term risk of inflammatory bowel disease (IBD) after an endoscopic biopsy with normal mucosa.

Methods and findings

In the present nationwide cohort study, we identified all individuals in Sweden with a lower or upper gastrointestinal (GI) biopsy of normal mucosa during 1965 to 2016 (exposed, n = 200,495 and 257,192 for lower and upper GI biopsy, respectively), their individually matched population references (n = 989,484 and 1,268,897), and unexposed full siblings (n = 221,179 and 274,529). Flexible parametric model estimated hazard ratio (HR) as an estimate of the association between a GI biopsy of normal mucosa and IBD as well as cumulative incidence of IBD, with 95% confidence interval (CI). The first 6 months after GI biopsy were excluded to avoid detection bias, surveillance bias, or reverse causation. During a median follow-up time of approximately 10 years, 4,853 individuals with a lower GI biopsy of normal mucosa developed IBD (2.4%) compared to 0.4% of the population references. This corresponded to an incidence rate (IR) of 20.39 and 3.39 per 10,000 person-years in the respective groups or 1 extra estimated IBD case among 37 exposed individuals during the 30 years after normal GI biopsy. The exposed individuals had a persistently higher risk of overall IBD (average HR = 5.56; 95% CI: 5.28 to 5.85), ulcerative colitis (UC, average HR = 5.20; 95% CI: 4.85 to 5.59) and Crohn’s disease (CD, average HR = 6.99; 95% CI: 6.38 to 7.66) than their matched population references. In the sibling comparison, average HRs were 3.27 (3.05 to 3.51) for overall IBD, 3.27 (2.96 to 3.61) for UC, and 3.77 (3.34 to 4.26) for CD. For individuals with an upper GI biopsy of normal mucosa, the average HR of CD was 2.93 (2.68 to 3.21) and 2.39 (2.10 to 2.73), compared with population references and unexposed full siblings, respectively. The increased risk of IBD persisted at least 30 years after cohort entry. Study limitations include lack of data on indications for biopsy and potential residual confounding from unmeasured risk or protective factors for IBD.

Conclusions

Endoscopic biopsy with normal mucosa was associated with an elevated IBD incidence for at least 30 years. This may suggest a substantial symptomatic period of IBD and incomplete diagnostic examinations in patients with early IBD.

Jiangwei Sun and colleagues investigate the long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa in a Swedish cohort.

Author summary

Why was this study done?

  • As the most frequent gastrointestinal (GI) histologic finding on endoscopy, a negative endoscopic result (i.e., normal mucosa) has been associated with a persistently decreased risk of colorectal cancer for up to 10 years. However, concerns have been raised about other long-term health outcomes among these individuals.

  • We aimed to explore the long-term risk of inflammatory bowel disease (IBD) after an endoscopic biopsy with normal mucosa.

What did the researchers find?

  • In this population-based, sibling-controlled cohort study, we identified individuals with a lower (n = 200,495) or upper (n = 257,192) GI biopsy of normal mucosa, their individually matched general population references (n = 989,484 and 1,268,897, respectively), and unexposed full siblings (n = 221,179 and 274,529, respectively).

  • Compared with population references and unexposed full siblings, individuals with a lower GI biopsy of normal mucosa had a persistently higher risk of overall IBD (average HR = 5.56 and 3.27, respectively), ulcerative colitis (UC, average HR = 5.20 and 3.27, respectively), and Crohn’s disease (CD, average HR = 6.99 and 3.77, respectively).

  • Individuals with an upper GI biopsy of normal mucosa were also at an increased risk of CD (average HR = 2.93 and 2.39, respectively).

  • The elevated risk of IBD persisted at least 30 years after a biopsy with normal mucosa.

What do these findings mean?

  • Our findings suggest a substantial symptomatic period before IBD diagnosis.

  • Clinicians should be aware of the long-term increased risk of IBD in those with symptoms requiring GI investigation but with a finding of histologically normal mucosa.

Introduction

Inflammatory bowel disease (IBD) is a chronic relapsing and remitting disease of the gastrointestinal (GI) tract [1]. Subtypes mainly encompass ulcerative colitis (UC), Crohn’s disease (CD), and IBD-unclassified (IBD-U) [2]. Lower quality of life and shorter life expectancy were reported in patients with IBD than the general population likely due to digestive symptoms, extra-intestinal manifestations [3], as well as the increased risks of various malignancies [4]. Both genetic and environmental risk factors play a role in the development of IBD [5].

Previous studies have indicated that IBD may have a symptomatic period before diagnosis [6]. Already in the 1980s, Kanof and colleagues noted that adolescents with CD had impaired growth before IBD diagnosis [7]. Blackwell and colleagues demonstrated that patients with IBD often had GI symptoms 5 years before diagnosis [8]. A recent Swedish study reported that proteins related to inflammation might be up-regulated 15 years before IBD diagnosis [9]. Other dysregulated biomarkers have also been identified even 5 to 10 years prior to IBD diagnosis [10–13]. Although current guidelines stipulate that patients with suspected IBD be referred for specialist review within short time after symptom onset [14], long diagnostic delay in the symptomatic period is still a concern, prohibiting appropriate treatment [15] and leading to an increased risk of colectomy [8] and other complications [16]. Therefore, a better knowledge about this symptomatic period might not only improve disease understanding but also identify individuals who are at high risk of developing IBD.

In clinical practice, the most frequent histologic finding on endoscopy is normal mucosa. Although evidence suggests a persistently decreased risk of colorectal cancer for up to 10 years among individuals with a negative endoscopic result [17–19], an endoscopic finding of normal mucosa has been linked to excess risks of adverse health outcomes, including neurological and psychiatric disorders [20–22], chronic obstructive pulmonary disease [23], non-GI cancers [24], and mortality [25]. Therefore, concerns have been raised about the long-term health outcomes among them. Moreover, we are unaware of any studies investigating the long-term risk of IBD in those with a negative endoscopic result, which might be helpful to understand the long symptomatic period in IBD.

Taking advantage of the Swedish national healthcare registers, we conducted a cohort study, based on the nationwide histopathology cohort ESPRESSO (Epidemiology Strengthened by histoPathology Reports in Sweden) [26] and explored the risk of IBD among individuals with a GI biopsy of normal mucosa, compared with matched references randomly selected from the general population. Furthermore, because IBD as well as its protective or risk factors are likely to cluster within families, which may exert a yet unknown effect on the association between normal GI mucosa and IBD, we also compared individuals with normal mucosa to their unexposed full siblings. We hypothesized that a GI biopsy of normal mucosa was associated with an increased risk of IBD. IBD was studied as the primary outcome, while UC and CD were studied as the secondary outcomes.

Methods

Study design and participants

ESPRESSO contains information on all computerized GI biopsy reports from 28 pathology departments during 1965 to 2016 in Sweden, including date of biopsy, topography (upper GI tract: T60-T65 and lower GI tract: T66-T69 or T6X), and morphology (through a Swedish version of the Systematized Nomenclature of Medicine (SNOMED) coding) [26]. The exposed individuals were identified as those with a first GI biopsy report of normal mucosa (SNOMED codes: M00100 and M00110) and without other aberrations earlier (such as a biopsy record of inflammation). Our definition of normal mucosa has been validated and has a positive predictive value (PPV) of >98% [22].

In a population-matched cohort, for each exposed individual, we randomly selected up to 5 reference individuals from the Swedish Total Population Register [27], who were individually matched to the exposed individual by birth year, sex, county of residence, and calendar period. Reference individuals should be alive and biopsy-naïve at date of selection. To assess the influence of residual confounding from genetics and early environmental factors shared within families [28], we also conducted a sibling cohort to compare the risk of IBD between the exposed individuals and their unexposed full siblings. We identified the biopsy-naïve full siblings of the exposed individuals from the Swedish Multi-Generation Register (a component of the Total Population Register) [29]. The full siblings had to be alive at the biopsy date of the exposed individual. Date of biopsy for the exposed individuals and date of selection for population references or unexposed full siblings were used as the index date. Individuals with an earlier diagnosis of IBD were excluded from all analyses. The prespecified analysis plan is presented in S1 Text.

Follow-up and ascertainment of outcome

Study participants were then linked to several Swedish national healthcare registers, using the unique personal identity number assigned to all residents in Sweden [30], with a virtually complete follow-up until an incident diagnosis of IBD, proctocolectomy (when UC was used as the outcome), emigration, death, or December 31, 2016, whichever occurred first. Some of reference individuals or biopsy-naïve full siblings were also censored when receiving a GI biopsy of normal mucosa during follow-up. Individual informed consent was waived due to the study’s register-based nature [31].

We identified newly diagnosed IBD as having 1 relevant International Classification of Disease (ICD) code for IBD in the National Patient Register (available from 1964 onward) and 1 biopsy record indicating IBD in the ESPRESSO (see Table A in S1 Appendix for the ICD codes and histopathology SNOMED codes). A validation study has suggested a PPV of 95% (95% CI: 89% to 99%) for this diagnostic approach [32]. IBD was divided into 3 subtypes (i.e., UC, CD, or IBD-U), as outlined by Forss and colleagues [33]. As earlier research has revealed a low PPV for IBD-U [34], associations between normal GI mucosa and risk of IBD-U were not explored. For normal upper GI mucosa, only the risk of CD was assessed, since UC is limited to the rectum and colon.

Covariates

The following covariates were considered when exploring IBD risk among individuals with a normal GI biopsy. We retrieved data on country of birth from the Total Population Register [27] (Nordic or others) and educational attainment from the Swedish Longitudinal Integrated Database for Health Insurance and Labour Market Studies [35] (available from 1990 onward; 4 groups: 0 to 9 years, 10 to 12 years, ≥13 years, and “missing,” as a proxy for socioeconomic status). Number of non-primary healthcare visits, as a proxy for regular healthcare seeking behavior, was defined as the number of healthcare visits between 2 years and 6 months before the index date from the National Patient Register [36] (4 groups: 0, 1, 2 to 3, and ≥4). Healthcare visits from 6 months before until the index date was not considered to avoid overadjustment. Charlson comorbidity index [37] before the index date, without considering ulcer disease and as a proxy for general health status, was calculated according to diagnoses from the National Patient Register (3 groups: 0, 1, and ≥2). Finally, we considered history of GI diseases before the index date according to the National Patient Register (yes/no, see Table B in S1 Appendix for ICD codes), because endoscopy is an integral part of the management of GI diseases [38].

Statistical analyses

Standardized difference was used to examine the balance of a covariate between the exposed and unexposed groups, and imbalance was defined as a standardized difference value greater than 0.2 [39]. We explored the risk of overall IBD, UC, and CD in individuals with a normal lower GI mucosa and risk of CD in those with a normal upper GI mucosa. Follow-up (i.e., date of cohort entry) was started 6 months after the index date to decrease potential detection bias (e.g., work up for GI biopsy increases the chance of diagnosing the other), surveillance bias (e.g., regular check-ups after a GI biopsy increase chance of early detection of IBD), and reverse causation. To estimate the average and temporal pattern of hazard ratio (HR), with 95% confidence interval (CI), comparing the exposed individuals to the matched population references and unexposed full siblings, flexible parametric survival model using the stpm2 command in Stata was applied to allow normal mucosa to vary over time (a time-varying effect) [40]. Standardized cumulative incidence of IBD was also estimated using such approach [41]. Time since date of cohort entry was used as the underlying time scale.

As the HR as well as cumulative incidence and its difference might vary with follow-up time, we presented these estimates at 6 months, 1 year, 5 years, 10 years, 20 years, and 30 years after cohort entry for each outcome. In the population-matched cohort, we conditioned the analyses on the matching variables (birth year, sex, county of residence, and calendar period) and additionally adjusted for country of birth, educational attainment, number of healthcare visits, Charlson comorbidity index, and history of GI disease. In the sibling cohort, we performed similar analyses and conditioned on family identifier as well as adjusted for birth year, sex, county of residence (collected before index date), calendar period, and the aforementioned covariables.

For normal lower GI mucosa, the risk among disease phenotypes, including CD location and UC extent, were also explored. Data on CD location and UC extent was collected at the date of IBD diagnosis and classified according to the Montreal classification [34]. CD location includes ileal (L1)/ileocolonic (L3)/unknown (LX) or colonic (L2). UC extent includes proctitis (E1)/left-sided colitis (E2), extensive colitis (E3), or extent not defined (EX) (see Table C in S1 Appendix for ICD codes).

Subgroup and sensitivity analyses

To assess whether the associations would differ in different subgroup populations, we stratified the analysis by sex (male or female), age at index date (<18 y, 18 to 39.9 y, 40 to 59.9 y, and ≥60 y), and calendar period at index date (1969 to 1989, 1990 to 1999, 2000 to 2009, and 2010 to 2016). The P value for interaction was calculated using the Wald test for the product terms between the exposure and subgroup variables.

A number of sensitivity analyses were performed to assess the robustness of our results to potential influence from comorbidity and healthcare utilization. We restricted these analyses to: (a) individuals with a Charlson comorbidity index of zero; (b) individuals without a healthcare visit between 2 years and 6 months before the index date; (c) individuals free of GI diseases before the index date; (d) individuals free of previous endoscopy (see Table B in S1 Appendix for relevant codes); and (e) individuals free of previous colectomy or proctocolectomy (see Table B in S1 Appendix for relevant codes). Given that the Prescribed Drug Register was available since July 2005, we restricted the analysis to individuals with an index date in January 2006 or later and without prior prescription of IBD medication (see Table D in S1 Appendix for relevant codes). To evaluate the robustness of our results to unmeasured confounding, we calculated the E-value to identify the minimum strength of the association an unmeasured confounder would need to have with both exposure and outcome to explain away the observed association [42].

Data analyses were performed using SAS version 9.4 (SAS Institute, Cary, North Carolina), Stata (version 16.1; StataCorp LP, College Station, Texas), and R version 3.6.0. A two-sided P ≤ 0.05 was considered statistically significant. This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline (S1 Checklist).

Ethics consideration

This study was approved by the Regional Ethics Review Board in Stockholm (2014/1287-31/4 and 2018/972-32).

Results

Lower GI biopsy and IBD

We identified 200,495 exposed individuals with a histologically normal lower GI mucosa and 989,484 matched reference individuals in the population-matched cohort, as well as 121,287 exposed individuals and their 221,179 unexposed full siblings in the sibling cohort (Table 1). Median age at index date was 43.1 years and 37.3 years for the exposed individuals of the 2 cohorts, respectively. Most exposed individuals were female (approximately 60%) and over two-thirds had been biopsied since the year 2000. The exposed individuals tended to have more healthcare visits, comorbidities, GI diseases, and endoscopies (e.g., colonoscopy and sigmoidoscopy) prior to the index date, as well as more colonoscopy during the follow-up than the population references and their unexposed full siblings (Table 1).

Table 1. Characteristics of individuals with a lower GI biopsy of normal mucosa and their matched population references and unexposed full siblings.

Population matched cohort, No. (%) Standardized difference f Sibling cohort, No. (%) Standardized difference f
Characteristics Normal mucosa (n = 200,495) References (n = 989,484) Normal mucosa (n = 121,287) Unexposed full siblings (n = 221,179)
Age at index date, years a
    Mean ± SD 43.7 ± 20.4 43.5 ± 20.3 0.010 38.3 ± 17.6 39.6 ± 17.8 −0.075
    Median (IQR) 43.1 (27.4–59.5) 42.9 (27.3–59.2) 37.3 (24.4–52.0) 39.6 (25.7–53.5)
    <18 y 18,132 (9.0) 90,711 (9.2) 0.000 13,442 (11.1) 26,406 (11.9) 0.102
    18–39.9 y 72,197 (36.0) 357,985 (36.2) 52,778 (43.5) 85,747 (38.8)
    40–59.9 y 61,392 (30.6) 303,944 (30.7) 38,768 (32.0) 76,364 (34.5)
    ≥60 y 48,774 (24.3) 236,844 (23.9) 16,299 (13.4) 32,662 (14.8)
Female 121,977 (60.8) 602,336 (60.9) −0.001 73,056 (60.2) 108,833 (49.2) 0.223
Born in Nordic country 181,367 (90.5) 868,132 (87.7) −0.087 118,457 (97.7) 214,564 (97.0) −0.041
Calendar period at index date a 0.000 0.072
    1969–1989 22,258 (11.1) 110,151 (11.1) 11,514 (9.5) 23,285 (10.5)
    1990–1999 41,613 (20.8) 205,595 (20.8) 23,807 (19.6) 44,981 (20.3)
    2000–2009 74,187 (37.0) 365,619 (37.0) 45,257 (37.3) 81,818 (37.0)
    2010–2016 62,437 (31.1) 308,119 (31.1) 40,709 (33.6) 71,095 (32.1)
Educational attainment 0.029 0.119
    0–9 y 39,593 (19.8) 199,031 (20.1) 19,525 (16.1) 41,800 (18.9)
    10–12 y 73,745 (36.8) 353,217 (35.7) 48,187 (39.7) 85,887 (38.8)
    ≥13 y 50,709 (25.3) 248,770 (25.1) 32,996 (27.2) 51,522 (23.3)
    Missing 36,448 (18.2) 188,466 (19.1) 20,579 (17.0) 41,970 (19.0)
History before the index date a
    Comorbidity≥1 b 46,317 (23.1) 139,377 (14.1) 0.233 23,086 (19.0) 30,283 (13.7) 0.145
    GI disease 101,318 (50.5) 148,984 (15.1) 0.816 61,008 (50.3) 46,516 (21.0) 0.642
    Endoscopy c 64,475 (32.2) 25,433 (2.6) 0.848 40,321 (33.2) 11,687 (5.3) 0.758
    EGD 26,710 (13.3) 23,095 (2.3) 0.418 15,911 (13.1) 9,254 (4.2) 0.322
    Colonoscopy 51,628 (25.8) 6,111 (0.6) 0.800 33,018 (27.2) 4,205 (1.9) 0.769
    Sigmoidoscopy 7,730 (3.9) 1,671 (0.2) 0.265 4,757 (3.9) 1,064 (0.5) 0.236
    Colectomy or proctocolectomy 221 (0.1) 157 (0.0) 0.038 134 (0.1) 124 (0.1) 0.019
With healthcare visit d 80,497 (40.2) 260,437 (26.3) 0.297 47,786 (39.4) 62,776 (28.4) 0.234
Colonoscopy during follow-up 20,811 (10.4) 42,410 (4.3) 0.235 12,599 (10.4) 12,478 (5.6) 0.175
Follow-up time, years
    Median (IQR) 9.7 (4.8–17.0) 10.1 (5.1–17.6) −0.041 10.2 (5.1–17.7) 10.8 (5.5–18.8) −0.075
    0.5–0.9 y e 6,634 (3.3) 27,009 (2.7) 0.034 2,978 (2.5) 4,107 (1.9) 0.098
    1–4.9 y 45,185 (22.5) 216,933 (21.9) 26,516 (21.9) 44,842 (20.3)
    5–9.9 y 50,529 (25.2) 245,337 (24.8) 30,257 (25.0) 53,565 (24.2)
    10–19.9 y 61,613 (30.7) 306,643 (31.0) 37,452 (30.9) 69,289 (31.3)
    20–29.9 y 27,241 (13.6) 144,388 (14.6) 17,841 (14.7) 36,113 (16.3)
    ≥30 y 9,293 (4.6) 49,174 (5.0) 6,243 (5.2) 13,263 (6.0)
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a Index date: date of first biopsy record for individuals with a gastrointestinal biopsy of normal mucosa, and date of selection for their matched population references or unexposed full siblings.

b Measured by the Charlson comorbidity index.

c One individual may have multiple records of endoscopy before the index date.

d Defined as the number of healthcare visits between 2 years and 6 months before the index date.

e Follow-up started 6 months after index date.

f Standardized difference defined as difference in means or proportions divided by standard deviation; a covariate with a standardized difference greater than 0.2 was considered imbalanced.

EGD, esophagogastroduodenoscopy; GI, gastrointestinal; IQR, interquartile range; SD, standard deviation.

During a median follow-up time of approximately 10 years, 4,853 exposed individuals developed IBD (2.4%) compared to 0.4% of the population references (Table E in S1 Appendix). This corresponded to an incidence rate (IR) of overall IBD as 20.39 versus 3.39 per 10,000 person-years, with an IR difference of 17.01 per 10,000 person-years. The IR difference for UC and CD was 8.01 and 6.57 per 10,000 person-years, respectively (Table E in S1 Appendix). In the sibling cohort, the IR differences of overall IBD, UC, and CD slightly decreased to 15.87, 7.69, and 6.11 per 10,000 person-years, respectively (Table F in S1 Appendix).

Compared with the population references, the exposed individuals were at increased risk of IBD (overall as well as UC and CD, Table 2 and Fig 1A–1C). The average HR (95% CI) of overall IBD, UC, and CD was 5.56 (95% CI: 5.28 to 5.85), 5.20 (4.85 to 5.59), and 6.99 (6.38 to 7.66), respectively. The highest HR was observed shortly after cohort entry and decreased over time, but remained increased still 30 years after cohort entry (for overall IBD: HR = 2.47 (2.16 to 2.83)). The temporal pattern of HR for UC and CD was similar to that of overall IBD, although the HR was generally greater for CD than UC. For example, there was a nearly 12-fold increase in the risk of CD (HR = 11.95 (9.76 to 14.63)) at 6 months after cohort entry, while the corresponding HR for UC was 6.40 (5.49 to 7.48) (Table 2). HR decreased to 3.27 (3.05 to 3.51) of overall IBD, 3.27 (2.96 to 3.61) of UC, and 3.77 (3.34 to 4.26) of CD in the sibling cohort (Table 2).

Table 2. IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa.

Outcomes Average HR (95% CI) Years since cohort entry
0.5 y 1 y 5 y 10 y 20 y 30 y
Compared with population references
Overall IBD
    Cumulative incidence difference (95% CI), % 0.13 (0.11–0.14) 0.23 (0.21–0.25) 0.82 (0.78–0.86) 1.39 (1.33–1.45) 2.19 (2.09–2.29) 2.71 (2.57–2.86)
    HR (95% CI) 5.56 (5.28–5.85) 8.03 (7.18–8.97) 7.24 (6.57–7.97) 5.29 (4.95–5.65) 4.22 (3.97–4.49) 3.01 (2.76–3.28) 2.47 (2.16–2.83)
UC
    Cumulative incidence difference (95% CI), % 0.05 (0.04–0.06) 0.10 (0.08–0.11) 0.39 (0.36–0.42) 0.69 (0.64–0.73) 1.11 (1.04–1.18) 1.32 (1.22–1.42)
    HR (95% CI) 5.20 (4.85–5.59) 6.40 (5.49–7.48) 6.06 (5.30–6.94) 5.38 (4.91–5.88) 4.45 (4.09–4.84) 2.88 (2.54–3.26) 2.15 (1.74–2.66)
CD
    Cumulative incidence difference (95% CI), % 0.06 (0.05–0.07) 0.11 (0.09–0.12) 0.32 (0.30–0.35) 0.52 (0.48–0.55) 0.76 (0.71–0.81) 0.94 (0.86–1.02)
    HR (95% CI) 6.99 (6.38–7.66) 11.95 (9.76–14.63) 10.10 (8.48–12.04) 6.00 (5.33–6.77) 4.51 (4.04–5.03) 3.53 (3.02–4.12) 3.08 (2.47–3.85)
Compared with unexposed full siblings
Overall IBD
    Cumulative incidence difference (95% CI), % 0.12 (0.10–0.14) 0.22 (0.20–0.25) 0.81 (0.75–0.87) 1.37 (1.28–1.46) 2.15 (2.01–2.29) 2.66 (2.45–2.87)
    HR (95% CI) 3.27 (3.05–3.51) 4.10 (3.51–4.79) 3.85 (3.37–4.41) 3.13 (2.85–3.44) 2.77 (2.55–3.01) 2.36 (2.09–2.67) 2.16 (1.80–2.59)
UC
    Cumulative incidence difference (95% CI), % 0.05 (0.04–0.06) 0.09 (0.08–0.11) 0.37 (0.33–0.42) 0.69 (0.63–0.76) 1.12 (1.02–1.22) 1.34 (1.20–1.48)
    HR (95% CI) 3.27 (2.96–3.61) 3.45 (2.77–4.30) 3.38 (2.79–4.10) 3.42 (2.99–3.90) 3.12 (2.78–3.50) 2.46 (2.05–2.95) 2.17 (1.66–2.84)
CD
    Cumulative incidence difference (95% CI), % 0.07 (0.05–0.08) 0.11 (0.09–0.13) 0.35 (0.31–0.38) 0.53 (0.48–0.59) 0.76 (0.68–0.84) 0.94 (0.82–1.05)
    HR (95% CI) 3.77 (3.34–4.26) 5.73 (4.37–7.51) 4.88 (3.85–6.19) 3.26 (2.76–3.84) 2.72 (2.35–3.15) 2.45 (1.98–3.04) 2.31 (1.72–3.11)
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Cumulative incidence difference and hazard ratio were estimated from the flexible parametric survival model, allowing normal mucosa to vary over time. When comparing with population references, models were conditioned on matching set (birth year, sex, county of residence, and calendar period) and further adjusted for country of birth, educational attainment, number of healthcare visits, Charlson comorbidity index, and history of GI diseases. When comparing with unexposed full siblings, models were conditioned on family identifier and additionally adjusted for the abovementioned covariates.

CD, Crohn’s disease; CI, confidence interval; GI, gastrointestinal; HR, hazard ratio; IBD, inflammatory bowel disease; UC, ulcerative colitis.

Fig 1. HR and 95% CI of IBD as a function of time since biopsy, comparing individuals with a GI biopsy result of normal mucosa with their matched population references.

Fig 1

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HR was estimated from the flexible parametric survival model conditioned on matching set (birth year, sex, county of residence, and calendar period) and further adjusted for country of birth, educational attainment, number of healthcare visits, Charlson comorbidity index, and history of GI diseases. Date of cohort entry was defined as 6 months after the index date. CD, Crohn’s disease; CI, confidence interval; GI, gastrointestinal; HR, hazard ratio; IBD, inflammatory bowel disease; UC, ulcerative colitis.

The cumulative incidence of IBD (overall as well as UC and CD) was higher in the exposed individuals than the population references (Table 2 and Table G in S1 Appendix and Fig 2) and the unexposed full siblings (Table 2 and Table H in S1 Appendix). The 30-year cumulative incidence of overall IBD was 3.67% (3.52% to 3.83%) in the exposed individuals and 0.96% (0.92% to 1.00%) in the matched population references (Table G in S1 Appendix), with a cumulative incidence difference of 2.71% (2.57% to 2.86%) (Table 2). Consequently, there is 1 extra estimated IBD case among 37 exposed individuals with a normal lower GI mucosa within 30 years after cohort entry. In the sibling cohort, the corresponding 30-year cumulative incidence was 4.22% and 1.56% in the exposed and unexposed siblings (Table H in S1 Appendix), respectively, with a cumulative incidence difference of 2.66% (Table 2).

Fig 2. Standardized cumulative incidence and 95% CI of IBD in individuals with a GI biopsy result of normal mucosa (pink) and their matched population references (blue).

Fig 2

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The cumulative incidence was estimated from the flexible parametric survival model conditioned on matching set (birth year, sex, county of residence, and calendar period) and further adjusted for country of birth, educational attainment, number of healthcare visits, Charlson comorbidity index, and history of GI diseases. Date of cohort entry was defined as 6 months after the index date. CD, Crohn’s disease; CI, confidence interval; GI, gastrointestinal; IBD, inflammatory bowel disease; UC, ulcerative colitis.

In subgroup analyses by sex, age, and calendar period, the HR for overall IBD was higher in males (average HR = 6.24 (5.77 to 6.76)) than females (5.11 (4.78 to 5.46)) and in individuals aged 18 to 39.9 years (6.38 (5.91 to 6.88)) than other age groups in the population-matched cohort (both P for interaction < 0.001, Table I in S1 Appendix for HR, Fig A in S1 Appendix for cumulative incidence). Compared with adults, children with a normal lower GI mucosa had a slightly different temporal pattern of HR, with a very high HR during follow-up (e.g., 15.79 (10.77 to 23.14) at 6 months and 12.49 (8.98 to 17.36) at 1 year) but a null HR at 30 years after cohort entry. This contrasted with adults who typically had a slightly lower HR (e.g., around 4 to 9 at 6 months) and then an HR around 2 to 3 at 30 years after cohort entry. In analyses of overall IBD in relation to calendar period of biopsy, the HR and cumulative incidence were lower during the latter 2 calendar periods (P for interaction < 0.001), for example, within 5 years after cohort entry (Fig 3 and Table I in S1 Appendix and Fig A in S1 Appendix), compared with earlier calendar periods. Slightly lower HRs, but with similar temporal pattern, were observed in the sibling cohort (Table J in S1 Appendix). Positive associations were also noted between normal lower GI mucosa and risk of different IBD phenotypes, such as colonic CD (average HR = 4.09 (3.24 to 5.17)) and UC with extensive colitis (average HR = 4.94 (4.09 to 5.96)) (Table K in S1 Appendix).

Fig 3. HR of overall IBD as a function of time since biopsy, comparing individuals with a lower GI biopsy result of normal mucosa with their matched population references, stratified by the calendar period at index date.

Fig 3

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Date of cohort entry was defined as 6 months after the index date. GI, gastrointestinal; HR, hazard ratio; IBD, inflammatory bowel disease.

Upper GI biopsy and CD

Table L in S1 Appendix presents the characteristics of the 257,192 individuals with a histologically normal upper GI mucosa and their 1,268,897 matched population references as well as 148,564 exposed individuals and their 274,529 unexposed siblings. During a median follow-up time of approximately 11 years, 1,048 exposed individuals developed CD (0.4%) compared to 0.1% of the population references (Table E in S1 Appendix). This corresponded to an IR difference of 2.39 per 10,000 person-years. In the sibling cohort, the IR difference of CD was 2.33 per 10,000 person-years (Table F in S1 Appendix).

Compared with the population references and unexposed full siblings, individuals with a normal upper GI mucosa had an increased risk of CD with an average HR of 2.93 (2.68 to 3.21) and 2.39 (2.10 to 2.73) (Table 3), respectively. The HR was highest shortly after cohort entry and remained stable 5 years after cohort entry (Table 3 and Fig 1D).

Table 3. CD during follow-up in individuals with an upper GI biopsy result of normal mucosa.

Comparison Average HR (95% CI) Years since cohort entry
0.5 y 1 y 5 y 10 y 20 y 30 y
Compared with population references
    Cumulative incidence difference (95% CI), % 0.02 (0.02–0.03) 0.04 (0.03–0.05) 0.11 (0.09–0.12) 0.17 (0.15–0.19) 0.29 (0.26–0.33) 0.42 (0.35–0.48)
    HR (95% CI) 2.93 (2.68–3.21) 4.50 (3.67–5.51) 3.47 (2.92–4.14) 2.36 (2.07–2.69) 2.34 (2.10–2.61) 2.47 (2.07–2.96) 2.49 (1.95–3.20)
Compared with unexposed full siblings
    Cumulative incidence difference (95% CI), % 0.03 (0.02–0.03) 0.04 (0.03–0.05) 0.12 (0.10–0.15) 0.19 (0.15–0.22) 0.31 (0.26–0.37) 0.43 (0.34–0.53)
    HR (95% CI) 2.39 (2.10–2.73) 3.85 (2.82–5.26) 3.07 (2.35–4.01) 1.90 (1.57–2.30) 1.90 (1.62–2.23) 2.09 (1.61–2.73) 2.17 (1.50–3.14)
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Cumulative incidence difference and hazard ratio were estimated from the flexible parametric survival model, allowing normal mucosa to vary over time. When comparing with population references, models were conditioned on matching set (birth year, sex, county of residence, and calendar period) and further adjusted for country of birth, educational attainment, number of healthcare visits, Charlson comorbidity index, and history of GI diseases. When comparing with unexposed full siblings, models were conditioned on family identifier and additionally adjusted for the abovementioned covariates.

CD, Crohn’s disease; CI, confidence interval; HR, hazard ratio.

Higher cumulative incidence of CD was observed in the exposed individuals than the population references (Table G in S1 Appendix and Fig 2D) and the unexposed full siblings (Table H in S1 Appendix). The 30-year cumulative incidence difference was 0.42% (0.35% to 0.48%) in the population-matched cohort (Table 3) that corresponds to 1 extra estimated CD case among 238 individuals with a normal upper GI mucosa at 30 years after cohort entry.

Sensitivity analyses

The sensitivity analyses showed consistently higher risk of overall IBD after having a normal lower GI mucosa (Table M in S1 Appendix). The corresponding average HR was 5.92 (5.61 to 6.24) after restricting the analysis to individuals with a Charlson comorbidity score of zero, 6.15 (5.81 to 6.51) in individuals without an earlier healthcare visit, 5.37 (5.04 to 5.73) in those without earlier record of GI diseases, 5.65 (5.36 to 5.97) in those without earlier record of endoscopy, 5.56 (5.29 to 5.86) in those without previous colectomy or proctocolectomy, and 3.06 (2.71 to 3.45) in those with index date in January 2006 or later and without prescription of IBD medications before index date. Of note, although the HRs decreased in magnitude over time in all sensitivity analyses, all HRs remained statistically significant during the entire follow-up.

As HRs of overall IBD as well as UC and CD decreased over time, we performed a sensitivity analysis to assess the minimum degree of unmeasured confounding needed to fully explain away the associations at 30 years since cohort entry. The HR of 2.15 between normal lower GI mucosa and UC could be explained by an unmeasured confounder that confers a 3.72-fold increase in normal lower GI mucosa and a 3.72-fold risk of overall IBD. As all other HRs reported in the present study are higher than 2.15, they would therefore require a higher E-value.

Discussion

In this nationwide population-based and sibling-controlled cohort study, we used 2 different measures (HR and cumulative incidence over time) to explore the temporal association between undergoing a GI diagnostic examination with normal mucosa (signaling no obvious macroscopic or microscopic aberrations) and later IBD. We found an elevated IBD incidence for at least 30 years after an endoscopic biopsy with normal mucosa. This is consistent with earlier observations of impaired growth in children and adolescents [43], up-regulated inflammatory proteins [9], dysregulated antibodies and proteins [12], and increased intestinal permeability [13] years before IBD diagnosis.

The rapid initial risk increase for IBD after cohort entry might be driven by detection bias, surveillance bias, and reverse causation (e.g., the missed IBD diagnosis at time of biopsy with normal mucosa due to incomplete endoscopies or inadequate bowel preparation). While the association between a GI biopsy with normal mucosa and IBD remained statistically significant throughout follow-up, the cumulative incidence difference increased continuously over time, leading to 1 extra estimated IBD case among 37 biopsied individuals at 30 years after cohort entry. Compared with UC (average HR = 5.20), the risk increase was greater for CD (average HR = 6.99); however, the cumulative incidence was lower for CD (1.20% versus 1.79% at 30 years after cohort entry, Table G in S1 Appendix). As consistent and robust results were noted in multiple subgroup and sensitivity analyses, our findings provide high-quality evidence for the long-term IBD risk of a histologically normal mucosa. Our findings may have important implications for both public health and clinical practice, as they might suggest a substantial symptomatic period before IBD diagnosis. The potential diagnostic delay during such symptomatic period might not only impair quality of life but also be the risk of complications and possibly the effectiveness of treatment [6]. Moreover, clinicians should be aware that individuals with GI symptoms requiring a GI endoscopy, although with a finding of histologically normal mucosa, are at an increased risk of future IBD, which in turn might probably help to identify individuals who are at high risk of developing IBD.

Our stratified findings indicated that the HR for overall IBD was stronger in males, in those aged 18 to 39.9 and 40 to 59.9 years, and in individuals undergoing biopsy in earlier calendar period. Of note, the magnitude of relative risk increase in IBD decreased in more recent calendar periods, probably reflecting the improved diagnosis of IBD [44,45]. For example, there has been increasing use of medical equipment with higher resolution and accuracy (e.g., capsule endoscopy, enteroscopy, and cross-sectional imaging). The endoscopic performance has also improved over time due to the training of gastroenterologist, endoscopist, or radiologist. Further, the societal awareness of IBD has also increased over time. Detection of less severe IBD in recent years, as opposed to earlier years when primarily those with severe symptoms were likely identified, has also been improved.

Strength and limitations

Strengths of the study include the nationwide population-based and sibling-controlled cohort design with large sample size and virtually complete follow-up due to linkages to the national healthcare registers, which allowed us to present the associations up to 30 years after cohort entry and to perform a number of informative subgroup and sensitivity analyses. Both normal mucosa and IBD were objectively ascertained through validated methods with high PPVs (both ≥95%) [22,32], which greatly minimized potential information biases commonly seen in observational studies. Moreover, sibling comparisons that yielded similar results helped allay concerns about potential residual confounding from genetics and early environmental factors shared within families.

Our study also has limitations. First, lack of data on indications for biopsy is a concern. Normal mucosa might be a surrogate of a diverse range of GI symptoms or diseases before biopsy, which may be linked to IBD. Indeed, we found that individuals with normal mucosa were more likely to have a diagnosis of functional GI diseases (e.g., irritable bowel syndrome or functional dyspepsia) 5 years before biopsy in a previous study [22]. This is however unlikely to explain our results, as we noted a constantly positive association between normal mucosa and IBD during the entire 30 years of follow-up. Moreover, such concern might be partly alleviated by adjusting for Charlson comorbidity index, number of healthcare visit, and history of GI diseases in the analyses and by the similar results obtained after excluding individuals with comorbidity or GI diseases before GI biopsy. Second, incomplete coverage of inpatient care data before 1987, absence of outpatient care data before 2001, and absence of primary care information in the National Patient Register would have underestimated the number of patients with less severe IBD. Therefore, our findings should only be interpreted in the context of relatively severe IBD. On the other hand, we used all biopsies recorded by Swedish pathology departments, which should also include those referred by general practitioners. However, we cannot rule out a small number of earlier biopsies, prior to computerization of Swedish pathology departments. Third, due to the register-based nature of the study, we did not have complete information on all protective or risk factors for IBD, including lifestyle factors (e.g., smoking, body mass index, and physical activity) [5,46], medical conditions (e.g., antibiotic exposure and vitamin D deficiency) [5,32], and genetic factors (e.g., ethnicity [1,47] and risk loci (e.g., NOD2 in European populations) [48,49]), leaving residual confounding as a concern. However, such concern might be relieved partly by the similar results from the sibling comparison. On the other hand, since genetic and early environmental factors as well as IBD are more likely to cluster within families [50], some siblings may also have undiagnosed IBD, making effect estimates in sibling comparison too conservative. Moreover, the E-value estimated in the sibling cohort after considering familial factors is also a conservative estimate and suggests that an unmeasured confounder would have to confer a 3.72-fold increase in risk of both normal mucosa and IBD to explain away the lowest HR observed at 30 years after cohort entry. Although a few microorganisms have been suggested to have such an effect on IBD (e.g., Mycobacterium avium paratuberculosis with CD) [5], none of the other environmental risk factors of IBD has such strong effect. Fourth, while our IBD definition has a high PPV of 95% [32], its sensitivity and specificity have not been estimated. Fifth, we lacked data on endoscopic quality, macroscopic appearance, and inflammatory markers (e.g., C-reactive protein or faecal calprotectin [51]). We therefore cannot rule out that the endoscopist might have missed a present IBD at first biopsy. However, this should not explain away our finding of a very long symptomatic period before IBD diagnosis.

In conclusion, we found that individuals with a GI biopsy of normal mucosa had an elevated risk of IBD compared with their matched population references and unexposed full siblings, with the highest risk increase noted shortly after biopsy. Although the HR decreased thereafter, it remained statistically significant throughout the 30 years of follow-up. This might suggest a substantial symptomatic period of IBD and incomplete diagnostic examinations in patients with early IBD.

Supporting information

S1 Checklist. STROBE Statement—checklist of items that should be included in reports of observational studies.

(DOC)

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S1 Appendix. Supplementary figures and tables.

Fig A. Standardized cumulative incidence and 95% CI of inflammatory bowel disease in individuals with a GI biopsy result of normal mucosa (pink) and their matched population references (blue), stratified by sex, age at index date, or calendar period at index date. Table A. ICD codes and SNOMED codes defining IBD. Table B. Definitions of endoscopy, colectomy, and proctocolectomy. Table C. ICD codes assigned for phenotypes of IBD. Table D. Anatomical Therapeutic Chemical codes representing IBD treatment. Table E. Incidence rate of IBD in individuals with a GI biopsy result of normal mucosa and their matched population references. Table F. Incidence rate of IBD in individuals with a GI biopsy result of normal mucosa and their unexposed full siblings. Table G. Cumulative incidence and 95% CI of IBD during follow-up in individuals with a GI biopsy result of normal mucosa, compared with their matched population references. Table H. Cumulative incidence and 95% CI of IBD during follow-up in individuals with a GI biopsy result of normal mucosa, compared with their unexposed full siblings. Table I. Subgroup analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their matched population references. Table J. Subgroup analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their unexposed full siblings. Table K. Associations between lower GI biopsy result of normal mucosa and risk of IBD phenotypes, compared with their matched population references. Table L. Characteristics of individuals with an upper GI biopsy of normal mucosa and their matched population references and unexposed full siblings. Table M. Sensitivity analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their matched population references.

(DOCX)

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S1 Text. Analysis plan.

(DOCX)

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Abbreviations

CD

Crohn’s disease

CI

confidence interval

EGD

esophagogastroduodenoscopy

ESPRESSO

Epidemiology Strengthened by histoPathology Reports in Sweden

GI

gastrointestinal

HR

hazard ratio

IBD

inflammatory bowel disease

IBD-U

IBD-unclassified

ICD

International Classification of Diseases

IR

incidence rate

PPV

positive predictive value

SNOMED

Systematized Nomenclature of Medicine

UC

ulcerative colitis

Data Availability

The data set cannot be shared directly under current legislation for data protection and must be requested directly from the respective registry holders, Statistics Sweden (information@scb.se) and the Swedish National Board of Health and Welfare (registerservice@socialstyrelsen.se), after approval by the Swedish Ethical Review Authority.

Funding Statement

This study was supported by the Swedish Research Council (grant No: 2020-01706 (JFL)), FORTE (JFL), the Karolinska Institutet (FF), and the Chinese Scholarship Council (JS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Beryne Odeny

21 Sep 2022

Dear Dr Sun,

Thank you for submitting your manuscript entitled "Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study" for consideration by PLOS Medicine.

Your manuscript has now been evaluated by the PLOS Medicine editorial staff and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Sep 23 2022 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pmedicine

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosmedicine@plos.org if you have any queries relating to your submission.

Kind regards,

Beryne Odeny

PLOS Medicine

Decision Letter 1

Callam Davidson

9 Nov 2022

Dear Dr. Sun,

Thank you very much for submitting your manuscript "Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study" (PMEDICINE-D-22-03064R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

[LINK]

In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

In revising the manuscript for further consideration, your revisions should address the specific points made by each reviewer and the editors. Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments, the changes you have made in the manuscript, and include either an excerpt of the revised text or the location (eg: page and line number) where each change can be found. Please submit a clean version of the paper as the main article file; a version with changes marked should be uploaded as a marked up manuscript.

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Your article can be found in the "Submissions Needing Revision" folder.

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

We look forward to receiving your revised manuscript.

Sincerely,

Callam Davidson,

PLOS Medicine

plosmedicine.org

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Comments from the reviewers:

Reviewer #1: Overview

The manuscript entitled "Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study" present interesting results describing the implication of normal mucosal biopsies and risk of IBD in a large Swedish population. Higher risk of IBD was observed in the population with a lower gastrointestinal (GI) biopsy of normal mucosa compared to a matched control population or compared to unexposed full siblings. Similar observation was obtained in individuals with an upper GI biopsy of normal mucosa. The authors suggest the existence of a substantial preclinical period of IBD and incomplete diagnostic examinations in patients with early IBD.

Introduction

-NA

Methods

-The authors mentioned that "For normal upper GI mucosa, only the risk of CD was assessed, since UC is limited to the rectum and colon." While true, the study missed the opportunity to demonstrate the negative predictive value of UC diagnosis of the International Classification of Disease coding system, which would overall reinforce the strength of this coding used in the study.

- It is unclear how the matching of the population of references was selected. A large amount of covariate are presented, and it's not clear if those covariate had equal weight in the matching process. Can you clarify?

-It seems that the author had access to the general population and not only the matched reference population. Generally speaking, a regression model applied to the entire cohort is often a more powerful tool in detecting a given effect as compared to a matched study (PMC4756459) Can the author justify the choice to select a match-control design?

-It seems that a " flexible parametric survival model" was used, then the authors mentioned that "n the population-matched cohort, we conditioned the analyses on the matching variables (birth year, sex, county of residence, and calendar period). It's not clear what package was used or if the analysis used Cox regression or if conditional logistic regression was used to account for the matching process. Can you clarify?

-For the Sibling comparison, one of the adjusted variables was the analysis on the county of residence. Since adult's sibling may not anymore live in the same household, what was the timepoint selected for this variable?

- It seems that the authors have access to pedigree data. Was family history of IBD available in this cohort? Since healthy subject with multiple family members affected with IBD are at higher risk to develop IBD, it remains possible that the increased risk of IBD for subject with normal mucosa is confounded by being part of a multiplex.

Results

-Table 1 would benefit from a statistical comparison between the unexposed full siblings and sibling with normal mucosa. Was there an age of diagnosis difference between the unexposed full siblings and sibling with normal mucosa?

-The comparison and results with the unexposed siblings are interesting. Did the authors compared if the age difference between sibling contribute to an increased risk of IBD? Recent publication from Spencer et al. 2020 suggests a "clustering" of IBD onset in sibling born close to each other. This study could confirm if this is the case here as well, especially given the large sample size available here.

-Was disease phenotype data available in the cohort? If yes it would be interesting to compare the different disease behavior and location.

-Was the time to disease onset similar across the unaffected sibling and the matched control population? I suspect full sibling to have more awareness of IBD and thus they might be more likely to have an earlier diagnosis than the general population.

Discussion

-When describing unmeasured risk factors, the authors mentioned that "Such concern however might be relieved partly by the similar results from the sibling comparison, as these factors are more likely to cluster within families." This not completely true for many of the described risk factor including oral contraceptive use (sibling might be of a different a different sex), or current lifestyle factors (the sibling may not be currently living in the same household).

Reviewer #2: The paper by Sun et al presents a retrospective cohort study of people who have had a GI biopsy, showing normal mucosa, matched to unexposed individuals both from the general population and from their siblings. The paper reports increased rates of IBD in the exposed group, with risk increased for as much as 30 years after the initial biopsy. This review considers the use of statistics in the paper.

The general approach of using flexible parametric survival models is appropriate, since it allows modelling of time-varying associations between exposures and outcomes, and extraction of hazard functions and ratios, and cumulative incidence, for specific subgroups. This works well for this paper. One comment: is it strictly necessary to include a six-month blanking period after the index date, if the model allows for time-varying hazards? Maybe it is - it's just a thought.

The following comments are quite minor.

The title mentions the use of sibling controls, but not the general population controls.

The methods section of the abstract talks only of lower GI biopsy, though the study does look at upper GI biopsy as well. This does not become clear until later in the paper.

The conclusion of the abstract talks of elevated risk for at least 30 years. This is conclusion is reasonable, give the results reported in the paper as a whole, but not based on the results reported in the abstract, which shows only average HRs.

In the abstract, and possibly in the paper as a whole, not much is made of the difference in results between using general population controls and sibling controls. I think it is important to note how much of the general population association can be explained by shared genetic and/or environmental factors, or whatever it is that is causing this difference.

I am sure this is the case, but I assume the Charlson scores were derived only from data available prior to the index date?

A number of subgroup analyses are performed, by stratifying the analysis. This is OK, but if possible, I prefer to see subgroup analyses in terms of a single model with an interaction between the subgroup and the exposure variables. Otherwise, the effects of other covariates can differ between the separate models, but these differences are not assessed - only the variation in association between exposure and outcome are reported. However, I accept there are different views on this topic, so it is not critical.

Would it be appropriate to cite the specific package(s) used to fit these models (if done in R)?

Reporting the number of additional cases per exposed individual is an interesting way to look at the data. Viewed in this way, how many of these could simply be thought of as false negatives amongst the original biopsies? Does this align with the reducing HRs over time, assuming that the false negative rate has improved?

Personally, I do not thing the E-value adds very much. It says how strong an association would need to be with a single covariate to explain the observed association, but in reality there may be many unknown confounders. I think most readers can interpret the magnitude of the observed association without this.

The language used throughout the paper implies that receiving a normal biopsy is a risk factor for future IBD. Whilst I am sure this is not the intention, this seems contradictory. Regardless of the causal process involved (and regardless of the E-value reported) there must be unmeasured factors that explain the observed association. For example, are the authors suggesting that if the people in the control groups of this analysis were given a biopsy (and received a normal result) at the same time as their matched exposed individual, then their risk of IBD would have been elevated? This could only be true if the act of doing a biopsy is a causal factor in the development of IBD.

On the selection of unexposed individuals, the methods section states that they were biopsy-naïve at the index date. Does this mean that a matched control could become an exposed individual at a later date? I think that would be important, to avoid an immortal time bias (if that is the correct term), and could be made more explicit if that is the case.

Reviewer #3: Thank you for inviting me to read this interesting paper on rates of IBD diagnosis following normal mucosal biopsy. It is in fact a study I would have liked to do myself if I had the time, so I was very interested to read your findings and I hope it is published after revision.

The authors used a histopathology database to identify individuals with normal colorectal mucosal biopsies and then followed them longitudinally to determine the subsequent incidence of IBD. This was 2.4% and is in itself an interesting finding. They then compared the incidence rate of IBD to a matched referent group and also a sibling cohort, and found relatively speaking that those with previous normal biopsies still had an increased risk of subsequent IBD.

Please see my comments listed below. I hope they will help improve the manuscript.

1. "Blackwell et al demonstrated that patients with IBD often had GI symptoms five years before biopsy" - I believe this study was from a large primary care dataset which did not have access to biopsy results and this should actually read "diagnosis" instead of "biopsy".

2. "an endoscopic finding of normal mucosa might still entail increased risk of adverse health outcomes, including neurological and psychiatric disorders, chronic obstructive pulmonary disease, non-GI cancers, and mortality." - "Entail increased risk" suggests having a biopsy causes that risk, rather than just being a non-causative association. This wording is too strong and needs to be changed. I assume this is only because of a bias in healthcare utilization among patients with more co-morbidites (sick patients see doctor for one problem and mention an unrelated problem which results in more healthcare utilization including colonoscopy).

This is also a potential weakness in this study - are these patients more likely be diagnosed with IBD because of a selection bias? They have already demonstrated they are more likely than average population to see a doctor? Are they by definition they are a population willing to undergo endoscopy, which in the referent group is not necessarily the case? Therefore are the results in part a reflection of increased utilization of healthcare?

3. "The exposed individuals were identified as those with a first GI biopsy report of normal mucosa"

Is there any way of ensuring this was the patient's first ever GI biopsy, not just their first recorded biopsy in Espresso?

My understanding is that Espresso is not a comprehensive database for Sweden as it covers only 28 departments. Google tells me there are 100 hospitals in Sweden.

It is therefore very possible some of the small number of patients who later develop IBD were previously diagnosed elsewhere, were then treated, moved to a different hospital. and had a surveillance colonoscopy after with normal biopsies.cThis means it is possible these normal biopsies do not reflect pre-inflammatory IBD.

If this limitation cannot be addressed it should be listed in the limitations.

4. Do you have access to the indications for the biopsies? If so, a sensitivity analysis excluding any with "surveillance" or a previous diagnosis of IBD would be useful.

5. Do you have access to prescriptions for these patients? If so, a sensitivity analysis excluding any patients with use of any IBD drugs (mesalazine, thiopurines, systemic corticosteroids etc) before first colonoscopy would be really valuable.

6. What proportion of the referent cohort had a colonoscopy during follow-up compared with those in the previous normal biopsy group?

7. Are the code definitions of IBD validated in this dataset? Could you expand on their sensitivity and specificity. Also do these improve in more recent era, as is seen in many datasets like this - which could affect your results. What are the chances of them not picking up a diagnosis of IBD (in which case some patients may have already been diagnosed with IBD before the index biopsy and then only got coded after this)?

8. If your referent group was individually matched by birth year and calendar period then why is the median age at index date different between the cohorts? Presumably not all exposed individuals had the same number of matched referent individuals?

9. 32.2% of the exposed cohort had previously had an endoscopy. Can you specify if this was a gastroscopy, sigmoidoscopy, or colonoscopy?

Had these patients not had a normal biopsy previously if the previous procedure was not counted as the index date?

10. I note the definition of endoscopy includes colonoscopy and sigmoidoscopy - this should be split from OGD so the reader has a better understanding of the data.

I think a sensitivity analysis, where all individuals with previous lower GI endoscopy are excluded, is necessary.

11. Why are patients with previous colectomy not excluded? Presumably the colon was removed for a reason and would not have been normal at that time so it could not be the index normal biopsy.

12. "the HR for overall IBD was higher in male (average HR=6.24 (5.77-6.76)) than female (5.11(4.78-5.46))" This comparison of Hazard Ratios is confusing to me. A ratio of ratios? I think it is better to just state the Hazard Ratios as they are without comparing them. It doesn't really mean anything to compare relative measures between them as there is no cross-over of the referent or exposed groups, they are distinct populations.

13. "in those without easier record of endoscopy" Typo - "earlier" I'm guessing?

14. "We observed that the risk increase for IBD was highest shortly after cohort entry, suggesting potential diagnostic delay." Can the authors comment on how this could have happened? Incomplete index colonoscopy but biopsies taken from normal area? Poor bowel preparation? Do they have data on these? Trainee endoscopist?

15. "Compared with UC (average HR=5.20), the risk increase was greater for CD (average HR=6.99); however, the cumulative incidence was lower for CD." This illustrates my earlier point that comparison of relative measures is not particularly helpful and the paper may represent the data better by focusing more on absolute measures like cumulative incidence.

As a patient or clinician I am much more interested in the cumulative incidence because that tells me after a normal biopsy there is a 2.4% chance it is worth reinvestigating for IBD. The comparison to the general population is of academic interest but little clinical relevance.

That said I found the HRs as a function of time in figure 1 interesting and useful to understand when these IBD diagnoses are more likely to occur.

16. Is "preclinical" the right word? To me preclinical implies a period where there is active disease but symptoms have not yet developed.

What the results suggest is actually the opposite - a period of being symptomatic before any histological change is detectable. I think this is really interesting but needs a different word.

Perhaps "substantial symptomatic period before IBD is detectable with current histological methods"? I am sure you will find a more eloquent way of expressing this.

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 2

Callam Davidson

10 Jan 2023

Dear Dr. Sun,

Thank you very much for re-submitting your manuscript "Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study" (PMEDICINE-D-22-03064R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by three reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

***Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.***

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If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.  

We look forward to receiving the revised manuscript by Jan 17 2023 11:59PM.   

Sincerely,

Callam Davidson,

Senior Editor 

PLOS Medicine

plosmedicine.org

------------------------------------------------------------

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Comments from Reviewers:

Reviewer #1: The response to the reviewer helps to better understand the matching process, but it's still not clear to me if birth year, sex, county of residence, and calendar period had equal weight for the matching. The authors have addressed all my other concerns appropriately.

Reviewer #2: Alex McConnachie, Statistical Review

I thank the authors for their consideration of my original comments.

Regarding my comment about the need for a 6-month blanking period, the authors do not really answer this point. The HR curves over time all show a similar pattern, with highest risks early on, but a residual increased risk for up to 30 years. Would including events in the first 6 months alter this pattern at all? Or would you just see a continuation of the current trend? Perhaps all that would happen is that you would capture and measure the very high risk in those first 6 months.

That said, would the most natural explanation for the high early risk that is currently seen, be one of reverse causation? I.e. some of these people actually had disease at the initial biopsy, but this was not diagnosed. You could extend the blanking period to a year, to try to avoid this, but ultimately, where do you draw the line?

However, given the type of model used, which allows the HR to vary over time, would you end up just "chopping off" the left-hand end of the curves in Figure 1? So, in the end, it makes no difference to the HR estimates in the long term, whether or not you include events early on.

What you could say is that the HR>1 early on is more likely to be due to some sort of reverse causality or other bias, whereas the HR>1 in later periods is less likely to be affected in this way.

It is notable that the main conclusion of the paper is the IBD may have a lengthy period where patients have symptoms, before a successful diagnosis is made. In other words, the association between having a biopsy with normal mucosa and later diagnosis is due to reverse causation. I.e. patients in the early stages of the disease who have symptoms, may not be detected at biopsy, and it can take many years before a diagnosis is made.

On other matters, I still feel that the paper tends to phrase things badly, in terms of implying that having normal mucosa puts patients at increased risk. For example, line 156 says "individuals with normal mucosa had an elevated subsequent risk"; line 221 "the effect of normal mucosa"; line 256 "association between normal mucosa and IBD"; Table 2 (and Table 3) footnote "the effect of normal mucosa"; line 372-3 "higher risk of overall IBD among individuals with a normal lower GI mucosa"; line 384 "HR of 2.15 for normal lower GI mucosa"… I am sure there are other similar instances.

My issue is that it is improbable that having normal mucosa is associated with outcomes. I presume that the vast majority of the unexposed groups also had normal mucosa. The difference is that the exposed group had symptoms, sufficient to warrant undergoing a biopsy.

Otherwise, I am happy with the authors' responses to my original comments. On rereading the paper, one or two other things came up.

Table 1 now includes p-values, many of which are highly statistically significant, due to the vary large sample size. The differences between groups are not necessarily vary large (e.g. for age, comparing the exposed group with the general population, the difference is 0.2 years, but the p-value is <0.001), and may not be clinically relevant. Is there any other way to compare the groups? Some sort of standardised mean difference, perhaps?

Were the exposed group more likely to undergo biopsies after the index procedure? How many did they have? Is it possible that if you have enough biopsies, you will get a diagnosis of something eventually?

Line 385-6 says "an unmeasured confounder that confers a 3.72-fold increase in normal lower GI mucosa". Does this make sense? How can you have a confounder that "causes" normal mucosa?

Line 433-4 says "Both normal mucosa and IBD were objectively ascertained through validated methods with high PPVs (both ≥95%)." This only gives the PPV. What about the NPV? Is it known, of those with a result of normal mucosa, how many would be expected to have IBD? To what extent does this explain the observed associations?

Reviewer #3: Thank you for addressing my comments. Congratulations on a very interesting paper. I have no further comments.

Any attachments provided with reviews can be seen via the following link:

[LINK]

Decision Letter 3

Callam Davidson

24 Jan 2023

Dear Dr Sun, 

On behalf of my colleagues and the Academic Editor, Professor Sanjay Basu, I am pleased to inform you that we have agreed to publish your manuscript "Long-term risk of inflammatory bowel disease after endoscopic biopsy with normal mucosa: A population-based, sibling-controlled cohort study in Sweden" (PMEDICINE-D-22-03064R3) in PLOS Medicine.

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Checklist. STROBE Statement—checklist of items that should be included in reports of observational studies.

    (DOC)

    Click here for additional data file. (94.5KB, doc)
    S1 Appendix. Supplementary figures and tables.

    Fig A. Standardized cumulative incidence and 95% CI of inflammatory bowel disease in individuals with a GI biopsy result of normal mucosa (pink) and their matched population references (blue), stratified by sex, age at index date, or calendar period at index date. Table A. ICD codes and SNOMED codes defining IBD. Table B. Definitions of endoscopy, colectomy, and proctocolectomy. Table C. ICD codes assigned for phenotypes of IBD. Table D. Anatomical Therapeutic Chemical codes representing IBD treatment. Table E. Incidence rate of IBD in individuals with a GI biopsy result of normal mucosa and their matched population references. Table F. Incidence rate of IBD in individuals with a GI biopsy result of normal mucosa and their unexposed full siblings. Table G. Cumulative incidence and 95% CI of IBD during follow-up in individuals with a GI biopsy result of normal mucosa, compared with their matched population references. Table H. Cumulative incidence and 95% CI of IBD during follow-up in individuals with a GI biopsy result of normal mucosa, compared with their unexposed full siblings. Table I. Subgroup analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their matched population references. Table J. Subgroup analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their unexposed full siblings. Table K. Associations between lower GI biopsy result of normal mucosa and risk of IBD phenotypes, compared with their matched population references. Table L. Characteristics of individuals with an upper GI biopsy of normal mucosa and their matched population references and unexposed full siblings. Table M. Sensitivity analyses of IBD during follow-up in individuals with a lower GI biopsy result of normal mucosa, compared with their matched population references.

    (DOCX)

    Click here for additional data file. (388.2KB, docx)
    S1 Text. Analysis plan.

    (DOCX)

    Click here for additional data file. (53KB, docx)
    Attachment

    Submitted filename: R1_letter to editor and reviewers_final.docx

    Click here for additional data file. (102KB, docx)
    Attachment

    Submitted filename: R2_letter to editor and reviewers_final.docx

    Click here for additional data file. (286.1KB, docx)

    Data Availability Statement

    The data set cannot be shared directly under current legislation for data protection and must be requested directly from the respective registry holders, Statistics Sweden (information@scb.se) and the Swedish National Board of Health and Welfare (registerservice@socialstyrelsen.se), after approval by the Swedish Ethical Review Authority.

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