SUMMARY
Background:
The optimal ulcerative colitis (UC) biopsy protocol is unclear.
Aims:
We evaluated the number of biopsies required to accurately assess histologic activity in UC.
Methods:
Patients with UC, ≥4 rectosigmoid biopsies, clinical and endoscopic data, were included from a prospective biobank. Histology and endoscopic videos were blindly read. A 4-biopsy Robarts Histopathology Index (RHI) reference score, consisting of the worst item-level ratings from 4 biopsies, was compared to 1-, 2-, and 3-biopsies. Agreement was determined using bivariate errors-in-variable regression analysis (acceptance interval: ±8.25). Endoscopic activity and disease location subgroup analyses were performed.
Results:
Forty-six patients were included. The 2-biopsy (tolerance interval [TI]: −7.66, 4.79) and 3-biopsy (TI: −4.86, 3.46) RHI scores demonstrated acceptable agreement with 4-biopsy scores. One-biopsy scores demonstrated unacceptable agreement (TI: −13.99, 7.78). Mean RHI scores using 2-, 3-, and 4-biopsy approaches were similar (6.1±9.6 p=0.36; 6.8±10.5, p=0.7; 7.5±11.2), whereas 1-biopsy estimates were lower (4.4±8.1, p=0.06). Histologic remission rates were identical for 2-, 3- and 4-biopsy methods (65.2%, p=1.0). Subgroup analysis demonstrated three biopsies were required in patients with endoscopically-active disease. Sampling additional colonic locations yielded lower histologic remission rates than rectosigmoid sampling alone (33.3% vs. 61.9%, p=0.1).
Conclusion:
Two biopsies were the minimum number needed to accurately assess UC disease activity, however, 3 biopsies were required to assess endoscopically-active patients. A conservative approach to assess histologic inflammation would require 3 biopsies from the most macroscopic abnormal area. However, further studies of endoscopically-active patients and for sampling multiple locations are needed prior to integration into trial protocols.
Keywords: Histopathology, Outcomes Research, Inflammatory Bowel Disease, Ulcerative Colitis
INTRODUCTION
Ulcerative colitis is a chronic inflammatory disease of the colon characterized by symptoms of rectal bleeding, diarrhoea, and abdominal cramps.1 Approximately 25% of patients develop severe disease that requires hospitalization, and nearly 30% undergo colectomy.2 Additionally, longstanding ulcerative colitis is associated with an increased risk of colorectal cancer.3
Although normalization of clinical symptoms is the historical treatment target for patient management, observational data indicate that assessment of more objective measures of inflammation, such as endoscopic or histologic remission, may result in better long term outcomes.4–6 In support of this concept, studies have shown that patients who achieve a Mayo Clinic Endoscopic Subscore (MCES) of 0 or 1 have reduced corticosteroid use and lower rates of hospitalization and surgery than those with persistent endoscopic inflammation.7 However, up to one-third of patients who achieve both clinical and endoscopic remission have residual colonic histologic activity, which is associated with higher rates of clinical relapse, corticosteroid requirements, colectomy, and dysplasia, compared to those in histologic remission.8–12
These findings have invigorated interest in histology as both an outcome measure in clinical trials and a therapeutic goal in clinical practice. The currently recruiting VERDICT trial aims to evaluate the optimum treatment target (i.e., clinical symptoms, endoscopic activity, histologic activity, or biomarkers) in ulcerative colitis (NCT04259138). Other studies have focused on assessing the operating properties of histologic indices.13–15 The Geboes score (GS), an empirically derived 7-item categorical index, has been historically used to classify microscopic disease severity in ulcerative colitis clinical trials; however, it was not specifically designed to measure post-treatment changes.15,16 Consequently, novel histologic indices, including the Robarts Histopathology Index (RHI)13 and Nancy Histological Index (NHI),15 have been developed and validated to identify therapeutic effects in clinical trials. The RHI is calculated by summing a number of weighted items from the GS to yield a score that ranges from 0 (no disease) to 33 (severe disease).13,17 The NHI is a 5-point categorical grading system which ranges from Grade 0 (no histologically significant disease) to Grade 4 (severely actively disease).15,18 The NHI is easily scored, and best suited for use in clinical practice. Conversely, the RHI can be employed as a continuous outcome measure which makes it an efficient tool for detecting treatment differences in small sample sizes.
While histology is routinely assessed in contemporary ulcerative colitis trials, uniform biopsy procurement protocols are lacking. Although the most widely accepted sampling convention in clinical trials is to obtain two biopsies from the most severely affected area of the rectosigmoid colon (typically 15 to 25 cm from the anal verge), this convention is not based upon scientific evidence and the optimum number of biopsies needed to accurately estimate histologic disease activity is unknown. Furthermore, it is unclear whether histologic activity in the rectosigmoid colon reliably reflects histologic disease activity in the right colon among patients with extensive colitis.
Given the increased importance of histologic assessment in drug development and its potential value in clinical practice, development of a standardized sampling procedure is a critical research priority. The primary aim of this study was to determine the minimum number of biopsies required to adequately assess histologic disease activity in ulcerative colitis patients with varying degrees of endoscopic disease severity. We also sought to determine whether histologic activity in the rectosigmoid colon is reflective of that in colonic segments proximal to the splenic flexure in a subgroup of patients with extensive disease. Finally, the construct validity of histologic scores from the rectosigmoid colon was also evaluated.
MATERIALS AND METHODS
Patient population and study samples
Ulcerative colitis biopsy fragments collected between July 15, 2014 and July 24, 2017 were retrospectively obtained from the University of California, San Diego (UCSD) IBD biobank using pre-specified inclusion criteria. For the primary analysis, we evaluated patients for whom a minimum of four rectosigmoid biopsies were available. The IBD biobank consists of DNA, blood, stool, and tissue samples, in addition to endoscopic video recordings, that are prospectively collected from IBD patients who have given informed consent. Clinical disease activity was assessed using the Partial Mayo Clinic Score (PMCS), stool frequency subscore, and rectal bleeding subscore. Endoscopic videos were recorded and stored using Robarts Central Image Management Solutions (CIMS). During endoscopy, biopsies were obtained from each patient in a standardized and prespecified manner, with two samples collected during each pass of the biopsy forceps. In patients undergoing colonoscopy or flexible sigmoidoscopy, biopsies were obtained from the most macroscopically inflamed area in the right colon, left colon, or the rectosigmoid colon (or the rectum in the case of isolated proctitis). Additional biopsies were collected from the transverse colon when colorectal cancer surveillance was carried out.
Biopsy fragments were processed as formalin-fixed paraffin-embedded tissue specimens using 10% neutral formalin and stained with hematoxylin and eosin. For all patients, biopsies were procured from a single endoscopic procedure.
Central reading and endpoint assessment
For the purposes of this study, a digital scan of each included biopsy was created using a Leica Aperio AT2 whole slide scanner. These images were stored in a WebMicroscope (Fimmic, Helsinki, Finland) database that was hosted on a secure remote server.
Histologic image quality was centrally-read by a blinded histopathologist (MV) using a global rating scale (i.e., optimal; adequate; poor but readable; poor, not readable) and any specific concerns (e.g., biopsy size, scan quality, slide staining, etc.) were noted. To assess histologic disease activity, a histopathologist (MV) blindly and independently read each biopsy using the RHI and GS. The central reader also provided a global assessment of histologic disease activity using a 10 cm visual analog scale (VAS), where 0 indicates completely normal and 10 indicates the worst disease possible.
Endoscopic videos from the UCSD biobank were made available for expert central reading through the secure CIMS server. Two blinded central readers (RB, JWDM) evaluated endoscopic video quality using a global rating scale (i.e., optimal, sub-optimal, not readable) and any specific concerns (e.g., rapid withdrawal, insufficient insufflation, inadequate washing, etc.) were noted. Each endoscopic video was blindly and independently assessed by two central readers (RB, JWDM) using the MCES and Ulcerative Colitis Endoscopic Index of Severity (UCEIS). Electronic case report forms were used to record central reading data.
Data Analysis and Statistical Methods
Minimum number of biopsies required to assess histologic inflammation
As noted above, data from patients who had at least four rectosigmoid biopsies stored in the UCSD biobank were analysed in the primary analysis. A sample of four biopsies was randomly selected using a pre-defined statistical algorithm if the patient had more than four rectosigmoid biopsies available.
The “4-biopsy reference score”, which is a composite of the worst item-level ratings across four separate biopsies, was calculated using the RHI, GS, and VAS. To give an example, if one biopsy had moderate lamina propria neutrophils (Geboes 2B.2) but this feature was absent in the other biopsies, the final lamina propria neutrophil score used in the Geboes 4-biopsy reference score would be 2B.2. This methodology was chosen because it reflects how histologic scoring is performed in clinical trials when multiple biopsies are available, and it provides the most conservative estimate.
Agreement between the 4-biopsy reference score and score estimates based on one, two, or three of the reference biopsies was evaluated. The 1-, 2-, 3-biopsy scores were calculated using the same methodology as the 4-biopsy reference score. Pre-defined subgroup analyses were conducted according to endoscopic disease activity (MCES=0 vs. MCES ≥1).
Agreement was evaluated using a bivariate errors-in-variable regression approach that generated tolerance intervals.19 The tolerance interval is defined as the prediction interval which contains 95% of the population differences. Agreement is satisfied if the tolerance interval is completely contained in the acceptance interval, which was defined as ± 0.25 standard deviation of the maximum reference score of each index. Thus, the acceptance intervals for the RHI, GS, and VAS were −8.25 to 8.25, −1.25 to 1.25, and −2.42 to 2.42, respectively.
In addition to evaluating agreement, we compared the rate of histologic remission (RHI score ≤3 with neither lamina propria nor epithelial neutrophils) and mean RHI scores with corresponding standard deviations (SD) in the 4-biopsy reference score group to the 1-, 2-, and 3-biopsy groups, respectively. Continuous data, categorical data, binary data with small cells, and non-normal continuous data were tested using the t-test, chi-square test, Fisher’s exact test, and Wilcoxon rank test, respectively.
We also evaluated the relationship between biopsy number and endoscopic (as measured by the MCES) and clinical (as measured by the PMCS) disease activity to explore whether the number of biopsies procured was influenced by disease activity. A linear relationship between the variables would indicate that disease activity influenced number of biopsies procured, whereas a lack of a linear relationship would suggest disease activity did not influence number of biopsies procured.
Effect of biopsy location on estimates of histologic inflammation
For disease location analyses, data from patients with extensive ulcerative colitis who had at least three biopsies collected from both the rectosigmoid colon and colonic segments proximal to the splenic flexure were used to evaluate whether biopsy location had an impact on estimates of histologic disease activity. For this analysis, a minimum of three, rather than four, biopsies was chosen to ensure an adequate sample size given that only a subset of biobank patients had samples taken from colonic segments proximal to the splenic flexure.
Accordingly, a “total reference score”, the composite of the worst item-level ratings across all available biopsies, was calculated for the rectosigmoid colon, and for colonic segments proximal to the splenic flexure, using the RHI, GS, and VAS. Agreement between the “total reference” score from the rectosigmoid colon and colonic segments proximal to the splenic flexure was assessed using the bivariate errors approach described above. The rate of histologic remission (RHI score ≤3 with neither lamina propria nor epithelial neutrophils) in the rectosigmoid-only biopsy group compared to the group with biopsies taken from all colonic segments was also evaluated using the methods described above.
Construct validity of histologic measures
The construct validity of the differing approaches to histologic scoring was compared by calculating the correlation between centrally-read histologic (i.e., the RHI, GS, and VAS) centrally-read endoscopic (i.e., centrally-read MCES and UCEIS), and clinical (i.e., PCMS, stool frequency subscore, rectal bleeding subscore) measures of disease activity. Validity was quantified based on the point estimates of the correlation coefficients and associated 95% confidence intervals, which were obtained using bootstrap methods. All of the study population was included in these analyses, and the results were stratified according to endoscopic (as measured by the MCES) and clinical (as measured by the PMCS and rectal bleeding subscore) disease activity.
Ethics
All patients included in the UCSD biobank provided written informed consent. The institutional review board at UCSD approved the study protocol and materials. All subject data were de-identified and anonymized prior to being uploaded for central reading. The study was conducted in compliance with the Declaration of Helsinki.
RESULTS
Patient characteristics and number of biopsies sampled
A total of 299 biopsies from 50 patients were included. The median patient age was 45.8 years (30.3–61.9) and 52.0% (n=26) were female (Table 1). A total of 60.0% (n=30) of patients had extensive disease, 24.0% (n=12) had left-sided disease, and 12.0% (n=6) had proctitis. The mean MCES was 1.23 (SD 1.13). At the time of biopsy collection, 36.0% (n=18) of patients were receiving immunosuppressants, and 64.0% (n=29) were receiving biologics. The median number of biopsies collected was 5.0 (interquartile range 4.0–7.75, Supplementary Table 1) and the mean RHI score was 4.22 (SD 7.68).
Table 1:
Patient characteristics
| N=50 | |
|---|---|
| Age at diagnosis, years | |
| Mean (SD) | 34.4 (17.0) |
| Age, years | |
| Median (IQR) | 45.8 (30.3–61.9) |
| Sex, female | |
| n (%) | 26 (52) |
| Disease duration, years | |
| Mean (SD) | 12.0 (10.4) |
| Disease extent* | |
| Proctitis, n (%) | 6 (12) |
| Left side colitis, n (%) | 12 (24) |
| Extensive colitis, n (%) | 30 (60) |
| MCES | |
| 0, n (%) | 16 (33.3) |
| 1, n (%) | 15 (31.3) |
| 2, n (%) | 7 (14.6) |
| 3, n (%) | 10 (20.8) |
| Immunosuppressive use† | |
| n (%) | 18(36) |
| Biologic use† | |
| n (%) | 29 (64) |
| Corticosteroid use† | |
| n (%) | 38 (76) |
Disease location data missing for two patients.
Current or previous use.
Abbreviations: IQR, Interquartile Range; MCES, Mayo Clinic Endoscopic Subscore; SD, Standard Deviation.
Minimum number of biopsies required to assess histologic inflammation
A minimum of four rectosigmoid biopsies were available for 46 patients. The RHI score generated from three biopsies met the pre-specified definition of acceptable agreement (i.e., the tolerance interval is completely contained in the acceptance interval, which was defined as ± 0.25 standard deviation of the maximum reference score of each index) compared to the 4-biopsy reference score (Table 2), with a tolerance interval of −4.86 to 3.46 and acceptance interval of −8.25 to 8.25. Similarly, the 2-biopsy RHI score also provided acceptable agreement to the 4-biopsy reference score (tolerance interval: −7.66 to 4.79). In contrast, the 1-biopsy RHI score did not meet the agreement threshold when compared to the 4-biopsy reference score (tolerance interval: −13.99 to 7.78). Analyses comparing the 4-biopsy reference score to scores based on fewer biopsies yielded the same conclusions for VAS (Table 2).
Table 2:
Comparison of 4-biopsy reference scores to 1-, 2- and 3-biopsy scores.
| RHI | VAS | GS | ||||
|---|---|---|---|---|---|---|
| Acceptance interval | Tolerance interval | Acceptance interval | Tolerance interval | Acceptance interval | Tolerance interval | |
| 1 vs 4 biopsies | −8.25, 8.25 | −13.99, 7.78 | −2.42, 2.42 | −3.99, 2.30 | −1.25,1.25 | −3.11, 1.63 |
| 2 vs 4 biopsies | −8.25, 8.25 | −7.66, 4.79 | −2.42, 2.42 | −1.86, 1.08 | −1.25,1.25 | −1.75, 1.10 |
| 3 vs 4 biopsies | −8.25, 8.25 | −4.86, 3.46 | −2.42, 2.42 | −0.94, 0.67 | −1.25,1.25 | −1.20, 0.90 |
Abbreviations: GS, Geboes Score; RHI, Robarts Histopathology Index; VAS, Visual Analogue Scale.
For the GS, acceptable agreement between the 3-biopsy and 4-biopsy approaches was observed (tolerance interval: −1.20 to 0.90; acceptance interval: −1.25 to 1.25). In contrast to the RHI and VAS, agreement was not detected when the 4-biopsy reference score was compared to the 1- and 2-biopsy estimates (tolerance level: −3.11 to 1.63 and −1.75 to 1.10, respectively).
In subgroup analysis evaluating the effect of endoscopic disease activity, acceptable agreement was observed between the 4-biopsy reference score and 3-biopsy score in both the endoscopically active and inactive groups for both the RHI score and VAS (Table 3). Similarly, in patients with endoscopically inactive disease, there was acceptable agreement between the 4-biopsy and 2-biopsy RHI scores. However, in patients with active endoscopic disease, agreement between the 4-biopsy reference score and the 2-biopsy score was not observed for the RHI. Acceptable agreement was not detected for any of the GS-based comparisons.
Table 3:
Comparison of 4-biopsy reference scores to score estimates based on fewer biopsies (stratified by endoscopic activity).
| Tolerance Interval | ||
|---|---|---|
| No Endoscopic Activity (MCES=0) | Active Endoscopic Activity (MCES>0) | |
| RHI (acceptance interval: −7.50, 7.50) | ||
| 1 vs 4-biopsies | −4.33, 2.86 | −15.36, 7.46 |
| 2 vs 4-biopsies | −1.44, 1.04 | −9.72, 5.58 |
| 3 vs 4-biospies | −1.28, 1.01 | −6.29, 4.15 |
| VAS (acceptance interval: −2.25, 2.25) | ||
| 1 vs 4-biopsies | −1.33, 0.80 | −3.99, 2.08 |
| 2 vs 4-biopsies | −0.48, 0.28 | −2.35, 1.24 |
| 3 vs 4-biospies | −0.32, 0.25 | −1.20, 0.82 |
| GS (acceptance interval: −1.25, 1.25) | ||
| 1 vs 4-biopsies | −2.73, 1.66 | −3.44, 1.79 |
| 2 vs 4-biopsies | −1.44, 1.04 | −2.04, 1.21 |
| 3 vs 4-biospies | −1.28, 1.01 | −1.30, 0.95 |
Abbreviations: GS, Geboes Score; MCES, Mayo Clinic Endoscopic Subscore; RHI, Robarts Histopathology Index; VAS, Visual Analogue Scale.
The proportion of patients in RHI-defined histologic remission was similar when the 4-biopsy reference score was compared to the 1-, 2-, and 3-biopsy scores. Specifically, the rate of histologic remission was the same (65.2%, n=30, p=0.99) when the 4-biopsy reference score was compared to the 2- and 3-biopsy scores. When comparing the 4-biopsy reference score to the 1-biopsy estimate, histologic remission rates were numerically lower, but not statistically different (65.2% vs. 71.7%, respectively; p=0.65).
There was no statistically significant difference when the mean 4-biopsy RHI score (7.5, SD 11.2) was compared to the mean 2-biopsy (6.1, SD 9.6, p=0.36) and 3-biopsy (6.8, SD 10.5, p=0.71) RHI estimates (Supplementary Figure 1). The 1-biopsy method resulted in a mean RHI estimate that was lower than that obtained with the 4-biopsy approach (4.4, SD 8.1 vs. 7.5, SD 11.2, p=0.06).
No linear relationship was observed between number of biopsies procured and disease activity when assessed by either the MCES or the PCMS (Supplementary Figure 2).
Effect of biopsy location on estimates of histologic inflammation
The total RHI reference score derived from rectosigmoid biopsies did not have acceptable agreement with the total RHI reference score derived from either the ascending colon (tolerance interval −28.9 to 25.0, acceptance interval −7.5 to 7.5) or transverse colon (tolerance interval −29.1 to 23.1) biopsies. Similar results were obtained for both the VAS and GS (Table 4).
Table 4:
Agreement between total reference scores based on rectosigmoid biopsies and total reference scores based on biopsies taken from locations proximal to the splenic flexure.
| Tolerance interval | |
|---|---|
| RHI (acceptance interval: −7.50, 7.50) | |
| Rectosigmoid vs ascending | − 28.94, 25.03 |
| Rectosigmoid vs transverse | −29.05, 23.08 |
| Rectosigmoid vs all locations | −27.63, 27.34 |
| VAS (acceptance interval: −2.25, 2.25) | |
| Rectosigmoid vs ascending | −9.10, 7.20 |
| Rectosigmoid vs transverse | −8.50, 6.49 |
| Rectosigmoid vs all locations | −8.45, 7.53 |
| GS (acceptance interval: −1.25, 1.25) | |
| Rectosigmoid vs ascending | −5.70, 5.80 |
| Rectosigmoid vs transverse | −5.44, 4.69 |
| Rectosigmoid vs all locations | −5.16, 6.02 |
Abbreviations: GS, Geboes Score; RHI, Robarts Histopathology Index; VAS, Visual Analogue Scale.
The proportion of patients in histologic remission as defined by RHI criteria was numerically lower when biopsies from all locations (i.e., the rectosigmoid, transverse, and ascending colon), as opposed to biopsies from the rectosigmoid colon alone, were used to calculate RHI scores (33.3% vs 61.9%, respectively; p=0.1). Mean RHI scores by location are provided in Supplementary Table 2.
Construct validity
A strong, statistically significant correlation was observed between the RHI and endoscopic scores (RHI vs. MCES r=0.81, 0.71–0.92; p<0.0001, and RHI vs. UCEIS 0.78 0.65–0.89, p<0.0001; Table 5). Additionally, moderate, statistically significant correlations were observed between the RHI score and the stool frequency (0.45, 0.30–0.76, p=0.002) and rectal bleeding (0.40, 0.15–0.75, p=0.006) subscores. However, the PMCS did not significantly correlate with the RHI. Similar conclusions regarding the construct validity of the VAS or GS were made.
Table 5:
Construct validity of histologic scores
| RHI | VAS | GS | ||||
|---|---|---|---|---|---|---|
| r (95% CI) | P value | r (95% CI) | P value | r (95% CI) | P value | |
| MCES | 0.81 (0.71, 0.92) |
<0.0001 | 0.8 (0.68, 0.91) |
<0.0001 | 0.81 (0.72, 0.92) |
<0.0001 |
| UCEIS | 0.78 (0.65, 0.89) |
<0.0001 | 0.74 (0.60, 0.86) |
<0.0001 | 0.79 (0.69, 0.88) |
<0.0001 |
| SF subscore | 0.45 (0.30, 0.76) |
0.002 | 0.38 (0.11, 0.64) |
0.011 | 0.47 (0.23, 0.73) |
0.001 |
| RB subscore | 0.40 (0.15, 0.75) |
0.006 | 0.33 (0.04, 0.63) |
0.025 | 0.44 (0.22, 0.67) |
0.002 |
| PMCS | 0.19 (−0.16, 0.53) |
0.214 | 0.21 (−0.12, 0.52) |
0.165 | 0.2 (−0.08, 0.49) |
0.188 |
Abbreviations: CI, confidence interval; GS, Geboes Score; MCES, Mayo Clinic Endoscopic Subscore; PMCS, Partial Mayo Clinic Score; RB, Rectal Bleeding; RHI, Robarts Histopathology Index; SF, Stool Frequency; UCEIS, Ulcerative Colitis Endoscopic Index of Severity; VAS, Visual Analogue Scale.
When RHI “total reference score” estimates were stratified by the MCES, PMCS, and rectal bleeding score, a positive correlation between the RHI, MCES, and rectal bleeding subscore was observed. However, this relationship was not observed for the PMCS (Supplementary Table 3).
DISCUSSION
Histologic remission is an emerging treatment target in ulcerative colitis that is associated with improved patient outcomes relative to symptom- and endoscopy-based targets. Accordingly, considerable interest exists for including biopsy protocols into clinical trials. The most common procedure consists of collecting two biopsies from the rectosigmoid colon at baseline with repeat sampling conducted at the end of the induction or maintenance periods. However, the optimum biopsy sampling method has not been adequately evaluated. Specifically, the ideal number of biopsies and the effects of sampling multiple colonic locations relative to sampling only the rectosigmoid have not been determined.
The primary finding of the current study is that two rectosigmoid biopsies is the minimum number of samples required for reliable histologic assessment when using the RHI and VAS. A pre-defined threshold for agreement was used, and this was met by both the 2- and 3-biopsy sampling methods, but not the 1-biopsy sampling method. In contrast, three rectosigmoid biopsies were required to reliably assess histologic inflammation when using the GS. We speculate that the lesser degree of agreement observed with the GS relative to the RHI may be because the instrument was not developed as an evaluative instrument. In contrast, the RHI was generated as a continuous outcome measure using accepted methodological principles of index development including specific item generation, item selection, and formal reliability, and responsiveness testing.
Exploratory subgroup analysis based upon endoscopic disease activity suggested that in patients with active disease (MCES ≥1), a minimum of three rectosigmoid biopsies may be required for reliable assessment of histologic disease activity when using the RHI and VAS. However, this finding should be interpreted with caution given the relative imprecision of the estimates and sample size.
Another secondary finding was that when the RHI reference score calculated using rectosigmoid biopsies was compared to the RHI reference score calculated using biopsies from the transverse or ascending colon, the threshold for acceptable agreement was not met. Furthermore, using all locations and the maximum number of biopsies available to assess histologic remission resulted in reduced histologic remission rates estimates compared to using the rectosigmoid colon alone. This observation, which may reflect variable colonic healing following treatment and sampling strategies, has potential implications for both clinical practice and drug development since the most common practice is to exclusively obtain rectosigmoid biopsies based upon the notion that disease activity in the rectosigmoid is a surrogate of the most severe disease activity elsewhere in the colon. Our results do not support this hypothesis and suggest that comprehensive sampling of more proximal colonic segments may be a more appropriate approach. However, the findings of this secondary analysis are limited by the sample size; of the patients with >3 rectosigmoid biopsies available, only 42% (21/50) and 32% (16/50) had biopsies collected from the ascending and transverse colon, respectively. Data from rigorously designed, adequately powered studies are required to definitively address the role of colonic location with respect to biopsy procurement strategies in ulcerative colitis. Furthermore, while previous studies have demonstrated that histologic remission is a predictor of reduced risk of relapse in ulcerative colitis, longitudinal studies are needed to determine whether proximal, compared to distal, biopsies are relatively more predictive of outcome.11
Construct validity for histological scores was analysed and significant correlations existed between histologic scores and rectal bleeding, stool frequency, and endoscopic scores. This was true for both the RHI and GS, which further validates these indices as endpoints in clinical trials.
A limitation of the current study is that it was based on a relatively small sample; 46 patients were included in the primary analysis. Moreover, due to the retrospective nature of the study design, we were unable to standardize the decision to perform either flexible sigmoidoscopy or colonoscopy and the biopsy sampling methods. Nevertheless, a substantial number of biopsies (n=287) were assessed, and blinded central reading was employed since unblinded evaluations have been demonstrated to affect both endoscopic and histologic disease activity estimates.20,21
Given that histologic remission has been independently associated with improved clinical outcomes and low placebo rates,9,12,20,22,23 the U.S. Food and Drug Administration has suggested redefining “mucosal healing” to include histologic remission.24 This has prompted the development of standardized histologic scoring systems and their routine inclusion in RCTs.25–29 Currently, evaluation of histology in ulcerative colitis clinical trials is exclusively based on biopsies taken from the rectosigmoid colon. While sampling all colonic segments for assessment of histologic inflammation may provide a more detailed evaluation of colonic remission rates than isolated sampling of the rectosigmoid alone, implementation of colonoscopy and segmental sampling in trials could have major consequences. Firstly, a significant burden of sequential colonoscopy on patients and investigators would exist. Ulcerative colitis trials currently have exceedingly low recruitment rates, which could be further negatively affected by such a policy change. Secondly, as our results show, while the specificity of histologic evaluation could be improved by segmental sampling, it is likely that the sensitivity for identification of a treatment effect would be reduced, unless complex methods are developed to interpret meaningful segmental improvement and to account for inter-segment correlations and clustering within patients. This is a substantial problem given that the most effective therapies currently available for the treatment of ulcerative colitis have relatively low rates of histologic remission even when disease evaluation is restricted exclusively to the rectosigmoid. Lastly, the clinical significance of achieving histologic remission in colonic segments proximal to the rectosigmoid colon requires further study. Our findings demonstrated numeric differences that were not statistically significant. Larger studies are required to confirm these findings.
In summary, this study demonstrates that compared to a 4-biopsy reference standard, at minimum two, and preferably three, biopsies are required to reliably assess histologic disease activity in a single colonic segment using the RHI. However, further studies are needed to confirm the number of biopsies needed in patients with active endoscopic disease and whether sampling from multiple colonic locations affects this endpoint.
Supplementary Material
GRANT SUPPORT:
NVC holds a Research Scholar Award from the American Gastroenterological Association (AGA). BSB is supported by K23DK123406. WJS is supported in part by the NIDDK-funded San Diego Digestive Diseases Research Center (P30 DK120515).
ABBREVIATIONS:
- CIMS
Central Image Management Solutions
- GS
Geboes Score
- MCES
Mayo Clinic Endoscopic Subscore
- NHI
Nancy Histological Index
- PMCS
Partial Mayo Clinic Score
- RHI
Robarts Histopathology Index
- UCEIS
Ulcerative Colitis Endoscopic Index of Severity
- UCSD
University of California, San Diego
- VAS
Visual Analogue Scale
Footnotes
STATEMENT OF INTERESTS:
RB has no conflicts of interest to declare.
NVC has received research grants from R-Biopharm; grants and personal fees from Takeda and UCB; and personal fees from Celltrion and Prometheus. These activities were all outside of the submitted work.
RKP has received consulting feels from AbbVie, Genentech, Eli Lilly, Allergan, and Robarts Clinical Trials Inc. (Alimentiv Inc.).
ZW is an employee of Alimentiv Inc.
GYZ is an employee of Alimentiv Inc.
JWDM has received consulting fees from Robarts Clinical Trials, Inc (Alimentiv Inc).
MD has received advisory fees from Echo Pharma and Robarts Clinical Trials Inc (Alimentiv Inc); speaker fees from Janssen, Merck & Co., INC, Pfizer, Takeda, and Tillotts Pharma; and nonfinancial support from Dr. Falk Pharma.
JJ is an employee of Alimentiv Inc.
CEP is an employee of Alimentiv Inc.
TV is an employee of Alimentiv Inc.
SAN is an employee of Alimentiv Inc.
BSB has received consulting fees from Pfizer and has a research grant from Prometheus Biosciences.
SS has received research grants from AbbVie and Janssen, and personal fees from Pfizer.
PSD has received research support and/or consulting from Takeda, Pfizer, Abbvie, Janssen, Prometheus, Buhlmann, and Polymedco.
MAV has received consulting fees from Robarts Clinical Trials Inc. (Alimentiv Inc.).
BGF has received grant/research support from AbbVie Inc., Amgen Inc., AstraZeneca/MedImmune Ltd., Atlantic Pharmaceuticals Ltd., Boehringer-Ingelheim, Celgene Corporation, Celltech, Genentech Inc/Hoffmann-La Roche Ltd., Gilead Sciences Inc., GlaxoSmithKline (GSK), Janssen Research & Development LLC., Pfizer Inc., Receptos Inc./Celgene International, Sanofi, Santarus Inc., Takeda Development Center Americas Inc., Tillotts Pharma AG, and UCB; consulting fees from Abbott/AbbVie, Akebia Therapeutics, Allergan, Amgen, Applied Molecular Transport Inc., Aptevo Therapeutics, Astra Zeneca, Atlantic Pharma, Avir Pharma, Biogen Idec, BioMx Israel, Boehringer-Ingelheim, Bristol-Myers Squibb, Calypso Biotech, Celgene, Elan/Biogen, EnGene, Ferring Pharma, Roche/Genentech, Galapagos, GiCare Pharma, Gilead, Gossamer Pharma, GSK, Inception IBD Inc, JnJ/Janssen, Kyowa Kakko Kirin Co Ltd., Lexicon, Lilly, Lycera BioTech, Merck, Mesoblast Pharma, Millennium, Nestle, Nextbiotix, Novonordisk, Pfizer, Prometheus Therapeutics and Diagnostics, Progenity, Protagonist, Receptos, Salix Pharma, Shire, Sienna Biologics, Sigmoid Pharma, Sterna Biologicals, Synergy Pharma Inc., Takeda, Teva Pharma, TiGenix, Tillotts, UCB Pharma, Vertex Pharma, Vivelix Pharma, VHsquared Ltd., and Zyngenia; speakers bureau fees from Abbott/AbbVie, JnJ/Janssen, Lilly, Takeda, Tillotts, and UCB Pharma; is a scientific advisory board member for Abbott/AbbVie, Allergan, Amgen, Astra Zeneca, Atlantic Pharma, Avaxia Biologics Inc., Boehringer-Ingelheim, Bristol-Myers Squibb, Celgene, Centocor Inc., Elan/Biogen, Galapagos, Genentech/Roche, JnJ/Janssen, Merck, Nestle, Novartis, Novonordisk, Pfizer, Prometheus Laboratories, Protagonist, Salix Pharma, Sterna Biologicals, Takeda, Teva, TiGenix, Tillotts Pharma AG, and UCB Pharma; and is the Senior Scientific Officer of Alimentiv Inc.
VJ has received consulting fees from AbbVie, Eli Lilly, GlaxoSmithKline, Arena Pharmaceuticals, Genetech, Pendopharm, Sandoz, Merck, Takeda, Janssen, Robarts Clinical Trials Inc (Alimentiv Inc.), Topivert, and Celltrion; and speaker’s fees from Takeda, Janssen, Shire, Ferring, Abbvie, and Pfizer.
WJS has received research grants from Atlantic Healthcare Limited, Amgen, Genentech, Gilead Sciences, Abbvie, Janssen, Takeda, Lilly, Celgene/Receptos,Pfizer, Prometheus Laboratories (now Prometheus Biosciences); consulting fees from Abbvie, Allergan, Amgen, Arena Pharmaceuticals, Avexegen Therapeutics, BeiGene, Boehringer Ingelheim, Celgene, Celltrion, Conatus, Cosmo, Escalier Biosciences, Ferring, Forbion, Genentech, Gilead Sciences, Gossamer Bio, Incyte, Janssen, Kyowa Kirin Pharmaceutical Research, Landos Biopharma, Lilly, Oppilan Pharma, Otsuka, Pfizer, Progenity, Prometheus Biosciences (merger of Precision IBD and Prometheus Laboratories), Reistone, Ritter Pharmaceuticals, Robarts Clinical Trials (Alimentiv Inc.; owned by Health Academic Research Trust, HART), Series Therapeutics, Shire, Sienna Biopharmaceuticals, Sigmoid Biotechnologies, Sterna Biologicals, Sublimity Therapeutics, Takeda, Theravance Biopharma, Tigenix, Tillotts Pharma, UCB Pharma, Ventyx Biosciences, Vimalan Biosciences, Vivelix Pharmaceuticals; and stock or stock options from BeiGene, Escalier Biosciences, Gossamer Bio, Oppilan Pharma, Prometheus Biosciences (merger of Precision IBD and Prometheus Laboratories), Progenity, Ritter Pharmaceuticals, Ventyx Biosciences, Vimalan Biosciences. Spouse: Iveric Bio - consultant, stock options; Progenity - consultant, stock; Oppilan Pharma - consultant, stock options; Escalier Biosciences – prior employee, stock options; Prometheus Biosciences (merger of Precision IBD and Prometheus Laboratories) - employee, stock options; Ventyx Biosciences – stock options; Vimalan Biosciences – stock options.
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