When a pathologist peers down a microscope at a suspicious growth in the wall of the gut, one of the most consequential questions is whether the lesion is a gastrointestinal stromal tumour, or GIST. These tumours arise predominantly from the interstitial cells of Cajal, the pacemaker cells embedded in the muscle layers of the bowel wall, and most present in the stomach or small intestine with either spindle cell or epithelioid morphology. The primary identifying feature in their diagnosis is immunohistochemical detection of CD117, also known as c-KIT, a tyrosine kinase receptor protein encoded by the KIT gene. Mutations in KIT drive the tumourigenesis of GISTs and explain why they so commonly over-express the protein. Getting that stain right is not merely academic: the introduction of tyrosine kinase inhibitors such as imatinib and sunitinib revolutionised the prognosis for patients with advanced unresectable or metastatic disease, with imatinib highly effective in approximately 85 percent of GISTs, so it is imperative that GISTs are correctly identified at primary diagnosis.
Despite this obvious importance, remarkably few papers have reported on CD117 stain quality in the demonstration of GIST. A 2006 study conducted by the College of American Pathologists found that most participating laboratories produced good quality staining, and in 2019 the NordiQC external quality assessment organisation published an analysis of their own CD117 quality assessments. Now, a team at the UK National External Quality Assessment Scheme for Immunocytochemistry and In-Situ Hybridisation, UK NEQAS ICC & ISH, has mined five years of its own assessment archives to produce what may be the most detailed real-world picture yet of how laboratories actually perform this test, and which of the antibodies in regular use deliver the best results. The work, published in the British Journal of Biomedical Science, analysed data from twenty assessment runs conducted between May 2020 and May 2025.
The design of the scheme’s Alimentary Tract Pathology EQA Programme is elegant in its simplicity. Each participating laboratory, drawn roughly equally from the UK and overseas and predominantly healthcare-based cellular pathology departments, was provided with a slide bearing unstained sections taken from formalin-fixed paraffin-embedded tissue samples. The samples comprised an appendix, useful for assessing staining of the interstitial cells of Cajal and mast cells, both of which were expected to show specific staining, and for assessing non-specific staining elsewhere; a GIST previously tested to ensure CD117 positivity; and a relevant alternative tumour such as gastric adenocarcinoma, leiomyosarcoma or desmoid tumour, used to detect non-specific staining. After undertaking immunohistochemical staining for CD117 using their in-house standard operating procedures, participants returned their stained slide to UK NEQAS ICC & ISH for collation and central visual assessment.
The quality assessment metrics are rigorous. Four expert assessors score each participant’s submitted stained slide concurrently but independently using a multi-header microscope, each awarding a score within a range of 1 to 5, with 1 being of no utility and 5 being excellent quality. These scores are summed for all four assessors to give a score in the range 4 to 20. Assessor scores were only allowed to vary by one mark, so scores of 3 and 4 might be awarded by individual assessors scoring the same preparation, but not scores of 3 and 5; when such variations did occur, the quality of staining was discussed and a consensus agreed. If an assessor wished to fail a participant’s submission, then all assessors must have agreed and awarded scores in the same range. Samples were assessed against expected expression levels established beforehand using preparations stained by the commercial suppliers of commonly used primary antibodies and in-house stained slides, and sections cut at the same time as those distributed to participants were stained on the slide return deadline date to ensure the stability of expression.
The headline numbers are striking. Total submissions across the twenty runs numbered 2,656, with a mean of 132.8 per run and a range of 125 to 140, and the number of participants rose from 125 to 140 over the five-year period, a 12 percent increase. The total number of submissions awarded a quality score indicating at least acceptable stain quality was 2,642, or 99.5 percent. Only 14 submissions failed outright, a mean proportion of 0.5 percent per run; 158 obtained a borderline score, a mean of 5.9 percent; 394 obtained an acceptable score, a mean of 14.8 percent; and 2,090 obtained a good or excellent score, a mean of 78.7 percent. But there was a strong trend for the proportion of submissions obtaining a good or excellent quality score to increase over time, rising from 65 percent at the first run in 2020 to 90 percent at the last in 2025. At one point in the study, only just over half of submissions demonstrated good or excellent staining, which is the standard all laboratories should be aiming to achieve.
Four primary antibody clones and one polyclonal antiserum dominated the field, supplied by the three main commercial suppliers, Agilent Dako, Leica Biosystems and Roche Diagnostics, with one notable exception in the YR145 clone supplied by Cell Marque. Historically, the polyclonal antiserum from Agilent Dako, product code A4502, dominated the field, being the first commercially available primary to be widely reported as effective in the demonstration of CD117 in formalin-fixed paraffin-embedded samples, and submissions using it represented just under half of the total. However, its proportional use showed a steady decline over the course of the study, from 58.7 percent in 2020 to 42.4 percent in 2025. In contrast, the rabbit monoclonal antibody EP10 saw a dramatic increase in use, with the Roche Diagnostics supplied reagent, EP10(RD), seeing a ten-fold increase in proportional use from 3.3 percent to 30.4 percent, such that it became the second-most commonly used primary antibody. For the most part, this increase was due to laboratories that had previously used the polyclonal antiserum switching to EP10(RD), particularly amongst laboratories using Roche Diagnostics automation. Taking EP10(RD) and the Leica Biosystems supplied reagent, EP10(LB), together, the proportion of EP10 users was larger than that of the polyclonal antiserum by the end of the survey period, 48 percent compared to 42.4 percent respectively. Meanwhile, the use of the Clone 9.7 antibody, also from Roche Diagnostics, declined from 12.4 percent to just 0.8 percent.
When the performance of the primaries was examined using higher quality score as the indicator of superior performance, the results were highly statistically significant. The EP10(LB) reagent achieved a mean quality score of 16.9, with 95 percent confidence intervals of 16.7 to 17.0, and no submission that used this primary failed at assessment, in contrast to all other primary reagents where there was at least one fail. Its performance was superior to that of all other primary reagents, with the difference being significant at P less than 0.05 when compared to YR145 and highly significant at P less than 0.0001 when compared to both EP10(RD) and the polyclonal antiserum. Submissions that used Clone 9.7 had the worst performance, with a mean quality score of 14.6, and there was a highly significant association between the use of this antibody clone and lower quality scores when its performance was compared to all other reagents. The YR145, EP10(RD) and polyclonal antiserum produced results that were not significantly different from one another. The overall mean quality score for the whole population was 16.3.
But the most contextually revealing finding concerned the relationship between primary antibody and automation supplier. For Roche Diagnostics there was an absolute association for both Clone 9.7 and EP10(RD), and for Leica Biosystems it was extremely high, at 94.9 percent, for the EP10(LB) reagent. In contrast, the polyclonal antiserum supplied by Agilent Dako was used on machines manufactured by the same supplier with a frequency of only 26.7 percent, being used more frequently on both Leica Biosystems, at 27.6 percent, and Roche Diagnostics supplied automation, at 45.8 percent. When the data was further examined to see if this affected performance results for the polyclonal antiserum, it did. The mean quality score was highest when the polyclonal primary was used on Agilent Dako machines, at 16.6, compared to 16.1 on Leica Biosystems machines and 16.2 on Roche Diagnostics machines, with the difference in quality score distributions statistically significant when comparing Agilent Dako and Leica Biosystems at P less than 0.0001, and Agilent Dako and Roche Diagnostics at P equal to 0.0013. A follow-on analysis looking at submissions which used primary antibody reagent and automation matched for supplier found that EP10(LB) on Leica Biosystems machines and the polyclonal antiserum on Agilent Dako machines were both associated with superior quality staining results compared to those produced by EP10(RD) on Roche Diagnostics machines, but they were not statistically different from each other. This indicates the importance of analysing the data in a context-sensitive way that reflects real-world usage.
The authors conclude that the demonstration of CD117 over-expression in GIST is undertaken to a very high standard of quality in the majority of laboratories that participate in regular scheduled EQA for this important predictive biomarker. Statistically tested and proven results indicate that for users of Agilent Dako supplied automation, the highest quality staining is produced by using the polyclonal antiserum from the same supplier. Users of Leica Biosystems and Roche Diagnostics automation should use the EP10 rabbit monoclonal primary, and should source that reagent from the same supplier that provides their automation. It is recommended that Clone 9.7 as supplied by Roche Diagnostics should not be used, as the results it produces are statistically significantly inferior to those obtained using all other surveyed primary antibodies. The choice of primary antibody should be guided by the type of automation available to the laboratory, and this work represents an advance in biomedical science because it uses a set of real-world data to produce evidence that can be used to assure laboratory testing quality and improve patient outcomes.
Subject of Research: External quality assessment of CD117 immunohistochemistry for gastrointestinal stromal tumours
Article Title: Immunohistochemical testing of GISTs using CD117 markers: the UK NEQAS ICC & ISH external quality assessment data show significant differences in the performance of methods in regular use
Article References: Dodson, A., & Parry, S. (2026). Immunohistochemical testing of GISTs using CD117 markers: the UK NEQAS ICC & ISH external quality assessment data show significant differences in the performance of methods in regular use. British Journal of Biomedical Science, 83, Article 16506. https://doi.org/10.3389/bjbs.2026.16506
Image Credits: AI Generated
Keywords: CD117, c-KIT, GIST, immunohistochemistry, UK NEQAS ICC & ISH, external quality assessment, primary antibody, EP10, polyclonal antiserum, Clone 9.7, automation, diagnostic quality
News Source: Ophelia Keating. (October 17, 2026). CD117 Staining for GIST: UK EQA Data Reveal Significant Antibody Performance Differences. Scienmag.
