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

DOI: 10.3389/bjbs.2026.16506

Keywords: CD117, c-KIT, GIST, immunohistochemistry, UK NEQAS ICC & ISH, external quality assessment, primary antibody, EP10, polyclonal antiserum, Clone 9.7, automation, diagnostic quality

The report, led by Freya Townsend of Wear Referrals Veterinary Hospital in Stockton-on-Tees, United Kingdom, describes three dogs in which metastatic mast cell tumours produced polyostotic vertebral lesions, meaning tumour deposits scattered across multiple vertebrae rather than a single site of bone destruction. According to the authors, only one previous report in the veterinary literature has documented polyostotic mast cell tumour metastasis affecting the vertebrae. That scarcity is precisely what makes the new series noteworthy: it suggests that mast cell tumours deserve a place on the differential diagnosis list for extradural and polyostotic vertebral disease, a consideration that could influence biopsy decisions, staging protocols and prognostic conversations with owners.

The first case involved an eight-year-old neutered male small crossbreed presented originally for a small, raised mass on the upper lip. Histopathology classified it as a grade II tumour under the traditional Patnaik system and low grade under the more prognostically stringent Kiupel system, with a low mitotic count of one mitosis per ten high-power fields. The mass was excised completely, albeit with a narrow deep margin. Seven months later, the right mandibular lymph node was enlarged and biopsy confirmed metastatic mast cell disease. Fine needle aspirates of the opposite lymph node, liver and spleen were reassuringly negative at that stage, and the dog was started on lomustine chemotherapy. When a subcutaneous nodule later appeared at the surgical site and cytology again confirmed metastasis, treatment was switched to toceranib phosphate before referral to a specialist oncology service.

At the referral hospital, computed tomography revealed enlargement of the right mandibular and bilateral medial retropharyngeal lymph nodes, and surgical removal of these nodes confirmed overt metastatic mast cell infiltration, graded HN3 under the Weishaar classification system for nodal mast cell metastasis. The dog then received vinblastine chemotherapy combined with prednisolone, followed by a five-day course of radiotherapy totalling 20 Gy to the surgical site. One week after radiotherapy ended, the dog became reluctant to exercise. Neurological examination revealed pain on palpation of the thoracolumbar region, and within another week the dog had developed a hunched spine and ambulatory paraparesis, with absent postural reactions in the pelvic limbs. Blood work showed elevated C-reactive protein alongside neutropenia, leukopenia and reduced platelets, consistent with both inflammatory disease and chemotherapy side effects.

The second case was a fifteen-year-old neutered female springer spaniel whose story began with removal of a two-centimetre subcutaneous mass from the right caudal mammary gland. That tumour carried a high mitotic count exceeding ten mitoses per ten high-power fields, and the regional lymph node was already invaded by sheets of hyperchromatic, intermediately differentiated mast cells, classified HN2. The mass tested negative for a c-KIT mutation. Seven months after surgery, a new groin mass and imaging findings of enlarged abdominal lymph nodes and liver nodules confirmed widespread metastasis, and vinblastine with prednisolone was initiated. Two months into chemotherapy, the dog developed ambulatory paraparesis that progressed to non-ambulatory paraparesis by the time of specialist assessment, with pain suspected on palpation of the cervical spine and neurological signs localising to the mid-thoracic spinal cord segments.

The third patient, a thirteen-year-old neutered male Nova Scotia Duck Tolling Retriever, had a long history of mast cell tumours, including a recurrent low-grade shoulder mass and, more recently, a twenty-millimetre high-grade Kiupel tumour on the mid back with narrow surgical margins. Abdominal ultrasound had already shown nodular changes in the liver and spleen. The dog completed a twelve-week course of vinblastine and prednisolone but skipped restaging, and seven months later returned with a one-week history of non-ambulatory paraparesis, a large thoracic wall mass in the armpit region, and pain on palpation of the cranial thoracic spine. Neurological deficits again localised to the T3-L3 spinal cord segments, while reduced withdrawal reflex in the right forelimb was attributed to the large mass itself.

Magnetic resonance imaging of the vertebral column in all three dogs revealed the series’ defining finding: multiple lesions across many vertebrae, ranging from well-defined nodules to ill-defined patches, accompanied by similar lesions in the iliac bones of all three dogs and, in some, the ribs, sternebrae and scapula. Technically, every vertebral lesion appeared mildly hyperintense to isointense relative to the spinal cord on both T2-weighted and T1-weighted sequences, hyperintense on short tau inversion recovery sequences, and showed moderate homogeneous enhancement after gadolinium contrast. Vertebral shape was preserved, but cortical osteolysis was present in all cases, and several lesions breached the cortical margins to invade the extradural space and perivertebral soft tissues, compressing the spinal cord from moderately to severely. All three dogs were humanely euthanised following imaging, at the request of their caregivers.

Post-mortem histopathology cemented the diagnosis. In the first two dogs, neoplastic round cells filled the intertrabecular spaces of affected vertebrae, effacing normal haematopoietic tissue and, in the first case, extending into adjacent skeletal muscle and the extradural space, with mitotic counts of 49 and 5 per ten high-power fields respectively. Immunohistochemistry proved decisive: the neoplastic cells stained positive for CD117, a marker of mast cells, with c-KIT staining patterns II and III, while negative CD3 and negative CD20 or CD79A staining excluded both T-cell and B-cell lymphoma. In the third dog, the vertebral body itself showed only inflammatory change, but the extradural mass at T5 displayed a neoplastic round cell population with a mitotic count of 30 per ten high-power fields and confirmatory CD117 positivity. Splenic and hepatic metastasis was confirmed in the first two dogs, underscoring that none of the three had isolated spinal disease.

The imaging signature carries practical weight. In a retrospective study of sixty dogs with vertebral column tumours, preservation of vertebral shape, homogeneous contrast enhancement and lesions centred on bone were features associated with round cell neoplasms, a group that includes mast cell tumours, lymphoma, multiple myeloma, plasma cell tumours and histiocytic sarcoma. The signal intensities reported here, iso- to mildly hyperintense on T1 and T2 weighting, resemble those described for vertebral multiple myeloma and lymphoma, meaning mast cell metastasis can mimic its more familiar round cell cousins. Notably, the authors highlight that their T1 and T2 findings differ from the only prior polyostotic case, in which lesions were T1 hypointense and T2 hyperintense, adding to the recognised variability of mast cell tumour metastases.

What distinguishes this series from earlier reports is the pattern of bone involvement. Previous descriptions of mast cell tumours invading bone have mostly involved local infiltration or lysis near the primary tumour, such as a subcutaneous tumour invading the stifle joint and adjacent tibial plateau, or a disseminated tumour eroding the sphenoid bone and causing blindness. Most reported spinal mast cell tumours have been extradural masses compressing the cord without touching the vertebrae at all. By contrast, the three dogs described here developed distant metastatic deposits as multiple focal lesions in separate vertebrae, a haematogenous spread pattern more reminiscent of carcinoma or multiple myeloma than of the typical mast cell tumour. The authors conclude that metastasis should be actively considered in any dog with a previously diagnosed mast cell tumour that develops spinal pain, and that mast cell disease belongs among the differentials for polyostotic, extradural vertebral lesions, a message that may prompt earlier biopsy and more complete staging in similar patients.

Subject of Research: Metastatic mast cell tumours causing polyostotic vertebral lesions in dogs

Article Title: Metastatic mast cell tumours causing polyostotic vertebral lesions in three dogs

Article References: Townsend, F., Colverde, A., Stabile, F., Ferreira, A., Herrmann, A., Peschard, L., & Néčová, S. (2025). Metastatic mast cell tumours causing polyostotic vertebral lesions in three dogs. Veterinary Oncology, 2(1), Article 34. https://doi.org/10.1186/s44356-025-00050-3

Image Credits: AI Generated

DOI: 10.1186/s44356-025-00050-3

Keywords: canine, mast cell tumour, metastasis, vertebral lesions, MRI, histopathology, veterinary oncology, spinal tumour, polyostotic, CD117, round cell tumour, paraparesis