Synaptophysin-Ki67 double stain: a novel technique that improves interobserver agreement in the grading of well-differentiated gastrointestinal neuroendocrine tumors

Abstract

A common problem in the assessment of Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors is distinguishing tumor from non-tumor. This is because background stromal lymphocytes, entrapped non-neoplastic glands, and the delicate vascular network characteristic of neuroendocrine tumors frequently contain a subset of proliferating cells. Furthermore, in small biopsies, crush and cautery artifact can alter the morphologic appearance of tumor cells, making the Ki67 proliferative index more difficult to assess. To address these issues, we developed a synaptophysin-Ki67 double stain using a commercially available immunohistochemistry kit, allowing simultaneous visualization of tumor and proliferating nuclei. To test this method, 3 gastrointestinal pathologists individually graded 50 gastrointestinal neuroendocrine tumors first using synaptophysin-Ki67 double-stained slides and then, after a wash-out period, using Ki67-only stained slides (along with routine hematoxylin and eosin-stained slides). Interobserver agreement on Ki67 proliferative index was moderate using the Ki67-only stained slides (intraclass correlation 0.51, 95% confidence interval 0.35-0.66) and improved using the synaptophysin-Ki67 double stain (intraclass correlation 0.79, 95% confidence interval 0.69-0.86). Similarly, interobserver agreement on tumor grade was fair with Ki67-only stained slides (kappa = 0.39, P<0.001) and improved with the double stain (kappa = 0.58, P<0.001). Analysis of individual pathologists' scores revealed that fewer total number of tumor cells counted correlated with higher grade designation and appeared to contribute to grade discordance. In tumors cited as particularly challenging to assess by pathologists, 3 of 4 tumors were grade discordant with the Ki67-only stain, whereas all 4 tumors were grade concordant with the synaptophysin-Ki67 stain. The synaptophysin-Ki67 double stain is the first technique to specifically address the histomorphologic challenges of evaluating Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors. Although further validation is needed, this study provides evidence that the synaptophysin-Ki67 double stain can improve interobserver agreement.

Evaluating the prognostic potential of the Ki67 proliferation index and tumour‐infiltrating lymphocytes in olfactory neuroblastoma

“…Among the many potential markers for ONB, the Ki67 proliferation index (PI) is of particular interest. The Ki67 PI is reproducible and is widely used in neuroendocrine tumours . Its prognostic value has been evaluated for ONBs in several studies, but no prognostic threshold could be determined, possibly because of a lack of power (limited number of patients) …”

Improving the accuracy of gastrointestinal neuroendocrine tumor grading with deep learning

“…All patients provided written informed consent, and basic demographic information was collected. All glass slides used in this study were previously produced for a prior study by Matsukuma et al 10 . For each case, the glass slides of the synaptophysin/Ki-67 DS section, the adjacent Ki-67-onlystained or SS section, and the adjacent H&E-stained section ( Supplementary Fig.…”

“…tion of Ki-67 index were previously described 10 . In this study, the data from the three gastrointestinal pathologists' individual assessment of the Ki-67 index based on the DS glass slides reviewed under the microscope were SKIE: computational pipeline.…”

“…cells, if included in the analysis, may introduce error in estimating the Ki-67 index 10 , thereby the estimated tumor grade possibly becomes error-prone. The difficulty in distinguishing tumor from non-tumor cells on standard immunostained sections also makes the assessment of the Ki-67 index prone to inconsistency between pathologists.…”

“…The goal of this study is to bridge the above gaps via machine learning, and to improve the accuracy of current GI-NET grading. To achieve this goal, we developed two automated computational pipelines for GI-NET grading based on analysis of WSIs double-immunostained (DS) for synaptophysin (a marker for NETs) and Ki-67 10 . First, we developed an integrated approach termed Synaptophyin-Ki-67 Index Estimator (SKIE) ( Fig.…”