Calculation of the Ki67 index in pancreatic neuroendocrine tumors: a comparative analysis of four counting methodologies

Abstract

Ki67 index is now an essential part of classification of pancreatic neuroendocrine tumors. However, its adaptation into daily practice has been fraught with challenges related to counting methodology. In this study, three reviewers used four counting methodologies to calculate Ki67 index in 68 well-differentiated pancreatic neuroendocrine tumors: (1) ‘eye-ball’ estimation, which has been advocated as reliable and is widely used; (2) automated counting by image analyzer; (3) manual eye-counting (eye under a microscope without a grid); and (4) manual count of camera-captured/printed image. Pearson’s correlation (R) was used to measure pair-wise correlation among three reviewers using all four methodologies. Average level of agreement was calculated using mean of R values. The results showed that: (1) ‘eye-balling’ was least expensive and fastest (average time <1 min) but had poor reliability and reproducibility. (2) Automated count was the most expensive and least practical with major impact on turnaround time (limited by machine and personnel accessibility), and, more importantly, had inaccuracies in overcounting unwanted material. (3) Manual eye count had no additional cost, averaged 6 min, but proved impractical and poorly reproducible. (4) Camera-captured/printed image was most reliable, had highest reproducibility, but took longer than ‘eye-balling’. In conclusion, based on its comparatively low cost/benefit ratio and reproducibility, camera-captured/printed image appears to be the most practical for calculating Ki67 index. Although automated counting is generally advertised as the gold standard for index calculation, in this study it was not as accurate or cost-effective as camera-captured/printed image and was highly operator-dependent. ‘Eye-balling’ produces highly inaccurate and unreliable results, and is not recommended for routine use.

Tissue heterogeneity contributes to suboptimal precision of WHO 2010 scoring criteria for Ki67 labeling index in a subset of neuroendocrine neoplasms of the pancreascriteria for Ki67 labeling index in a subset of neuroendocrine neoplasms of the pancreas

“…Analytical validity is a sine qua non condition for any tumour biomarker which is intended to be used in clinical practice and to influence management of the patients [18]. Selected aspects of analytical validity of Ki67 LI in NEN were previously studied [19,20,21,22,23,24,25,26,27,28].…”

“…Of note, this approach is a reference technique of Ki67 LI assessment in pancreatic NEN [21]. Cells were considered Ki67-positive if they showed immunoreactivity in nucleus or in a mitotic figure, irrespective of the stain intensity [22,39], although it was recognized that some investigators ignored slightly stained nuclei for Ki67 LI assessment in NEN [21,40]. In each HS and CS, 2500 cells were counted in 25 por-tions of 100 cells each.…”

“…Counting was performed by manual marking of tumoral cells with and without Ki67 expression with color pens [21,38]. Of note, this approach is a reference technique of Ki67 LI assessment in pancreatic NEN [21]. Cells were considered Ki67-positive if they showed immunoreactivity in nucleus or in a mitotic figure, irrespective of the stain intensity [22,39], although it was recognized that some investigators ignored slightly stained nuclei for Ki67 LI assessment in NEN [21,40].…”

“…Prints were coded and randomly used for manual counting of Ki67 LI. Counting was performed by manual marking of tumoral cells with and without Ki67 expression with color pens [21,38]. Of note, this approach is a reference technique of Ki67 LI assessment in pancreatic NEN [21].…”

Ki-67 expression in pulmonary tumors

“…Currently, immunohistochemical staining for Ki-67 is a widely accepted method for evaluating proliferative activity in various tumor types (13)(14)(15)(16)(17)(18)(19), but in clinical practice is only incorporated in the diagnostic algorithms of neuroendocrine tumors of gastrointestinal tract.…”