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Impact of tissue sampling on accuracy of Ki67 immunohistochemistry evaluation in breast cancer

Besusparis, Justinas; Plancoulaine, Benoit; Rasmusson, Allan; Augulis, Renaldas; Green, Andrew R.; Ellis, Ian O.; Laurinaviciene, Aida; Herlin, Paulette; Laurinavicius, Arvydas


Justinas Besusparis

Benoit Plancoulaine

Allan Rasmusson

Renaldas Augulis

Aida Laurinaviciene

Paulette Herlin

Arvydas Laurinavicius


Background: Gene expression studies have identified molecular subtypes of breast cancer with implications to chemotherapy recommendations. For distinction of these types, a combination of immunohistochemistry (IHC) markers, including proliferative activity of tumor cells, estimated by Ki67 labeling index is used. Clinical studies are frequently based on IHC performed on tissue microarrays (TMA) with variable tissue sampling. This raises the need for evidence-based sampling criteria for individual IHC biomarker studies. We present a novel tissue sampling simulation model and demonstrate its application on Ki67 assessment in breast cancer tissue taking intratumoral heterogeneity into account.
Methods: Whole slide images (WSI) of 297 breast cancer sections, immunohistochemically stained for Ki67, were subjected to digital image analysis (DIA). Percentage of tumor cells stained for Ki67 was computed for hexagonal tiles super-imposed on the WSI. From this, intratumoral Ki67 heterogeneity indicators (Haralick’s entropy values) were extracted and used to dichotomize the tumors into homogeneous and heterogeneous subsets. Simulations with random selection of hexagons, equivalent to 0.75 mm circular diameter TMA cores, were performed. The tissue sampling requirements were investigated in relation to tumor heterogeneity using linear regression and extended error analysis.
Results: The sampling requirements were dependent on the heterogeneity of the biomarker expression. To achieve a coefficient error of 10 %, 5–6 cores were needed for homogeneous cases, 11–12 cores for heterogeneous cases; in mixed tumor population 8 TMA cores were required. Similarly, to achieve the same accuracy, approximately 4,000 nuclei must be counted when the intratumor heterogeneity is mixed/unknown. Tumors of low proliferative activity would require larger sampling (10–12 TMA cores, or 6,250 nuclei) to achieve the same error measurement results as for highly proliferative tumors.
Conclusions: Our data show that optimal tissue sampling for IHC biomarker evaluation is dependent on the heterogeneity of the tissue under study and needs to be determined on a per use basis. We propose a method that can be applied to determine the sampling strategy for specific biomarkers, tissues and study targets. In addition, our findings highlight the benefit of high-capacity computer-based IHC measurement techniques to improve accuracy of the testing.


Besusparis, J., Plancoulaine, B., Rasmusson, A., Augulis, R., Green, A. R., Ellis, I. O., …Laurinavicius, A. (2016). Impact of tissue sampling on accuracy of Ki67 immunohistochemistry evaluation in breast cancer. Diagnostic Pathology, 11(1), Article 82.

Journal Article Type Article
Acceptance Date Jul 31, 2016
Online Publication Date Aug 30, 2016
Publication Date Dec 30, 2016
Deposit Date Oct 15, 2018
Publicly Available Date Oct 15, 2018
Journal Diagnostic Pathology
Electronic ISSN 1746-1596
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 11
Issue 1
Article Number 82
Public URL
Publisher URL
Additional Information OA (CCBY)