A novel model for Ki67 assessment in breast cancer

Abstract

Background: Ki67 is currently the proliferation biomarker of choice, with both prognostic and predictive value in breast cancer. A lack of consensus regarding Ki67 use in pre-analytical, analytical and post-analytical practice may hinder its formal acceptance in the clinical setting.

Methods: One hundred breast cancer samples were stained for Ki67. A standard estimation of Ki67 using fixed denominators of 200, 400 and 1 000 counted tumor cells was performed, and a cut-off at 20% was applied, Ki67static. A novel stepwise counting strategy for Ki67 estimation, Ki67scs, was developed based on rejection regions derived from exact two-sided binomial confidence intervals for proportions. Ki67scs was defined by the following parameters: the cut-off (20%), minimum (50) and maximum (400) number of tumor cells to count, increment (10) and overall significance level of the test procedure (0.05). Results from Ki67scs were compared to results from the Ki67static estimation with fixed denominators.

Results: For Ki67scs, the median number of tumor cells needed to determine Ki67 status was 100; the average, 175. Among 38 highly proliferative samples, the average Ki67scs fraction was 45%. For these samples, the fraction decreased from 39% to 37% to 35% with static counting of 200, 400 and 1 000 cells, respectively. The largest absolute difference between the estimation methods was 23% (42% (Ki67scs) vs. 19% (Ki67static)) and resulted in an altered sample classification. Among the 82 unequivocal samples, 74 samples received the same classification using both Ki67scs and Ki67static. Of the eight disparate samples, seven were classified highly proliferative by Ki67static when 200 cells were counted; whereas all eight cases were classified as low proliferative when 1 000 cells were counted.

Conclusions: Ki67 estimation using fixed denominators may be inadequate, particularly for tumors demonstrating extensive heterogeneity. We propose a time saving stepwise counting strategy, which acknowledges small highly proliferative hot spots.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3588156111195336.

Figures

Figure 1

The stepwise counting procedure in tabular form. Start by counting 50 cells in a hot spot. If 0-2 cells are positive, declare the sample as Ki67-negative, and if 19-50 cells are positive, declare as Ki67-positive. If the number of positive cells is in the 3-18 range, count another 10 cells. If the null hypothesis was not rejected in the first step, the number of positive cells out of 60 will vary between 3+0 = 3 and 18+10 = 28. The three possible decisions based on 60, 70, …, 400 cells are listed in the table. The color-coding is green for low Ki67, red for high Ki67 and yellow for equivocal Ki67 status.

Figure 2

Five sequences of cumulative Ki67 fractions simulated under the null hypothesis of homogeneity and probability 0.20 of a positive cell.

Figure 3

One hundred sequences of cumulative Ki67 fractions simulated under the null hypothesis of homogeneity and probability 0.20 of a positive cell. The red curves correspond to the upper and lower rejection boundaries based on 99.0% exact two-sided binomial confidence intervals. The five sequences that cross a boundary are highlighted in green, whereas the remaining 95 are shown in black.

Figure 4

A graphical presentation of the step-wise procedure for determination of Ki67 status. The black jagged line corresponds to the null hypothesis of probability 0.20 of a positive cell, the cut-off. The dark region covers the counts for which the null hypothesis cannot be rejected by a two-sided binomial test at the 1.0% significance level. If the estimate falls in this region, another 10 cells are counted and a new test is performed. If 400 cells have been counted without reaching the upper or lower rejection regions (light regions), the Ki67 status of the sample is considered equivocal. If the upper or lower rejection region is reached for a sample, the counting is stopped, and the Ki67-status determined.

Figure 5

Digital pictures at x10 magnification of four breast cancer samples stained for Ki67. A: A highly proliferative and relatively homogenous case. B: A highly proliferative and heterogeneous case. C: A low proliferative and relatively homogenous case. D: A low proliferative and heterogeneous case.

Figure 6

Demonstration of the stepwise counting strategy used to determine the Ki67 status of the four cases presented in Figure3.

Figure 7

Cumulative Ki67 estimate based on 50 to 400 tumor cells in steps of 10 for the sample (a core biopsy) showing the largest absolute and relative difference between the model-based estimate and an estimate based on the fixed counting of 200 cells. The shaded region is a 99.0% point-wise confidence interval corresponding to the step-wise test procedure.