Anatomical pathologyValidity and reliability of Ki-67 assessment in oestrogen receptor positive breast cancer
Introduction
Kiel-67 antigen (Ki-67) is a well-studied prognostic and predictive biomarker in breast cancer. It is a nuclear protein expressed in all phases of the cell cycle except G0, and therefore indicates which cells are proliferating.1, 2 Ki-67 expression is assessed by immunohistochemistry as the Ki-67 labelling index (LI), which refers to the percentage of positively stained tumour cell nuclei.3 The test is inexpensive and readily available in diagnostic pathology laboratories, allowing for rapid turnaround times to facilitate clinical decision-making.
Numerous clinical applications for Ki-67 have been proposed. The prognostic value of Ki-67 for disease-free survival and overall survival in early oestrogen receptor (ER) positive breast cancer has been consistently demonstrated.4, 5, 6 A role for the prediction and monitoring of neoadjuvant therapy is also emerging, particularly for endocrine therapy.7, 8, 9 The importance of Ki-67 expression has been supported by gene expression profiling studies. For example, expression of MKI67 (the gene encoding the Ki-67 protein) and other proliferation-associated genes helps to distinguish between the luminal A and luminal B intrinsic subtypes of breast cancer.10, 11 Moreover, MKI67 expression is included in gene expression profiling-based tests which predict benefit from chemotherapy, such as Oncotype DX, Genomic Grade Index and PAM50.12, 13, 14 However, because of the high cost of these tests (e.g., Oncotype DX costs $4000 in Australia15), there is interest in the use of immunohistochemical profiling, including measurement of Ki-67, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) as cost-effective surrogate markers for identifying intrinsic subtypes and calculating recurrence risk.16, 17
However, there is concern about the analytical validity of Ki-67,18 and there is a lack of consensus regarding its measurement. Variability in pre-analytic, analytic and scoring protocols makes it difficult to implement the cut-off values for clinical decision-making proposed in the literature.1, 2, 3, 19 Furthermore, the relationship between Ki-67 LI assessed on whole slides and tissue microarrays (TMA) is not well studied, despite the widespread use of cut-off values established by studies which assessed Ki-67 LI on TMA.16 Although the International Ki-67 in Breast Cancer Working Group has published consensus guidelines for the assessment of Ki-67,3 these recommendations have not been widely implemented.20 Of note, the proposed gold standard is manual counting of at least 1000 cells at high power,3 which is labour intensive and may imply a false sense of precision. Visual estimation has been proposed as a rapid alternative, but its validity and reliability are disputed.21, 22, 23 Digital image analysis (DIA) is emerging as a highly reproducible technique, but is not yet widely adopted.21, 22, 23, 24, 25
We performed a concordance study to compare five different methods of Ki-67 assessment, including the gold standard and different methods of visual estimation and DIA. We assessed validity, intra- and inter-observer reproducibility and reporting time to determine which is most appropriate for use at our institution. We also compared the use of TMA with whole slides.
Section snippets
Ethics and patients
The study was performed on a random series of 71 invasive breast cancers diagnosed at the Austin Hospital, Melbourne, Australia. Data were extracted from the Kestral pathology database to identify cases of breast cancer diagnosed between 1 January 2005 and 12 December 2010. Eligibility criteria were: invasive carcinoma of no special type, ER positive. Tumour grade (Elston-Ellis modification of Scarff-Bloom-Richardson grade; BRE), ER, PR and HER2 characteristics were extracted from routine
Patient and tumour characteristics
The median patient age at diagnosis was 53 years. All patients were female and all the tumours were ER positive; most (90%) were also PR positive. A minority were HER2-amplified (10%). There were 16 BRE grade 1 tumours, 28 grade 2 tumours and 27 grade 3 tumours. The median Ki-67 LI was 29% on whole slides and 17% on TMA, as assessed by manual counting. Ki-67 LI was significantly correlated with BRE grade (Spearman rank correlation=0.57, p<0.001), particularly the mitotic count component
Discussion
In our study, all methods tested demonstrated a high degree of validity and reliability for Ki-67 assessment on TMA, particularly manual counting and semi-manual DIA. However, manual counting proved to be highly labour intensive. On whole slides, both visual estimation with the aid of reference photographs and semi-manual DIA demonstrated high validity and reliability. The use of reference photographs significantly improved validity and inter-observer reproducibility on whole slides compared to
Conclusions
Standardisation of Ki-67 assessment is essential in order to translate studies of the prognostic and predictive use of Ki-67 into clinical practice. Our study validated the use of semi-manual DIA for the assessment of Ki-67 and established parameters for the use of semi-manual DIA using the Aperio platform at our institution. Whilst DIA algorithms require tuning in order to account for inter-laboratory variations in immunohistochemistry and slide scanning protocols, we have demonstrated that
Acknowledgements
Many thanks to the laboratory staff at the Department of Anatomical Pathology, Austin Hospital.
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Assessment of Ki67 immunohistochemical expression as a prognostic marker in breast carcinoma
2020, Akusherstvo i Ginekologiya (Russian Federation)