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The role of Ki-67 in Asian triple negative breast cancers: a novel combinatory panel approach

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Abstract

The proliferation marker Ki-67 is frequently used to assess aggressiveness in the pathological evaluation of cancer, but its role remains uncertain in triple-negative breast cancer (TNBC). We aimed to quantify and localize Ki-67 expression in both epithelial and immune compartments in TNBC and investigate its association with clinicopathological parameters and survival outcomes. A total of 406 TNBC cases diagnosed between 2003 and 2015 at Singapore General Hospital were recruited. Using state-of-the-art, 7-colour multiplex immunofluorescence (mIF) tissue microarrays (TMAs) were stained to assess the abundance, density and spatial distribution of Ki-67-positive tumour cells and immune cells co-decorated with cytokeratin (CK) and leukocyte common antigen (CD45) respectively. Furthermore, MKI67 mRNA profiles were analysed using NanoString technology. In multivariate analysis adjusted for tumour size, histologic grade, age at diagnosis, and lymph node stage, a high Ki-67 labelling index (LI) > 0.3% was associated with improved disease-free survival (DFS; HR = 0.727; p = 0.027). High Ki-67-positive immune cell count per TMA was a favourable prognostic marker for both DFS (HR = 0.379; p = 0.00153) and overall survival (OS; HR = 0.473; p = 0.0482). The combination of high Ki-67 LI and high MKI67 expression was associated with improved DFS (HR = 0.239; p = 0.00639) and OS (HR = 0.213; p = 0.034). This study is among the first to highlight that Ki-67 is associated with favourable prognosis in an adjuvant setting in TNBC, and the mIF-based evaluation of Ki-67 expression on both tumour and immune cells represents a novel prognostic approach.

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Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ALN:

axillary lymph node

CD45:

leukocyte common antigen

CK:

cytokeratin

CRC:

colorectal cancer

DFS:

disease-free survival

ER:

oestrogen receptor

HER2:

human epidermal growth factor receptor 2

LI:

labelling index

mIF:

multiplex immunofluorescence

OS:

overall survival

PR:

progesterone receptor

TIL:

tumour infiltrating lymphocytes

TMA:

tissue microarray

TNBC:

triple negative breast cancer

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Funding

This article was funded by the A*STAR Biomedical Research Council, National Medical Research Council Stratified Medicine Programme Office (SMPO201302) awarded to Dr. PH Tan. Dr. Jabed Iqbal is a recipient of the Transition Award from the Singapore National Medical Research Council (NMRC/TA/0041/2015).

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Author notes

  1. An Sen Tan and Chi Peng Timothy Lai contributed equally to this work.

Authors and Affiliations

  1. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

    An Sen Tan & Chi Peng Timothy Lai

  2. Division of Pathology, Singapore General Hospital, 20 College Road, Academia, Level 7, Diagnostics Tower, Singapore, 169856, Singapore

    Joe Poe Sheng Yeong, Chong Hui Clara Ong, Jeffrey Chun Tatt Lim, Aye Aye Thike, Jabed Iqbal & Puay Hoon Tan

  3. Singapore Immunology Network (SIgN), Agency of Science, Technology and Research (A*STAR), Singapore, Singapore

    Joe Poe Sheng Yeong & Bernett Lee

  4. National Cancer Centre Singapore, Singapore, Singapore

    Rebecca Alexandra Dent & Elaine Hsuen Lim

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  1. An Sen Tan
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Contributions

PT and JY conceived and directed the study. PT and JY supervised the research. JL constructed TMAs, performed IHC, prepared samples for NanoString and collated data. BL performed bioinformatics analysis. AT, JY and TL performed immunohistochemical scoring, interpreted the data and performed biostatistical analysis. CO constructed TMAs, performed IHC and collated data. TP, AT, JI, RD and EL contributed to the scientific content of the study. AT, JY and TL drafted the manuscript with the assistance and final approval of all authors.

Corresponding author

Correspondence to Puay Hoon Tan.

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Ethics approval and consent to participate

The SingHealth Centralized Institutional Review Board (CIRB) approved the authors’ request for waiver of informed consent based on ethical consideration (Ref: 2011/433/F). The SingHealth CIRB operates in accordance with the ICH/Singapore Guideline for Good Clinical Practices and with the applicable regulatory requirement(s).

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The authors declare that they have no competing interests.

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Electronic supplementary material

Supplementary Figure 1

Kaplan-Meier analysis of disease-free survival, with previously reported Ki-67 LI cut-offs. (JPG 1116 kb)

Supplementary Figure 2

Kaplan-Meier analysis of overall survival, with previously reported Ki-67 LI cut-offs. (JPG 1126 kb)

Supplementary Figure 3

Kaplan-Meier analysis of (A) overall survival and (B) disease free survival outcomes in women with high compared with low total Ki-67 cell count (both cancer and immune cells) per high-power field in the cohort (JPG 477 kb)

Supplementary Table 1

Summary of studies investigating the prognostic value of Ki-67 in TNBC. (DOCX 15 kb)

Supplementary Table 2

Tumour subtypes represented in the study population. (DOCX 13 kb)

Supplementary Table 3

mIF antibody details (DOCX 13 kb)

Supplementary Table 4

Summary of variables and cut-offs presented (DOCX 13 kb)

Supplementary Table 5

Multivariate analysis of the prognostic value of total Ki-67 cell count (both cancer and immune cells) for DFS, with various additional cut-offs. (DOCX 13 kb)

Supplementary Table 6

Multivariate analysis of survival outcomes using total Ki-67 cell count (both cancer and immune cells). (DOCX 13 kb)

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Tan, A.S., Yeong, J.P.S., Lai, C.P.T. et al. The role of Ki-67 in Asian triple negative breast cancers: a novel combinatory panel approach. Virchows Arch 475, 709–725 (2019). https://doi.org/10.1007/s00428-019-02635-4

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