Evaluation of phospho-histone H3 in Asian triple-negative breast cancer using multiplex immunofluorescence

  • Chi Peng Timothy Lai
  • Joe Poh Sheng Yeong
  • An Sen Tan
  • Chong Hui Clara Ong
  • Bernett Lee
  • Jeffrey Chun Tatt Lim
  • Aye Aye Thike
  • Jabed Iqbal
  • Rebecca Alexandra Dent
  • Elaine Hsuen Lim
  • Puay Hoon TanEmail author
Preclinical study



We used multiplex immunofluorescence (mIF) to determine whether mitotic rate represents an independent prognostic marker in triple-negative breast cancer (TNBC). Secondary aims were to confirm the prognostic significance of immune cells in TNBC, and to investigate the relationship between immune cells and proliferating tumour cells.


A retrospective Asian cohort of 298 patients with TNBC diagnosed from 2003 to 2015 at the Singapore General Hospital was used in the present study. Formalin-fixed, paraffin-embedded breast cancer samples were analysed on tissue microarrays using mIF, which combined phospho-histone H3 (pHH3) expression with cytokeratin (CK) and leukocyte common antigen (CD45) expression to identify tumour and immune cells, respectively.


Multivariate analysis showed that a high pHH3 index was associated with significantly improved overall survival (OS; p = 0.004), but this was not significantly associated with disease-free survival (DFS; p = 0.22). Similarly, multivariate analysis also revealed that a pHH3 positive count of > 1 cell per high-power field in the malignant epithelial compartment was an independent favourable prognostic marker for OS (p = 0.033) but not for DFS (p = 0.250). Furthermore, a high CD45 index was an independent favourable prognostic marker for DFS (p = 0.018), and there was a significant positive correlation between CD45 and pHH3 index (Spearman rank correlation coefficient, 0.250; p < 0.001).


Mitotic rates as determined by pHH3 expression in epithelial cells are significantly associated with improved survival in TNBC. mIF analysis of pHH3 in combination with CK and CD45 could help clinicians in prognosticating patients with TNBC.


pHH3 TNBC Immune Proliferation Prognosis Breast cancer 



Axillary lymph node


Leukocyte common antigen




Disease-free survival


Oestrogen receptor


Formalin-fixed, paraffin-embedded


Human epidermal growth factor receptor 2


Multiplex immunofluorescence


Overall survival


Phospho-histone H3


Progesterone receptor


Tumour-infiltrating lymphocytes


Tissue microarray


Triple-negative breast cancer


Author 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.


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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 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). This article does not contain any studies with animals performed by any of the authors.

Informed consent

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).

Supplementary material

10549_2019_5396_MOESM1_ESM.doc (54 kb)
Supplementary material 1 (DOC 54 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Chi Peng Timothy Lai
    • 1
  • Joe Poh Sheng Yeong
    • 2
    • 3
  • An Sen Tan
    • 1
  • Chong Hui Clara Ong
    • 2
  • Bernett Lee
    • 3
  • Jeffrey Chun Tatt Lim
    • 2
    • 4
  • Aye Aye Thike
    • 2
  • Jabed Iqbal
    • 2
    • 6
  • Rebecca Alexandra Dent
    • 5
  • Elaine Hsuen Lim
    • 5
  • Puay Hoon Tan
    • 2
    • 6
    Email author
  1. 1.Lee Kong Chian School of MedicineNanyang Technological UniversitySingaporeSingapore
  2. 2.Division of PathologySingapore General HospitalSingaporeSingapore
  3. 3.Singapore Immunology Network (SIgN), Agency of ScienceTechnology and Research (A*STAR)SingaporeSingapore
  4. 4.Institute of Molecular Cell Biology (IMCB), Agency of ScienceTechnology and Research (A*STAR)SingaporeSingapore
  5. 5.National Cancer Centre SingaporeSingaporeSingapore
  6. 6.Duke-NUS Medical SchoolSingaporeSingapore

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