Prognostic value of tumor cell DNA content determined by flow cytometry using formalin-fixed paraffin-embedded breast cancer tissues

  • Hiroki Kusama
  • Masafumi ShimodaEmail author
  • Tomohiro Miyake
  • Tomonori Tanei
  • Naofumi Kagara
  • Yasuto Naoi
  • Kenzo Shimazu
  • Seung Jin Kim
  • Shinzaburo Noguchi
Preclinical study



The use of formalin-fixed paraffin-embedded (FFPE) tumor tissues in flow cytometry (FCM)-based determination of tumor cell DNA content is more complicated than the use of fresh-frozen tissues. This study aimed to accurately measure tumor cell DNA content from FFPE tissues by separating tumor cells from stromal cells through FCM and investigating its prognostic impact.


We separately measured the DNA contents of tumor cells and stromal cells by gating with pan-cytokeratin and vimentin (FCMC/V). We evaluated tumor cell DNA contents [DNA index (DI)] of 290 FFPE tumor tissues and classified them into low and high DI groups, using a cutoff DI value determined through an unbiased computational method.


The distribution of DI was bimodal, and a cutoff value was determined at a DI of 1.26. The high-DI tumors were associated with aggressive phenotypes and had significantly worse distant recurrence-free intervals (DRFI) than low-DI tumors. Multivariate analysis revealed that lymph node metastasis, Ki67, and DI were independent factors affecting DRFI. Accordingly, patients with low-DI/low-Ki67 tumors had excellent outcomes compared with other tumor types. Multiploid tumors were associated with increased lymphocytic infiltration and aggressive phenotypes.


The DI of FFPE tumors could be precisely determined through FCMC/V. A combination of DI and Ki67 analyses may be able to predict the prognoses of breast cancer patients with greater accuracy.


Breast neoplasms Ploidies Keratins Vimentin Prognosis Paraffin-embedding Ki-67 antigen 



We thank Dr. Jun-ichiro Ikeda (Department of Pathology, Osaka University Hospital) for pathological evaluation and Dr. Wataru Kikuchi (Nittobo Medical co., ltd.) for technical assistance. This study was supported in part by the research funding from Nittobo Medical, Tokyo, Japan.


This study was supported in part by Japan Agency for Medical Research and Development under Grant Number JP17he1302014.

Compliance with ethical standards

Conflict of interest

Noguchi S. has been an adviser for Taiho, AstraZeneca and Novartis and has received honoraria and research funding for the other studies from AstraZeneca, Pfizer, Novartis, Chugai, Takeda, Nippon-Kayaku, Nittobo, and Sysmex.

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.

Supplementary material

10549_2019_5222_MOESM1_ESM.pdf (480 kb)
Supplementary material 1 (PDF 480 kb) Figure S1. (a) Classification of breast tumors into high-DI and low-DI tumors by FCMC/V and conventional FCM without gating. Migration from high-DI (FCMC/V) to low-DI (conventional FCM) was observed in 41 tumors and that from low-DI to high-DI in 8 tumors. (b) DRFI of patients with breast cancer of which DI was determined by the conventional FCM. Log-rank P values are shown
10549_2019_5222_MOESM2_ESM.pdf (441 kb)
Supplementary material 2 (PDF 440 kb) Figure S2. Kaplan–Meier curves of DRFI of patients with HR +/HER2−, node-negative breast cancer receiving no chemotherapy. Log-rank P values are shown
10549_2019_5222_MOESM3_ESM.docx (14 kb)
Supplementary material 3 (DOCX 14 kb) Table S1. Regimens for adjuvant systemic therapy
10549_2019_5222_MOESM4_ESM.docx (26 kb)
Supplementary material 4 (DOCX 26 kb) Table S2. Univariate and multivariate analyses of clinicopathological factors including the combination of DI and Ki67 in association with DRFI of all patients
10549_2019_5222_MOESM5_ESM.docx (25 kb)
Supplementary material 5 (DOCX 24 kb) Table S3. Univariate and multivariate analyses of clinicopathological factors including the combination of DI and Ki67 in association with DRFI of the hormone receptor-positive and HER2-negative subset
10549_2019_5222_MOESM6_ESM.docx (26 kb)
Supplementary material 6 (DOCX 26 kb) Table S4. Univariate and multivariate analyses of clinicopathological factors including the combination of DI and ploidy in association with DRFI of all patients
10549_2019_5222_MOESM7_ESM.docx (26 kb)
Supplementary material 7 (DOCX 25 kb) Table S5. Univariate and multivariate analyses of clinicopathological factors including the combination of DI and ploidy in association with DRFI of the hormone receptor-positive and HER2-negative subset


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Copyright information

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

Authors and Affiliations

  1. 1.Department of Breast and Endocrine SurgeryOsaka University Graduate School of MedicineSuitaJapan

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