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Kallikrein-related peptidase 6 (KLK6) expression differentiates tumor subtypes and predicts clinical outcome in breast cancer patients

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Abstract

Novel molecular markers that address the heterogeneity of breast cancer (BC) and provide meaningful prognostic information for BC patients are needed. Kallikrein-related peptidase 6 (KLK6) is aberrantly expressed and functionally implicated in BC and, like other members of the KLK family, may prove a useful molecular tool for clinical management. Our objective was to assess, for the first time, the clinical relevance of KLK6 mRNA expression in BC. Total RNA was isolated from 165 breast tumors, as well as 100 adjacent non-cancerous tumor specimens. After cDNA synthesis, and following quality control, quantitative real-time PCR for KLK6 expression analysis took place. Receiver operating characteristic curves were constructed in order to assess the ability of KLK6 mRNA expression levels to differentiate between molecular BC subtypes. Survival analyses, using DFS as endpoint, were performed at the univariate and multivariate levels. Publicly available BC databases and online survival analysis tools were used to validate our findings. A significant downregulation of KLK6 mRNA expression was observed in BC tissue sections compared to the non-cancerous component (P < 0.001). The expression of KLK6 is positively associated with tumor grade (P = 0.038) and is overexpressed in TNBC and HER2-positive tumors (P < 0.001). Aberrant KLK6 expression predicts the clinical outcome of BC patients in terms of DFS, independently of currently used prognostic markers (HR = 7.11, 95% CI = 1.19–42.45). The differential expression of KLK6 and its association with unfavorable outcome in BC patients was validated via in silico analyses. Although an independent external cohort is necessary to confirm our findings, we proved for the first time that KLK6 can provide independent prognostic information for BC patients.

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Abbreviations

AUC:

Area under the curve

BC:

Breast cancer

CI:

Confidence interval

Ct:

Threshold cycle

DFS:

Disease-free survival

DMFS:

Distant metastasis-free survival

ECM:

Extracellular matrix

EGFR:

Epidermal growth factor receptor

EMT:

Epithelial to mesenchymal transition

ER:

Estrogen receptor

HER2:

Human epidermal growth factor receptor 2

HPRT1 :

Hypoxanthine phosphoribosyltransferase 1

IHC:

Immunohistochemistry

KLK:

Kallikrein-related peptidase

PARs:

Protease-activated receptors

PR:

Progesterone receptor

ROC:

Receiver operating characteristic

RQ units:

Relative quantification units

r s :

Spearman correlation coefficient

RSSPC:

Robust single sample predictor classification

RT-qPCR:

Quantitative real-time PCR

SSPs:

Single sample predictors

TNBC:

Triple-negative breast cancer

TNM:

Tumor–node–metastasis

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Authors and Affiliations

  1. The Breast Clinic, Saint Savvas Anticancer Hospital, 171, Alexandras Avenue, 11522, Athens, Greece

    Christoforos Haritos & Ioannis Missitzis

  2. Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701, Athens, Greece

    Kleita Michaelidou, Konstantinos Mavridis & Andreas Scorilas

  3. First Department of Oncology, Saint Savvas Anticancer Hospital, 171, Alexandras Avenue, 11522, Athens, Greece

    Alexandros Ardavanis

  4. First Department of Surgery, Medical School, National and Kapodistrian University of Athens, Laiko Hospital, 17 Agiou Thoma Street, 11527, Athens, Greece

    John Griniatsos

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  1. Christoforos Haritos
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  2. Kleita Michaelidou
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Correspondence to Andreas Scorilas.

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The authors declare that they have no conflict of interest.

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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.

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Informed consent was obtained from all individual participants included in the study.

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10238_2018_487_MOESM1_ESM.tif

Quality control of the developed qPCR assay for quantification of KLK6 expression. Dissociation curves of (A) HPRT1 and (B) KLK6 amplicons. (C) Corresponding 3.0% w/v agarose gel electrophoresis of the RT-qPCR products of randomly selected breast tissue samples. (D) Standard curves for HPRT1 and KLK6, constructed using serial dilutions of calibrator cDNA, covering several orders of magnitude. M: molecular weight marker; PC: positive control, NC: negative control. (TIFF 9682 kb)

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Haritos, C., Michaelidou, K., Mavridis, K. et al. Kallikrein-related peptidase 6 (KLK6) expression differentiates tumor subtypes and predicts clinical outcome in breast cancer patients. Clin Exp Med 18, 203–213 (2018). https://doi.org/10.1007/s10238-018-0487-4

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  • DOI: https://doi.org/10.1007/s10238-018-0487-4

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