Diagnostic and Prognostic Value of SHOX2 and SEPT9 DNA Methylation and Cytology in Benign, Paramalignant and Malignant Pleural Effusions

Dimo Dietrich,Maria Jung,Svenja Puetzer,Philipp Schatz,Glen Kristiansen,Annette Leisse,Barbara Uhl,Sebastian Meller,Emily Eva Holmes,Claudia Ivascu,Goli Samimi

doi:10.1371/journal.pone.0084225

Abstract

Pleural effusions (PE) are a common clinical problem. The discrimination between benign (BPE), malignant (MPE) and paramalignant (PPE) pleural effusions is highly important to ensure appropriate patient treatment. Today, cytology is the gold standard for diagnosing malignant pleural effusions. However, its sensitivity is limited due to the sometimes low abundance of tumor cells and the challenging assessment of cell morphology in cytological samples. This study aimed to develop and validate a diagnostic test, which allows for the highly specific detection of malignant cells in pleural effusions based on the DNA methylation biomarkers SHOX2 and SEPT9. A quantitative real-time PCR assay was developed which enabled the accurate and sensitive detection of SHOX2 and SEPT9 in PEs. Cytological and DNA methylation analyses were conducted in a case control study comprised of PEs from 114 patients (58 cases, 56 controls). Cytological analysis as well as SHOX2 and SEPT9 methylation resulted in 100% specificity. 21% of the cases were cytologically positive and 26% were SHOX2 or SEPT9 methylation positive. The combined analysis of cytology and DNA methylation resulted in an increase of 71% positively classified PEs from cancer patients as compared to cytological analysis alone. The absolute sensitivity of cytology and DNA methylation was not determinable due to the lack of an appropriate gold standard diagnostic for distinguishing between MPEs and PPEs. Therefore, it was unclear which PEs from cancer patients were malignant (containing tumor cells) and which PEs were paramalignant and resulted from benign conditions in cancer patients, respectively. Furthermore, DNA methylation analysis in PEs allowed the prognosis of the overall survival in cancer patients (Kaplan-Meier analysis, log rank test, p = 0.02 (SHOX2), p = 0.02 (SEPT9)). The developed test may be used as a diagnostic and prognostic adjunct to existing clinical and cytopathological investigations in patients with PEs of unclear etiology.

Highlights

Pleural effusions (PEs) represent a common clinical complication
Reliable figures regarding the relative number of paramalignant pleural effusions (PPEs) and Malignant pleural effusions (MPEs) cannot be found in the literature due to the lack of an accurate gold standard method to discriminate between PPEs and MPEs
Quantification of total DNA content was based on the b-actin gene (ACTB) in a methylation independent manner

Summary

Introduction

Pleural effusions (PEs) represent a common clinical complication. Most commonly, PEs are caused by cardiac failure, pneumonia, pulmonary embolism and malignant neoplasm [1,2]. The presence of MPEs in cancer patients is associated with an overall poor prognosis leading to an overall survival of less than one year [8,9,10]. Treatment of these patients is always palliative [11,5]. Not all cancer patients with a PE appear to have a MPE, but show paramalignant pleural effusions (PPEs). These MPEs occur since cancer patients frequently develop comorbidities such as heart failure, pneumonia, or pulmonary embolism, each of them possibly causing an effusion [11]. The discrimination between MPE and PPE might direct a decision towards curative or palliative treatment [11] and is considered for accurate tumor staging according to the TNM-classification

Objectives

Methods

Results