A Multiplexed Cytokeratin Analysis Using Targeted Mass Spectrometry Reveals Specific Profiles in Cancer-Related Pleural Effusions

Abstract

Pleural effusion (PE), excess fluid in the pleural space, is often observed in lung cancer patients and also forms due to many benign ailments. Classifying it quickly is critical, but this remains an analytical challenge often lengthening the diagnosis process or exposing patients to unnecessary risky invasive procedures. We tested the analysis of PE using a multiplexed cytokeratin (CK) panel with targeted mass spectrometry–based quantitation for its rapid classification. CK markers are often assessed in pathological examinations for cancer diagnosis and guiding treatment course. We developed methods to simultaneously quantify 33 CKs in PE using peptide standards for increased analytical specificity and a simple CK enrichment method to detect their low amounts. Analyzing 121 PEs associated with a variety of lung cancers and noncancerous causes, we show that abundance levels of 10 CKs can be related to PE etiology. CK-6, CK-7, CK-8, CK-18, and CK-19 were found at significantly higher levels in cancer-related PEs. Additionally, elevated levels of vimentin and actin differentiated PEs associated with bacterial infections. A classifier algorithm effectively grouped PEs into cancer-related or benign PEs with 81% sensitivity and 79% specificity. A set of undiagnosed PEs showed that our method has potential to shorten PE diagnosis time. For the first time, we show that a cancer-relevant panel of simple-epithelial CK markers currently used in clinical assessment can also be quantitated in PEs. Additionally, while requiring less invasive sampling, our methodology demonstrated a significant ability to identify cancer-related PEs in clinical samples and thus could improve patient care in the future.

An MRM‐Based Cytokeratin Marker Assay as a Tool for Cancer Studies: Application to Lung Cancer Pleural Effusions

“…Classifying PEs is currently a diagnostic challenge. We previously detected a set of CKs in PEs of lung cancer patients using nanoLC‐MRM assays, corroborating what has been observed in excised tumors obtained with invasive surgical methods . This analysis of CK levels was performed on PE samples, which are obtained in a less‐invasive manner (thoracentesis), and was able to identify cancer‐related PEs .…”

“…We previously detected a set of CKs in PEs of lung cancer patients using nanoLC‐MRM assays, corroborating what has been observed in excised tumors obtained with invasive surgical methods . This analysis of CK levels was performed on PE samples, which are obtained in a less‐invasive manner (thoracentesis), and was able to identify cancer‐related PEs . In this work, a standard‐LC‐MRM CK assay using SIS peptides was developed and characterized on the basis of endogenous target detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility, and peptide stability.…”

“…The study was approved by the Bioethical Committee at the Regional Medical Chamber in Warsaw (KB/928/14). One hundred and eighteen PE samples were collected over a 36‐month period and processed as described previously . MRM analysis was performed as described below.…”

Discerning the Primary Carcinoma in Malignant Peritoneal and Pleural Effusions Using Imaging Mass Spectrometry—A Feasibility Study

“…Most proteomic applications in the field of cytology, however, do not use the cellular material but the cell free supernatant instead and are focused on discerning malignant from benign effusions ,– . Using a label‐free quantitative proteomic approach, Liu et al.…”

“…In the validation set ( n = 18), sensitivity and specificity decreased to 75%. Keratins have also been used in a multiplexed cytokeratin (CK) panel with targeted MS–based quantitation to discriminate cancer‐related from benign pleural effusions with a sensitivity and specificity of 81% and 79%, respectively …”