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Pleural effusion (PE) is excess fluid in the pleural cavity that stems from lung cancer, other diseases like extra-pulmonary tuberculosis (TB) and pneumonia, or from a variety of benign conditions. Diagnosing its cause is often a clinical challenge and we have applied targeted proteomic methods with the aim of aiding the determination of PE etiology. We developed a mass spectrometry (MS)-based multiple reaction monitoring (MRM)-protein-panel assay to precisely quantitate 53 established cancer-markers, TB-markers, and infection/inflammation-markers currently assessed individually in the clinic, as well as potential biomarkers suggested in the literature for PE classification. Since MS-based proteomic assays are on the cusp of entering clinical use, we assessed the merits of such an approach and this marker panel based on a single-center 209 patient cohort with established etiology. We observed groups of infection/inflammation markers (ADA2, WARS, CXCL10, S100A9, VIM, APCS, LGALS1, CRP, MMP9, and LDHA) that specifically discriminate TB-PEs and other-infectious-PEs, and a number of cancer markers (CDH1, MUC1/CA-15-3, THBS4, MSLN, HPX, SVEP1, SPINT1, CK-18, and CK-8) that discriminate cancerous-PEs. Some previously suggested potential biomarkers did not show any significant difference. Using a Decision Tree/Multiclass classification method, we show a very good discrimination ability for classifying PEs into one of four types: cancerous-PEs (AUC: 0.863), tuberculous-PEs (AUC of 0.859), other-infectious-PEs (AUC of 0.863), and benign-PEs (AUC: 0.842). This type of approach and the indicated markers have the potential to assist in clinical diagnosis in the future, and help with the difficult decision on therapy guidance.

Purpose The goal of this work was to develop an LC-MRM assay for the quantitative analysis of a set of established and diagnostically important cytokeratin (CK) markers used in cancer diagnosis, prognosis, and therapy monitoring. Second, the potential of this assay in lung cancer diagnosis through pleural effusion (PE) analysis was examined. Experimental design A multiplexed MRM assay was developed for 17 CKs and their select caspase-cleaved fragments. Isotope-labeled standard peptides were used for high assay specificity and absolute peptide quantitation; with robust standard-flow LC coupled to a latest-generation triple-quadrupole instrument for high sensitivity. The potential clinical applicability was demonstrated by the analysis of 118 PE samples. Results The MRM assay was evaluated for endogenous detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility and peptide stability, and is generally applicable to any epithelial cancer study. A set of 118 patients with known pathologies allowed us to define the range of CK levels in clinical PE samples. Specific CKs were able to differentiate cancer-related PEs from those caused by benign ailments. In addition, they allowed to differentiate between PEs from subjects with small cell lung cancer versus non-small cell lung carcinoma, and to further differentiate the latter into its two subtypes, adenocarcinoma and squamous cell carcinoma. Conclusion and clinical relevance An MRM-based CK assay for carcinoma studies can differentiate between the three lung cancer histological types using less-invasive PE sampling providing potential therapy-guiding information on patients that are inoperable.

A 51-year-old man, heavy cigarette smoker, a homeless alcoholic, with disseminated lesions in lungs and with ulceration and infiltration of the tongue is presented. Treatment with antibiotics was ineffective. He was admitted to the otolaryngological department because of suspicion of the tongue cancer. The histological examination of the tongue biopsy revealed tuberculous granuloma. In the pulmonological department, on admission the patient was cachectic, with massive oedema and ulceration of the tongue, and enlargement of the cervical lymph nodes. He was fed through the gastric tube. He had severe pain of the tongue demanding treatment with opiates analgesics. Chest x-ray revealed disseminated lesions in lungs. Antituberculous therapy was administered because of suspicion of tuberculosis of the tongue and lungs. During the treatment clinical improvement was observed. Tubercule bacilli were grown in the sputum culture after 6 weeks of observation. After 8 weeks of antituberculous therapy regression of lung lesions and healing of the tongue were observed. The patient continued treatment for 6 months in the Lung Disease Outpatient Clinic.