Immunofluorescent and molecular characterization of effusion tumor cells reveal cancer site‐of‐origin and disease‐driving mutations

Abstract

Background Effective cancer treatment relies on precision diagnostics. In cytology, an accurate diagnosis facilitates the determination of proper therapeutics for patients with cancer. Previously, the authors developed a multiplexed immunofluorescent panel to detect epithelial malignancies from pleural effusion specimens. Their assay reliably distinguished effusion tumor cells (ETCs) from nonmalignant cells; however, it lacked the capacity to reveal specific cancer origin information. Furthermore, DNA profiling of ETCs revealed some, but not all, cancer‐driver mutations. Methods The authors developed a new multiplex immunofluorescent panel that detected both malignancy and pulmonary origin by incorporating the thyroid transcription factor‐1 (TTF‐1) biomarker. Evaluation for TTF‐1–positive ETCs (T‐ETCs) was performed on 12 patient samples. T‐ETCs and parallel ETCs from selected patients were collected and subjected to DNA profiling to identify pathogenic mutations. All samples were obtained with Institutional Review Board approval. Results Malignancy was detected in all samples. T‐ETCs were identified in 9 of 10 patients who had clinically reported TTF‐1 positivity (90% sensitivity and 100% specificity). Furthermore, DNA profiling of as few as five T‐ETCs identified pathogenic mutations with equal or greater sensitivity compared with profiling of ETCs, both of which showed high concordance with clinical findings. Conclusions The findings suggest that the immunofluorescent and molecular characterization of tumor cells from pleural effusion specimens can provide reliable diagnostic information, even with very few cells. The integration of site‐specific biomarkers like TTF‐1 into ETC analysis may facilitate better refined diagnosis and improve patient care.

Comprehensive epithelial biomarker analysis of malignant mesothelioma: EpCAM positivity is a potential diagnostic pitfall

“…By using the four MOC31 IHC‐positive MM specimens and five other MM specimens, we evaluated for membranous EpCAM protein expression using IF. We previously established a multiplexed IF panel to distinguish epithelial malignancy from background mesothelial cells on ThinPrep slides 8,20 . When visualizing the staining patterns of multiplex biomarkers (cytokeratin/CK, CD45, and EpCAM), mesothelial cells consistently display positive CK, negative CD45, and negative EpCAM staining, reliably distinguishing them from epithelial and hematopoietic cells 8,20 …”

“…We previously established a multiplexed IF panel to distinguish epithelial malignancy from background mesothelial cells on ThinPrep slides 8,20 . When visualizing the staining patterns of multiplex biomarkers (cytokeratin/CK, CD45, and EpCAM), mesothelial cells consistently display positive CK, negative CD45, and negative EpCAM staining, reliably distinguishing them from epithelial and hematopoietic cells 8,20 …”

Detection of effusion tumor cells under different storage and processing conditions

“…Additionally, multiplex immunofluorescence facilitates detection and quantitation of useful site-of origin markers on malignant cells. 18 ETCs can not only be identified via multiplexed IF assay, but also be detected from RNA-ISH assay with probes detecting/targeting EpCAM and cancer type-specific biomarkers. 23 Next-generation DNA sequencing can detect pathogenic mutations with good concordance to clinical findings using as few as five malignant cells.…”

“…17 Further incorporation of cancer-specific immunofluorescent biomarkers also reveal tumor site-of-origin information from ThinPrep slides. 18 To broaden the potential of using this platform in both clinical and research settings, we evaluated its performance on different slide preparation, staining, and fixation conditions and after short-and long-term storage.…”

As few as five TTF1 positive cells in pleural effusions may provide comprehensive genomic information for single cell sequencing

“…The International System for Reporting Serous Fluid Cytopathology3 does not specify a number of malignant cells to be present for diagnosis 3 , and the current article by Zhu and colleagues 4 in this issue of Cancer Cytopathology 4 confirms that even a small number of cells may provide adequate information for confident clinical management.…”