Identification and characterization of effusion tumor cells (ETCs) from remnant pleural effusion specimens

Abstract

BACKGROUND:Cancer is a leading cause of death worldwide, and patients may have advanced disease when diagnosed.Targeted therapies guided by molecular subtyping of cancer can benefit patients significantly. Pleural effusions are frequently observed in patients with metastatic cancer and are routinely removed for therapeutic purposes; however, effusion specimens have not been recognized as typical substrates for clinical molecular testing because of frequent low tumor cellularity. METHODS: Excess remnant pleural effusion samples (N = 25) from 21 patients with and without suspected malignancy were collected at Stanford Health Care between December 2019 and November 2020. Samples were processed into ThinPrep slides and underwent novel effusion tumor cell (ETC) analysis. The ETC results were compared with the original clinical diagnoses for accuracy. A subset of confirmed ETCs was further isolated and processed for molecular profiling to identify cancer driver mutations. All samples were obtained with Institutional Review Board approval. RESULTS: The authors established novel quantitative standards to identify ETCs and detected epithelial malignancy with 89.5% sensitivity and 100% specificity in the pleural effusion samples. Molecular profiling of confirmed ETCs (pools of 5 cells evaluated) revealed key pathogenic mutations consistent with clinical molecular findings. CONCLUSIONS: In this study, the authors developed a novel ETC-testing assay that detected epithelial malignancies in pleural effusions with high sensitivity and specificity.Molecular profiling of 5 ETCs showed promising concordance with the clinical molecular findings. To promote cancer subtyping and guide treatment, this ETC-testing assay will need to be validated in larger patient cohorts to facilitate integration into cytologic workflow.

Beyond Circulating Tumor Cell Enumeration: Cell-Based Liquid Biopsy to Assess Protein Biomarkers and Cancer Genomics Using the RareCyte® Platform

“…These small-volume samples can be difficult to thoroughly evaluate by traditional methods that require multiple slides for immunohistochemistry or sufficient material for sequencing. But since they are similar to buffy coat smears, they could be analyzed using the liquid biopsy methods described here for multi-protein biomarkers and target cell sequencing (Zhu, et al, 2021). An APC nonsense mutation (R283*) was identified in three of the CTCs, with variant allele frequencies ranging from 22 to 37%.…”

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

“…By comparison, the previously described ETC panel has separate CK and EpCAM channels as positive markers, CD45 to exclude leukocytes, and a nuclear marker (0700-MA CTC Panel). 8 Parallel model cancer cells and patient sample TP slides were fixed in 10% neutralized buffered formalin for 20 min and then incubated at 100°C in 1 × Thermo Scientific Citrate Buffer for Heat-Induced Epitope Retrieval, pH 6.0, for 10-15 min. Pretreated slides were then incubated with anti-TTF-1 antibody at different concentrations (0.33, 0.67, and 1 μg/mL; Ventana SP141) and a fluorophore-conjugated secondary antibody (AlexaFluor 488; 0.1 μg/mL), followed by multiplexed panel staining.…”

“…To obtain high-quality DNA libraries of T-ETCs and parallel ETCs, we collected three representative patient samples from the recently processed samples, as described in our previous study. 8 This time, we also collected one sample of CD45-positive cells from each patient as an internal negative control. In brief, the immunofluorescentstained patient slides were de-coverslipped, and single T-ETCs or ETCs were retrieved using the CyteFinder II HT Instrument with the integrated CytePicker Retrieval Module.…”

“…6,7 Previously, we reported the development of an epithelial malignancy-detection panel examining pleural effusion specimens (an alternative source of tumor material) with the capacity to perform molecular profiling from rare tumor cells. 8 This effusion tumor cell (ETC) assay segregated carcinoma cells from background leukocytes and mesothelial cells with high fidelity, but it lacked the capacity to provide tumor site-of-origin information. Because most of the cases we obtained were diagnosed with lung ACAs, we sought to incorporate a pulmonaryspecific biomarker as our pilot additional marker to expand the capacity of our previous ETC panel.…”

“…Furthermore, the molecular profiling results from our previous study exhibited only 33%-60% accuracy in reporting pathogenic mutations and requires further improvement. 8 In the current study, we successfully incorporated the lung ACA-specific biomarker TTF-1 into a new multiplexed immunofluorescent panel to provide both malignancy and tumor origin information. New T-ETC analysis and original ETC analysis were performed in parallel on all samples and were compared with clinical diagnoses.…”