Circulating Tumor Cells in Diagnosing Lung Cancer: Clinical and Morphologic Analysis Alfonso Fiorelli, MD, PhD, Marina Accardo, MD, Emanuele Carelli,

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Image 3 Cytomorphologic criteria for circulating nonhematologic cells with benign features obtained by the isolation by size of epithelial tumor cell method.

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Circulating Tumor Cells in Diagnosing Lung Cancer: Clinical and Morphologic Analysis Alfonso Fiorelli, MD, PhD, Marina Accardo, MD, Emanuele Carelli, MD, Denise Angioletti, MD, Mario Santini, MD, Marina Di Domenico, MD The Annals of Thoracic Surgery Volume 99, Issue 6, Pages 1899-1905 (June 2015) DOI: 10.1016/j.athoracsur.2014.11.049 Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) CNHC-MF isolated from patient with squamous carcinoma (clinical stage II). Cells on isolation by size of tumor cells membrane stained with hematoxylin and eosin (H&E) stain showed anisonucleosis (*); nuclei larger than 3 times the calibrated 7.5-μm pore (**) size of the membrane; irregular nuclear outline (black arrow); and high nuclear to cytoplasm ratio. (B) Clusters of circulating non-hematologic cells (CNHC) with malignant features (CNHC-MF), named as circulating tumor microemboli, isolated from patient with adenocarcinoma (clinical stage IV); H&E showed 3 or more distinct nuclei (*). (C) CNHC with uncertain malignant features (CNHC-UMF) isolated from patient with squamous carcinoma (clinical stage I); the H&E showed the tridimensional cellular sheets. The presence of this criteria did not allow a definitive diagnosis of malignancy. (**) The calibrated 7.5-μm pore size of the membrane. (D) CNHC with benign features (CNHC-BF) from a patient with fibrotic lesion; H&E showed no malignant feature. (Magnification for images A, B, C, D is 400×.) The Annals of Thoracic Surgery 2015 99, 1899-1905DOI: (10.1016/j.athoracsur.2014.11.049) Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Flowchart of study population. (CNHC = circulating non-hematologic Cells; CNHC-MF = CNHC with malignant features; CNHC-UMF = CNHC with uncertain malignant features; pts = patients.) The Annals of Thoracic Surgery 2015 99, 1899-1905DOI: (10.1016/j.athoracsur.2014.11.049) Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 (A) Receiver operating characteristic curve showed a sensitivity, specificity, positive predictive value, and negative predictive, of 89%; 100%; 100%, respectively; and 81% for a circulating non-hematologic cells with malignant features (CNHC-MF) count greater than 25 (area under the curve, 0.9; standard error: 0.02; 95% confidence interval 0.869% to 0.985%; p < 0.0001). (B) A significant difference (p < 0.05) of CNHCs-MF count was seen between the different clinical stages (analysis of variance test). (C) CNHCs-MF counts were significantly correlated with tumor size (r = 0.5; p = 0.001; Spearman rank correlation test) in stage I lung cancer patients and with standard uptake value (SUV) value (r = 0.6; p < 0.0001; Spearman rank correlation test) in all lung cancer patients (D). The Annals of Thoracic Surgery 2015 99, 1899-1905DOI: (10.1016/j.athoracsur.2014.11.049) Copyright © 2015 The Society of Thoracic Surgeons Terms and Conditions