Novel approach for detection of isolated tumor cells in pulmonary vein using negative selection method: morphological classification and clinical implications

Objective: The presence of isolated tumor cells (ITCs) in the pulmonary vein (PV) of a lung resected for lung cancer has been reported to be a prognostic factor. Previous investigations noted correlations between prognosis and the presence or amount of ITCs, although few studies have investigated the clinical implications of the morphological characteristics of those cells. We assessed the clinical implications of ITCs in the PV using a novel enrichment approach that maintained their morphological characteristics.

Methods: Ninety-four consecutive patients with primary non-small-cell lung cancer (NSCLC) without preoperative chemo- and/or radiation therapy (p-stage I in 75, II in 13, III or IV in six) were studied. Blood samples were drawn from the PV draining the lung just after pulmonary resection, and ITCs were enriched using a CD45-negative selection method and density-gradient centrifugation, followed by Papanicolaou staining using 1ml of PV blood and immunohistochemical staining for cytokeratin in cases with an additional available blood sample. The ITCs were classified into four types based on patterns of cluster formation: no tumor cells (N), singular tumor cells (S), clustered cells (≤ 0.2mm) (CSs), and bulky clustered cells (> 0.2mm) (BCSs). We evaluated the correlations between ITC morphology and clinical results.

Results: ITCs were detected in 68 of 94 patients (72%), of which the BCS type was observed in two, CS in 33, S in 33, and N in 26. Over a median follow-up period of 13 months (range 6-22 months), cancer recurrence occurred in 16 cases (17%): 14 in the combined CS/BCS group, one in S, and one in N. Log-rank analysis revealed that the disease-free survival rate was exclusively worse in patients with clustered ITCs as compared with the other two groups (p < 0.01).

Conclusions: The present method was useful to detect and enrich ITCs from the PV, and showed the clinical relevance of their morphology in lung cancer cases. The presence of ITC clusters may be a prognostic biomarker for patients with resected NSCLC.