Patterns of circulating tumor cells identified by CEP8, CK and CD45 in pancreatic cancer.

To improve the identification for CTCs with weak or negative CK and diploid CTCs in pancreatic cancer, we combined immune-staining of CK, CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome 8 (CEP8) probe method. CTCs in 3.75 mL of blood were depleted for CD45 positive cells with anti-CD45 antibodies and identified by combining CK, CD45, DAPI and CEP8 in 61 cases including 22 pancreatic cancers, 3 borderline pancreatic solid pseudopapillary tumors, 6 pancreatic benign tumors, and 30 healthy individuals. We found that enriched cells could be classified into 5 patterns: CK+CD45-DAPI+CEP8=2 (2 hybridization signals), CK+CD45-DAPI+CEP8>2 (>2 hybridization signals), CK-CD45-DAPI+CEP8>2, CK-CD45-DAPI+CEP8=2, and CK+/-CD45+DAPI+CEP8=2 or >2. Among 22 pancreatic cancers, CK+CD45-DAPI+CEP8=2 and CK+CD45-DAPI+CEP8>2 patterns were identified in two cases, and CK-CD45- DAPI+CEP8>2 pattern was identified in 16 cases. CK-CD45-DAPI+CEP8=2 and CK+/-CD45+DAPI+CEP8=2 or >2 patterns were detected in pancreatic cancers, other pancreatic diseases and healthy individuals. Among the five patterns, CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2 and CK-CD45-DAPI+CEP8>2 were considered as CTCs, while CK-CD45-DAPI+CEP8=2 and CK+/-CD45+DAPI+CEP8=2 or >2 were considered as indeterminate cells. When the cutoff value was set as 2 cells/3.75 mL based on ROC curve, the sensitivity and specificity in the diagnosis of pancreatic cancer was 68.18 and 94.87%, respectively. Dynamically monitoring CTCs changes prior to and after surgery in pancreatic patients revealed that CTCs count decreased in 3 days after surgery, but increased in 10 days after surgery in most patients. During our one and a half year follow-up, CTCs positive patients showed metastasis and worse survival rate.