Importance of circulating tumor cells in newly diagnosed colorectal cancer

Background: The presence of circulating tumor cells (CTC) is associated with poor prognosis in patients with metastatic colorectal cancer (CRC). This study was conducted to determine if the presence of CTC prior to surgery and during follow-up in patients with newly diagnosed non-metastatic CRC identifies patients who are at risk for disease recurrence. Methods: In a prospective single center study 183 patients with newly diagnosed non-disseminated CRC scheduled for surgery were enrolled from 2003 till 2008 and followed up for a median of 5.1 years. CTC were enumerated with the CellSearch System in 4 aliquots of 7.5 ml of peripheral blood before and after surgery (1-26 weeks), after adjuvant therapy and 1, 2, 3 and 4 years after surgery. Findings: ≥1 CTC/ 30ml of blood were detected in 44 (24%) patients before surgery. CTC frequency did not change significantly at the time points after surgery. Patients with CTC before surgery had a significant decrease in Recurrence Free Survival (RFS, logrank test p=0.014) and Colon Cancer Related Survival (CCRS, p=0.002). Five year RFS dropped from 75% to 61% and five year CCRS from 83% to 69% for patients with CTC before surgery. In a multivariate analysis of CTC, T-Stage and N-stage, the presence of CTC and N-stage remained as significant factors for RFS and CCRS. Surprisingly the presence of CTC after surgery was not significantly associated with RFS and CCRS whereas CTC 2-3 years after surgery was again significantly associated with RFS and CCRS. Interpretation: The presence of CTC in patients with stage I-III CRC before surgery is associated with a significant reduction RFS and CCRS. Although similar amounts of CTC were detected within 3 months after surgery they were not associated with RFS or CCRS. In contrast CTC were again highly significant for RFS and CCRS 2-3 years after surgery. These findings suggest a role of CTC detection, to assess which patients need adjuvant treatment. To implement CTC detection in the non-metastatic setting a validated CTC detection technology is needed with increased sensitivity and specificity

Evaluation of HER2 expression in urothelial carcinoma cells as a biomarker for circulating tumor cells

Background Detection of circulating tumor cells (CTC) by techniques based on epithelial cell adhesion molecule (EpCAM) is suboptimal in urothelial carcinoma (UC). As HER2 is thought to be broadly expressed in UC, we explored its utility for CTC detection. Methods HER2 and EpCAM expression was analyzed in 18 UC cell lines (UCCs) by qRT-PCR, western blot and fluorescence-activated cell scanning (FACS) and compared to the strongly HER2-expressing breast cancer cell line SKBR3 and other controls. HER2 expression in UC patient tissues was measured by qRT PCR and correlated with data on survival and risk for metastasis. UCCs with high EpCAM and variable HER2 expression were used for spike-in experiments in the CellSearch system. Twenty-one blood samples from 13 metastatic UC patients were analyzed for HER2-positive CTCs with CellSearch. Results HER2 mRNA and protein were broadly expressed in UCC, with some heterogeneity, but at least 10-fold lower than in the HER-2+ SKBR3 cells. Variations were unrelated to cellular phenotype or clinicopathological characteristics. EpCAM expression was essentially restricted to UCCs with epitheloid phenotypes. Heterogeneity of EpCAM and HER2 expression was observed also in spike-in experiments. The 7 of 21 blood samples from 6 of 13 patients were enumerated as CTC positive via EpCAM, but only one sample stained weakly positive (1+) for HER2. Conclusions Detection rate of CTCs by EpCAM in UC is poor, even in metastatic patients. Because of its widespread expression, particularly in patients with high risk of metastasis, detection of HER2 could improve identification of UC CTCs, which is why combined detection using antibodies for EpCAM and HER2 may be beneficial

Classification of Cells in CTC-Enriched Samples by Advanced Image Analysis

In the CellSearch® system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4′6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 ± 74,948, as compared to 359 control samples with an average and standard deviation of 4191 ± 4463 (p < 0.001). In patients, only 18% ± 21% and in controls 23% ± 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% ± 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells

Quantifying HER-2 expression on circulating tumor cells by ACCEPT

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch (R) and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches