Sjoerd Ligthart

Challenges in the enumeration and phenotyping of CTC

Purpose: Presence of circulating tumor cells (CTC) in metastatic carcinoma is associated with poor survival. Phenotyping and genotyping of CTC may permit “real-time” treatment decisions, provided CTCs are available for examination. Here, we investigate what is needed to detect CTC in all patients. Experimental Design: CTCs enumerated in 7.5 mL of blood together with survival from 836 patients with metastatic breast, colorectal, and prostate cancer were used to predict the CTC concentration in the 42% of these patients in whom no CTCs were found and to establish the relation of concentration of CTCs with survival. Influence of different CTC definitions were investigated by automated cell recognition and a flow cytometric assay without an enrichment or permeabilization step. Results: A log-logistic regression of the log of CTC yielded a good fit to the CTC frequency distribution. Extrapolation of the blood volume to 5 L predicted that 99% of patients had at least one CTC before therapy initiation. Survival of patients with EpCAM+, cytokeratin+, CD45− nucleated CTCs is reduced by 6.6 months for each 10-fold CTC increase. Using flow cytometry, the potential three-fold recovery improvement is not sufficient to detect CTC in all patients in 7.5 mL of blood. Conclusions: EpCAM+, cytokeratin+, CD45− nucleated CTCs are present in all patients with metastatic breast, prostate, and colorectal cancer and their frequency is proportional to survival. To serve as a liquid biopsy for the majority of patients, a substantial improvement of CTC yield is needed, which can only be achieved by a dramatic increase in sample volume. Clin Cancer Res; 18(20); 5711–8. ©2012 AACR.

Circulating Tumor Cells Count and Morphological Features in Breast, Colorectal and Prostate Cancer

Background Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters. Methodology Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival. Results Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected. Significance of the study CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer.

Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non-metastatic breast cancer

Background Circulating tumor cells (CTCs) can provide the basis for a liquid biopsy and may guide the use of targeted therapies. We report on unbiased quantification of Her-2 protein expression of CTCs. Patients and methods Her-2 assessment of CTCs was carried out using the CellSearch(®) system in 103 metastatic (M1) and 88 non-metastatic (M0) breast-cancer patients. Expression of Her-2 on CTCs was determined by a manual review and an automated algorithm using Her-2- fluorescein isothiocyanate (FITC) fluorescence of leukocytes to determine the Her-2-expression threshold in each sample. Results Her-2 expression of CTCs varied greatly within and among patients compared with Her-2 expression of leukocytes. In M1 patients, a threshold of 75% of Her-2 positive CTCs in patients with ≥5 CTCs was set. Applying this threshold, 9% of M1 patients with Her-2-negative primary tumors had Her-2-positive CTC status and 29% of M1 patients with Her-2-positive primary tumors had Her-2-negative CTC status. No Her-2 discrepancy was observed between CTCs and primary tumors in M0 patients. Conclusions Our findings demonstrate that Her-2 expression is heterogeneous among CTCs within each patient. We show the feasibility of unbiased quantitative and reproducible assessment of treatment targets on CTCs, opening a path towards personalized treatment.

Unbiased and Automated Identification of a Circulating Tumour Cell Definition That Associates with Overall Survival

Circulating tumour cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC however remains subjective, as they are morphologically heterogeneous. We acquired digital images, using the CellSearch™ system, from blood of 185 castration resistant prostate cancer (CRPC) patients and 68 healthy subjects to define CTC by computer algorithms. Patient survival data was used as the training parameter for the computer to define CTC. The computer-generated CTC definition was validated on a separate CRPC dataset comprising 100 patients. The optimal definition of the computer defined CTC (aCTC) was stricter as compared to the manual CellSearch CTC (mCTC) definition and as a consequence aCTC were less frequent. The computer-generated CTC definition resulted in hazard ratios (HRs) of 2.8 for baseline and 3.9 for follow-up samples, which is comparable to the mCTC definition (baseline HR 2.9, follow-up HR 4.5). Validation resulted in HRs at baseline/follow-up of 3.9/5.4 for computer and 4.8/5.8 for manual definitions. In conclusion, we have defined and validated CTC by clinical outcome using a perfectly reproducing automated algorithm.

Automated identification of circulating tumor cells by image cytometry

Presence of circulating tumor cells (CTC), as detected by the CellSearch® System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 40×/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright‐field images. Improved detection efficiency for CD45‐APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated.

CellTracks TDI: an Image Cytometer for Cell Characterization.

Characterization of rare cells usually requires high sensitivity quantification of multiple parameters. Detection of morphological features of these cells is highly desired when routinely identifying circulating tumor cells (CTC) in blood of patients. We have designed an image cytometer intended for fast and sensitive routine analysis of CTC. After an initial scan, prospective events can be revisited for more detailed analysis. The image cytometer features: 375, 491, and 639 nm laser lines, a 40×/0.6NA objective, a CCD camera operating in TDI mode, servo stages to move the sample in two dimensions and a piëzo microscope objective positioner to move the objective in the third dimension. ImageJ is used for dedicated image analysis. A homogeneous illumination area, measuring 180 × 180 μm2, was created by the use of a rotating diffuser in combination with two micro‐lens arrays. For feed‐forward automatic focusing of the sample during a scan, a 3D spline was fitted through 30 predetermined focus positions before scanning the sample. Continuous signal acquisition is made possible by using a CCD operating in TDI mode synchronized to the movement of two servo scan stages. The limit of fluorescence sensitivity is 120 PE molecules on a bead with a diameter of 6.8 μm, at a scanning speed of 1.0 mm s−1. The resolution of the imaging system is 0.76 μm in the TDI scan direction at a wavelength of 580 nm. Identification of cells is facilitated by scatter plots of the fluorescent parameters in which each individual event can be viewed for its morphological features by fluorescence as well as bright field. The image cytometer measures quantitative fluorescence and morphological features at a high sensitivity, high resolution, and with minimal overhead time. It has the ability torelocate events of interest for further detailed analysis. The system can be used for routine identification and characterization of rare cells.

Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non metastatic breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Circulating tumor cells (CTC) can provide the basis for a real-time liquid biopsy and may guide the use of targeted therapies. We report on unbiased quantification of Her-2 protein expression of CTC. Her-2 assessment of CTC was performed using the CellSearch® system in 103 metastatic (M1) and 88 non metastatic (M0) breast-cancer patients. Digital images of Cytokeratin-PE, DAPI, CD45-APC, and Her-2-FITC from these samples were stored. Expression of Her-2 on CTC was determined by manual review and an automated algorithm using Her-2-FITC fluorescence of leukocytes to determine the Her-2-expression threshold in each sample. Her-2 expression of CTC varied greatly within and between patients compared to Her-2 expression of leukocytes. In M1 patients, a threshold of 75% of Her-2 positive CTC in patients with >5 CTC showed a relatively low discrepancy rate between the primary tumor and CTC Her-2 status. Applying this threshold, 9% of M1 patients with Her-2 negative primary tumors had Her-2 positive CTC status and 29% of M1 patients with Her-2 positive primary tumors had Her-2 negative CTC status. No Her-2 discrepancy was observed between CTC and primary tumor in M0 patients. Our findings demonstrate the feasibility of real-time quantitative and reproducible assessment of treatment targets on CTC, opening a path towards personalized treatment. Her-2 expression is heterogeneous among CTC within each patient. Overall, M1 patients with Her-2 positive primary tumors exhibited Her-2 negative CTC frequently, whereas discrepancies in Her-2 status were limited in other clinical settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2368. doi:1538-7445.AM2012-2368

FISH Probe Counting in Circulating Tumor Cells

Presence of tumor cells in blood of patients with metastatic carcinomas has been associated with poor progression free and overall survival (Cohen et al., 2008; Cristofanilli et al., 2004; de Bono et al., 2008). Assessment of treatment targets on circulating tumor cells (CTC) before initiation of therapy may provide a means to guide therapy (Attard et al., 2009; de Bono et al., 2007; Hayes et al., 2002; Meng et al., 2006; Meng et al., 2004; Smirnov et al., 2005; Swennenhuis et al., 2009). Characterization of CTC can be performed by Fluorescence In Situ Hybridization (FISH) which has been used to prove that CTC are indeed malignant (Fehm et al., 2002; Swennenhuis, et al., 2009), and that gene amplifications, deletions and translocations related to certain therapies can be detected (Attard, et al., 2009; Meng, et al., 2006; Meng, et al., 2004).

Abstract 2391: All patients with metastatic breast, colorectal and prostate carcinoma have circulating tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Presence of EpCAM+ cytokeratin+ nucleated circulating tumor cells (CTC) in metastatic carcinoma patients is associated with poor survival and may be used to guide treatment. Assessment of treatment targets on these CTC holds the promise of a liquid biopsy. However the proportion of patients in which a sufficient number of CTC are detected in 7.5 mL of blood is not sufficient. In this study we estimate the number of CTC in blood of patients with metastatic disease, explore the relationship with survival and determine whether assay modifications and/or increases in sample volume are needed to achieve the required increase in number of CTC detected. Methods: EpCAM+CK+DNA+CD45- CTC enumeration was performed with the CellSearch® system in 7.5 mL of blood of 836 patients with metastatic breast, colorectal and prostate cancer patients (Cristofanilli, NEJM 2004, Cohen, JCO 2008, De Bono, CCR 2008). EpCAM+, CD45-, Nucleic acid+ CTC were enumerated in 100ul of NH4CL lysed blood from 140 metatatic cancer patients and compared to CTC as enumerated with the CellSearch system. Image analysis for automated CTC enumeration and modeling of CTC frequencies was performed in Matlab. Results: The median number of CTC in 7.5mL of blood metastatic breast, colorectal and prostate is 5 and in 39% of patients no CTC are detected. The best fit of the CTC frequency distribution was used to extrapolate the sample volume to 5 liters of blood and predicted that 99% (95% confidence interval, CI, 95-99.8%) of patients had at least 1 CTC before initiation of therapy, which decreased to 97% (95% CI 87-99.5%) after the first cycles of therapy. One hundred CTC per liter of blood are present in ∼80% of patients, 1 CTC per ml of blood in ∼40% of patients and 10 CTC per ml of blood in ∼20% of patients. The median survival of patients with CTC is reduced by 6.6 months for each tenfold CTC increase. CTC definitions that do not include a requirement for presence of DNA, EpCAM, cytokeratin 8,18,19 (CK) or absence of CD45 are less predictive of survival, than the strictest definition requiring a CTC to be EpCAM+CK+DNA+CD45-. Comparison of CTC detected by flow cytometry to the CellSearch CTC method on 140 patients shows that improvements in EpCAM recovery may increase the average number of detected CTC by 6.5 fold (95% CI 5.6-7.3), yet the fits predict that this will only reduce the number of patients with 0 CTC from 39% to 21% (95% CI 13-31%). Conclusions: EpCAM+CK+DNA+CD45- CTC are present in blood of all patients with metastatic breast, colorectal and prostate cancer. The predicted strong relation with survival suggests the importance of this phenotype for metastasis. To use CTC as a liquid biopsy for the majority of patients, the CTC yield needs to be improved 100-1000 fold. This requires a dramatic increase in sample volume which may be achieved by in vivo flow cytometry, or through processing an apheresis product. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2391. doi:1538-7445.AM2012-2391

Abstract 4175: Automated classification of circulating tumor cells optimized using clinical outcome of castration resistant prostate cancer patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Circulating tumor cells (CTC) in patients with metastatic carcinomas are associated with poor survival and can be used to guide therapy. Classification of CTC can however be subjective as they are morphologically heterogeneous. Method: Digital images from 175 castration resistant prostate cancer (CRPC) patients and 68 healthy donors acquired using the CellSearch(TM) system during the multicenter prospective study IMMC38 (de Bono JS, et al. ClinCanRes 2008;14(19):6302-09) were used to define CTC with algorithms developed in Matlab 2009a. Objects were segmented in the images of Cytokeratin-PE, DAPI, CD45-APC, a control channel, PE+DAPI, and the maximum intensity profile of the channels. The patients were dichotomized on the median number of objects. The Cytokeratin-PE channel returned the highest Cox Hazard Ratio (HR) and was chosen for feature extraction. Features were measured from objects in each channel. For each feature, the patients were again dichotomized on the median number of objects included after thresholding. Using only samples before initiation of treatment (baseline), the standard deviation of Cytokeratin-PE signal, the size of the objects, the peak value of the DAPI signal and the peak value of CD45-APC signal had the highest impact on the HR and a low correlation with each other. They were chosen as the features for CTC classification. Classifiers representing different morphological CTC definitions were created from these features and tested using different value ranges. Bootstrap aggregating was used to determine the robustness of the classifier. To arrive at the optimal classifier the first samples after initiation of therapy from patients (follow-up) and control samples were included in the analysis. CTC definitions to be selected were to have the most statistically significant association with overall survival (HR), and a low total count in the control samples. Results: Programmed automated computerized CTC image analyses generated CTC definitions that resulted in a HR of 3.1 for baseline and 5.2 for follow-up samples. These results were equivalent to the laborious and time-consuming manual identification of CellSearch CTC by trained operators which had resulted in HR of 3.2 and 4.6 respectively. Overall, median CTC count was 7 for baseline and 3 for follow-up samples. A total of 9 CTC were found in 68 control samples. Critically, processing of a CTC blood sample was very rapid, taking approximately 1 minute per patient. Conclusions: We have automated CTC counting utilizing a classification that optimally dichotomized castration resistant prostate cancer patient based on clinical outcome. This method is reproducible and rapid, allowing standardization of CTC counting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2011-4175