Arjan G.J. Tibbe

Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases

Purpose: The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. Experimental Design: The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. Results: Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of ≥85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had ≥2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 ± 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had ≥2 CTCs. Detection of ≥2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. Conclusions: The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

Statistical considerations for enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) in patients with carcinomas are extremely rare. In metastatic breast cancer, the presence of ≥5 CTCs in 7.5 ml of blood has been associated with short survival. As this threshold has clinical implications, it is important to recognize the limitations associated with the detection and enumeration of CTCs.

Optical tracking and detection of immunomagnetically selected and aligned cells.

We have developed a platform for cell analysis based on immunomagnetic selection and magnetic alignment of cells in combination with an epi-illumination tracking and detection system. Whole blood was labeled with ferromagnetic nanoparticles and fluorescent probes, and placed in a magnetic field in a chamber. Cells labeled with ferromagnetic nanoparticles moved upward and aligned along ferromagnetic lines deposited by lithographic techniques on an optically transparent surface of the chamber. An epi-illumination system using a 635 nm laser diode as a light source scanned the lines and measured signals obtained from the aligned cells. The cell counts per unit of blood volume obtained with the system correlated well with those obtained from the counts from a standard hematology analyzer and flow cytometer. The cell analysis platform is significantly less complex and more sensitive than current cell analysis equipment and provides additional functionality through its ability to subject the cells to repeated and varied analyses while they remain in a natural environment (i.e., whole blood).

Circulating tumor cells in small-cell lung cancer: a predictive and prognostic factor

BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. Patients and methods In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14,040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.BACKGROUND Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. PATIENTS AND METHODS In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch® systems. RESULTS At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14 040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.

Comparison of Two Methods for Enumerating Circulating Tumor Cells in Carcinoma Patients

Monitoring of circulating tumor cells (CTCs) in blood of carcinoma patients treated with novel compounds may be a measurement of treatment effectiveness. Before it can be used clinically, a reliably method is needed to enumerate CTCs. We compared two methods for CTC enumeration, OnkoQuick and the CellSearch system.

Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer.

IntroductionThe presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free survival and breast cancer-related death (BRD) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence-free survival (RFS) and BRD.MethodsIn a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 602 patients before undergoing surgery for breast cancer. There were 97 patients with a benign tumor, 101 did not meet the inclusion criteria of which there were 48 patients with DCIS, leaving 404 stage I to III patients. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC = 0) prognostic groups.Results≥1 CTC in 30 ml blood was detected in 15 (15%) benign tumors, in 9 DCIS (19%), in 28 (16%) stage I, 32 (18%) stage II and in 16 (31%) patients with stage III. In stage I to III patients 76 (19%) had ≥1 CTC of whom 16 (21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four-year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (P = 0.038). A total of 25 patients died of breast cancer-related causes and 11 (44%) had ≥1 CTC. BRD was 4.3% for favorable and 14.5% for unfavorable CTC (P = 0.001). In multivariate analysis ≥1 CTC was associated with distant disease-free survival, but not for overall recurrence-free survival. CTC, progesterone receptor and N-stage were independent predictors of BRD in multivariate analysis.ConclusionsPresence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for breast cancer-related death.

Characterization of circulating tumor cells by fluorescence in situ hybridization

Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearch™ system. After enumeration of Cytokeratin+, CD45−, nucleated cells, the cells are fixed in the cartridge while maintaining their original position. Cartridges were hybridized with FISH probes against the centromeric regions of chromosome 1, 7, 8, and 17. Next fluorescence images of the FISH probes of the previous identified CTC were acquired. Leukocytes surrounding the CTC were used as internal controls. The number of copies of chromosome 1, 7, 8, and 17 could be determined in 118 CTC containing blood samples from 59 metastatic prostate cancer patients. The samples contained a total of 21,751 CTC (mean 184, median 16, SD 650). Chromosome counts were obtained in 61% of the relocated CTC. On an average, these CTC contained 2.8 copies of chromosome 1, 2.7 copies of chromosome 7, 3.1 copies of chromosome 8, and 2.3 copies of chromosome 17. CTC in which no chromosome count was obtained most likely underwent apoptosis indicated by the expression of M30. In 6/59 patients only diploid CTC were detected these samples, however, only contained 1–5 CTC. Heterogeneity in the chromosomal abnormalities was observed between CTC of different patients as well as among CTC of the same patient. Cytogenetic composition of CTC can be reliably assessed after they have been identified by the CellSearch™ system. The majority of CTC in hormone refractory prostate cancer are aneuploid confirming that they indeed are cancer cells. An extensive heterogeneity in the copy number of each of the chromosomes was observed.

The detection of EpCAM + and EpCAM - circulating tumor cells

EpCAM expressing circulating tumor cells, detected by CellSearch, are predictive of short survival in several cancers and may serve as a liquid biopsy to guide therapy. Here we investigate the presence of EpCAM+ CTC detected by CellSearch and EpCAM– CTC discarded by CellSearch, after EpCAM based enrichment. EpCAM– CTC were identified by filtration and fluorescent labelling. This approach was validated using different cell lines spiked into blood and evaluated on blood samples of 27 metastatic lung cancer patients. The majority of spiked EpCAM+ cells could be detected with CellSearch, whereas most spiked cells with EpCAMlow or EpCAM– expression were detected using filtration. Five or more CTC were detected in 15% of the patient samples, this increased to 41% when adding the CTC detected in the discarded blood. The number of patients with CTC and the number of CTC detected were doubled by the presence of EpCAM– CTC. In this pilot study, the presence of EpCAM+ CTC was associated with poor outcome, whereas the EpCAM– CTC were not. This observation will need to be confirmed in larger studies and molecular characterization needs to be conducted to elucidate differences between EpCAM– and EpCAM+ CTC.

A single platform image cytometer for resource-poor settings to monitor disease progression in HIV infection

For resource‐poor countries, affordable methods are required for enumeration of CD4+ T lymphocytes of HIV‐positive patients. For infants, additional determination of CD4/CD8 ratio is needed.

Cell Analysis System Based on Immunomagnetic Cell Selection and Alignment Followed by Immunofluorescent Analysis Using Compact Disk Technologies

BACKGROUND Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.