Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting

Fehm, Tanja; Meier-Stiegen, F; Driemel, C; Jäger, Bernadette; Reinhardt, F; Naskou, Johanna; Franken, A; Neubauer, Hans; Neves, Rui P.; Dalum, Guus van; Ruckhäberle, Eugen; Niederacher, Dieter; Rox, Jutta M.; Fischer, Johannes C.; Stoecklein, Nikolas H.

doi:10.1002/cyto.a.23669

Cited by 64 publications

(85 citation statements)

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“…Enumeration of CTCs in humans with OSA using a combination of leukocyte depletion, immunochemistry, and fluorescent in situ hybridization identified significant differences in progression-free survival among patients with ≤2 versus >2 CTC/7.5 ml of blood (30). Diagnostic leukapheresis of 60 ml of blood with continuous centrifugation and return of all blood elements, except the mononuclear the cell fraction including carcinoma CTCs, was recently described, and dramatically increased the number of CTCs detected, and therefore the repeatability of enumeration (32). Diagnostic leukapheresis of 60 ml of blood with continuous centrifugation and return of all blood elements, except the mononuclear the cell fraction including carcinoma CTCs, was recently described, and dramatically increased the number of CTCs detected, and therefore the repeatability of enumeration (32).…”

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“…Furthermore, using quantitative PCR techniques, detection of osteospecific mRNA in blood of humans with OSA was significantly associated with the development of metastases during the subsequent 12-months period (31). Diagnostic leukapheresis of 60 ml of blood with continuous centrifugation and return of all blood elements, except the mononuclear the cell fraction including carcinoma CTCs, was recently described, and dramatically increased the number of CTCs detected, and therefore the repeatability of enumeration (32).…”

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Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

et al. 2019

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Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumorbearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/10 6 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/10 6 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. OSTEOSARCOMA (OSA) is a primary bone cancer that is common in middle-aged to older, large breed dogs, and less common in humans, where children and young adults are most often affected. Therapy consisting of limb amputation or limbsparing tumor resection followed by chemotherapy (typically platinum or doxorubicin-based) is the gold standard, but the clinical outcome of OSA in dogs and humans remains poor (1-3). Dogs with OSA of the appendicular skeleton treated with amputation and chemotherapy had median survival times (STs) of 235-540 days (4), while in humans 5-year survival has been stagnant at about 60% (5). Although the median ST is relatively homogeneous, the range of ST in dogs with OSA treated identically varies from 2 to >30 months, illustrating that there is extensive heterogeneity in tumor biology and/or host response (6-8). Metastatic disease is the usual cause of spontaneous death or euthanasia, but fewer than 10% of dogs have radiographic evidence of metastasis at the time of diagnosis (2).Computed tomography also lacks sensitivity for identifying metastases, and histologic grading schemes for canine OSA poorly predict biological behavior (9-11).

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Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

et al. 2019

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“…The use of apheresis was introduced to enable processing of the mononuclear cell fraction of large blood volumes (34). In this issue, clinical results and recommendations for standardized reporting of DLA results are presented (37). However, at present the available technologies are not yet capable of isolating CTC from the complete DLA volume (34)(35)(36).…”

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“…However, at present the available technologies are not yet capable of isolating CTC from the complete DLA volume (34)(35)(36). In this issue, clinical results and recommendations for standardized reporting of DLA results are presented (37). To provide direction to the CTC and tdEV field we show in figure 2 the frequency, size and density of the different cells detected in blood.…”

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CTC Technologies and Tools

Coumans

Dalum

Terstappen³

2018

Cytometry Pt A

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CANCER cells can enter the blood stream. Some of these circulating tumor cells (CTC) extravasate and form distant metastases which ultimately lead to the death of the patient. CTC have been observed quite some time ago (1-3). The major difficulty for the identification of CTC is their extremely low frequency (~1/ml in metastatic patients). When no CTC were detected in a tube of blood of a patient with metastases one always wonders whether they were not present or whether they were missed by the detection technology. Another hurdle is the extensive heterogeneity of the physical and immunological appearance of CTC within and between patients.Tumor cell lines play a pivotal role in both informing technology developers of the probable physical and immunological properties of CTC, as well as in the determination of the analytical accuracy, reproducibility, and linearity of any developed technology. However, tumor cell lines are not CTC. Excellent performance on tumor cell lines does not warrant that (1) the developed technology will actually identify CTC in blood samples of cancer patients nor that (2) these CTC relate to clinical outcome nor that (3) information relevant for treatment can be extracted.At the start of the trajectory toward the FDA cleared CellSearch system very little was known about the CTC frequency and their physical and immunological appearance. CTC enrichment in 10 ml of blood was performed by manual immunomagnetic enrichment targeting EpCAM using the GA73.3 antibody. The enriched cell suspension was fluorescently labeled with the PE-labeled CAM5.2 antibody recognizing cytokeratins 7 and 8, a PerCP-labeled antibody recognizing CD45 and a nucleic acid dye that could be measured in the FITC channel of a flowcytometer. Using this approach, it was first shown that CTC indeed could be detected in 10 ml blood of cancer patients with metastatic disease and at a lesser frequency in patients with no detectable metastasis while few "CTC" were detected in healthy controls (4-8). These results were sufficiently encouraging to further develop the CTC technology and design and conduct clinical studies to explore the clinical utility of CTC. The commercial CellSearch system that was ultimately developed included (1) a blood collection tube to preserve the fragile CTC for a period of up to 96 h, (2) an automated sample preparation system in which (3) the EpCAM antibody was replaced by VU1D9, (4) CAM5.2 cytokeratin antibody was replaced by C11 and A.53B/A2 to increase the range of cytokeratins, (5) the CD45-PerCP was switched to CD45-APC, (6) the nucleic acid dye was switched to DAPI, and (7) the flow cytometer replaced by a semi-automated fluorescence microscope with dedicated software to present CTC candidates to a reviewer. Thanks to an excellent team of reagent developers and engineers the system was reliable and provided accurate results over a large range of spiked tumor cells in blood with a high sensitivity and specificity. Whereas few "CTC" were detected in healthy donors and patients with benign disease...

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“…664), and their focus is to improve instruments that can detect “circulating tumor cells” (CTCs). CTCs have been a focus in cytometry for several years and although they are defined differently depending on the clinical case, we continue to see contributions that describe efforts toward CTC measurements in vivo . In fact, we recently published an entire issue focused on CTC detection (volume 93, issue 12).…”

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Apoptosis and autophagy

Houston

2019

Cytometry Pt A

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IN the March issue of this year, a collection of articles on cancer and cytometry (1-3) were published together and highlighted by Editor in Chief, Prof. Attila Tárnok. Our particular focus on those published articles made obvious the fact that flow cytometry continues to play an essential role in the fundamental understanding of cancer progression. The publications had foci that ranged from studies of cancer at a fundamental level to use of mass and imaging cytometry for clinical/therapeutic outcomes. Yet beyond that single issue and our journal as a whole, there exists a massive assemblage of published work (see Fig. 1) that chronicle the salient partnership between cancer and cytometry. Therefore, it is unsurprising that several more articles on the subject of cancer can again be found in the current issue of Cytometry Part A. Hence, we found it not only appropriate to compile a special section related to cancer this month, but also consequential, because of the particular commonality in the research presented. That is, each paper included in our special section demonstrates a new approach for understanding and/or detecting intracellular mechanisms that malfunction during cancer progression-of course, when revealed by flow cytometry.We draw your attention to a group of four original articles on apoptosis and autophagy. These regulatory processes are often studied in cancer cells because they require staged intracellular mechanisms, which if fully understood can be exploited as therapeutic targets. Cytometry as a multiparametric tool has been employed for decades to study these mechanisms (see a few recent publications on these subjects in References 4-8), and the publications we highlight herein exemplify modern approaches toward that end.Four papers in this issue focus on evaluating differences in cells that are either treated with compounds to initiate apoptosis, autophagy, or that circulate in vivo after intravenous injection into anesthetized mice. In the latter case, two articles are featured in which cell counting instruments are designed to detect fluorescence from cells (apoptotic and nonapoptotic) in vivo, as they move through the blood stream after injection. Many of these reports present multiparametric measurements from either in vivo events or from treated cell suspensions, and in doing so demonstrate unique advances to how cytometry can be exploited for diagnosis of cancer or identification of functional traits that occur at the onset of regulated cell death.The paper by Halicka and colleagues (this issue, pp. 683) discusses how multiparametric cytometry (side scatter and fluorescence) is used on apoptosis-induced cells to quantify function of transglutaminase 2 (TG2) and the activation of lysosomal proton pumps as a result of cell treatment by a variety of compounds known to induce DNA damage or cytotoxic outcomes. When detecting the lack of accumulation of acridine orange in compromised lysosomes and protein crosslinking activity of TG2 via scattered light signals, both phenomena can be measure...

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Diagnostic leukapheresis for CTC analysis in breast cancer patients: CTC frequency, clinical experiences and recommendations for standardized reporting

Cited by 64 publications

References 18 publications

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

CTC Technologies and Tools

Apoptosis and autophagy

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