Prognostic Role Of Circulating Tumor Cells Research Articles

Longitudinal Assessment of Circulating Tumor Cells and Outcome in Small Cell Lung Cancer: A Sub-Study of RASTEN-A Randomized Trial with Low Molecular Weight Heparin.

Circulating tumor cells (CTCs) may provide a liquid biopsy approach to disease monitoring in small cell lung cancer (SCLC), a particularly aggressive tumor subtype. Yet, the prognostic role of CTCs during and after treatment in relation to baseline remains ill-defined. Here, we assessed the value of longitudinal CTC analysis and the potential of low-molecular-weight heparin (LMWH) to reduce CTC abundance in SCLC patients from a randomized trial (RASTEN). Blood samples were collected at baseline, before chemotherapy Cycle 3, and at 2-month follow-up from 42 patients in total, and CTCs were quantified using the FDA-approved CellSearch system. We found a gradual decline in CTC count during and after treatment, independently of the addition of LMWH to standard therapy. Detectable CTCs at baseline correlated significantly to reduced survival compared to undetectable CTCs (unadjusted hazard ratio (HR) of 2.75 (95% CI 1.05-7.20; p = 0.040)). Furthermore, a persistent CTC count at 2-month follow-up was associated with a HR of 4.22 (95% CI 1.20-14.91; p = 0.025). Our findings indicate that persistently detectable CTCs during and after completion of therapy offer further prognostic information in addition to baseline CTC, suggesting a role for CTC in the individualized management of SCLC.

CK+/CD45+ (dual-positive) circulating cells are associated with prognosis in patients with advanced breast cancer.

1093 Background: Circulating tumor cells (CTCs) expressing epithelial markers (EPCAM, cytokeratin (CK)) and lacking CD45 (a leukocyte marker) have been associated with poor outcome in many cancer types. Nonetheless, the presence of cells expressing both CK and CD45 (CK+/CD45+), circulating in the blood of cancer patients (pts) have also been reported, but not widely investigated. Early evidence indicates that circulating dual-positive cells (DPcells) are hybrids deriving from the fusion of tumor cells and macrophages. We previously reported that it is possible to detect DPcells in the blood of pts with metastatic breast cancer (BC) and that they are associated with shorter progression-free survival (PFS), in pts with <5 CK+/CD45- CTCs. Here, we investigated the impact of DPcells on overall survival (OS) in pts with advanced BC (aBC). Methods: Blood samples (7.5 ml) were collected from aBC pts before starting a new therapy and processed with the FDA-approved CellSearch platform for CTCs and DPcells enumeration. The prognostic role of CTCs and DPcells was assessed through the Kaplan-Meier method using the log-rank test. Single DPcells were isolated using the DEPArray platform and underwent whole genome amplification and lowpass whole genome sequencing (Ampli1 WGA and Ampli1 Lowpass kits). Results: Blood samples from 341 pts with luminal (n=168), HER2+ (n=76) and triple negative (n=88) BC were analyzed. Of these, 131 samples (38.4%) contained ≥5 CTCs (CTCpos), whereas DPcell were detected in 152 samples (44.6%, range 0-53), of which 66 (43.4%) were CTCpos and 86 (56.6%) CTCneg. Overall, DPcells were associated with a shorter OS: median OS 24.5 vs 35.0 months, p=0.046. However, when analyzing CTCpos and CTCneg separately, only the latter group showed a difference in OS according to DPcells presence. In particular, among CTCneg pts, those with ≥4 DPcells showed a 2.3-fold shorter OS (26.7 vs 60.6 months, p=0.025). Moreover, pts with ≥4 DPcells were less likely to experience a 6-months PFS clinical benefit (p=0.015). Interestingly, in the analysis by BC subtype, DPcells were confirmed to be associated with worse OS only in pts with triple negative BC (median OS 11.5 vs 16.9, p=0.048). To explore the exiology of DPcells, 2 out of 3 cells analyzed after single-cell isolation from 1 patient were confirmed to have copy number alterations (CNA) consistent with malignant cells. CNA and mutational profiling of additional single DPcells and CTCs are ongoing. Conclusions: DPcells are associated with worse OS in aBC pts, with the prognostic impact primarily in pts with <5 CTCs and triple negative BC. This suggests that DPcells might be an alternative way of tumor dissemination in specific pts, in which CK+/CD45- CTCs are less prevalent. More studies are needed to better elucidate DPcell clinical significance in BC, and to confirm their fusion-hybrid origin.

Abstract 584: Circulating tumor cells as prognostic biomarkers in glioblastoma

Abstract Background: Glioblastoma (GBM) is the most common and aggressive type of tumor arising from the central nervous system. GBM remains an incurable disease despite current advancement in therapies, with survival rates of approximately 15 months. Recent literature has highlighted that GBM can release tumoral content which crosses the blood-brain barrier (BBB) and as a consequence can be detected in patients' blood, such as circulating tumor cells (CTCs). CTCs carry tumor information and have shown promise as prognostic and predictive biomarkers in other cancer types. Currently, the prognostic utility of CTCs in GBM is not well understood. Methods: Blood samples from GBM patients (n=18) attending a tertiary referral hospital were collected before and after surgery to investigate the prognostic role of CTCs. CTCs were isolated using spiral microfluidic technology and characterized using immunofluorescence for GFAP, cell surface vimentin and CD45, in addition to fluorescence in situ hybridization (FISH) to detect EGFR amplification. CTC counts of patients with poor outcomes (recurrence or death) versus patients with good outcomes (stable disease and no recurrence) were compared using a negative binomial distribution. Results: CTCs were found in 12 out of 18 patients (66.7%), 8/18 before surgery and 10/17 after surgery. CTC clusters were found in 2 patients. CTCs counts before surgery were significantly higher for patients with poor clinical outcome (p=0.0396). Conclusion: Our preliminary data support the use of this spiral technology to isolate CTCs. CTCs and CTC clusters can be used as prognostic biomarkers for managing GBM patients. Citation Format: Juliana Müller Bark, Arutha Kulasinghe, Paul Leo, Gunter Hartel, Majid E. Warkiani, Rosalind L. Jeffree, Benjamin Chua, Bryan W. Day, Chamindie Punyadeera. Circulating tumor cells as prognostic biomarkers in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 584.

Abstract A03: Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers

Abstract Metastasis in head and neck cancer (HNC) patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in HNC. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. n=300 head and neck cancer patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples, which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification). The presence of CTC clusters was associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression-free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence interval [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof-of-principle study, we were able to demonstrate for the first time short-term patient-derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggest that PD-L1 is frequently expressed on CTCs in HNC and an immunoscore may be able to identify patients likely to benefit from immunotherapy—a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression, and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean Paul Thiery, Ken O’Byrne, Chamindie Punyadeera. Isolation, characterization, and expansion of circulating tumor cells in head and neck cancers [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A03.

Abstract IA09: Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells

Abstract Detection of circulating tumor cells (CTCs) has been established as a blood-based biomarker of poor prognosis in most types of epithelial cancer. The observation of increased CTC detection rates in patients with metastatic disease supported the hypothesis that CTCs might represent the precursor cells of distant metastases. Experimental proof for this hypothesis was provided by studies in experimental mouse models in which single metastasis-initiating cells within the total pool of CTCs could be causally linked to the formation of distant metastases. In addition, a potential of CTCs of reseeding at the primary site has been reported, suggesting that CTCs could also play a role in local tumor recurrence. Enumeration and molecular characterization of CTCs might thus be clinically exploited for improving the clinical management of epithelial cancers both in the locally advanced and recurrent/metastatic disease setting. Numerous studies reported on the presence of CTCs in peripheral blood samples of head and neck squamous cell carcinoma (HNSCC) patients. Significant associations with clinical high-risk parameters such as advanced stage, high tumor burden, nodal disease, and distant metastasis were reported. Accordingly, CTC detection was correlated with increased risk of local and/or distant recurrence, progression-free and overall survival. Current data in HNSCC are not yet robust enough for the implementation of CTC detection in clinical routine. In addition, the mere enumeration of CTCs has proven insufficiently informative to prompt widespread clinical adoption. There is accumulating evidence that more extended phenotyping of CTCs might be necessary for improving their diagnostic value. Indeed, despite the estimated daily shedding of millions of tumor cells from the bulk tumor tissue, only a small subpopulation of CTCs is able to reach and survive in the blood circulation and to contribute to the formation of distant metastasis. Identification of the true metastasis-inducing subclones within the bulk CTC population remains a challenge but is imperative in order to improve the diagnostic potential of CTCs. In addition, improved knowledge of the relevant biologic mechanisms endowing CTCs with the potential to emigrate from the primary site to blood circulation, to survive their journey, and to reseed at distant organs will support the development of novel antimetastatic therapies. Several technologies are currently available that allow multiparametric CTC phenotyping at the single-cell level. These platforms include imaging flow cytometry (IFC), by which multispectral images from a large number of cells acquired in suspension can be analyzed. By combining the features of flow cytometry and fluorescent microscopy, information on the cell morphology together with the extent of target protein expression and its subcellular localization can be collected. This provides a unique opportunity for statistical analysis of large populations, but also helps in avoiding artifacts. In the talk, the evidence of a prognostic role of CTCs in HNSCc will be summarized. Results from recent studies reporting on multiparametric CTC phenotyping at the single-cell level will be presented. Examples of CTC-based biomarkers that could be exploited for the optimization of current molecular treatment strategies in HNSCC will be discussed. Citation Format: Ingeborg Tinhofer. Beyond counting: Imaging flow cytometry-based detection of therapeutic molecular targets in circulating tumor cells [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA09.

Abstract 1333: Isolation, characterization and expansion of circulating tumor cells in solid cancers

Abstract Metastasis in cancer patients is reflected by measurable levels of circulating tumor cells (CTCs) in the blood of cancer patients. CTCs represent cancer cells from the primary and metastatic sites, thereby providing a comprehensive representation of the tumor burden of an individual patient. Recent advancements have shown that PD-1/PD-L1 immune checkpoint therapies have durable responses in a number of solid tumor types. Our study was designed to use multiple CTC enrichment platforms for the capture of CTCs and novel culture formulations for the ex vivo expansion of CTCs. Head and Neck cancer (n=300) and lung cancer (n=80) patients were recruited to investigate the prognostic role of CTCs. We evaluated multiple CTC isolation technologies (CellSearch, filtration, CD45 depletion, inertial microfluidics) using matched patient samples which showed that epitope-independent CTC isolation captured a greater proportion of CTCs. Molecular alterations present in the primary tissue were confirmed in the CTCs by 3D-DNA FISH (EGFR-amplification, ALK-translocations). In HNC, the presence of CTC clusters associated with the development of distant metastatic disease (P=0.0313). HNC CTC-positive patients had shorter progression free survival (PFS) (Hazard ratio [HR]: 4.946; 95% confidence internal [CI]:1.571-15.57; P=0.0063) and PD-L1-positive CTCs were found to be significantly associated with worse outcome ([HR]:5.159; 95% [CI]:1.011-26.33; P=0.0485). In a proof of principle study, we were able to demonstrate for the first time, short-term patient derived CTC cultures outside the patient’s body from 7/18 HNC samples (4/7 HPV-positive). Recently, we have preliminary data that suggests that PD-L1 is frequently expressed on CTCs in HNC and lung cancer and an immunoscore may be able to identify patients likely to benefit from immunotherapy-a current unmet clinical need. Expanding CTCs outside the patient’s body allows for the recapitulation of the molecular diversity present within the tumor, understanding of the disease progression and testing of therapies. Citation Format: Arutha Kulasinghe, Joanna Kapeleris, Liz Kenny, Majid Warkiani, Ian Vela, Jean-Paul Thiery, Ken O'Byrne, Chamindie Punyadeera. Isolation, characterization and expansion of circulating tumor cells in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1333.

Prognostic impact of baseline circulating tumor cells (CTCs) detected by the isolation by size of epithelial tumor cells (ISET) in locally advanced head and neck squamous cell carcinoma (LAHNSCC): Results of a prospective study.

6061 Background: The prognostic impact of CTCs in LAHNSCC is yet to be determined, with conflicting results in previous trials, the majority utilizing cytokeratin dependent techniques for identification and counting of CTCs. The primary objective of this study is to determine the detection rates using the ISET method, and the prognostic role of CTCs in LAHNSCC patients (pts) treated with a curative intent. Methods: In this prospective study, peripheral blood samples of pts with non-metastatic LAHNSCC, stages III/IV, were analyzed for CTCs using the ISET method, in two scenarios: curative surgical resection and adjuvant radiotherapy (CTCs before RT) and candidates for a non-surgical strategy (unresectable or organ preservation) either with upfront RT concurrent with chemotherapy (CT) or cetuximab (CTCs before RT), or preceded by induction CT (CTCs before ICT). Results: Eighty-three pts were included, the majority males (83%), with oropharynx primary (50%) and submitted to ICT (40%). The detection rate of baseline CTCs was 94% (78/83), and CTCs counts were significantly correlated with survival. For each increase of 1 CTC at baseline there was a relative increase of 18% in the risk of death (HR = 1.18; 95%CI: 1.06-1.31; p < 0.001), 16% in the risk of progression (HR = 1.16; 95%CI: 1.04-1.28; p = 0.004) and a reduction of 26% in the odds of complete response to treatment (non-surgical group only - OR = 0.74; 95%CI: 0.58-0.95; p = 0.022). Using the maximum of the standardized log-rank statistic proposed by Lausen and Schumacher we establish cut off points for overall survival (OS) and progression-free survival (PFS). Pts with CTCs < 6.5/ml had an estimated 2y OS of 85.6% versus 22.9% for CTCs ≥ 6.5/ml (HR = 0.18; 95%CI: 0.06-0.49; P < 0.0001) and pts with CTCs ≤ 3.8/ml had an estimated 2y PFS of 71.8% versus 37% for CTCs > 3.8/ml (HR = 0.32; 95%CI:0.15-0.67; p = 0.001). Conclusions: The detection rate of baseline CTCs using the ISET method was very high in LAHNSCC and the counts of CTCs were strongly correlated with survival and response to treatment.

Portal vein-circulating tumor cells predict liver metastases in patients with resectable pancreatic cancer.

Pancreatic cancer patients underwent surgical resection often present distant metastases early after surgery. Detection of circulating tumor cells (CTCs) has been correlated to a worse oncological outcome in patients with advanced pancreatic cancer. The objective of this pilot study is to investigate the possible prognostic role of CTCs in patients undergoing surgery for pancreatic cancer. In 20 patients undergoing pancreatic resection, 10 mL blood sample was collected intraoperatively from both systemic circulation (SC) and portal vein (PV). Blood sample was analyzed for CTCs with CellSearch® system. All patients underwent an oncologic follow-up for at least 3 years, quarterly. CTCs were detected in nine (45%) patients: five patients had CTCs in PV only, three patients in both SC and PV, and one patient in SC only. CTC-positive and CTC-negative patients were similar for demographics and cancer stage pattern. No significant differences were found in both overall and disease-free survival between CTC-positive and CTC-negative patients. At 3-year follow-up, portal vein CTC-positive patients presented a higher rate of liver metastases than CTC-negative patients (53 vs. 8%, p = 0.038). CTCs were found in 45% of the patients. No correlation between CTCs and survival was found. The presence of CTCs in portal vein has been associated to higher rate of liver metastases after surgery.

Minimal residual cancer and its clinical relevance

About one third of axillary node-negative breast cancer patients will develop distant metastases, even if there was no evidence of tumor spread beyond the breast at the time of primary diagnosis. Several studies support the hypothesis that the presence of hematogenous disseminated tumor cells (DTC) in the bone marrow of cancer patients, a condition known as minimal residual disease (MRD), can be regarded as a precursor of clinically manifest distant metastases. MRD can be detected either as DTC in the bone marrow or as circulating tumor cells (CTC) in the peripheral blood. Bone marrow and peripheral blood represent mesenchymal compartments with rare prevalence of epithelial cells without evidence of malignant disease. There are two main approaches for the detection of DTC and/or CTC: 1) immunologic assays using monoclonal antibodies against epithelium-specific proteins, and 2) polymerase chain reaction-based molecular methods detecting tissue-specific transcripts. Although data on the prognostic relevance of DTC in bone marrow are vast, data on the prognostic role of CTC are much less convincing. Sampling peripheral blood instead of bone marrow, however, might enhance the therapeutic implications of detecting MRD in breast cancer, a topic that is examined in this article. This article also examines the major quality criteria for the transfer of new markers into the clinical routine in relation to MRD and reviews whether and in what detail MRD in bone marrow meets these criteria.