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Background: Research on circulating tumor cells (CTCs) offers the opportunity to better understand the initial steps of blood-borne metastasis as main cause of cancer-related deaths. Here, we have used the colon cancer CTC-MCC-41 and breast cancer CTC-ITB-01 lines, which were both established from human CTCs as permanent cell lines as models to further study CTC biology with special emphasis on anchorage-independent survival and growth., Methods and Results: Both cell lines showed a marked intrinsic plasticity to switch between suspension and adherent in vitro growth, in 2D adherent culture conditions, and established an equilibrium of both growth patterns with predominant adherent cells in the CTC-MCC-41 line (77%) and suspension cells in the CTC-ITB-01 line (85%). Western blot analysis revealed a higher expression of pERK1/2 in CTC-ITB-01 adherent cells compared to the suspension counterpart that suggested the involvement of kinases in this process. Subsequent functional kinome profiling identified several serine/threonine as well as tyrosine kinases that were differentially regulated in adherent and suspension CTCs. In the adherent cells of the breast cancer line CTC-ITB-01 the activity of MSK1, Src family kinases and the PKG family was increased compared to the suspension counterpart. In adherent cells of the colorectal CTC-MCC-41 line, an increased activity of TYRO3 and JAK2 was detected, whereas p38 MAPK was strongly impaired in the suspension CTC-MCC-41 cells. Some of the regulated kinases, which include the Src family, TYRO3, MSK1, JAK2 and p38 MAPK, have been associated with crucial cellular processes including proliferation, migration and dormancy in the past., Conclusions: The investigated CTC lines exhibit a high plasticity, similar to the concept of 'adherent-to-suspension transition (AST)' that was recently suggested as a new hallmark of tumor biology by Huh et al. Moreover, we identified differentially regulated kinome profiles that may represent potential targets for future studies on therapeutic interventions., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Background: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor., Methods: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging., Results: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC 50 MK2206: 148 nM, IC 50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction., Conclusions: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The concept of liquid biopsy analysis has been established more than a decade ago. Since the establishment of the term, tremendous advances have been achieved and plenty of methods as well as analytes have been investigated in basic research as well in clinical trials. Liquid biopsy refers to a body fluid-based biopsy that is minimal-invasive, and most importantly, allows dense monitoring of tumor responses by sequential blood sampling. Blood is the most important analyte for liquid biopsy analyses, providing an easily accessible source for a plethora of cells, cell-derived products, free nucleic acids, proteins as well as vesicles. More than 12,000 publications are listed in PubMed as of today including the term liquid biopsy. In this manuscript, we critically review the current implications of liquid biopsy, with special focus on circulating tumor cells, and describe the hurdles that need to be addressed before liquid biopsy can be implemented in clinical standard of care guidelines., (© 2024. The Author(s).)

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis., (© 2024. The Author(s).)

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2) -negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC 50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2 -negative, RAS wild-type mGAC patients.

Background: Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA's full potential, this study introduces a novel approach for CTC enrichment from DLAs., Methods: DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations., Results: Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes., Conclusions: In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions., (© 2024. The Author(s).)

Over the past decade, novel methods for enrichment and identification of cancer cells circulating in the blood have been established. Blood-based detection of cancer cells and other tumor-associated products can be summarized under the term of Liquid Biopsy. Circulating tumor cells (CTCs) have been used for diagnosis, risk stratification and treatment selection as well as treatment monitoring in several studies over the past years, thus representing a valuable biomarker for cancer patients. A plethora of methods to enrich, detect and analyze CTCs has been established. In contrast to other liquid biopsy analytes (e.g. ctDNA), CTCs represent a viable analyte that provides a unique opportunity to understand the underlaying biology of cancer and the metastatic cascade on the molecular level. In this review, we provide an overview on the current methods used for enrichment, detection, molecular and functional characterization of CTCs., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Cancer of unknown primary (CUP) is defined as a primary metastatic malignancy, in which the primary tumor remains elusive in spite of a comprehensive diagnostic workup. The frequency and prognostic value of circulating tumor cells (CTCs), which are considered to be the source of metastasis, has not yet been systematically evaluated in CUP., Methods: A total of 110 patients with a confirmed diagnosis of CUP according to the European Society for Medical Oncology (ESMO) guidelines, who presented to our clinic between July 2021 and May 2023, provided blood samples for CTC quantification using CellSearch methodology. CTC counts were correlated with demographic, clinical, and molecular data generated by comprehensive genomic profiling of tumor tissue., Results: CTCs were detected in 26% of all patients at initial presentation to our department. The highest CTC frequency was observed among patients with unfavorable CUP (35.5%), while patients with single-site/oligometastatic CUP harbored the lowest CTC frequency (11.4%). No statistically significant association between CTC positivity and the number of affected organs (P = 0.478) or disease burden (P = 0.120) was found. High CTC levels (≥5 CTCs/7.5 mL; 12/95 analyzed patients) predicted for adverse overall survival compared to negative or low CTC counts (6-months overall survival rate 90% vs 32%, log-rank P < 0.001; HR 5.43; 95% CI 2.23-13.2). CTC dynamics were also prognostic for overall survival by landmark analysis (log-rank P < 0.001, HR 10.2, 95% CI 1.95-52.9)., Conclusions: CTC frequency is a strong, independent predictor of survival in patients with CUP. CTC quantification provides a useful prognostic tool in the management of these patients., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The phenotypes of tumor cells change during disease progression, but invasive rebiopsies of metastatic lesions are not always feasible. Here we aimed to determine whether initially HER2-negative metastatic breast cancer (MBC) patients with HER2-positive circulating tumor cells (CTCs) benefit from a HER2-targeted therapy., Methods: The open-label, interventional randomized phase III clinical trial (EudraCT Number 2010-024238-46, CliniclTrials.gov Identifier: NCT01619111) recruited from March 2012 until September 2019 with a follow-up duration of 19.5 months. It was a multicenter clinical trial with 94 participating German study centers. A total of 2137 patients with HER2-negative MBC were screened for HER2-positive CTCs with a final modified intention-to-treat population of 101 patients. Eligible patients were randomized to standard therapy with or without lapatinib. Primary study endpoints included CTC clearance (no CTCs at the end of treatment) and secondary endpoints were progression-free survival, overall survival (OS), and safety., Results: In both treatment arms CTC clearance at first follow-up visit-although not being significantly different for both arms at any time point-was significantly associated with improved OS (42.4 vs 14.1 months; P = 0.002). Patients treated additionally with lapatinib had a significantly improved OS over patients receiving standard treatment (20.5 vs 9.1 months, P = 0.009)., Conclusions: DETECT III is the first clinical study indicating that phenotyping of CTCs might have clinical utility for stratification of MBC cancer patients to HER2-targeting therapies. The OS benefit could be related to lapatinib, but further studies are required to prove this clinical observation. ClinicalTrials.gov Registration Number: NCT01619111., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Metastasis is the leading cause of cancer-related deaths. Most studies have focused on the primary tumor or on overt metastatic lesions, leaving a significant knowledge gap concerning blood-borne cancer cell dissemination, a major step in the metastatic cascade. Circulating tumor cells (CTCs) in the blood of patients with solid cancer can now be enumerated and investigated at the molecular level, giving unexpected information on the biology of the metastatic cascade., Content: Here, we reviewed recent advances in basic and translational/clinical research on CTCs as key elements in the metastatic cascade., Summary: Findings from translational studies on CTCs have elucidated the complexity of the metastatic process. Fully understanding this process will open new potential avenues for cancer therapeutic and diagnostic strategies to propose precision medicine to all cancer patients., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The proliferation marker Ki-67 is a major pathological feature for the description of the state of disease in breast cancer. It helps to define the molecular subtype and to stratify between therapy regimens in early breast cancer and helps to assess the therapy response. Circulating tumor cells (CTCs) are a negative prognostic biomarker for progression free (PFS) and overall survival (OS) in patients with metastatic breast cancer. Therefore, the CTC count is often described as surrogate for the tumor burden. Both, decrease of Ki-67 and CTC count are considered as evidence for therapy response. The presented work analyzed the correlation between the Ki-67 indices of metastatic tissue biopsies and CTC counts in biopsy time-adjacent peripheral blood samples., Patients and Methods: Blood samples from 70 metastatic breast cancer patients were obtained before the start of a new line of systemic therapy. CTCs were enumerated using CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) whereas intact CTCs (iCTCs) and non-intact or apoptotic CTCs (aCTCs) were distinguished using morphologic criteria. The proportion of cells expressing Ki-67 was evaluated using immunohistochemistry on biopsies of metastases obtained concurrently with CTC sampling before the start of a new line of systemic therapy., Results: 65.7% of patients had a Ki-67 index of > 25%. 28.6% of patients had ≥ 5, 47.1% ≥ 1 iCTCs. 37.1% had ≥ 5, 51.4% ≥ 1 aCTCs. No correlation was shown between Ki-67 index and iCTC and aCTC count (r = 0.05 resp. r = 0.05, Spearman's correlation index). High CTC-counts did not coincide with high Ki-67 index. High Ki-67, ≥ 5 iCTCs and aCTCs are associated with poor progression free (PFS) and overall survival (OS)., Conclusion: CTCs and Ki-67 are independent prognostic markers in metastatic breast cancer. High Ki-67 in metastatic tumor tissue is not correlated to high iCTC or aCTC counts in peripheral blood., (© 2023. The Author(s).)

The analysis of tumor cells circulating in the blood or of products of tumor cells circulating in other body fluids has gained increasing attention in recent years and is summarized under the term liquid biopsy (LB). LB includes the analysis of circulating tumor cells, cell-free circulating tumor-associated nucleic acids, extracellular vesicles, proteins, or other products that are released into the peripheral bloodstream by the primary or metastatic tumor. For a huge number of solid tumor entities, LB has already been successfully applied in preclinical and clinical studies for the detection, risk stratification, treatment monitoring and relapse detection. LB provides valuable real-time information on tumor cell development, therapeutic targets, and mechanisms of therapy resistance using a non-invasive peripheral blood test. In this article, the most important LB analytes and the current state of research are presented. In addition, the remaining obstacles and the diverse efforts to implement LB in clinical routine are critically discussed., Competing Interests: K.P. erhielt Forschungsförderung durch EU/IMI CANCER-ID EFPIA sowie Honorare von BMS, Agena, Menarini, Novartis, Sanofi, Illumina, Abcam, MSD, Boehringer Ingelheim, Eppendorf und Hummingbird. Die übrigen Autoren erklären, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Hepatocellular carcinoma (HCC) has high recurrence rates exceeding 50% despite curative resection. The serum biomarker alpha-fetoprotein (AFP) is a well-known prognostic marker for HCC. EpCAM-positive circulating tumor cells (CTC) have a high predictive value for early HCC recurrence after curatively intended resection, most likely indicating micro-metastases at the time of resection. However, sensitivity remains low. The objective of this study was to evaluate a composite test comprising both CTC and AFP to identify patients at high risk for early HCC recurrence. We prospectively enrolled 58 patients undergoing curative intended resection for HCC at a tertiary referral center. Blood specimens were obtained prior to resection and analyzed for EpCAM-positive CTC and serum AFP levels. A positive result was defined as either detection of CTC or AFP levels ≥ 400 ng/ml. Eight patients tested positive for CTC, seven for AFP, and two for both markers. A positive composite test was significantly associated with shorter early recurrence-free survival (5 vs. 16 months, p = 0.005), time to recurrence (5 vs. 16 months, p = 0.011), and overall survival (37 vs. not reached, p = 0.034). Combining CTC and AFP identified patients with poor outcome after surgical resection, for whom adjuvant or neoadjuvant therapies may be particularly desirable., (© 2023. The Author(s).)

Background: Obesity and the presence of circulating tumor cells (CTCs) before and/or after chemotherapy are associated with poor outcome in breast cancer (BC) patients. The activation of oncogenic pathways in fatty tissue leads to cell proliferation, suggesting a possible link between obesity and CTCs., Materials and Methods: In the phase III SUCCESS A trial, 3754 patients with early BC were randomized to 3 cycles of fluorouracil, epirubicin and cyclophosphamide followed by 3 cycles of docetaxel with or without gemcitabine. Data of 1088 patients with CTC assessments (CellSearch-System; Menarini Silicon Biosystems, Italy) and body mass index (BMI) measurements both before and after chemotherapy were available. Patients were classified according to the WHO's international definitions as underweight, normal weight, overweight, or obese, and according to their weight-change during chemotherapy into a weight-loss group (> 5% decrease), stable-weight group (≤ 5% weight-change) or weight-gain group (>5% increase). Associations between CTC positivity and, BMI or weight-change group were analyzed using frequency-table methods., Results: At study entry, 47.4% patients were underweight or normal weight, 33.6% were overweight and 18.9% were obese. Before and after chemotherapy, CTCs were detected in 20.1% and 22.6% of patients, respectively. There was no association between CTC positivity and BMI before (P = 0.104) or after (P = 0.051) chemotherapy. Furthermore, there was no association between weight-change group and CTC status before/after chemotherapy (P = 0.332)., Conclusions: According to our analysis, the risk factors obesity and prevalence of CTCs are not associated and may represent independent prognostic factors., Competing Interests: Disclosure TWP: Personal fees from Lilly and Novartis. FS: Grants from AstraZeneca, Roche and Karyopharm. Personal fees from Roche, Pfizer, Novartis, AstraZeneca, GSK, MSD, Clovis and Eisai. TF: Personal fees from Medconcept, Onkowissen. KP: Grants from: EU/IMI CANCER-ID EFPIA, Personal fees from: BMS, Agena, Menarini, Novartis, Sanofi, Illumina, Abcam, MSD, Boehringer Ingelheim, Eppendorf and Hummingbird. AS: Grants from Celgene, Roche and AbbVie. Personal fees from Celgene, Roche, Pfizer, AstraZeneca, Novartis, MSD, Tesaro, Lilly, Seagen, Gilead, GSK, Bayer, Amgen and Pierre Fabre. HT: Personal fees from Novartis, Roche, GSK, Seagan, Pfizer, Lilly, Astra Zeneca, Daiichi, Exact Science, Vifor and Seagan. Shares/stocks from CHOP GmbH, VISION MED GmbH, Care and Coach GmbH, Onco Medical Consult GmbH. VA-F: Honoraria from Novartis. PF: Grants from Pfizer, Cepheid and Biontech. Personal fees from Novartis Daiichi-Sankyo, Pfizer Astra Zeneca, Eisai, Merck Sharp & Dohme, Lilly, Pierre Fabre, SeaGen, Roche, Agendia, Sanofi Aventis, Gilead, Mylan and Menarini., WJ: honoraria from Sanofi-Aventis, Novartis and Pfizer. BR: honoraria from AstraZeneca, Chugai, Lilly, Novartis, Sanofi-Aventis, and Jannssen-Cilag. All other authors have no conflicts of interest., (Copyright © 2023. Published by Elsevier Inc.)

Background: We explored the interrelation between prostate-specific membrane antigen (PSMA) expression on circulating tumor cells (CTCs) and that of solid metastatic lesions as determined by whole-body PSMA-targeted positron emission tomography (PET) to refine the prediction of response to subsequent PSMA-targeted radioligand therapy (RLT)., Methods: A prospective study was performed in 20 patients with advanced mCRPC. Of these, 16 underwent subsequent RLT with [ 177 Lu]Lu-PSMA-617 at a dose of 7.4 GBq every 6-8 weeks. PSMA expression on CTCs using the CellSearch system was compared to clinical and serological results, and to marker expression in targeted imaging and available histological sections of prostatectomy specimens (19% of RLT patients). Clinical outcome was obtained after two cycles of RLT., Results: Marked heterogeneity of PSMA expression was observed already at first diagnosis in available histological specimens. Targeted whole-body imaging also showed heterogeneous inter- and intra-patient PSMA expression between metastases. Heterogeneity of CTC PSMA expression was partially paralleled by heterogeneity of whole-body tumor burden PSMA expression. Twenty percent of CTC samples showed no PSMA expression, despite unequivocal PSMA expression of solid metastases at PET. A high fraction of PSMA-negative CTCs emerged as the sole predictor of poor RLT response (odds ratio [OR]: 0.9379 [95% confidence interval, CI, 0.8558-0.9902]; p = 0.0160), and was prognostic for both shorter progression-free survival (OR: 1.236 [95% CI, 1.035-2.587]; p = 0.0043) and overall survival (OR: 1.056 [95% CI, 1.008-1.141]; p = 0.0182)., Conclusion: This proof-of-principle study suggests that liquid biopsy for CTC PSMA expression is complementary to PET for individual PSMA phenotyping of mCRPC., (© 2023 The Authors. The Prostate published by Wiley Periodicals LLC.)

Given that cancer mortality is usually a result of late diagnosis, efforts in the field of early detection are paramount to reducing cancer-related deaths and improving patient outcomes. Increasing evidence indicates that metastasis is an early event in patients with aggressive cancers, often occurring even before primary lesions are clinically detectable. Metastases are usually formed from cancer cells that spread to distant non-malignant tissues via the blood circulation, termed circulating tumour cells (CTCs). CTCs have been detected in patients with early stage cancers and, owing to their association with metastasis, might indicate the presence of aggressive disease, thus providing a possible means to expedite diagnosis and treatment initiation for such patients while avoiding overdiagnosis and overtreatment of those with slow-growing, indolent tumours. The utility of CTCs as an early diagnostic tool has been investigated, although further improvements in the efficiency of CTC detection are required. In this Perspective, we discuss the clinical significance of early haematogenous dissemination of cancer cells, the potential of CTCs to facilitate early detection of clinically relevant cancers, and the technological advances that might improve CTC capture and, thus, diagnostic performance in this setting., (© 2023. Springer Nature Limited.)

Background: Circulating tumour cells (CTCs) are mainly enriched based on the epithelial cell adhesion molecule (EpCAM). Although it was shown that an EpCAM low-expressing CTC fraction is not captured by such approaches, knowledge about its prognostic and predictive relevance and its relation to EpCAM-positive CTCs is lacking., Methods: We developed an immunomagnetic assay to enrich CTCs from metastatic breast cancer patients EpCAM independently using antibodies against Trop-2 and CD-49f and characterised their EpCAM expression. DNA of single EpCAM high expressing and low expressing CTCs was analyzed regarding chromosomal aberrations and predictive mutations. Additionally, we compared CTC-enrichment on the CellSearch system using this antibody mix and the EpCAM based enrichment., Results: Both antibodies acted synergistically in capturing CTCs. Patients with EpCAM high-expressing CTCs had a worse overall and progression-free survival. EpCAM high- and low-expressing CTCs presented similar chromosomal aberrations and mutations indicating a close evolutionary relationship. A sequential enrichment of CTCs from the EpCAM-depleted fraction yielded a population of CTCs not captured EpCAM dependently but harbouring predictive information., Conclusions: Our data indicate that EpCAM low-expressing CTCs could be used as a valuable tumour surrogate material-although they may be prognostically less relevant than EpCAM high-expressing CTCs-and have particular benefit if no CTCs are detected using EpCAM-dependent technologies., (© 2023. The Author(s).)

Alternative sources of tumour information need to be explored in patients with non-small cell lung cancer (NSCLC). Here, we compared programmed cell death ligand 1 (PD-L1) expression on cytology imprints and circulating tumour cells (CTCs) with PD-L1 tumour proportion score (TPS) from immunohistochemistry staining of tumour tissue from patients with NSCLC. We evaluated PD-L1 expression using a PD-L1 antibody (28-8) in representative cytology imprints, and tissue samples from the same tumour. We report good agreement rates on PD-L1 positivity (TPS ≥ 1%) and high PD-L1 expression (TPS ≥ 50%). Considering high PD-L1 expression, cytology imprints showed a PPV of 64% and a NPV of 85%. CTCs were detected in 40% of the patients and 80% of them were PD-L1 + . Seven patients with PD-L1 expression of < 1% in tissue samples or cytology imprints had PD-L1 + CTCs. The addition of PD-L1 expression in CTCs to cytology imprints markedly improved the prediction capacity for PD-L1 positivity. A combined analysis of cytological imprints and CTCs provides information on the tumoural PD-L1 status in NSCLC patients, which might be used when no tumor tissue is available., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The analysis of tumor cells circulating in the blood or of products of tumor cells circulating in other body fluids has gained increasing attention in recent years and is summarized under the term liquid biopsy (LB). LB includes the analysis of circulating tumor cells, cell-free circulating tumor-associated nucleic acids, extracellular vesicles, proteins, or other products that are released into the peripheral bloodstream by the primary or metastatic tumor. For a huge number of solid tumor entities, LB has already been successfully applied in preclinical and clinical studies for the detection, risk stratification, treatment monitoring and relapse detection. LB provides valuable real-time information on tumor cell development, therapeutic targets, and mechanisms of therapy resistance using a non-invasive peripheral blood test. In this article, the most important LB analytes and the current state of research are presented. In addition, the remaining obstacles and the diverse efforts to implement LB in clinical routine are critically discussed., Competing Interests: K.P. erhielt Forschungsförderung durch EU/IMI CANCER-ID EFPIA sowie Honorare von BMS, Agena, Menarini, Novartis, Sanofi, Illumina, Abcam, MSD, Boehringer Ingelheim, Eppendorf und Hummingbird. Die übrigen Autoren erklären, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

Circulating tumor cells (CTCs) that are detached from the tumor can be precursors of metastasis. The majority of studies focus on enumeration of CTCs from patient blood to predict recurrence and therapy outcomes. Very few studies have managed to expand CTCs to investigate their functional dynamics with respect to genetic changes, tumorigenic potential, and response to drug treatment. A growing amount of evidence based on successful CTC expansion has revealed novel therapeutic targets that are associated with the process of metastasis. In this review, we summarize the successes, challenges, and limitations that collectively contribute to the better understanding of metastasis using patient-derived CTCs as blood-borne seeds of metastasis. The roadblocks and future avenues to move CTC-based scientific discoveries forward are also discussed., Competing Interests: Declaration of interests All authors declare no potential conflicts of interest. All authors reviewed and approved the manuscript., (Published by Elsevier Inc.)

Androgen deprivation therapy has a central role in the treatment of advanced prostate cancer, often causing initial tumour remission before increasing independence from signal transduction mechanisms of the androgen receptor and then eventual disease progression. Novel treatment approaches are urgently needed, but only a fraction of promising drug candidates from the laboratory will eventually reach clinical approval, highlighting the demand for critical assessment of current preclinical models. Such models include standard, genetically modified and patient-derived cell lines, spheroid and organoid culture models, scaffold and hydrogel cultures, tissue slices, tumour xenograft models, patient-derived xenograft and circulating tumour cell eXplant models as well as transgenic and knockout mouse models. These models need to account for inter-patient and intra-patient heterogeneity, the acquisition of primary or secondary resistance, the interaction of tumour cells with their microenvironment, which make crucial contributions to tumour progression and resistance, as well as the effects of the 3D tissue network on drug penetration, bioavailability and efficacy., (© 2022. Springer Nature Limited.)

Cancer-related deaths are mainly caused by metastatic spread of tumor cells from the primary lesion to distant sites via the blood circulation. Understanding the mechanisms of blood-borne tumor cell dissemination by the detection and molecular characterization of circulating tumor cells (CTCs) in the blood of patients with cancer has opened a new avenue in cancer research. Recent technical advances have enabled a comprehensive analysis of the CTCs at the genome, transcriptome and protein level as well as first functional studies using patient-derived CTC cell lines. In this review, we describe and discuss how research on CTCs has yielded important insights into the biology of cancer metastasis and the response of patients with cancer to therapies directed against metastatic cells. Future investigations will show whether CTCs leaving their primary site are more vulnerable to attacks by immune effector cells and whether cancer cell dissemination might be the 'Achilles heel' of metastatic progression. Here, we focus on the lessons learned from CTC research on the biology of cancer metastasis in patients with particular emphasis on the interactions of CTCs with the immune system. Moreover, we describe and discuss briefly the potential and challenges for implementing CTCs into clinical decision-making including detection of minimal residual disease, monitoring efficacies of systemic therapies and identification of therapeutic targets and resistance mechanisms., Competing Interests: Competing interests: KP and CA-P have received honoraria from Menarini and both authors have patent applications related to CTC technologies., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Metastasis formation is the main cause of cancer-related death in patients with solid tumours. At the beginning of this process, cancer cells escape from the primary tumour to the blood circulation where they become circulating tumour cells (CTCs). Only a small subgroup of CTCs will survive during the harsh journey in the blood and colonise distant sites. The in-depth analysis of these metastasis-competent CTCs is very challenging because of their extremely low concentration in peripheral blood. So far, only few groups managed to expand in vitro and in vivo CTCs to be used as models for large-scale descriptive and functional analyses of CTCs. These models have shown already the high variability and complexity of the metastatic cascade in patients with cancer, and open a new avenue for the development of new diagnostic and therapeutic approaches., (© 2022. The Author(s).)

Circulating tumor cells (CTCs) are the seeds of distant metastasis, and the number of CTCs detected in the blood of cancer patients is associated with a worse prognosis. CTCs face critical challenges for their survival in circulation, such as anoikis, shearing forces, and immune surveillance. Thus, understanding the mechanisms and interactions of CTCs within the blood microenvironment is crucial for better understanding of metastatic progression and the development of novel treatment strategies. CTCs interact with different hematopoietic cells, such as platelets, red blood cells, neutrophils, macrophages, natural killer (NK) cells, lymphocytes, endothelial cells, and cancer-associated fibroblasts, which can affect CTC survival in blood. This interaction may take place either via direct cell-cell contact or through secreted molecules. Here, we review interactions of CTCs with blood cells and discuss the potential clinical relevance of these interactions as biomarkers or as targets for anti-metastatic therapies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The liquid biopsy concept has been introduced for circulating tumor cells more than 10 years ago (Pantel & Alix-Panabieres, 2010) and rapidly extended to cell-free DNA released from tumor cells (ctDNA; Lo et al, 2021) and other tumor-derived products such as circulating cell-free RNA (noncoding and messenger RNA), extracellular vesicles, or tumor-educated platelets (Alix-Panabières & Pantel, 2021). In this issue of EMBO Molecular Medicine, the report of Sauer et al (2022) demonstrates the feasibility of longitudinal monitoring of disease burden and response using ctDNA from dried blood spots in xenograft models., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Despite the strong prognostic stratification of circulating tumor cells (CTCs) enumeration in metastatic breast cancer (MBC), current clinical trials usually do not include a baseline CTCs in their design. This study aimed to generate a classifier for CTCs prognostic simulation in existing datasets for hypothesis generation in patients with MBC. A K-nearest neighbor machine learning algorithm was trained on a pooled dataset comprising 2436 individual MBC patients from the European Pooled Analysis Consortium and the MD Anderson Cancer Center to identify patients likely to have CTCs ≥ 5/7 mL blood (StageIVaggressive vs StageIVindolent). The model had a 65.1% accuracy and its prognostic impact resulted in a hazard ratio (HR) of 1.89 (Simulatedaggressive vs SimulatedindolentP < .001), similar to patients with actual CTCs enumeration (HR 2.76; P < .001). The classifier's performance was then tested on an independent retrospective database comprising 446 consecutive hormone receptor (HR)-positive HER2-negative MBC patients. The model further stratified clinical subgroups usually considered prognostically homogeneous such as patients with bone-only or liver metastases. Bone-only disease classified as Simulatedaggressive had a significantly worse overall survival (OS; P < .0001), while patients with liver metastases classified as Simulatedindolent had a significantly better prognosis (P < .0001). Consistent results were observed for patients who had undergone CTCs enumeration in the pooled population. The differential prognostic impact of endocrine- (ET) and chemotherapy (CT) was explored across the simulated subgroups. No significant differences were observed between ET and CT in the overall population, both in terms of progression-free survival (PFS) and OS. In contrast, a statistically significant difference, favoring CT over ET was observed among Simulatedaggressive patients (HR: 0.62; P = .030 and HR: 0.60; P = .037, respectively, for PFS and OS)., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer., Methods: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes., Results: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1., Conclusions: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells., (© American Association for Clinical Chemistry 2022.)

Understanding cancer heterogeneity is essential to finding diverse genetic mutations in metastatic cancers. Thus, it is critical to isolate all types of CTCs to identify accurate cancer information from patients. Moreover, full automation robustly capturing the full spectrum of CTCs is an urgent need for CTC diagnosis to be routine clinical practice. Methods: Here we report the full capture of heterogeneous CTC populations using fully automated, negative depletion-based continuous centrifugal microfluidics (CCM). Results: The CCM system demonstrated high performance (recovery rates exceeding 90% and WBC depletion rate of 99.9%) across a wide range of phenotypes (EpCAM(+), EpCAM(-), small-, large-sized, and cluster) and cancers (lung, breast, and bladder). Applied in 30 lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations, the system isolated diverse phenotypes of CTCs in marker expression and size, implying the importance of unbiased isolation. Genetic analyses of intra-patient samples comparing cell-free DNA with CCM-isolated CTCs yielded perfect concordance, and CTC enumeration using our technique was correlated with clinical progression as well as response to EGFR inhibitors. Conclusion: Our system also introduces technical advances which assure rapid, reliable, and reproducible results, thus enabling a more comprehensive application of robust CTC analysis in clinical practice., Competing Interests: Competing Interests: S.-H.K., J.L., H.Y.S. and M.S.K. have financial interests in CTCELLS, Inc. All other authors declare that they have no competing interests., (© The author(s).)

Purpose: Low-dose computed tomography (LDCT) screening of high-risk patients decreases lung cancer-related mortality. However, high false-positive rates associated with LDCT result in unnecessary interventions. To distinguish non-small-cell lung cancer (NSCLC) from benign nodules, in the present study, we integrated cellular liquid biomarkers in patients with suspicious lung nodules (lung cancer screening reporting and data system [Lung-RADS] 4)., Methods: Prospectively, 7.5 mL of blood was collected from 221 individuals (training set: 90 nonscreened NSCLC patients, 74 high-risk screening patients with no/benign nodules [Lung-RADS 1-3], and 20 never smokers; validation set: 37 patients with suspicious nodules [Lung-RADS 4]). Circulating tumor cells (CTCs), CTC clusters, and tumor-macrophage fusion (TMF) cells were identified by blinded analyses. Screening patients underwent a median of two LDCTs (range, 1-4) with a median surveillance time of 30 (range, 11-50) months., Results: In the validation set of 37 Lung-RADS 4 patients, all circulating cellular biomarker counts ( P < .005; Wilcoxon test) and positivity rates were significantly higher in 23 biopsy-proven NSCLC patients (CTCs: 23 of 23 [100%], CTC clusters: 6 of 23 [26.1%], and TMF cells: 15 of 23 [65.2%]) than in 14 patients with biopsy-proven benign nodules (6 of 14 [42.9%], 0 of 14 [0%], and 2 of 14 [14.3%]). On the basis of cutoff values from the training set, logistic regression with receiver operating characteristic and area under the curve analyses demonstrated that CTCs (sensitivity: 0.870, specificity: 1.0, and area under the curve: 0.989) and TMF cells (0.652; 0.880; 0.790) complement LDCT in diagnosing NSCLC in Lung-RADS 4 patients., Conclusion: Cellular liquid biomarkers have a potential to complement LDCT interpretation of suspicious Lung-RADS 4 nodules to distinguish NSCLC from benign lung nodules. A future prospective, large-scale, multicenter clinical trial should validate the role of cellular liquid biomarkers in improving diagnostic accuracy in high-risk patients with Lung-RADS 4 nodules., Competing Interests: Kevin F. Staveley-O'CarrollHonoraria: AstraZeneca Klaus PantelHonoraria: Agena Bioscience, Novartis, Roche, Medac, Impulze, Ipsen, Sanofi, Merck KGaA, MSD, Beiersdorf, Galderma, Hummingbird, Illumina, Hello Healthcare, Menarini Silicon Biosystems, Abcam, Atheneum, CureVac, DeciBio, Inflection Biosciences, Molecular Health, TacticsConsulting or Advisory Role: Sanofi, Agena Bioscience, Hummingbird Diagnostics, Menarini Silicon BiosystemsResearch Funding: Janssen Diagnostics, Cancer-ID (Inst)Patents, Royalties, Other Intellectual Property: Application No. WO2016 128125A1, application No. 17157020.3—1405, application No. 10004180.5, application No. 07825055.2, application No. 95108760.7Travel, Accommodations, Expenses: Agena Bioscience Guangfu LiPatents, Royalties, Other Intellectual Property: Ceramide nanoliposomes as a method and device for immunotherapy (Inst) Aadel A. ChaudhuriLeadership: Droplet BiosciencesStock and Other Ownership Interests: Geneoscopy, Droplet BiosciencesHonoraria: RocheConsulting or Advisory Role: Geneoscopy, Roche, Tempus, AstraZeneca/Daiichi SankyoPatents, Royalties, Other Intellectual Property: US Patent No. US8685727B2Travel, Accommodations, Expenses: Roche, Foundation MedicineOther Relationship: Roche Jussuf T. KaifiPatents, Royalties, Other Intellectual Property: Cancer Biomarker detectionNo other potential conflicts of interest were reported.

Background: Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa)., Methods: In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining., Results: All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection., Conclusions: In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies.

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. There is an urgent need to identify prognostic markers for patients undergoing curative resection of CRC. The detection of circulating tumor cells in peripheral blood is a promising approach to identify high-risk patients with disseminated disease in colorectal cancer. This study aims to evaluate the prognostic relevance of preoperative CTCs using the Cellsearch® system (CS) in patients, who underwent resection with curative intent of different stages (UICC I-IV) of colorectal cancer. Out of 91 Patients who underwent colorectal resection, 68 patients were included in this study. CTC analysis was performed in patients with CRC UICC stages I-IV immediately before surgery. Data were correlated with clinicopathological parameters and patient outcomes. One or more CTCs/7.5 mL were detected in 45.6% (31/68) of patients. CTCs were detected in all stages of the Union of International Cancer Control (UICC), in stage I (1/4, 25%), in stage II (4/12, 33.3%), in stage III (5/19, 26.3%) and in stage IV (21/33, 63.6%). The detection of ≥ 1 CTCs/ 7.5ml correlated to the presence of distant overt metastases (p = 0.014) as well as with shorter progression-free (p = 0.008) and overall survival (p = 0.008). Multivariate analyses showed that the detection of ≥ 1 CTCs/ 7.5ml is an independent prognostic indicator for overall survival (HR, 3.14; 95% CI, 1.18-8.32; p = 0.021). The detection of CTCs is an independent and strong prognostic factor in CRC, which might improve the identification of high-risk patients in future clinical trials., Competing Interests: The authors have declared that no competing interests exist.

Circulating tumor cells (CTCs) play a crucial role in metastasis and became an emerging topic in today's cancer research. In addition, the analysis of CTCs in liquid biopsies will be a valuable tool for prognosis prediction and real time therapy monitoring. The characterization of CTCs may open up a new field of treatment strategy to prevent metastasis or maintain a stable disease. In 2013, the first cell cultures of CTCs have been established in vitro. Additionally, functional studies have been successfully performed over the last years. Meanwhile, more than 300 short-term CTC cultures and 42 long-term CTC cultures from a variety of tumor entities have been described. More than 45 inhibitors have already been tested for their efficacy to target CTCs in several studies in vitro as well as in xenograft mouse models in vivo. Here, we summarize the currently available data of these inhibition experiments and their effects in targeting CTCs. The results suggest that CTCs may be useful for individualized drug susceptibility testing., (Copyright © 2021. Published by Elsevier Inc.)

Background: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model., Methods: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested., Results: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods., Conclusions: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS)., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Aim of this prospective study was to evaluate the prognostic significance of disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) in 1 cohort of patients with esophageal cancer (EC)., Background: Hematogenous tumor cell dissemination is a key event in tumor progression, and clinical significance of DTCs and CTCs are controversially discussed in the literature. However, evaluation of both biomarker in 1 patient's cohort has not been described before., Methods: In this prospective, single-center study, 76 patients with preoperatively nonmetastatic staged EC were included. The CellSearch system was used to enumerate CTCs. Bone marrow was aspirated from the iliac crest and cells were enriched by Ficoll density gradient centrifugation. DTCs were immunostained with the pan-keratin antibody A45-B/B3., Results: Fifteen of 76 patients (19.7%) harbored CTCs, whereas in 13 of 76 patients (17.1%), DTCs could be detected. In only 3 patients (3.9%), CTCs and DTCs were detected simultaneously, whereas concordant results (DTC/CTC negative and DTC/CTC positive) were found in 54 patients (71.1%). Surprisingly, only patients with CTCs showed significant shorter overall and relapse-free survival (P = 0.038 and P = 0.004, respectively). Multivariate analyses revealed that only the CTC status was an independent predictor of overall and relapse-free survival (P = 0.007 and P < 0.001, respectively)., Conclusions: This is the first study analyzing CTC and DTC status in 1 cohort of nonmetastatic patients with EC. In this early disease stage, only the CTC status was an independent, prognostic marker suitable and easy to use for clinical staging of patients with EC., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

The analysis of tumor cells or tumor cell products obtained from blood or other body fluids ("liquid biopsy" [LB]) provides a broad range of opportunities in the field of oncology. Clinical application areas include early detection of cancer or tumor recurrence, individual risk assessment and therapy monitoring. LB allows to portray the entire disease as tumor cells or tumor cell products are released from all metastatic or primary tumor sites, providing comprehensive and real-time information on tumor cell evolution, therapeutic targets and mechanisms of resistance to therapy. Here, we focus on the most prominent LB markers, circulating tumor cells (CTCs) and circulating tumor-derived DNA (ctDNA), in the blood of patients with breast, prostate, lung and colorectal cancer, as the four most frequent tumor types in Europe. After a brief introduction of key technologies used to detect CTCs and ctDNA, we discuss recent clinical studies on these biomarkers for early detection and prognostication of cancer as well as prediction and monitoring of cancer therapies. We also point out current methodological and biological limitations that still hamper the implementation of LB into clinical practice., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

While the majority of patients with advanced testicular germ cell tumors (GCT) achieve complete responses after chemotherapy and if indicated after postchemotherapy resection of residual lesions, about 20% of patients have incomplete responses or show relapses. Moreover, toxicity of chemotherapy is high, and severe adverse chronic effects have been described. Therefore, there is an urgent need for biomarkers that could help to improve tumor staging, and support decision-making, ideally including monitoring of therapy response and prediction of relapse. Besides the well-established serum markers lactate dehydrogenase, α-fetoprotein, and β-subunit of human chorionic gonadotropin, during recent years new noninvasive liquid biopsy markers have been investigated in GCT, including cell-free nucleic acids like microRNAs, and circulating tumor cells (CTCs).Prognostic relevance has been demonstrated for circulating tumor cells (CTCs) in patients with different cancers. However, little is known in GCT patients. Histologically, GCT are a very heterogeneous group of tumors comprising pure seminomas (consisting of cells that remember primordial germ cells) and nonseminomas, which are either undifferentiated (embryonal carcinoma) or differentiated, exhibiting different degrees of embryonic (teratoma) or extraembryonic (yolk sac tumor and choriocarcinoma) differentiation. This heterogeneity hampers capture and detection of CTCs deriving from those tumors using a single method or a single antibody. To date, label-independent capture methods that enrich tumor cells according to the density of GCT cells, which is similar to that of mononuclear cells, have been successfully applied. Since testicular GCT might also express epithelial proteins, methods based on enrichment of CTCs using epithelial markers are promising to detect CTCs in certain subgroups of patients with GCTs as well.Here, we describe and discuss a combination of methods to capture and detect GCT cells with epithelial and germ cell characteristics in blood.

Background: The prognostic value of circulating tumor cells (CTCs) in patients with hormone-naïve oligometastatic prostate cancer (HNoMPC) undergoing cytoreductive radical prostatectomy (CRP) is unknown., Objective: To determine the pre- and postoperative prognostic value of CTC enumeration in patients undergoing CRP., Design, Setting, and Participants: Thirty-three patients with HNoMPC from the prospective, single-arm ProMPT trial who underwent CRP between 2014 and 2015 at the Martini-Klinik were evaluated. Follow-up visits for all patients were conducted every 6 mo up to 36 mo after CRP and included serial detection of CTCs in 7.5 ml blood samples using the CellSearch system., Intervention: CRP., Outcome Measurements and Statistical Analysis: CTC enumerations before and after CRP, and their prognostic value on metastatic castration-resistant prostate cancer-free survival and overall survival (OS) were analyzed using Kaplan-Meier plots and univariable Cox-regression analysis., Results and Limitations: Sixteen patients (48.5%) had positive CTCs prior to CRP. A CTC count of ≥2 before or 6 mo after CRP was a prognostic factor for worse oncologic outcome. Compared with other biomarkers (prostate-specific antigen, lactate dehydrogenase, and bone-specific alkaline phosphatase), the prognostic value of CTCs was highest using Harrell's C for OS (0.69), while the highest C-index could be achieved for a combination of conventional markers and CTC count (0.74). After progression to metastatic castration-resistant prostate cancer, CTC enumeration of ≥5 was prognostic for OS. The main limitation is the small sample size., Conclusions: CTC enumeration contributes to prognostic information, which might help select HNoMPC patients who might benefit most from CRP., Patient Summary: In this report, we looked at the value of circulating tumor cell (CTC) determination in patients undergoing radical prostatectomy for oligometastatic prostate cancer. We could show that the number of CTCs was a prognostic factor at all analyzed time points and was more closely associated with prognosis than other biomarkers commonly used in daily clinical practice., (Copyright © 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

The promise of precision medicine as a model to customize health care to the individual patient is heavily dependent upon new genetic tools to classify and characterize diseases and their hosts. Liquid biopsies serve as a safe alternative to solid biopsies and are thus a useful and critical component to fully realizing personalized medicine. The International Liquid Biopsy Standardization Alliance (ILSA) comprises organizations and foundations that recognize the importance of working towards the global use of liquid biopsy in oncology practice to support clinical decision making and regulatory considerations and seek to promote it in their communities. This manuscript provides an overview of the independent liquid biopsy- and standardization-based programs engaged with ILSA, their objectives and progress to date, and the tools and resources each is developing to contribute to the field. It also describes the unique areas of effort as well as synergy found within the group., (Copyright © 2020. Published by Elsevier B.V.)

Background: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination., Methods: EGFR overexpression (EGFR over ) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices., Results: EGFR over was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFR over correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFR over was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness., Conclusions: EGFR over is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.

Circulating tumor cells (CTCs) are cells shed from the primary tumor into the bloodstream. While many studies on solid tumor cells exist, data on CTCs are scarce. The mortality of cancer is mostly associated with metastasis and recent research identified CTCs as initiators of metastasis. The PI3K/AKT/mTOR signaling pathway is an intracellular pathway that regulates essential functions including protein biosynthesis, cell growth, cell cycle control, survival and migration. Importantly, activating oncogenic mutations and amplifications in this pathway are frequently observed in a wide variety of cancer entities, underlining the significance of this signaling pathway. In this study, we analyzed the functional role of the PI3K/AKT/mTOR signaling pathway in the CTC-MCC-41 line, derived from a patient with metastatic colorectal cancer. One striking finding in our study was the strong sensitivity of this CTC line against AKT inhibition using MK2206 and mTOR inhibition using RAD001 within the nanomolar range. This suggests that therapies targeting AKT and mTOR could have been beneficial for the patient from which the CTC line was isolated. Additionally, a dual targeting approach of AKT/mTOR inside the PI3K/AKT/mTOR signaling pathway in the colorectal CTCs showed synergistic effects in vitro. Depending on the phenotypical behavior of CTC-MCC-41 in cell culture (adherent vs. suspension), we identified altered phosphorylation levels inside the PI3K/AKT/mTOR pathway. We observed a downregulation of the PI3K/AKT/mTOR signaling pathway, but not of the RAS/RAF/MAPK pathway, in CTCs growing in suspension in comparison to adherent CTCs. Our results highlight distinct functions of AKT isoforms in CTC-MCC-41 cells with respect to cell proliferation. Knockdown of AKT1 and AKT2 leads to significantly impaired proliferation of CTC-MCC-41 cells in vitro. Therefore, our data demonstrate that the PI3K/AKT/mTOR signaling pathway plays a key role in the proliferation of CTC-MCC-41.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER + ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER + in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER + breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Introduction: Various subtypes of circulating cancer-associated cells in the blood are described. A unique circulating, large, and polymorphonuclear cell with a dual epithelial and myeloid phenotype has been suggested as a tumor-macrophage fusion cell (TMF). The goal of the study was to identify the impact of distinct TMFs on survival among patients with NSCLC., Methods: In this prospective trial, 7.5 mL of whole blood sample was collected. After microfilter enrichment, immunofluorescent staining was performed, identifying TMFs as greater than or equal to 30 μm in size and dual epithelial (cytokeratin 8, 18, or 19-, or epithelial cell adhesion molecule-positive) and myeloid- or macrophage-positive (CD14- or CD45-positive) cells with at least one 4',6-diamidino-2-phenylindole+ nucleus., Results: Circulating TMFs were identified in 88 of 115 patients (76.5%) with NSCLC (mean 3.052 [SEM ± 0.306]; median 2 [range 0-17]) but were rare in long-term smokers without cancer (6 of 87 [6.9%]; 0.081 [±0.034]; 0 [0-2]), and absent in 20 healthy controls. Comparing the presence of TMFs in patients with NSCLC versus smokers without cancer, specificity was 93.1% (95% confidence interval: 85.6-97.4%) and sensitivity 76.5% (95% confidence interval: 67.7%-83.9%). TMF counts correlated with American Joint Committee on Cancer tumor stages. More importantly, more than one TMF and giant TMFs sizes greater than or equal to 50 μm were associated with statistically significantly shorter overall and cancer-specific disease-free (p < 0.05) survival after curative resection for stage I to IIIA. Giant TMFs greater than or equal to 50 μm size were an independent survival predictor by multivariate analysis., Conclusions: Circulating, in particular, giant TMFs are associated with aggressive clinical behavior in surgically treated patients with NSCLC. The biological role of unique TMFs will need to be further investigated, as these may have a potential impact on immune responses toward cancer., (Published by Elsevier Inc.)

Purpose: As an independent, negative-prognostic biomarker for progression-free survival (PFS) and overall survival (OS), circulating tumor cells (CTCs) constitute a promising component for developing a liquid biopsy for patients with metastatic breast cancer (MBC). The effects of HER2-targeted therapy such as trastuzumab, pertuzumab, T-DM1, and lapatinib on CTC status and longitudinal enumeration were assessed in this trial., Methods: CTC status of 264 patients with MBC was analyzed prior to and after 4 weeks of a new line of palliative systemic therapy. CTCs were assessed using CellSearch®. Three groups were compared: patients with HER2-positive MBC receiving ongoing HER2-targeted therapy (n = 28), patients with de novo HER2-positive MBC and no HER2-targeted therapy in the last 12 months prior to enrollment and start of HER2-targeted therapy (n = 15), and patients with HER2-nonamplified disease and no HER2-targeted therapy (n = 212)., Results: Positive CTC status (≥ 5 CTC/7.5 ml blood) at enrollment was observed in the 3 groups for 17.9, 46.7, and 46.2% (p = 0.02) of patients, respectively. At least one CTC/7.5 ml was seen in 28.6, 53.3, and 67.0% (p < 0.001) of these patients. Furthermore, 3.6, 40.0, and 3.3% (p < 0.001) of the patients had at least one HER2-positive CTC. After 4 weeks of therapy 7.1, 0.0, and 31.1% (p = 0.001) of patients had still a positive CTC status (≥ 5 CTC/7.5 ml blood). At least one CTC/7.5 ml was still observed in 25.0, 20.0, and 50.5% (p = 0.004) of the patients. Furthermore, 7.1, 0.0, and 1.9% (p = 0.187) had at least one HER2-positive CTC. After 3 months of therapy, 35.7, 20.0, and 28.3% (p = 0.536) showed disease progression., Conclusions: HER2-targeted therapy seems to reduce the overall CTC count in patients with MBC. This should be taken into account when CTC status is used as an indicator for aggressive or indolent metastatic tumor disease.

Background: Solid epithelial tumors like breast cancer are the most frequent malignancy in women. Circulating tumor cells (CTCs) are frequently released from hypoxic areas into the blood, where CTCs face elevated oxygen concentrations. This reoxygenation might challenge the use of CTCs for liquid biopsy., Methods: We modeled this situation in vitro using the breast cancer cell lines-MCF-7, MDA-MB-468, MDA-MB-231-and the cell line BC-M1 established from DTCs in the bone marrow. Cells were cultured under hypoxia, followed by a reoxygenation pulse for 4 h, reflecting the circulation time of CTCs. Analyzed were gene products like EGFR, ErbB-2, EpCAM, PD-L1 on mRNA and protein level., Results: mRNAs of erbb2 or pdl1 and protein levels of PD-L1 displayed significant changes, whereas ErbB-2 protein levels remained constant. The strongest discrepancy between protein and mRNA levels under hypoxia was observed for EGFR, supporting the idea of cap-independent translation of egfr mRNA. Analyses of the phosphorylation of AKT, Erk 1/2, and Stat3 revealed strong alterations after reoxygenation., Conclusions: CTCs reaching secondary sites faster than reoxygenation could alter the mRNA and protein levels in the cells. CTC and DTC with high PD-L1 levels might become quiescent under hypoxia but were easily reactivated by reoxygenation.

Keratins are essential elements of the cytoskeleton of normal and malignant epithelial cells. Because carcinomas commonly maintain their specific keratin expression pattern during malignant transformation, keratins are extensively used as tumor markers in cancer diagnosis including the detection of circulating tumor cells in blood of carcinoma patients. Interestingly, recent biological insights demonstrate that epithelial keratins should not only be considered as mere tumor markers. Emerging evidence suggests an active biological role of keratins in tumor cell dissemination and metastasis. In this review, we illustrate the family of keratin proteins, summarize the latest biological insights into keratin function related to cancer metastasis and discuss the current use of keratins for detection of CTCs and other blood biomarkers used in oncology., (Copyright © 2019. Published by Elsevier Ltd.)

Immune checkpoint inhibition (ICI) of the PD-1/PD-L1 axis shows durable responses in a subset of patients with metastatic urothelial carcinoma (UC). However, PD-L1 expression in tumor biopsies does not necessarily correlate with response to PD-1/PD-L1 inhibitors. Thus, a reliable predictive biomarker is urgently needed. Here, the expression of PD-L1 on circulating tumor cells (CTCs) in blood from patients with advanced UC was analyzed. For this purpose, an assay to test PD-L1 expression on CTCs using the CellSearch® system was established using cells of five UC cell lines spiked into blood samples from healthy donors and applied to a heterogeneous cohort of UC patients. Enumeration of CTCs was performed in blood samples from 49 patients with advanced UC. PD-L1 expression in ≥1 CTC was found in 10 of 16 CTC-positive samples (63%). Both intra- and inter-patient heterogeneity regarding PD-L1 expression of CTCs were observed. Furthermore, vimentin-expressing CTCs were detected in 4 of 15 CTC-positive samples (27%), independently of PD-L1 analysis. Both CTC detection and presence of CTCs with moderate or strong PD-L1 expression correlated with worse overall survival. Analyses during disease course of three individual patients receiving ICI suggest that apart from CTC numbers also PD-L1 expression on CTCs might potentially indicate disease progression. This is the first study demonstrating the feasibility to detect CTC-PD-L1 expression in patients with advanced UC using the CellSearch® system. This assay is readily available for clinical application and could be implemented in future clinical trials to evaluate its relevance for predicting and monitoring response to ICI., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)