Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma.

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.

Diagnostic leukapheresis reveals distinct phenotypes of NSCLC circulating tumor cells.

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.

Circulating Tumor Cells and Thromboembolic Events in Patients with Glioblastoma.

Patients with glioblastoma (GBM) are at increased risk for arterial and venous thromboembolism (TE). Risk factors include surgery, the use of corticosteroids, radiation, and chemotherapy, but also prothrombotic characteristics of the tumor itself such as expression of tissue factor, vascular endothelial growth factor, or podoplanin. Although distant metastases are extremely rare in this tumor entity, circulating tumor cells (CTCs) have been detected in a significant proportion of GBM patients, potentially linking local tumor growth characteristics to systemic hypercoagulability. We performed post hoc analysis of a study, in which GBM patients had been investigated for CTCs. Information on TE was retrieved from electronic patient charts. In total, 133 patients (median age, 63 years; interquartile range, 53-70 years) were analyzed. During follow-up, TE was documented in 14 patients (11%), including 8 venous and 6 arterial events. CTCs were detected in 26 patients (20%). Four (15%) patients with CTCs had a TE compared with 10 (9%) patients without CTCs. There was no difference in the frequency of TE events between patients with and those without detectable CTCs (p = 0.58). In summary, although our study confirms a high risk of TE in GBM patients, it does not point to an obvious association between CTCs and vascular thrombosis.

Limited prognostic role of routine serum markers (AP, CEA, LDH and NSE) in oligorecurrent prostate cancer patients undergoing PSMA-radioguided surgery.

INTRODUCTION: We evaluated the prognostic role of pre-salvage prostate-specific membrane antigen-radioguided surgery (PSMA-RGS) serum levels of alkaline phosphatase (AP), carcinoembryonic antigen (CEA), lactate dehydrogenase (LDH), and neuron-specific enolase (NSE). MATERIALS AND METHODS: Patients who consecutively underwent PSMA-RGS for prostate cancer (PCa) oligorecurrence between January 2019 and January 2022 were selected. Biomarkers were assessed one day before surgery. Cox regression and logistic regression models tested the relationship between biochemical recurrence-free survival (BFS), 6- and 12-month biochemical recurrence (BCR), and several independent variables, including biomarkers. RESULTS: 153 consecutive patients were analyzed. In the univariable Cox regression analysis, none of the biomarkers achieved predictor status (AP: hazard ratio [HR] = 1.03, 95% CI 0.99, 1.01; p = 0.19; CEA: HR = 1.73, 95% CI 0.94, 1.21; p = 0.34; LDH: HR = 1.01, 95% CI 1.00, 1.01; p = 0.05; NSE: HR = 1.02, 95% CI 0.98, 1.06; p = 0.39). The only independent predictor of BFS was the number of positive lesions on PSMA PET (HR = 1.17, 95% CI 1.02, 1.30; p = 0.03). The number of positive lesions was confirmed as independent predictor for BCR within 6 and 12 months (BCR < 6 months: odds ratio [OR] = 1.1, 95% CI 1.0, 1.3; p = 0.04; BCR < 12 months: OR = 1.1, 95% CI 1.0, 1.3; p = 0.04). CONCLUSION: The assessment of AP, CEA, LDH, and NSE before salvage PSMA-RGS showed no prognostic impact. Further studies are needed to identify possible predictors that will optimize patient selection for salvage PSMA-RGS.

Frequency and Prognostic Value of Circulating Tumor Cells in Cancer of Unknown Primary.

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.

Efficacy of Lapatinib in Patients with HER2-Negative Metastatic Breast Cancer and HER2-Positive Circulating Tumor Cells-The DETECT III Clinical Trial.

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.

Relationship of Ki-67 index in biopsies of metastatic breast cancer tissue and circulating tumor cells (CTCs) at the time of biopsy collection.

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.

Protocol of the IntenSify-Trial: An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy.

Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2 ) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2 ) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.

EpCAM-positive circulating tumor cells and serum AFP levels predict outcome after curative resection of hepatocellular carcinoma.

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.

First-line treatment of unresectable or metastatic HER2 positive esophagogastric adenocarcinoma: liquid biomarker analysis of the phase 2 INTEGA trial.

BACKGROUND: The addition of nivolumab to trastuzumab and chemotherapy in first-line unresectable or metastatic HER2 positive esophagogastric adenocarcinoma (HER2+ EGA) results in long progression-free and overall survival as shown by the INTEGA (ipilimumab or FOLFOX in combination with nivolumab and trastuzumab in HER2 positive esophagogastric adenocarcinoma) trial. This trial suggested that the chemotherapy backbone is needed in an unselected HER2+ patient population. Yet, it remains an open question if there are specific patient subsets that may benefit from an enhanced immunotherapeutic but chemotherapy-free approach. METHODS: We analyzed blood T cell repertoire metrics determined by next-generation sequencing, circulating tumor cell (CTC) counts detected by CellSearch and their expression of HER2 and PD-L1 as potential liquid biomarkers predicting outcomes on ipilimumab versus FOLFOX (folinic acid, FOL, fluorouracil, F, oxaliplatin, OX) chemotherapy added to a backbone of trastuzumab and nivolumab in patients with HER2+ EGA in the INTEGA trial population. RESULTS: Patients with two out of three baseline-determined liquid biomarkers-high T cell repertoire richness, absence of CTCs or HER2-expression on CTCs-made up approximately 44% of HER2+ EGA cases and did not show compromise in efficacy if treated with a chemotherapy-free regimen. Long-term responders showing a progression-free survival of >12 months were enriched in this biomarker triad, especially if treated on the chemotherapy-free arm. CONCLUSION: Prospective validation of this liquid biomarker triad is needed to molecularly define HER2+ EGA patient subsets with different needs in the first-line systemic treatment setting.

PSMA-heterogeneity in metastatic castration-resistant prostate cancer: Circulating tumor cells, metastatic tumor burden, and response to targeted radioligand therapy.

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.

A phase I prospective, non-randomized trial of autologous dendritic cell-based cryoimmunotherapy in patients with metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 108) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

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.

Comparative analysis of EpCAM high-expressing and low-expressing circulating tumour cells with regard to their clonal relationship and clinical value.

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.

Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Circulating tumor cells as a preoperative risk marker for occult metastases in patients with resectable cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive tumor associated with a high rate of recurrence after resection. An important risk factor for recurrence is the presence of occult metasta-ses, which are not radiologically detectable at the time of diagnosis. There are currently no biomarkers for the preoperative assessment of micrometastases. A previous study demonstrated the prognostic relevance of circulating tumor cells (CTC) in patients with advanced CCA but the potential of CTCs as a preoperative marker for detecting occult metastases has not been investigated so far. In this two-phase study, we first recruited a cohort of 27 patients with histologically proven, metastatic CCA or gallbladder cancer (GBCA) to assess feasibility (feasibility cohort, FC). CTCs were measured in the peripheral blood using the CellSearch System (CSS) between October 2012 and January 2017. Subsequently, in 11 patients undergoing curative-intended resection for CCA (intrahepatic CCA: n =4; extrahepatic CCA n= 6; gallbladder cancer: n=1), peripheral and central venous blood specimens were obtained to improve detection rate by simultaneous measurement and to elucidate distribution of CTCs in different venous compartments. Presence of CTCs detection was correlated with postoperative TNM-status. In the FC, CTCs (range 1-3 cells, median: 1) were detected in 40% (11/27) patients and were signifi-cantly associated with worse overall survival (hazard ratio: 3.59; 95% CI: 1.79- 7.1; p = 0.04). By combined peripheral and central measurement, CTC detection was increased to 54% (6/11) in the resection cohort (RC) and was associated with metastases that were only identified during the surgical procedure (peritoneal carcinoma: n = 1; infiltration of the duodenum: n = 1) or immediately after surgery (evidence of pulmonary metastases by CT scan two days after resection, not evident on initial tumor staging prior resection). Taken together, in this single center pilot study, we demonstrated that CTCs are detectable in CCA patients and are associated with significantly impaired survival in patients at metastatic stage. Detection rate prior to surgery was improved to >50% by combined peripheral and central measurement. Moreover, preoperative CTC detection may indicate existing metastases and could help to stratify patients more accurately.

Circulating tumor cells and extracellular vesicles as liquid biopsy markers in neuro-oncology: prospects and limitations.

For many tumor entities, tumor biology and response to therapy are reflected by components that can be detected and captured in the blood stream. The so called "liquid biopsy" has been stratified over time into the analysis of circulating tumor cells (CTC), extracellular vesicles (EVs), and free circulating components such as cell-free nucleic acids or proteins. In neuro-oncology, two distinct areas need to be distinguished, intrinsic brain tumors and tumors metastatic to the brain. For intrinsic brain tumors, specifically glioblastoma, CTCs although present in low abundance, contain highly relevant, yet likely incomplete biological information for the whole tumor. For brain metastases, CTCs can have clinical relevance for patients especially with oligometastatic disease and brain metastasis in cancers like breast and lung cancer. EVs shed from the tumor cells and the tumor environment provide complementary information. Sensitive technologies have become available that are able to detect both, CTCs and EVs in the peripheral blood of patients with intrinsic and metastatic brain tumors despite the blood brain barrier. In reference to glioblastoma EVs, being shed by tumor cells and microenvironment and being more diffusible than CTCs may yield a more complete reflection of the whole tumor compared to low-abundance CTCs representing only a fraction of the multiclonal tumor heterogeneity. We here review the emerging aspects of CTCs and EVs as liquid biopsy biomarkers in neuro-oncology.

Detection and Characterization of Estrogen Receptor α Expression of Circulating Tumor Cells as a Prognostic Marker.

CTCs have increasingly been used as a liquid biopsy analyte to obtain real-time information on the tumor through minimally invasive blood analyses. CTCs allow for the identification of proteins relevant for targeted therapies. Here, we evaluated the expression of estrogen receptors (ER) in CTCs of patients with metastatic breast cancer. From sixty metastatic breast cancer patients who had ER-positive primary tumors (range of 1−70% immunostaining) at initial cancer diagnosis, 109 longitudinal blood samples were prospectively collected and analyzed using the CellSearch System in combination with the ERα monoclonal murine ER-119.3 antibody. Prolonged cell permeabilization was found to be required for proper staining of nuclear ER in vitro. Thirty-one cases were found to be CTC-positive; an increased number of CTCs during endocrine and chemotherapy was correlated with disease progression, whereas a decrease or stable amount of CTC number (<5) during treatment was correlated with a better clinical outcome. Survival analyses further indicate a positive association of CTC-status with progression-free survival (HR, 66.17; 95%CI, 3.66−195.96; p = 0.0045) and overall survival (HR, 6.21; 95%CI, 2.66−14.47; p < 0.0001). Only one-third of CTC-positive breast cancer patients, who were initially diagnosed with ER-positive primary tumors, harbored ER-positive CTCs at the time of metastasis, and even in those patients, both ER-positive and ER-negative CTCs were found. CTC-positivity was correlated with a shorter relapse-free survival. Remarkably, ER-negative CTCs were frequent despite initial ER-positive status of the primary tumor, suggesting a switch of ER phenotype or selection of minor ER-negative clones as a potential mechanism of escape from ER-targeting therapy.

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Modeling the Prognostic Impact of Circulating Tumor Cells Enumeration in Metastatic Breast Cancer for Clinical Trial Design Simulation.

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).