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.

Liquid biopsy for monitoring of tumor dormancy and early detection of disease recurrence in solid tumors.

Cancer is one of the three leading causes of death worldwide. Even after successful therapy and achieving remission, the risk of relapse often remains. In this context, dormant residual cancer cells in secondary organs such as the bone marrow constitute the cellular reservoir from which late tumor recurrences arise. This dilemma leads the term of minimal residual disease, which reflects the presence of tumor cells disseminated from the primary lesion to distant organs in patients who lack any clinical or radiological signs of metastasis or residual tumor cells left behind after therapy that eventually lead to local recurrence. Disseminated tumor cells have the ability to survive in a dormant state following treatment and linger unrecognized for more than a decade before emerging as recurrent disease. They are able to breakup their dormant state and to readopt their proliferation under certain circumstances, which can finally lead to distant relapse and cancer-associated death. In recent years, extensive molecular and genetic characterization of disseminated tumor cells and blood-based biomarker has contributed significantly to our understanding of the frequency and prevalence of tumor dormancy. In this article, we describe the clinical relevance of disseminated tumor cells and highlight how latest advances in different liquid biopsy approaches can be used to detect, characterize, and monitor minimal residual disease in breast cancer, prostate cancer, and melanoma patients.

Circulating Tumor Cells: From Basic to Translational Research.

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.

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.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

BACKGROUND: More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. METHODS: In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. RESULTS: The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. CONCLUSIONS: This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

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.

Differential predictive value of tissue-specific PD-L1 expression scores in adjuvant immunotherapy of melanoma.

BACKGROUND: Adjuvant treatment of stage II-IV melanoma with PD-1-based immune checkpoint inhibitors (ICI) has improved relapse-free survival (RFS) and has therefore become a standard-of-care treatment option. Approximately 25%-30% of patients still recur within 1 year. Predictive biomarkers reflecting real-world data are desired. The predictive relevance of tumour tissue PD-L1 expression in the adjuvant setting remains inconclusive. OBJECTIVES: This retrospective, observational study was conducted to evaluate the value of PD-L1 expression scores in different tumour tissue locations in predicting response towards adjuvant immunotherapeutic treatment. METHODS: Tumour tissue taken prior to anti-PD-1 adjuvant ICI in 243 stage II-IV melanoma patients was collected at University Skin Cancer Center Hamburg. PD-L1 expression was evaluated on immune cells (ICS), tumour cells (TPS) and combined (CPS). Scores were determined by independent pathological physician quantification and correlated with therapy outcome at different cut-off (CO) levels (relapse-free survival, RFS) for different tumour tissue locations (primary tumour, metastases). RESULTS: A total of 104 patients were eligible for analysis. Positivity of ICS, TPS and CPS showed no predictive RFS outcome association at different CO levels when analysed irrespective of tissue origin. In primary tumours, ICS at CO 1% showed a significantly improved RFS upon positivity (HR 0.22). In contrast, positivity to TPS (CO 1%) correlated significantly and independently with improved RFS when evaluated in metastatic tumour tissue specimens (HR 0.37). CONCLUSIONS: PD-L1 tumour tissue expression may serve as a predictive biomarker for adjuvant ICI treatment response stratification in melanoma, but caution should be spent on the origin of tumour tissue analysed. The cell-type relevant for the predictive value of PD-L1 expression is tissue-specific with immune cells being important in primary tumours while tumour cells are key in metastases. The present results should be validated in a multicentre cohort.

C-reactive protein flare predicts response to checkpoint inhibitor treatment in melanoma.

BACKGROUND: The treatment of melanoma has been revolutionized by the use of immune checkpoint inhibition (ICI), but many patients do not benefit. Furthermore, immune-related adverse events may occur during therapy. A predictive biomarker is needed to reliably identify patients benefitting. In lung, renal cell and bladder cancer early C-reactive protein (CRP) kinetics were shown to be a predictive biomarker for ICI. OBJECTIVE: Here, we investigate early CRP kinetics as predictive biomarker for ICI in melanoma patients. METHODS: Two independent prospectively collected cohorts were analysed: Cohort 1 (n = 87) with advanced and Cohort 2 (n = 99) with completely resected melanoma. Patients were stratified by in the dynamics of CRP after ICI initiation: A doubling of baseline CRP within 30 days followed by at least a 30% drop within 3 months was classified as a CRP flare. If no doubling of CRP was reported, but a 30% drop within 3 months, patients were classified as CRP responders and all others as CRP non-responders. Analysed factors included clinical characteristics like S100B and LDH. Median follow-up was 1.5 and 1.7 years for Cohorts 1 and 2. RESULTS: In Cohort 1 CRP flare (n = 12), CRP responders (n = 43) and CRP non-responders (n = 32) with a progression-free survival (PFS) of 0.7, 0.6 and 0.2 years (p = 0.017) and an overall survival (OS) of 2.2, 1.5 and 1.0 years (p = 0.014), respectively. Multivariable Cox analysis showed an independent risk reduction of progression for CRP responders by 62% compared to CRP non-responders (p = 0.001). In Cohort 2 CRP flare (n = 13), CRP responders (n = 70) and CRP non-responders (n = 16) the log-rank analysis showed a significant difference between OS and recurrence-free survival (RFS) curves (p = 0.046 and p = 0.049). CONCLUSION: Early CRP kinetics could indicate a response to ICI with improved OS and RFS/PFS. CRP flare and CRP response indicating significantly improved outcomes compared to CRP non-responders.

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.

EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

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 (IC50 = 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.

Expression profiling by high-throughput sequencing reveals GADD45, SMAD7, EGR-1 and HOXA3 activation in Myostatin (MSTN) and GDF11 treated myoblasts.

Growth differentiation factor 11 (GDF11) and myostatin (MSTN/GDF8) are closely related members of the transforming growth factor ß (TGFß) superfamily, sharing structural homology. Despite these structural similarities, recent research has shed light on the distinct roles these ligands play within muscle tissue. This study aims to uncover both the differences and similarities in gene expression at the transcriptome level by utilizing RNA sequencing. We conducted experiments involving five distinct groups, each with three biological replicates, using C2C12 cell cultures. The cells were subjected to high-throughput profiling to investigate disparities in gene expression patterns following preconditioning with either GDF11 or MSTN at concentrations of 1 nM and 10 nM, respectively. In addition, control groups were established. Our research revealed concentration-dependent gene expression patterns, with 38 genes showing significant differences when compared to the control groups. Notably, GADD45, SMAD7, EGR-1, and HOXA3 exhibited significant differential expression. We also conducted an over-representation analysis, highlighting the activation of MAPK and JNK signaling pathways, along with GO-terms related to genes that negatively regulate metabolic processes, biosynthesis, and protein phosphorylation. This study unveiled the activation of several genes not previously discussed in existing literature whose full biological implications are yet to be determined in future research.

[Liquid Biopsy - A new diagnostic concept in oncology]. / Liquid Biopsy ­ Ein neues diagnostisches Konzept in der Onkologie.

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.

Clinical management and biology of tumor dormancy in breast cancer.

Clinical tumor dormancy is specified as an extended latency period between removal of the primary tumor and subsequent relapse in a cancer patient who has been clinically disease-free. In particular, patients with estrogen receptor-positive breast cancer can undergo extended periods of more than five years before they relapse with overt metastatic disease. Recent studies have shown that minimal residual disease in breast cancer patients can be monitored by different liquid biopsy approaches like analysis of circulating tumor cells or cell-free tumor DNA. Even though the biological principles underlying tumor dormancy in breast cancer patients remain largely unknown, clinical observations and experimental studies have identified emerging mechanisms that control the state of tumor dormancy. In this review, we illustrate the latest discoveries on different molecular aspects that contribute to the control of tumor dormancy and distant metastatic relapse, then discuss current treatments affecting minimal residual disease and dormant cancer cells, and finally highlight how novel liquid biopsy based diagnostic methodologies can be integrated into the detection and molecular characterization of minimal residual disease.

Metabolic regulation of prostate cancer heterogeneity and plasticity.

Metabolic reprogramming is one of the main hallmarks of cancer cells. It refers to the metabolic adaptations of tumor cells in response to nutrient deficiency, microenvironmental insults, and anti-cancer therapies. Metabolic transformation during tumor development plays a critical role in the continued tumor growth and progression and is driven by a complex interplay between the tumor mutational landscape, epigenetic modifications, and microenvironmental influences. Understanding the tumor metabolic vulnerabilities might open novel diagnostic and therapeutic approaches with the potential to improve the efficacy of current tumor treatments. Prostate cancer is a highly heterogeneous disease harboring different mutations and tumor cell phenotypes. While the increase of intra-tumor genetic and epigenetic heterogeneity is associated with tumor progression, less is known about metabolic regulation of prostate cancer cell heterogeneity and plasticity. This review summarizes the central metabolic adaptations in prostate tumors, state-of-the-art technologies for metabolic analysis, and the perspectives for metabolic targeting and diagnostic implications.

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.

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.

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.

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.

Peripheral and Portal Venous KRAS ctDNA Detection as Independent Prognostic Markers of Early Tumor Recurrence in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: KRAS circulating tumor DNA (ctDNA) has shown biomarker potential for pancreatic ductal adenocarcinoma (PDAC) but has not been applied in clinical routine yet. We aim to improve clinical applicability of ctDNA detection in PDAC and to study the impact of blood-draw site and time point on the detectability and prognostic role of KRAS mutations. METHODS: 221 blood samples from 108 PDAC patients (65 curative, 43 palliative) were analyzed. Baseline peripheral and tumor-draining portal venous (PV), postoperative, and follow-up blood were analyzed and correlated with prognosis. RESULTS: Significantly higher KRAS mutant detection rates and copy numbers were observed in palliative compared to curative patients baseline blood (58.1% vs 24.6%; P = 0.002; and P < 0.001). Significantly higher KRAS mutant copies were found in PV blood compared to baseline (P < 0.05) samples. KRAS detection in pre- and postoperative and PV blood were significantly associated with shorter recurrence-free survival (all P < 0.015) and identified as independent prognostic markers. KRAS ctDNA status was also an independent unfavorable prognostic factor for shorter overall survival in both palliative and curative cohorts (hazard ratio [HR] 4.9, P = 0.011; HR 6.9, P = 0.008). CONCLUSIONS: KRAS ctDNA detection is an independent adverse prognostic marker in curative and palliative PDAC patients-at all sites of blood draw and a strong follow-up marker. The most substantial prognostic impact was seen for PV blood, which could be an effective novel tool for identifying prognostic borderline patients-guiding future decision-making on neoadjuvant treatment despite anatomical resectability. In addition, higher PV mutant copy numbers contribute to an improved technical feasibility.

Tumor cell E-selectin ligands determine partialefficacy of bortezomib on spontaneous lung metastasis formation of solid human tumors in vivo.

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.