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.

High Serum Levels of CCL20 Are Associated with Recurrence and Unfavorable Overall Survival in Advanced Melanoma Patients Receiving Immunotherapy.

BACKGROUND: Immune checkpoint inhibition has revolutionized melanoma therapy, but many patients show primary or secondary resistance. Biomarkers are, therefore, urgently required to predict response prior to the initiation of therapy and to monitor disease progression. METHODS: In this prospective study, we analyzed the serum C-C motif chemokine ligand 20 (CCL20) concentration using an enzyme-linked immunosorbent assay. Blood was obtained at baseline before the initiation of immunotherapy with anti-PD-1 monotherapy or Nivolumab and Ipilimumab in advanced melanoma patients (stages III and IV) enrolled at the University Medical Center Hamburg-Eppendorf. The CCL20 levels were correlated with clinico-pathological parameters and disease-related outcomes. RESULTS: An increased C-C motif chemokine ligand 20 (CCL20) concentration (≥0.34 pg/mL) at baseline was associated with a significantly impaired progression-free survival (PFS) in the high-CCL20 group (3 months (95% CI: 2-6 months) vs. 11 months (95% CI: 6-26 months)) (p = 0.0033) and could be identified as an independent negative prognostic factor for PFS in univariate (Hazard Ratio (HR): 1.98, 95% CI 1.25-3.12, p = 0.004) and multivariate (HR: 1.99, 95% CI 1.21-3.29, p = 0.007) Cox regression analysis, which was associated with a higher risk than S100 (HR: 1.74). Moreover, high CCL20 levels were associated with impaired overall survival (median OS not reached for low-CCL20 group, p = 0.042) with an HR of 1.85 (95% CI 1.02-3.37, p = 0.043) in univariate analysis similar to the established prognostic marker S100 (HR: 1.99, 95% CI: 1.02-3.88, p = 0.043). CONCLUSIONS: CCL20 may represent a novel blood-based biomarker for the prediction of resistance to immunotherapy that can be used in combination with established strong clinical predictors (e.g., ECOG performance score) and laboratory markers (e.g., S100) in advanced melanoma patients. Future prospective randomized trials are needed to establish CCL20 as a liquid biopsy-based biomarker in advanced melanoma.

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis.

BACKGROUND: Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the "metastasis-suppressor" effect of HERC5, with a focus on mitochondrial metabolism pathways. METHODS: We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. RESULTS: Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. CONCLUSIONS: Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

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.

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.

Circulating tumor cells as liquid biopsy markers in cancer patients.

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.

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.

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.

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.

Clinical applications of circulating tumor cells in patients with solid tumors.

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.

[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.

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.

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.

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.

RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer.

Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat due to the lack of targeted therapies. Cancer stem cells (CSCs) are strongly enriched in TNBC lesions and are responsible for the rapid development of chemotherapy resistance and metastasis. Ubiquitin-based epigenetic circuits are heavily exploited by CSCs to regulate gene transcription and ultimately sustain their aggressive behavior. Therefore, therapeutic targeting of these ubiquitin-driven dependencies may reprogram the transcription of CSC and render them more sensitive to standard therapies. In this work, we identified the Ring Finger Protein 40 (RNF40) monoubiquitinating histone 2B at lysine 120 (H2Bub1) as an indispensable E3 ligase for sustaining the stem-cell-like features of the growing mammary gland. In addition, we found that the RNF40/H2Bub1-axis promotes the CSC properties and drug-tolerant state by supporting the glycolytic program and promoting pro-tumorigenic YAP1-signaling in TNBC. Collectively, this study unveils a novel tumor-supportive role of RNF40 and underpins its high therapeutic value to combat the malignant behavior of TNBC.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

Circulating tumour cells for early detection of clinically relevant cancer.

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.

Association Between Obesity and Circulating Tumor Cells in Early Breast Cancer Patients.

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.

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.