Prognostic impact of circulating tumor cells assessed with the CellSearch System (TM) and AdnaTest Breast (TM) in metastatic breast cancer patients: the DETECT study

Introduction: There is a multitude of assays for the detection of circulating tumor cells (CTCs) but a very limited number of studies comparing the clinical relevance of results obtained with different test methods. The DETECT trial for metastatic breast cancer patients was designed to directly compare the prognostic impact of two commercially available CTC assays that are prominent representatives of immunocytochemical and RT-PCR based technologies. Methods: In total, 254 metastatic breast cancer patients were enrolled in this prospective multicenter trial. CTCs were assessed using both the AdnaTest Breast Cancer and the CellSearch system according to the manufacturers' instructions. Results: With the CellSearch system, 116 of 221 (50%) evaluable patients were CTC-positive based on a cut-off level at 5 or more CTCs. The median overall survival (OS) was 18.1 months in CTC-positive patients. (95%-CI: 15.1-22.1 months) compared to 27 months in CTC-negative patients (23.5-30.7 months; p<0.001). This prognostic impact for OS was also significant in the subgroups of patients with triple negative, HER2-positive and hormone receptor-positive/HER2-negative primary tumors. The progression free survival (PFS) was not correlated with CTC status in our cohort receiving different types and lines of systemic treatment (p = 0.197). In multivariate analysis, the presence of CTCs was an independent predictor for OS (HR: 2.7, 95%-CI: 1.6-4.2). When the AdnaTest Breast was performed, 88 of 221 (40%) patients were CTC-positive. CTC-positivity assessed by the AdnaTest Breast had no association with PFS or OS. Conclusions: The prognostic relevance of CTC detection in metastatic breast cancer patients depends on the test method. The present results indicate that the CellSearch system is superior to the AdnaTest Breast Cancer in predicting clinical outcome in advanced breast cancer

Functional characterization of circulating tumor cells (CTCs) from metastatic ER+/HER2− breast cancer reveals dependence on HER2 and FOXM1 for endocrine therapy resistance and tumor cell survival: Implications for treatment of ER+/HER2− breast cancer

Mechanisms of acquired endocrine resistance and late recurrence in patients with ER+/HER2− breast cancer are complex and not fully understood. Here, we evaluated mechanisms of acquired resistance in circulating tumor cells (CTCs) from an ER+/HER2− breast cancer patient who initially responded but later progressed under endocrine treatment. We found a switch from ERα-dependent to HER2-dependent and ERα-independent expression of FOXM1, which may enable disseminated ER+/HER2− cells to re-initiate tumor cell growth and metastasis formation in the presence of endocrine treatment. Our results also suggest a role for HER2 in resistance, even in ER+ breast cancer cells that have neither HER2 amplification nor activating HER2 mutations. We found that NFkB signaling sustains HER2 and FOXM1 expression in CTCs in the presence of ERα inhibitors. Inhibition of NFkB signaling blocked expression of HER2 and FOXM1 in the CTCs, and induced apoptosis. Thus, targeting of NFkB and FOXM1 might be an efficient therapeutic approach to prevent late recurrence and to treat endocrine resistance. Collectively our data show that CTCs from patients with endocrine resistance allow mechanisms of acquired endocrine resistance to be delineated, and can be used to test potential drug regimens for combatting resistance

The impact of HER2 phenotype of circulating tumor cells in metastatic breast cancer: a retrospective study in 107 patients

Background: In metastatic breast cancer (MBC), antigen profiles of metastatic tissue and primary tumor differ in up to 20 % of patients. Reassessment of predictive markers, including human epidermal growth factor receptor 2 (HER2) expression, might help to optimize MBC treatment. While tissue sampling is invasive and often difficult to repeat, circulating tumor cell (CTC) analysis requires only a blood sample and might provide an easy-to-repeat, real-time “liquid biopsy” approach. The present retrospective study was conducted to compare HER2 expression in primary tumors, metastatic tissue, and circulating tumor cells (CTCs) from MBC patients and to analyze the potential impact of HER2 overexpression by CTCs on progression-free (PFS) and overall survival (OS) in MBC. Methods: CTC-positive (five or more CTCs/7.5 mL blood; CellSearch®, Janssen Diagnostics) MBC patients starting a new line of systemic treatment were eligible for the study. HER2 status of CTCs was determined by immunofluorescence (CellSearch®). HER2 status of primary (PRIM) and metastatic (MET) tumor tissue was determined by immunohistochemistry. Data were analyzed using descriptive statistics and Kaplan–Meier plots. Results: One hundred seven patients (median age (range) 57 (33–81) years) were included. 100/107 (93 %) patients were followed-up for a median [95 % confidence interval (CI)] of 28.5 [25.1–40.1] months. Of 37/107 (35 %) CTC-HER2-positive patients only 10 (27 %) were PRIM-HER2-positive. 6/46 (13 %) patients were MET-HER2-positive; only 2/10 (20 %) CTC-HER2-positive patients were MET-HER2-positive. Overall accuracy between CTC-HER2 expression and PRIM-HER2 and MET-HER2 status was 69 % and 74 %, respectively. Kaplan–Meier plots of PFS and OS by CTC-HER2 status revealed significantly longer median [95 % CI] PFS of CTC-HER2-positive versus CTC-HER2-negative patients (7.4 [4.7–13.7] versus 4.34 [3.5–5.9] months; p = 0.035). CTC-HER2-positive status showed no significant difference for OS (13.7 [7.7–30.0] versus 8.7 [5.9–15.3] months; p = 0.287). Conclusions: HER2 status can change during the course of breast cancer. CTC phenotyping may serve as an easy-to-perform “liquid biopsy” to reevaluate HER2 status and potentially guide treatment decisions. Further, prospective studies are needed

Characterization of different CTC subpopulations in non-small cell lung cancer

Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features

Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing

Background Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively. Methods We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC). Results The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone. Conclusions The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'

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.</p

Quantitative High-Resolution Genomic Analysis of Single Cancer Cells

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics

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.publishedVersio

International study on inter-reader variability for circulating tumor cells in breast cancer

Introduction Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. Methods CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. Results For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). Conclusions The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are require