A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

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

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

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

Real-world use and acceptance of rituximab biosimilars in non-Hodgkin lymphoma in an oncologist network in Germany.

Aim: Present real-world data for rituximab (biosimilar and reference)-containing regimens in extrapolated indications in non-Hodgkin lymphoma (NHL)/chronic lymphocytic leukemia (CLL). Patients & methods: Data collected from office-based oncologic practices in Germany (July 2017-June 2019). Results: Of 1741 patients, 1241 had NHL; 500 had CLL. Of 7595 therapy cycles, 28.3% used reference rituximab; 55.2% used rituximab biosimilars; 2.0% used subcutaneous rituximab; 14.5% used rituximab, not otherwise specified. Rituximab biosimilars were used across all indications; 57.3% of cycles were administered in extrapolated indications. Over 24 months, the proportion of rituximab prescriptions that were for biosimilars increased from 12.0 to 83.0%. Conclusion: Our real-world data in NHL and CLL depicts increasing use of rituximab biosimilars across multiple treatment protocols, including extrapolated indications.

Tumor-Associated Release of Prostatic Cells into the Blood after Transrectal Ultrasound-Guided Biopsy in Patients with Histologically Confirmed Prostate Cancer.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS) is a standard procedure for prostate cancer diagnosis. Because prostate cancer is a multifocal disease in many patients, multiple sampling (n ≥ 10) is required, which may bear the risk of systemic spread of cancer cells. DESIGN: Using the standardized CellSearch® system that allows for the detection of single epithelial cell adhesion molecule-positive circulating tumor cells (CTCs) in blood, we investigated whether prostate biopsy is associated with release of prostatic tumor cells into the circulation. Peripheral blood was obtained before and within 30 min after performing prostate biopsy from 115 men with increased serum prostate-specific antigen. RESULTS: The number of CTCs significantly increased after biopsy in men with histologically confirmed prostate cancer (odds ratio, 7.8; 95% CI, 4.8-12.8), whereas no biopsy-related changes could be detected in men without confirmed prostate cancer. Multivariable analysis showed that biopsy-related increase of CTCs was significantly correlated with a worse progression-free survival (hazard ratio, 12.4; 95% CI, 3.2-48.6) within the median follow-up of 41 months. CONCLUSIONS: Prostate biopsies may lead to a tumor-associated release of CTCs into the blood circulation. Larger confirmatory trials with longer follow-up periods are required before any change in clinical practice can be recommended.

In Vivo Detection of Circulating Tumor Cells in High-Risk Non-Metastatic Prostate Cancer Patients Undergoing Radiotherapy.

High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique; further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%; p = 0.024) and yielded significantly higher CTC numbers (range: 0-15 vs. 0-5; p = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Catch and Release: rare cell analysis from a functionalised medical wire.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single-cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Improved detection of circulating tumor cells in non-metastatic high-risk prostate cancer patients.

The relevance of blood-based assays to monitor minimal residual disease (MRD) in non-metastatic prostate cancer (PCa) remains unclear. Proving that clinically relevant circulating tumor cells (CTCs) can be detected with available technologies could address this. This study aimed to improve CTC detection in non-metastatic PCa patients by combining three independent CTC assays: the CellSearch system, an in vivo CellCollector and the EPISPOT. Peripheral blood samples from high-risk PCa patients were screened for CTCs before and three months after radical prostatectomy (RP). Combining the results of both time points, CTCs were detected in 37%, 54.9% and 58.7% of patients using CellSearch, CellCollector and EPISPOT, respectively. The cumulative positivity rate of the three CTC assays was 81.3% (87/107) with 21.5% (23/107) of patients harboring ≥5 CTCs/7.5 ml blood. Matched pair analysis of 30 blood samples taken before and after surgery indicated a significant decrease in CTCs captured by the CellCollector from 66% before RP to 34% after therapy (p = 0.031). CTC detection by EPISPOT before RP significantly correlated with PSA serum values (p < 0.0001) and clinical tumor stage (p = 0.04), while the other assays showed no significant correlations. In conclusion, CTC-based liquid biopsies have the potential to monitor MRD in patients with non-metastatic prostate cancer.

Accession of Tumor Heterogeneity by Multiplex Transcriptome Profiling of Single Circulating Tumor Cells.

BACKGROUND: Transcriptome analysis of circulating tumor cells (CTCs) holds great promise to unravel the biology of cancer cell dissemination and identify expressed genes and signaling pathways relevant to therapeutic interventions. METHODS: CTCs were enriched based on their EpCAM expression (CellSearch®) or by size and deformability (ParsortixTM), identified by EpCAM and/or pan-keratin-specific antibodies, and isolated for single cell multiplex RNA profiling. RESULTS: Distinct breast and prostate CTC expression signatures could be discriminated from RNA profiles of leukocytes. Some CTCs positive for epithelial transcripts (EpCAM and KRT19) also coexpressed leukocyte/mesenchymal associated markers (PTPRC and VIM). Additional subsets of CTCs within individual patients were characterized by divergent expression of genes involved in epithelial-mesenchymal transition (e.g., CDH2, MMPs, VIM, or ZEB1 and 2), DNA repair (RAD51), resistance to cancer therapy (e.g., AR, AR-V7, ERBB2, EGFR), cancer stemness (e.g., CD24 and CD44), activated signaling pathways involved in tumor progression (e.g., PIK3CA and MTOR) or cross talks between tumors and immune cells (e.g., CCL4, CXCL2, CXCL9, IL15, IL1B, or IL8). CONCLUSIONS: Multimarker RNA profiling of single CTCs reveals distinct CTC subsets and provides important insights into gene regulatory networks relevant for cancer progression and therapy.

Effects of environmental variation on host-parasite interaction in three-spined sticklebacks (Gasterosteus aculeatus).

Recent research provides accumulating evidence that the evolutionary dynamics of host-parasite adaptations strongly depend on environmental variation. In this context, the three-spined stickleback (Gasterosteus aculeatus) has become an important research model since it is distributed all over the northern hemisphere and lives in very different habitat types, ranging from marine to freshwater, were it is exposed to a huge diversity of parasites. While a majority of studies start from explorations of sticklebacks in the wild, only relatively few investigations have continued under laboratory conditions. Accordingly, it has often been described that sticklebacks differ in parasite burden between habitats, but the underlying co-evolutionary trajectories are often not well understood. With the present review, we give an overview of the most striking examples of stickleback-parasite-environment interactions discovered in the wild and discuss two model parasites which have received some attention in laboratory studies: the eye fluke Diplostomum pseudospathacaeum, for which host fish show habitat-specific levels of resistance, and the tapeworm Schistocephalus solidus, which manipulates immunity and behavior of its stickleback host to its advantage. Finally, we will concentrate on an important environmental variable, namely temperature, which has prominent effects on the activity of the immune system of ectothermic hosts and on parasite growth rates.

Heterogeneous PSMA expression on circulating tumor cells: a potential basis for stratification and monitoring of PSMA-directed therapies in prostate cancer.

The prostate specific membrane antigen (PSMA) is the only clinically validated marker for therapeutic decisions in prostate cancer (PC). Characterization of circulating tumor cells (CTCs) obtained from the peripheral blood of PC patients might provide an alternative to tissue biopsies called "liquid biopsy". The aim of this study was to develop a reliable assay for the determination of PSMA on CTCs. PSMA expression was analyzed on tissue samples (cohort one, n = 75) and CTCs from metastatic PC patients (cohort two, n = 29). Specific signals for the expression of PSMA could be seen for different prostate cancer cell line cells (PC3, LaPC4, 22Rv1, and LNCaP) by Western blot, immunohistochemistry (IHC), immunocytochemistry (ICC), and FACS. PSMA expression was found to be significantly increased in patients with higher Gleason grade (p = 0.0011) and metastases in lymph nodes (p = 0.0000085) or bone (p = 0.0020) (cohort one). In cohort two, CTCs were detectable in 20 out of 29 samples (69 %, range from 1 - 1000 cells). Twelve out of 20 CTC-positive patients showed PSMA-positive CTCs (67 %, score 1+ to 3+). We found intra-patient heterogeneity regarding the PSMA status between CTCs and the corresponding primary tumors. The results of our study could help to address the question whether treatment decisions based on CTC PSMA profiling will lead to a measurable benefit in clinical outcome for prostate cancer patients in the near future.

Heat and immunity: an experimental heat wave alters immune functions in three-spined sticklebacks (Gasterosteus aculeatus).

Global climate change is predicted to lead to increased temperatures and more extreme climatic events. This may influence host-parasite interactions, immunity and therefore the impact of infectious diseases on ecosystems. However, little is known about the effects of rising temperatures on immune defence, in particular in ectothermic animals, where the immune system is directly exposed to external temperature change. Fish are ideal models for studying the effect of temperature on immunity, because they are poikilothermic, but possess a complete vertebrate immune system with both innate and adaptive immunity. We used three-spined sticklebacks ( Gasterosteus aculeatus) originating from a stream and a pond, whereby the latter supposedly were adapted to higher temperature variation. We studied the effect of increasing and decreasing temperatures and a simulated heat wave with subsequent recovery on body condition and immune parameters. We hypothesized that the immune system might be less active at low temperatures, but will be even more suppressed at temperatures towards the upper tolerable temperature range. Contrary to our expectation, we found innate and adaptive immune activity to be highest at a temperature as low as 13 °C. Exposure to a simulated heat wave induced long-lasting immune disorders, in particular in a stickleback population that might be less adapted to temperature variation in its natural environment. The results show that the activity of the immune system of an ectothermic animal species is temperature dependent and suggest that heat waves associated with global warming may immunocompromise host species, thereby potentially facilitating the spread of infectious diseases.