Showing papers by "Klaus Pantel published in 2017"

Circulating and disseminated tumour cells - mechanisms of immune surveillance and escape.

Abstract: Metastatic spread of tumour cells is the main cause of cancer-related deaths. Understanding the mechanisms of tumour-cell dissemination has, therefore, become an important focus for cancer research. In patients with cancer, disseminated cancer cells are often detectable in the peripheral blood as circulating tumour cells (CTCs) and in the bone marrow or lymph nodes as disseminated tumour cells (DTCs). The identification and characterization of CTCs and DTCs has yielded important insights into the mechanisms of metastasis, resulting in a better understanding of the molecular alterations and profiles underlying drug resistance. Given the expanding role of immunotherapies in the treatment of cancer, interactions between tumour cells and immune cells are the subject of intense research. Theoretically, cancer cells that exit the primary tumour site - leaving the protection of the typically immunosuppressive tumour microenvironment - will be more vulnerable to attack by immune effector cells; thus, the survival of tumour cells after dissemination might be the 'Achilles' heel' of metastatic progression. In this Review, we discuss findings relating to the interactions of CTCs and DTCs with the immune system, in the context of cancer immuno-editing, evasion from immune surveillance, and formation of metastases.

Liquid Biopsy: Current Status and Future Perspectives.

Abstract: Cancer patients usually receive therapies according to their primary tumor's molecular traits. These characteristics may change during the molecular evolution of distant metastases as the leading cause of cancer deaths. Primary tumor tissue, if accessible at all, does not always provide enough information to stratify individual patients to the most promising therapy. Re-analysis of metastatic lesions by needle biopsy is possible but invasive, and limited by the known intra-patient heterogeneity of individual lesions. These hurdles might be overcome by analyzing tumor cells or tumor cell products in blood samples (liquid biopsy), which in principle might reflect all subclones present at that specific time point and allow sequential monitoring of disease evolution. Liquid biopsies inform on circulating tumor cells as well as tumor-derived cell-free nucleic acids, exosomes and platelets. Here, we introduce the different approaches of blood-based liquid biopsies and discuss the clinical applications in oncology.

Epithelial-mesenchymal plasticity in circulating tumor cells

Abstract: Epithelial-to-Mesenchymal Transition (EMT) is a complex process that supports the migratory capacity of epithelial tumor cells and is thought to play a crucial role in promoting cancer metastasis. Despite the wealth of experimental data, the exact role of EMT in cancer patients remains more controversial. Over the past 10 years, sensitive technologies that allow the detection and molecular characterization of circulating tumor cells (CTCs) in the peripheral blood of tumor patients have been developed. These analyses help to shed new light into the importance of EMT for human tumor cell dissemination. CTCs with mesenchymal features can be attributed in some clinical studies (in particularly on breast cancer) to higher disease stages, presence of metastases, and even to therapy response and worse outcome. However, the published studies addressing the impact of mesenchymal-like CTCs show heterogeneity with regard to assay specificity, size of cancer and control groups, and endpoint parameters. In the present review, we present the key features of the biology of CTCs in relation to epithelial-to-mesenchy-mal plasticity, describe the current technologies for enrichment and detection of CTCs with high epithelial-mesenchymal plasticity, and discuss the clinical studies that have assessed the relevance of mesenchymal CTCs in carcinoma patients.

Prognostic Impact of Circulating Tumor Cells for Breast Cancer Patients Treated in the Neoadjuvant "Geparquattro" Trial

Abstract: Purpose: This study aimed to evaluate the prognostic impact of circulating tumor cells (CTC) detected in patients with operable or locally advanced breast cancer before and after neoadjuvant therapy (NT) within the clinical trial GeparQuattro. Experimental Design: Data on CTCs enumerated with the CellSearch system were available for 213 and 207 patients before and after NT, respectively. Associations of CTCs with disease-free survival (DFS) and overall survival (OS) were analyzed by nonparametric Kaplan–Meier estimates and parametric Cox regression. Results: After a median follow-up of 67.1 months, the detection of ≥1 CTC/7.5 mL and ≥2 CTCs/7.5 mL before NT was associated with reduced DFS (P = 0.031 and P Conclusions: Detection of CTCs before NT is an independent prognostic factor of impaired clinical outcome, and combined with pCR, it could be helpful to stratify breast cancer patients for therapeutic interventions. Clin Cancer Res; 23(18); 5384–93. ©2017 AACR.

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells.

Abstract: // Anna Babayan 1 , Malik Alawi 2, 3 , Michael Gormley 4 , Volkmar Muller 5 , Harriet Wikman 1 , Ryan P. McMullin 6 , Denis A. Smirnov 4 , Weimin Li 4 , Maria Geffken 7 , Klaus Pantel 1 and Simon A. Joosse 1 1 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Bioinformatics Core, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology (HPI), Hamburg, Germany 4 Janssen Research and Development, Spring House, PA, USA 5 Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 6 LabConnect LLC, Seattle, WA, USA 7 Department of Transfusion Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Klaus Pantel, email: pantel@uke.de Simon A. Joosse, email: s.joosse@uke.de Keywords: NGS, WGA, SNP, allelic dropout, CellSave Received: April 06, 2016 Accepted: June 09, 2016 Published: July 19, 2016 ABSTRACT BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS: We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION: We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

Mast cells decrease efficacy of anti-angiogenic therapy by secreting matrix-degrading granzyme B

Abstract: Resistance towards VEGF-centered anti-angiogenic therapy still represents a substantial clinical challenge. We report here that mast cells alter the proliferative and organizational state of endothelial cells which reduces the efficacy of anti-angiogenic therapy. Consequently, absence of mast cells sensitizes tumor vessels for anti-angiogenic therapy in different tumor models. Mechanistically, anti-angiogenic therapy only initially reduces tumor vessel proliferation, however, this treatment effect was abrogated over time as a result of mast cell-mediated restimulation of angiogenesis. We show that mast cells secrete increased amounts of granzyme b upon therapy, which mobilizes pro-angiogenic laminin- and vitronectin-bound FGF-1 and GM-CSF from the tumor matrix. In addition, mast cells also diminish efficacy of anti-angiogenic therapy by secretion of FGF-2. These pro-angiogenic factors act beside the targeted VEGFA–VEGFR2-axis and reinduce endothelial cell proliferation and angiogenesis despite the presence of anti-angiogenic therapy. Importantly, inhibition of mast cell degranulation with cromolyn is able to improve efficacy of anti-angiogenic therapy. Thus, concomitant mast cell-targeting might lead to improved efficacy of anti-angiogenic therapy. Resistance towards VEGF-centered anti-angiogenic therapy is an important clinical challenge. Here, the authors show that mast cells mediate resistance to anti-angiogenetic inhibitors by altering the proliferative and organizational state of endothelial cells through mobilization of FGF-1 and GM-CSF from the tumor matrix and secretion of FGF-2.

Circulating tumor cells as liquid biomarker for high HCC recurrence risk after curative liver resection.

Abstract: // Johann von Felden 1, * , Kornelius Schulze 1, * , Till Krech 2 , Florian Ewald 3 , Bjorn Nashan 3 , Klaus Pantel 4 , Ansgar W. Lohse 1 , Sabine Riethdorf 4 and Henning Wege 1 1 Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 2 Institute of Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 3 Department for Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 4 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany * These authors have contributed equally to this work Correspondence to: Henning Wege, email: h.wege@uke.de Keywords: hepatocellular carcinoma, CTC, BCLC A, liquid biopsy, precision medicine Received: July 13, 2017 Accepted: July 31, 2017 Published: September 23, 2017 ABSTRACT Background: Early hepatocellular carcinoma (HCC) has a limited prognosis due to recurrence rates of more than 50% after liver resection. Recurrence within two years is believed to be caused by untraceable micro metastases at the time of resection. The objective of this study was to investigate EpCAM-positive circulating tumor cells (CTC) as liquid biomarker to identify patients with high risk of recurrence after liver resection. Methods: 61 patients undergoing resection between 2011 and 2015 were consecutively enrolled. Blood specimens were obtained prior to surgery and processed with the CellSearch TM system, detecting EpCAM-positive CTC. The primary endpoint was recurrence-free survival (RFS). Results: 13 women and 44 men (63.6 ± 11.1 years) were finally evaluated. CTC-positive patients had a significantly higher risk of recurrence with a hazard ratio (HR) of 2.3 (p=0.027), and a shorter RFS compared to CTC-negative patients (5.0 ± 1.5 vs. 12.0 ± 2.5 months, p=0.039). As expected, incomplete resection (R1) was also associated with shorter RFS (HR=2.6, p=0.035), but vascular invasion was not. However, the predictive power of CTC status was independent of R1. Conclusion: Bloodstream detection of CTC prior to curative-intended liver resection discloses an elevated risk of HCC recurrence and could identify patients, who might benefit from adjuvant treatment.

Liquid biopsy in 2016: Circulating tumour cells and cell-free DNA in gastrointestinal cancer

Abstract: The challenge to obtain needle biopsy samples from patients with cancer has steered the development of new blood-based diagnostics called 'liquid biopsy'. In 2016, major advances have been made in the use of circulating tumour cells and cell-free DNA for monitoring tumour evolution in patients with cancer of the gastrointestinal tract, with a focus on colorectal cancer.

Plasma microRNA signature is associated with risk stratification in prostate cancer patients.

Abstract: The aim of the current study was to establish a unique expression profile of circulating cell-free microRNAs (miRNAs) capable of differentiating between prostate cancer (PCa) patients with high risk and intermediate risk Gleason scores MiRNA expression profiles were determined in plasma samples from 79 treatment-naive PCa patients, 1-2 follow-up samples after radical prostatectomy (RP) from 51 out of the 79 PCa patients, and 33 healthy men, using a quantitative real time PCR based array containing 48 selected miRNAs We identified 27 up- and 2 downregulated plasma miRNAs in PCa patients compared with healthy men Most of the upregulated miRNAs levels were also associated with increasing PSA levels and Gleason scores Particularly, the levels of miR-16 (p=0002), miR-148a (p=0006) and miR-195 (p=0006) significantly correlated with high-risk Gleason scores, whereby miR-148a (p=0003) was also significantly associated with increasing PSA values The high miRNA levels before RP remained increased in the postsurgical plasma samples Our findings show a network of deregulated plasma miRNAs In particular, miR-16, miR-148a and miR-195 are involved in the regulation of the PI3K/Akt signaling pathway These miRNAs may be promising therapeutic targets for high-risk PCa stratification This article is protected by copyright All rights reserved

Axl Blockade by BGB324 Inhibits BCR-ABL Tyrosine Kinase Inhibitor–Sensitive and -Resistant Chronic Myeloid Leukemia

Abstract: Purpose:BCR-ABL kinase inhibitors are employed successfully for chronic myeloid leukemia (CML) treatment. However, resistant disease and persistence of BCR-ABL1-independent leukemia stem and progenitor cells (LSPC) remain clinical challenges. The receptor tyrosine kinase Axl can mediate survival and therapy resistance of different cancer cells. We investigated the therapeutic potential of Axl inhibition in CML. Experimental Design:We used primary cells from CML patients and TKI-sensitive and -resistant BCR-ABL1+ CML cell lines and a novel Ponatinib-resistant cell line KCL-22 PonR. We analyzed the effects of genetic and pharmacologic Axl blockade by the small molecule Axl inhibitor BGB324 in vitro and in vivo. In BCR-ABL1 unmutated cells we also investigated BGB324 in combination with imatinib. Results:We demonstrate overexpression of Axl receptor tyrosine kinase in primary cells of CML patients compared to healthy individuals, and a further increase of Axl expression in BCR-ABL TKI-resistant patients. We show that Axl blockage decreased growth of BCR-ABL TKI-sensitive CML cells including CD34+ cells and exerts additive effects with imatinib via inhibition of Stat5 activation. BGB324 also inhibits BCR-ABL TKI-resistant cells, including T315I-mutated and ponatinib-resistant primary cells. BGB324 exerted therapeutic effects in BCR-ABL1 T315I-mutated and ponatinib-resistant preclinical mouse models. Notably, BGB324 does not inhibit BCR-ABL1 and consequently inhibits CML independent of BCR-ABL1 mutational status. Conclusions:Our data show that Axl inhibition has therapeutic potential in BCR-ABL TKI-sensitive as well as resistant CML and support the need for clinical trials.

Tumour microenvironment: informing on minimal residual disease in solid tumours.

Abstract: Patients with resectable solid tumours can harbour minimal residual disease (MRD) after initial treatment, which is a potential source for subsequent metastatic relapse. The interaction between disseminated tumour cells (DTCs) and the new microenvironment in which they reside determines whether DTCs remain dormant or progress into overt metastases. We highlight the promise of liquid biopsies to inform on MRD.

The HER2 phenotype of circulating tumor cells in HER2-positive early breast cancer: A translational research project of a prospective randomized phase III trial.

Abstract: Background HER2 is one of the predominant therapeutic targets in breast cancer. The metastatic selection process may lead to discrepancies between the HER2 status of the primary tumor and circulating tumor cells (CTCs). This study analyzed the HER2 status of CTCs in patients with HER2-positive primary breast cancer at the time of diagnosis. Aim of the study was to assess potential discordance of HER2 status between primary tumor and CTCs, as this may have important implications for the use of HER2-targeted therapy. Methods The number and HER2 status of CTCs out of 30ml peripheral blood were assessed in 642 patients using the CellSearch System (Janssen Diagnostics, USA). The cutoff for CTC positivity was the presence of at least 1 CTC, and the cutoff for HER2 positivity of CTCs was the presence of at least 1 CTC with a strong HER2 staining. Results 258 (40.2%) of the 642 patients were positive for CTCs (median 2; range 1–1,689). 149 (57.8%) of these 258 patients had at least 1 CTC with strong HER2 staining. The presence of HER2-positive CTCs was not associated with tumor size (p = 0.335), histopathological grading (p = 0.976), hormone receptor status (ER: p = 0.626, PR: p = 0.263) or axillary lymph node involvement (p = 0.430). Overall, 83 (32.2%) of the CTC-positive patients exclusively had CTCs with strong HER2 staining, whereas 31 (12.0%) had only CTCs with negative HER2 staining. Within-sample variation in the HER2 status of CTCs was found in 86 (57.8%) of the 149 patients with more than 1 CTC. Conclusion This study demonstrated that discordance between the HER2 expression of CTCs and that of the primary tumor frequently occurs in early breast cancer. Future follow-up evaluation will assess whether this discrepancy may contribute to trastuzumab resistance.

Circulating Tumour Cell Release after Cement Augmentation of Vertebral Metastases

Abstract: Cement augmentation via percutaneous vertebroplasty or kyphoplasty for treatment of spinal metastasis is a well-established treatment option. We assessed whether elevated intrametastatic pressure during cement augmentation results in an increased dissemination of tumour cells into the vascular circulation. We prospectively collected blood from patients with osteolytic spinal column metastases and analysed the prevalence of circulating tumour cells (CTCs) at three time-points: preoperatively, 20 minutes after cement augmentation, and 3–5 days postoperatively. Enrolling 21 patients, including 13 breast- (61.9%), 5 lung- (23.8%), and one (4.8%) colorectal-, renal-, and prostate-carcinoma patient each, we demonstrate a significant 1.8-fold increase of EpCAM+/K+ CTCs in samples taken 20 minutes post-cement augmentation (P < 0.0001). Despite increased mechanical CTC dissemination due to cement augmentation, follow-up blood draws demonstrated that no long-term increase of CTCs was present. Array-CGH analysis revealed a specific profile of the CTC collected 20 minutes after cement augmentation. This is the first study to report that peripheral CTCs are temporarily increased due to vertebral cement augmentation procedures. Our findings provide a rationale for the development of new prophylactic strategies to reduce the increased release of CTC after cement augmentation of osteolytic spinal metastases.

A nonrandomized, prospective, clinical study on the impact of circulating tumor cells on outcomes of urothelial carcinoma of the bladder patients treated with radical cystectomy with or without adjuvant chemotherapy.

Abstract: To investigate outcomes of urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC) according to the presence of circulating tumor cells (CTC) and the administration of adjuvant chemotherapy (AC). We prospectively enrolled 226 UCB patients treated with RC without neoadjuvant chemotherapy at our institution between 2007 and 2013. Blood samples were obtained from all patients preoperatively and analyzed for CTC using the CellSearch® system. Platinum-based AC was administered in 50 patients (27.0%). Cox regression models evaluated the association of CTC with disease recurrence, cancer-specific and overall mortality according to AC administration. 185 patients were available for analyses. CTC were present in 41 patients (22.2%). Patients with presence of CTC received AC more frequently, compared to patients without CTC (p = 0.027). At a median follow-up of 31 months, the presence of CTC was associated with disease recurrence, cancer-specific and overall mortality (p-values < 0.001) in patients without AC administration. In patients who received AC, there was no difference in either endpoint between patients with or without presence of CTC. In multivariable analysis of patients without AC administration, the presence of CTC was an independent predictor for disease recurrence (HR: 4.9; p < 0.001), cancer-specific (HR: 4.2; p = 0.003) and overall mortality (HR: 4.2; p = 0.001). The CTC status may be implemented in decision-making regarding AC administration in UCB patients following RC. CTC measurement should be implemented in future UCB studies on systemic chemotherapy to validate our findings.

Expression of Epithelial Mesenchymal Transition and Cancer Stem Cell Markers in Circulating Tumor Cells.

Abstract: The characterization of circulating tumor cells (CTC) has the potential not only to provide important insights into molecular alterations of advanced tumor disease but also to facilitate risk prediction. Epithelial mesenchymal transition (EMT) has been discovered as important process for the development of metastases and the dissemination of tumor cells into the blood stream. In different tumor types, CTC with a mesenchymal phenotype have been reported that have presumably underwent EMT. Moreover, CTC with stem-cell like characteristics have been postulated as important drivers of tumor progression. Different platforms have been introduced to allow CTC enrichment independent of expression of epithelial antigens, as these may be downregulated in EMT- or stem-cell-like CTC. Both for CTCs with EMT- or stem-cell features different markers have been proposed. However, there is still a lack of evidence on the association of these markers with functional features and characteristics for stem cells and cells undergoing EMT.

PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.

Abstract: // Ina Hohensee 1 , Han-Ning Chuang 2, * , Astrid Grottke 3, * , Stefan Werner 1 , Alexander Schulte 4 , Stefan Horn 5 , Katrin Lamszus 4 , Kai Bartkowiak 1 , Isabell Witzel 6 , Manfred Westphal 4 , Jakob Matschke 7 , Markus Glatzel 7 , Manfred Jucker 3 , Tobias Pukrop 2, 8 , Klaus Pantel 1 , Harriet Wikman 1 1 Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, 20246, Germany 2 Department of Hematology/Oncology, University Medical Center Gottingen, 37099 Gottingen, Germany 3 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 4 Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 5 Bone Marrow Transplantation Unit, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 6 Department of Gynecology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 7 Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf, 20246 Hamburg, Germany 8 Department of Medicine III, University Medical Center Regensburg, 93053 Regensburg, Germany * Shared senior author Correspondence to: Harriet Wikman, email: h.wikman@uke.de Keywords: brain metastases, breast cancer, PTEN, microenvironment, astrocytes Received: August 04, 2016 Accepted: December 13, 2016 Published: December 20, 2016 ABSTRACT Despite improvement of therapeutic treatments for breast cancer, the development of brain metastases has become a major limitation to life expectancy for many patients. Brain metastases show very commonly alterations in EGFR and HER2 driven pathways, of which PTEN is an important regulator. Here, we analyzed PTEN expression in 111 tissue samples of breast cancer brain metastases (BCBM). Loss of PTEN was found in a substantial proportion of BCBM samples (48.6%) and was significantly associated with triple-negative breast cancer (67.5%, p = 0.001) and a shorter survival time after surgical resection of brain metastases ( p = 0.048). Overexpression of PTEN in brain-seeking MDA-MB-231 BR cells in vitro reduced activation of the AKT pathway, notably by suppression of Akt1 kinase activity. Furthermore, the migration of MDA-MB-231 BR cells in vitro was promoted by co-culturing with both astrocytes and microglial cells. Interestingly, when PTEN was overexpressed the migration was significantly inhibited. Moreover, in an ex vivo organotypic brain slice model, PTEN overexpression reduced invasion of tumor cells. This was accompanied by reduced astrocyte activation that was mediated by autocrine and paracrine activation of GM-CSF/ CSF2RA and AKT/ PTEN pathways. In conclusion, loss of PTEN is frequently detected in triple-negative BCBM patients and associated with poor prognosis. The findings of our functional studies suggest that PTEN loss promotes a feedback loop between tumor cells and glial cells, which might contribute to disease progression.

Clinical prospects of liquid biopsies

Abstract: The clinical utility of diagnostic markers extracted from liquid biopsies from cancer patients is hampered by knowledge gaps in biological understanding but can be aided by technological developments.

Biology and clinical significance of circulating tumor cell subpopulations in lung cancer

Abstract: By identifying and tracking genetic changes in primary tumors and metastases, patients can be stratified for the most efficient therapeutic regimen by screening for known biomarkers. However, retrieving tissues biopsies is not always feasible due to tumor location or risk to patient. Therefore, a liquid biopsies approach offers an appealing solution to an otherwise invasive procedure. The rapid growth of the liquid biopsy field has been aided by improvements in the sensitivity and specificity of enrichment assays for isolating circulating tumor cells (CTCs) from normal surrounding blood cells. Furthermore, the identification and molecular characterization of CTCs has been shown in numerous studies to be of diagnostic and prognostic relevance in breast, prostate and colon cancer patients. Despite these advancements, and the highly metastatic nature of lung cancer, it remains a challenge to detect CTCs in advanced non-small cell lung cancer (NSCLC). It may be that loss of epithelial features, in favor of a mesenchymal phenotype, and the highly heterogeneous nature of NSCLC CTCs contribute to their evasion from current detection methods. By identifying a broader spectrum of biomarkers that could better differentiate the various NSCLC CTCs subpopulations, it may be possible to not only improve detection rates but also to shed light on which CTC clones are likely to drive metastatic initiation. Here we review the biology of CTCs and describe a number of proteins and genetic targets which could potentially be utilized for the dissemination of heterogenic subpopulations of CTCs in NSCLC.

Copy number variations of circulating, cell-free DNA in urothelial carcinoma of the bladder patients treated with radical cystectomy: a prospective study.

Abstract: // Armin Soave 1 , Felix K.-H. Chun 1 , Timo Hillebrand 2 , Michael Rink 1 , Lars Weisbach 1 , Bettina Steinbach 3 , Margit Fisch 1 , Klaus Pantel 3 and Heidi Schwarzenbach 3 1 Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 AJ Innuscreen GmbH, Berlin, Germany 3 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Heidi Schwarzenbach, email: hschwarzenbach@uke.de Keywords: urothelial carcinoma of the bladder, copy number variation, multiplex ligation-dependent probe amplification (MLPA), cell-free DNA (cfDNA), chromosomal region Received: March 21, 2017 Accepted: April 26, 2017 Published: May 07, 2017 ABSTRACT The aim of the present study was to establish a rapid profiling method using multiplex ligation-dependent probe amplification (MLPA) and characterize copy number variations (CNV) in circulating, cell-free DNA (cfDNA) in 85 urothelial carcinoma of the bladder (UCB) patients treated with radical cystectomy (RC). MLPA was tested for the use of cfDNA extracted from serum and plasma by various commercial extraction kits. Eighteen probes served as reference to control denaturation, ligation and amplification efficiency. MLPA was exclusively suitable for cfDNA extracted from serum. Serum from 72 patients (84.7%) could be analyzed. Thirty-five patients (48.6%) had presence of CNV in cfDNA. The median CNV count in patients with presence of CNV was 2. Predominantly, CNV were located in the genes CDH1, ZFHX3, RIPK2 and PTEN in 15 patients (20.8%), 12 patients (16.7%), 9 patients (12.5%) and 7 patients (9.7%), respectively. CNV in TSG1, RAD21, KIAA0196, ANXA7 and TMPRSS2 were associated with presence of variant UCB histology ( p = 0.029, 0.029, 0.029, 0.029, 0.043, respectively). Furthermore, CNV in miR-15a, CDH1 and ZFHX3 were associated with presence of incidental prostate cancer ( p = 0.023, 0.003, 0.025, respectively). Patients with CNV in KLF5, ZFHX3 and CDH1 had reduced cancer-specific survival, compared to patients without CNV in these genes (pairwise p = 0.028, 0.026, 0.044, respectively). MLPA represents an efficient method for the detection of CNV among numerous genes on various chromosomal regions. CNV in specific genes seem to be associated with aggressive UCB biologic features and presence of incidental prostate cancer, and may have a negative impact on cancer-specific survival.

Detection and oncological effect of circulating tumour cells in patients with variant urothelial carcinoma histology treated with radical cystectomy.

Abstract: Objectives To investigate for the presence of circulating tumour cells (CTC) in patients with variant urothelial carcinoma of the bladder (UCB) histology treated with radical cystectomy (RC), and to determine their impact on oncological outcomes. Patients and methods We prospectively collected data of 188 patients with UCB treated with RC without neoadjuvant chemotherapy. Pathological specimens were meticulously reviewed for pure and variant UCB histology. Preoperatively collected blood samples (7.5 mL) were analysed for CTC using the CellSearch® system (Janssen, Raritan, NJ, USA). Results Variant UCB histology was found in 47 patients (25.0%), most frequently of squamous cell differentiation (16.5%). CTC were present in 30 patients (21.3%) and 12 patients (25.5%) with pure and variant UCB histology, respectively. At a median follow-up of 25 months, the presence of CTC and non-squamous cell differentiation were associated with reduced recurrence-free survival (RFS) and cancer-specific survival (pairwise P ≤ 0.016). Patients without CTC had better RFS, independent of UCB histology, than patients with CTC with any UCB histology (pairwise P < 0.05). In multivariable analyses, the presence of CTC, but not variant UCB histology, was an independent predictor for disease recurrence [hazard ratio (HR) 3.45; P < 0.001] and cancer-specific mortality (HR 2.62; P = 0.002). Conclusion CTC are detectable in about a quarter of patients with pure or variant UCB histology before RC, and represent an independent predictor for outcomes, when adjusting for histological subtype. In addition, our prospective data confirm the unfavourable influence of non-squamous cell-differentiated UCB on outcomes.

Evaluation of serum epidermal growth factor receptor (EGFR) in correlation to circulating tumor cells in patients with metastatic breast cancer

Abstract: Overexpression of epidermal growth factor receptor in breast cancer is associated with estrogen receptor negativity, higher histological grade and larger tumors. The aim of the present study was to evaluate the clinical significance of serum EGFR (sEGFR) in relation to circulating tumor cells (CTCs) in metastatic breast cancer. 252 patients were enrolled in this prospective multicentre study. Blood was drawn before start of a new line of therapy. sEGFR was determined using a sandwich-type ELISA. CTCs were detected using CellSearch. sEGFR was determined in 48 healthy controls and 252 patients, with no significant differences between the two groups. Clinical-pathological parameters did not correlate with sEGFR, irrespective of the cutoff chosen. Patients with sEGFR levels above the 50th and 75th percentile were more likely to present with <5 CTCs per 7.5 ml blood (p = 0.007; p = 0.003). Patients with sEGFR ≥73 ng/ml had significantly longer overall survival than those with sEGFR <73 ng/ml (19.7 vs. 15.2 months; p = 0.007). In the multivariate analysis, presence of ≥5 CTCs, higher grading and higher line of therapy remained independent predictors of shorter OS, while only higher line of therapy and presence of ≥5 CTCs were independent predictors of shorter PFS.

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

Abstract: 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.

Clinical Relevance of Serum HER2 and Circulating Tumor Cell Detection in Metastatic Breast Cancer Patients.

Abstract: Background/Aim: Presence of circulating tumor cells (CTCs) is associated with impaired survival in metastatic breast cancer (MBC). This study was designed to evaluate whether assessment of serum HER2 (sHER2) levels provide additional prognostic information in MBC. Materials and Methods: Two hundred and fifty-three MBC patients were enrolled in this multicentre trial. CTCs were detected before the start of first-or later-line treatment using the CellSearch system. sHER2 was determined using ELISA. Results: >= 5 CTCs were detected in 122 of 245 evaluable patients (49.8%). One hundred and nineteen of 251 patients (47%) had sHER2 levels above 15 ng/ml. Median overall survival (OS) was 16.3 months in patients with elevated sHER2;median OS in patients with non-elevated sHER2 has not been reached (p=0.001). Patients with >= 5 CTCs were more likely to present with elevated sHER2 (61% vs. 33% in those with <5 CTC;p<0.001). In patients with HER2-negative tumors, elevated sHER2 was associated with shorter OS and PFS;in HER2-positive patients with OS only. Including sHER2, CTC status and established prognostic factors into a multivariate analysis, only the presence of CTCs and higher-line of therapy remained independent predictors of OS. Conclusion: Elevated levels of sHER2 are associated with worse survival, irrespective of the HER2 status of the tumor. However, sHER2 does not provide additional prognostic information in patients with known CTC status.

Abstract S3-01: IMENEO: International MEta-analysis of circulating tumor cell detection in early breast cancer patients treated by NEOadjuvant chemotherapy

Abstract: Background We performed an international meta-analysis of individual patient data to assess the clinical validity of circulating tumor cell (CTC) count in non-metastatic breast cancer (BC) patients (pts) treated by neoadjuvant chemotherapy (NCT). Methods A protocol pre-specified the study objectives. We performed a literature & abstracts search up to Dec 2014, then contacted all centers deemed to have eligible data (published or not): early BC pts treated with NCT with CTC count by CellSearch®. The primary endpoint was overall survival (OS); secondary endpoints included distant disease-free survival (DDFS), locoregional relapse-free interval (LRFI) and pathological complete response (pCR). Non-overlapping CTC time points were: baseline (5-0 weeks before NCT), 1-8 weeks after NCT start, 5-0 weeks before surgery and 1-52 weeks after surgery. We used Cox regression models, stratified by study, and the landmark method to establish the prognostic value of CTC count/changes during treatment and survival. Results We collected 2,156 individual pt data from 21 studies and 16 centers worldwide. With ≥1/≥2/≥5 CTC/7.5ml as thresholds, CTC positivity rate was 25/13/6% at baseline, 17/6/3% after NCT start, 15/5/1% before surgery and 11/4/1% after surgery (decrease, p 301, 418 and 157 events were reported for OS, DDFS and LRFI, respectively. In univariate analyses, ≥1 CTC at baseline was a prognostic factor for OS (HR=2.6 [1.9-3.4], p Finally, in multivariate analyses, baseline CTC detection (whatever the CTC threshold used : ≥1/≥2/≥5 CTC) was an independent prognostic factor for OS, DDFS and LRFI, together with pCR, cT, cN and tumor subtype, (e.g. for OS: CTC≥2 HR=4.2 [3.0-5.9] p Conclusions Our study demonstrates with the highest level of evidence that CTCs are a prognostic biomarker in early BC treated by NCT. This impact was independent to that of pCR and was observed on OS, DDFS and also -for the first time- on LRFI. CTC count can usefully complement standard prognostic factors and pCR to improve the prognostication of early BC pts. Citation Format: Bidard F-C, Michiels S, Mueller V, Riethdorf S, Esserman LJ, Lucci A, Naume B, Horiguchi J, Gisbert-Criado R, Sleijfer S, Toi M, Garcia-Saenz JA, Hartkopf A, Generali D, Rothe F, Smerage J, Muinelo L, Stebbing J, Viens P, Magbanua M, Hall CS, Engebratenm O, Takata D, Vidal-Martinez J, Onstenk W, Fujisawa N, Diaz-Rubio E, Taran F-A, Cappelletti MR, Ignatiadis M, Name N, Proudhon C, Wolf D, Bowman Bauldry J, Borgen E, Nagaoka R, Caranana V, Kraan J, Maestro M, Brucker SY, Weber K, Reyal F, Amara D, Gopalkrishna Karhade M, Ruud Mathiesen R, Tokiniwa H, Llombart-Cussac A, D9Hollander K, Cottu P, Park JW, Loibl S, Pierga J-Y, Pantel K. IMENEO: International MEta-analysis of circulating tumor cell detection in early breast cancer patients treated by NEOadjuvant chemotherapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S3-01.

Biological labels: Here comes the spaser.

Abstract: Plasmonic nanolasers embedded in living cells and animal tissues are shown to emit strongly, making them promising potential biocompatible probes.

Hamburg-Glasgow classification: preoperative staging by combination of disseminated tumour load and systemic inflammation in oesophageal carcinoma

Abstract: The aim of this study was to establish a new preoperative staging classification and evaluate its comparability to the post-operative tumour stage, lymph node invasion and metastasis (TNM) classification. To date, adequate, preoperative staging in patients with oesophageal carcinoma (EC) is still missing but urgently needed. Systemic inflammation and disseminated tumour load have a pivotal role in recurrence and oncological outcome. To improve the clinical staging, we merged the Glasgow Prognostic Score (GPS) and disseminated tumour cells (DTC) into a new sufficient preoperative staging classification, the Hamburg-Glasgow classification (HGC). In this prospective, single-centre study, 326 patients following curative oesophagectomy were included. From all patients preoperative bone marrow was aspirated from the iliac crest to detect DTCs by immunostaining with the pan-keratin antibody A45-B/B3. HGC was subdefined into four prognostic groups on the basis of C-reactive protein (CRP), albumin and DTC. The three prognostic groups of the GPS were supplemented by DTC detection status. Results were correlated with clinicopathological parameters and clinical outcome. Increasing HGC significantly correlated with lymph node invasion (P=0.022), post-operative pathohistological TNM staging (P=0.001) and tumour recurrence (P=0.001). The four HGC prognostic groups displayed a gradual decrease in overall as well as disease-free survival (P<0.001, each). Hamburg-Glasgow classification was a strong, significant independent predictor of overall survival and disease-free survival (P<0.001, both) in multivariate analysis. Hamburg-Glasgow classification seems to be a promising preoperative additive staging classification for accurate and simple outcome stratification.