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The prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer–derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer–derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer–derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694–704. ©2014 AACR.

PDF file - 360KB, aCGH analysis of chromosomal aberrations in single CRC-derived CTC and corresponding tumor tissue (S1); An alternative aCGH algorithm confirms the tumor cell identity of the examined single CTC (S2); Validation of the reproducibility of low cell number real-time PCR (S3); Establishment and performance of single cell realtime PCR analysis (S4); Picture of a cluster of 3 CTC from patient HD-2095 (S5).

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker‐positive cells were isolated by micromanipulation followed by single‐cell whole‐genome amplification and metaphase‐based comparative genomic hybridization (mCGH) to determine genome‐wide copy number alterations. The findings were correlated with clinical parameters and follow‐up data. We detected chromosomal aberrations in KRT18‐ and EpCAM‐positive cells from both compartments; BM‐derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow‐up. Genomic profiling of BM‐DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker‐positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM‐DTCs with high‐resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer., We isolated disseminated tumor cells from lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma and comprehensively characterized them genetically at the single cell level. While the cells in the lymph nodes are harder to identify, those in the bone marrow showed up as highly aberrant subclones that could possibly become the starting point for distant metastases.

Background: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof‐of‐concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. Methods: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. Results: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient’s CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. Conclusion: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens., Cancers, 13 (23), ISSN:2072-6694

INTRODUCTIONCirculating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients.METHODSDLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture.RESULTSViable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture.CONCLUSIONSThe increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Here, we found that radioresistant PCa cells and prostate cancer stem cells (CSCs) have a high glutamine demand. Glutaminase (GLS)-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as glutaminase (GLS) and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

The progression of colorectal cancer (CRC) is supposedly driven by cancer stem cells (CSC) which are able to self-renew and simultaneously fuel bulk tumour mass with highly proliferative and differentiated tumour cells. However, the CSC-phenotype in CRC is unstable and dependent on environmental cues. Fibroblast growth factor 2 (FGF2) is essential and necessary for the maintenance of self-renewal in adult and embryonic stem cells. Investigating its role in self-renewal in advanced CRC patient-derived organoids, we unveiled that FGF-receptor (FGFR) inhibition prevents organoid formation in very early expanding cells but induces cyst formation when applied to pre-established organoids. Comprehensive transcriptome analyses revealed that the induction of the transcription factor activator-protein-1 (AP-1) together with MAPK activation was most prominent after FGFR-inhibition. These effects resemble mechanisms of an acquired resistance against other described tyrosine kinase inhibitors such as EGF-receptor targeted therapies. Furthermore, we detected elevated expression levels of several self-renewal and stemness-associated genes in organoid cultures with active FGF2 signalling. The combined data assume that CSCs are a heterogeneous population while self-renewal is a common feature regulated by distinct but converging pathways. Finally, we highlight FGF2 signalling as one of numerous components of the complex regulation of stemness in cancer.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

Background Mortality rates of pancreatic cancer remain high, which is mainly due to advanced disease and metastasis. We hypothesized that genomic copy number alterations are enriched in metastatic cells compared to autologous primary tumors, which may inform on cancer-related pathways possibly serving as potential targets for specific therapies. We investigated 18 pancreatic ductal adenocarcinomas, including 39 lymph node and 5 distant metastases after surgical resection. Analysis was performed with array-based comparative genomic hybridization (aCGH). Results Metastases acquire a higher frequency of copy number alterations with the highest in distant metastasis (median = 42, lymph node metastases: median = 23, primary tumors: median = 17). In lymph node metastases, gains were prevalent on chromosome bands 8q11.23-q24.3, 12q14.1, 17p12.1, 21q22.12, and losses on 3p21.31, 4p14, 8p23.3-p11.21,17p12-11.2. Genes on amplified regions are involved in cancer-related pathways such as WNT-signaling, also involved in metastasis. Conclusions Pancreatic cancers show a high degree of intratumor heterogeneity, which could lead to resistance of chemotherapy and worse outcome. ACGH analysis reveals regions preferentially gained or lost in synchronous metastases encoding for genes involved in cancer-related pathways, which could lead to novel therapeutic opportunities. Electronic supplementary material The online version of this article (10.1186/s13104-017-2886-0) contains supplementary material, which is available to authorized users.

BACKGROUND Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearchTM cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter–linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R2 = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.

The prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer–derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer–derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer–derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694–704. ©2014 AACR.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

Background: Solid tumors are constantly releasing circulating tumor cells (CTCs) into the circulatory system. They are genotypically and phenotypically different from the primary tumor. Especially viable CTCs can be of high interest to obtain further therapeutically relevant information. Their extremely low frequency is one of the main limiting factors to use them for further characterization. To overcome this challenge we tested to isolate viable CTCs from diagnostic leukapharesis (DLA) products obtained from breast cancer patients. Material/methods: 16 DLA samples and matched peripheral blood (PB) samples of metastasized breast cancer patients were collected and CTC numbers were determined by CellSearch® analysis. Viable cells were enriched from DLA product with Parsortix system. Genomic DNA of single cultured CTCs was isolated and amplified by whole genome amplification. Array-based comparative genomic hybridization of single cells was performed to analyze for genomic aberrations. Resulting profiles were compared to genomic aberration profiles of CTCs isolated before in vitro culture. Results: CTCs were detected in ten PB and matched DLA samples and in three more DLA samples whose corresponding blood samples were CTC-negative. CTC number per ml was increased by an average of 22.5 fold. Parsortix was optimized to enrich tumor cells from DLA product while keeping them best viable. An enrichment rate of 67.7% ± 11.3% and a harvesting rate of 65.3% ± 9.7% were determined with spiked MCF7 cells. We could grow viable CTCs from three out of eight CTC positive DLA samples from metastasized patients as determined by immune fluorescence analysis. The growing cells harbor genomic anomalies confirming their malignant origin. Most aberrations are widely identical to the aberrations detected in uncultured CTCs. Conclusions: DLA provides greater numbers of viable CTCs which can be enriched with Parsortix system in order to enable their in vitro cultivation. This workflow will allow functional studies. Citation Format: Neubauer H, Franken A, Driemel C, Lampignano R, Behrens B, Reinhardt F, Niederacher D, Stoecklein NH, Fischer JC, Fehm T. Isolation of viable CTCs from leukapharesis product with parsortix system enables subsequent culture [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-07.

Circulating tumor cells (CTCs) are believed to be metastatic precursors of most solid epithelial tumors. Therefore, characterization of their genomic, transcriptomic, and proteomic features may improve cancer diagnosis and prognosis as well as treatment selection and monitoring. CTCs are mostly detected by systems utilizing expression of the epithelial cell adhesion molecule (EpCAM) on CTCs. However, CTCs which have acquired mesenchymal-like features are overlooked although they are believed to be the most malignant cells. Moreover, due to inter cellular heterogeneity single cell isolation by e.g. micromanipulation is required to achieve cellular resolution. In this project we established a workflow combining the Parsortix™ system to enrich for EpCAMlow/negative CTCs by size and deformability and the CellCelector™ micromanipulator to singularize them for further molecular characterizations. First, our workflow was validated by cell-line spiking experiments into blood from healthy subjects (MCF7). Captured cells were stained in situ for nuclei, cytokeratin and leukocyte markers, were harvested and then micromanipulated as single cells reaching a capturing rate of 62.66% (± 17.79) and a harvesting rate of 72% (± 29.61). Then this workflow was applied to a cohort of metastatic breast cancer blood samples which were previously depleted for EpCAMhigh cells. In 18 out of 22 samples 541 EpCAMhigh CTCs and in 16 samples 257 EpCAMlow/negative potential CTCs were found. Of these 156 EpCAMlow/negative and comparable numbers of EpCAMhigh cells were successfully isolated and lysed for whole genome amplification (Ampli1™). Successful genome-wide amplification was observed in 6.5% of EpCAMlow/negative cells vs. 30% of EpCAMhigh cells indicating lower quality of genomic DNA in the first subpopulation. Sanger sequencing of PCR-amplified exons 9 and 20 of PIK3CA gene revealed heterogeneous mutation status in EpCAMlow/negative cells from the same patient. High quality WGA products were also processed for array-based comparative genomic hybridization (aCGH). Comparison of resulting profiles to 267 archived aCGH profiles from breast cancer single cells identified similar somatic copy number alterations: i.e. chromosome 1p loss and 1q gain confirming the tumor origin of captured EpCAMlow/negative cells. In addition we could also identify some different aberrations which need to be further investigated. In summary, our workflow combining size based CTC enrichment followed by in situ labeling and micromanipulation is suitable for single tumor cell analysis. Citation Format: Neubauer HJ, Lampignano R, Neumann MHD, Behrens B, Franken A, Stoecklein N, Niederacher D, Fehm TN. A new workflow comprising size based CTC enrichment followed by in situ labeling and micromanipulation with CellCelector™ enables label-free enrichment, isolation and characterization of circulating tumor cells in breast cancer [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 P1-01-13.

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

Abstract 1437 Introduction: Genetic heterogeneity is common not only in solid tumors, but also in leukemias. The analysis of genetic heterogeneity among single cancer cells is vital for a better understanding of cancer evolution and therapeutic failure of systemic cancer therapy. So far, comprehensive genome-wide single cell studies were limited by many technical difficulties. Here, we present a novel approach, combining adapter-linker PCR based whole genome amplification (WGA) with 2nd generation sequencing, that enables comprehensive and comparative genome-wide analysis of single leukemic cells. Methods: WGA, based on adapter-linker PCR (Klein et al PNAS 1999, Stoecklein et al Cancer Cell 2008), of three individually picked cells of the permanent leukemia cell line REH was performed. WGA products, subsequently fragmented to 100 bp or 250 bp, were used for library preparation. After loading one amplified single cell genome per flowcell, DNA was sequenced with paired end (PE) reads (2× 75bp or 2× 100 bp respectively) on a Genome Analyzer IIx or a HiSeq 2000 (Illumina). After alignment with Burrows-Wheeler Aligner (BWA), removal of duplicate read pairs, and identification of SNPs by the Genome Analysis Toolkit (GATK), copy number variants (CNV), loss of heterozygosity (LOH) and allele dropout rates were analyzed, based on the human reference genome (hg19/GRCh37). Results were compared to data obtained by hybridizing pooled gDNA of REH cells of the same passage to a SNP 6.0 array (Affymetrix). Interchromosomal translocations were determined in single cells of the same passage of REH cells by spectral karyotyping (SKY) and compared to sequencing data, analyzed by Geometric Analysis of Structural Variants (GASV). Results: With our approach we obtained up to 600 mio mappable reads per run, evenly spread over the genome, which led to a sequence coverage of up to 67%, with an even higher coverage of coding sequence (76%) and a sequence depth of 16x. Comparison of SNP arraydata with PE sequencing data showed, that they are highly overlapping (99,3%) regarding the detection of normal copy numbers. But also for copy number alterations, consistency between both methods was observed in detecting losses (94.1%) or gains (77.1%) of genomic material (figure 1). Up to 97% of regions of LOH detected by sequencing, were also detected by the SNP array, when analyzed in a resolution of 500K bp. By analyzing the data with higher resolutions of up to 10K bp, an increasing amount of regions of LOH could be detected. However, decreased correlation between SNP array and sequencing data (max. 74.5%) was observed, with high correlation between the sequencing runs (85%). This indicates increased detection of false positive LOH regions by the SNP array and the sequencing approach to be superior in this high resolution. To assess the allele dropout rate as a quality control for the PCR based WGA method, the heterozygous SNPs detected by PE sequencing were compared to those called by the SNP array. High consistency (95%) indicates an allele dropout rate of only 5%. To analyze the accuracy of our approach in detecting genetic heterogeneity between single cells, we assessed the variability in the SNP profile between the three individual cells. As they are derived from a permanent cell line, they are expected to be highly similar. In fact, the SNPs, that were covered in all three sequencing runs showed a variation of less than 0,1% among the single REH cells. As the SNP array is not applicable to asses copy number neutral variations as translocations, the karyotype of REH cells was assessed by SKY, confirming the predescribed translocations t(4;12), t(4;16), t(5;12), t(16;21) and t(12;21). Breakpoint regions comparable to those defined by SKY, were identified for all 5 translocations by analysis of discordant read pairs with GASV. The detection of additional, exclusively by sequencing identified breakpoints, is currently under intensified investigation, to confirm potentially newly discovered breakpoints and reliably rule out false positive results. Conclusion: Our approach provides a powerful tool to achieve an unprecedented genome-wide overview on genomic variations of single cells. The robustness of our single cell approach in comparison to the data acquired with pooled gDNA and the homogeneity of our results in the permanent REH cell line clearly shows the reliability of our approach to assess single cell heterogeneity in primary leukemic samples. Disclosures: No relevant conflicts of interest to declare.

Background: Genetic heterogeneity is a hallmark of cancer and significant evolution occurs locally and at metastatic sites with disease progression. This heterogeneity might contribute significantly to therapy resistance. Since systematic in depth analyses are lacking in head and neck squamous cell carcinomas (HNSCC), we established and tested a protocol that enables comprehensive single cell analyses of cancer cells from primary tumors and metastasis. Material and Methods: From matched primary tumors and lymph node metastases of two HNSCC patients, single cell suspensions were generated and sedimented on positively charged glass slides at low density. Epithelial tumor cells were identified with double-immunofluorescence staining for CD44v6 and CK5/14. Twelve to 14 double-positive cells were isolated via micromanipulation from each tumor and an adapter-linker PCR was used for whole genome amplification of single cells. Sequencing of TP53 was performed on genomic DNA from primary tumors as well on single cell amplification products. An allele-drop-out experiment was performed to control for artificial allelic loss. Fluorescence-in-situ-hybridization (FISH) was used to determine the copy number of chromosome 17 and TP53-gene loci, respectively. Results: Two different heterozygous TP53 mutations were detected in both cases. On the single cell level, we observed an unexpected variation of wild-types, heterozygous mutations and homozygous mutations on the investigated mutation sites. This mutational distribution was significantly different from the allele-drop-out experiment (drop-out rate: 8%). In one we found an expansion of mutated clones in metastasis, and in the other patient a transmission of the genetic changes between the primary tumor and the lymph node metastasis. Interestingly, our preliminary data indicate a polyclonal origin of the metastasis. Conclusion: Our protocol is feasible to study comprehensively genetic heterogeneity between primary tumors and metastases on a single-cell level. The single cell amplification products tested for TP53 mutations can be used for several other genetic studies, including genome-wide methods such as CGH. Our initial data revealed an unexpected intratumoral genetic heterogeneity in primaries and metastases of HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4823. doi:10.1158/1538-7445.AM2011-4823