Cancer Circulating Tumor Cells Research Articles

Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.

For solid tumors, extravasation of cancer cells and their survival in circulation represents a critical stage of the metastatic process that lacks complete understanding. Gaining insight into interactions between circulating tumor cells (CTCs) and other peripheral blood mononuclear cells (PBMCs) may provide valuable prognostic information. The purpose of this study was to use single-cell RNA-sequencing (scRNA-seq) of liquid biopsies from breast cancer patients to begin defining intravascular interactions. We captured CTCs from the peripheral blood of breast cancer patients using size-exclusion membranes followed by scRNA-seq of enriched CTCs and carry-over PBMCs. Transcriptome analysis identified two populations of CTCs: one enriched for transcripts indicative of estrogen responsiveness and increased proliferation and another enriched for transcripts characteristic of reduced proliferation and epithelial-mesenchymal transition (EMT). We applied interactome and pathway analysis to determine interactions between CTCs and other captured cells. Our analysis predicted for enhanced immune evasion in the CTC population with EMT characteristics. In addition, PD-1/PD-L1 pathway activation and T cell exhaustion were predicted in T cells isolated from breast cancer patients compared with normal T cells. We conclude that scRNA-seq of breast cancer CTCs generally stratifies them into two types based on their proliferative and epithelial state and differential potential to interact with PBMCs. Better understanding of CTC subtypes and their intravascular interactions may help design treatments directed against CTCs with high metastatic and immune-evasive competence.

Comparative Analysis of Blood and Bone Marrow for the Detection of Circulating and Disseminated Tumor Cells and Their Prognostic and Predictive Value in Esophageal Cancer Patients.

Hematogenic tumor cell spread is a key event in metastasis. However, the clinical significance of circulating tumor cells (CTC) in the blood and disseminated tumor cells (DTC) in bone marrow is still not fully understood. Here, the presence of DTC and CTC in esophageal cancer (EC) patients and its correlation with clinical parameters was investigated to evaluate the CTC/DTC prognostic value in EC. This study included 77 EC patients with complete surgical tumor resection. CTC and DTC were analyzed in blood and bone marrow using nested CK20 reverse transcription-nested polymerase chain reaction (RT-PCR) and findings were correlated with clinical data. Twenty-seven of 76 patients (36.5%) showed CK20 positivity in the blood, 19 of 61 patients (31.1%) in bone marrow, and 40 (51.9%) of 77 patients were positive in either blood or bone marrow or both. In multivariate analyses, only the DTC status emerged as independent predictor of overall and tumor specific survival. Our study revealed that, while the presence of CTC in blood is not associated with a worse prognosis, DTC detection in the bone marrow is a highly specific and independent prognostic marker in EC patients. Larger cohort studies could unravel how this finding can be translated into improved therapy management in EC.

Abstract 2603: Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract Introduction: Circulating tumor cells (CTCs) mediate metastases, which account for 90% of solid tumor-related mortality. Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival in breast cancer (BC). We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding, and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery, 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation, and serve as a novel therapeutic target for BC metastasis. Methods: Using single cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of BC patient-derived xenografts. We identified genes specifically expressed in the lung metastases and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. We performed proteomic and transcriptomic analyses as well as machine learning to elucidate the downstream signaling pathways and protein structural basis involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30-60 fold higher expression of CD34, CD36, ICAM1, VCAM1, ZEB1, ALDH1A1, TGFBR2 and TSPAN8. Analysis of patient blood samples (N=40) revealed that CD44 and many of these new candidate proteins were enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules (e.g. ICAM1) significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of ICAM1+/CD44+ tumor cells had increased stemness and tumor growth upon orthotopic implantation in vivo. Knockdown of ICAM1 dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusions: We identified new molecular mediators of CTC aggregation and lung metastasis in BC. We anticipate that specific blockade of tumor clustering could decrease cancer progression and improve survival of BC patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Youbin Zhang, Andrew Davis, Carolina Reduzzi, Yue Cao, Yang Shen, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Single cell RNA sequencing-based identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2603.

Abstract 3535: Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile

Abstract Advances in the development of precision medicine for prostate cancer patients rely on accurate identification of the genomic features underlying a patient's tumor. Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity, however for the use of CTCs as a less-invasive sampling of prostate cancer and as biomarkers for patient stratification and selection of targeted therapy, complex platforms are necessary to ensure an efficient enrichment (isolation), detection (identification-imaging) and characterization strategies (molecular profiles). In this study, we combined a filtration-based CTC isolation technology with prostate cancer cell-specific antibodies to identify the prostate cancer CTCs. After identification, we performed the 3D telomere profiling prior to laser microdissection for whole genome sequencing of singles CTCs from 11 localized high-risk prostate cancer patient samples (36 CTCs in total). We identified unique and common somatic single nucleotide variants (SSNVs) and copy number alterations (CNAs) that can be used to predict high-risk lethal prostate cancer and treatment response in patients with clinically localized high- risk prostate cancers. The distribution of telomeres intensities show a range between 0 to 190.000 (a.u) and telomeres parameters were similar as described previously for high risk patients by our group. We analyzed 36 CTCs from 11 different patient samples and compared with normal lymphocytes from the same patient using whole genome sequencing (WGS). We identified 184 common mutated genes in all CTCs and two mutated genes with the same base pair (bp) mutation (PMS2 and EIF2AK4). We also identified 503 consensus mutations found in all patient but not in all CTCs and 1728 unique mutations found in all or any CTCs of only one patient, showing the heterogeneity that arises in single cells. The list of genes affected are all involved in potential pathways for tumor development such as immune system, signal transduction, homeostasis, gene expression, chromatin organization, DNA repair, cell cycle, programmed cell death, cell-cell communication and others. MCPH1, also known as BRIT1, is a key regulatory gene of the DNA damage response pathway and was mutated in all CTCs from prostate patients. MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. The PMS2 gene is a member of a set of genes known as the mismatch repair (MMR) genes which promotes also genomic Instability. Those mutations can be closely linked with the genomic instability we found assessing the telomere profiles. The CDH17 gene is a member of the cadherin superfamily and in contrast to classic cadherins, such as E- and N-cadherin, LI-cadherin possess biological functions distinct from classic cadherins and reduced expression of LI-cadherin is closely associated with tumor progression and lymph node metastasis of human colorectal carcinoma. These mutations were also found in all CTCs. All this information is needed for individualized therapy and predict drug resistance, phenotypic transition which is heterogeneous from cell to cell from patient to patient. Citation Format: Aline Rangel Pozzo. Whole genome sequencing of single circulating tumor cells from localized high-risk prostate cancer patients: Laser microdissection and 3Dtelomere profile [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3535.

Genomic Analysis of Localized High-Risk Prostate Cancer Circulating Tumor Cells at the Single-Cell Level.

Accurate risk classification of men with localized high-risk prostate cancer directly affects treatment management decisions and patient outcomes. A wide range of risk assessments and classifications are available. However, each one has significant limitations to distinguish between indolent and aggressive prostate cancers. Circulating tumor cells (CTCs) may provide an alternate additional source, beyond tissue biopsies, to enable individual patient-specific clinical assessment, simply because CTCs can reveal both tumor-derived and germline-specific genetic information more precisely than that gained from a single diagnostic biopsy. In this study, we combined a filtration-based CTC isolation technology with prostate cancer CTC immunophenotyping to identify prostate cancer CTCs. Next, we performed 3-D telomere profiling prior to laser microdissection and single-cell whole-exome sequencing (WES) of 21 CTCs and 4 lymphocytes derived from 10 localized high-risk prostate cancer patient samples. Localized high-risk prostate cancer patient CTCs present a high number of telomere signals with lower signal intensities (short telomeres). To capture the genetic diversity/heterogeneity of high-risk prostate cancer CTCs, we carried out whole-exome sequencing. We identified 202,241 single nucleotide variants (SNVs) and 137,407 insertion-deletions (indels), where less than 10% of these genetic variations were within coding regions. The genetic variation (SNVs + indels) and copy number alteration (CNAs) profiles were highly heterogeneous and intra-patient CTC variation was observed. The pathway enrichment analysis showed the presence of genetic variation in nine telomere maintenance pathways (patients 3, 5, 6, and 7), including an important gene for telomere maintenance called telomeric repeat-binding factor 2 (TRF2). Using the PharmGKB database, we identified nine genetic variations associated with response to docetaxel. A total of 48 SNVs can affect drug response for 24 known cancer drugs. Gene Set Enrichment Analysis (GSEA) (patients 1, 3, 6, and 8) identified the presence of CNAs in 11 different pathways, including the DNA damage repair (DDR) pathway. In conclusion, single-cell approaches (WES and 3-D telomere profiling) showed to be useful in unmasking CTC heterogeneity. DDR pathway mutations have been well-established as a target pathway for cancer therapy. However, the frequent CNA amplifications found in localized high-risk patients may play critical roles in the therapeutic resistance in prostate cancer.

Simultaneous Single Cell Gene Expression and EGFR Mutation Analysis of Circulating Tumor Cells Reveals Distinct Phenotypes in NSCLC.

While cancer cell populations are known to be highly heterogeneous within a tumor, the current gold standard of tumor profiling is through a tumor biopsy. These biopsies are invasive and prone to missing these clones due to spatial heterogeneity, and this bulk analysis approach can miss information from rare subpopulations. To noninvasively investigate tumor cell heterogeneity, a streamlined workflow is developed to scrutinize rare cells, such as circulating tumor cells (CTCs), for simultaneous analysis of mutations and gene expression profiles at the single cell level. This powerful workflow overcomes low-input limitations of single cell analysis techniques. The utility of this multiplexed workflow to unravel inter- and intra-patient heterogeneity is demonstrated using non-small-cell lung cancer (NSCLC) CTCs (n = 58) from six epidermal growth factor receptor (EGFR) mutant positive NSCLC patients. CTCs are isolated using a high-throughput microfluidic technology, the Labyrinth, and their EGFR mutation status and gene expression profiles are characterized.

A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer.

The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases.

Abstract IA19: Multiple uses of circulating tumor cells in lung cancer?

Abstract Liquid biopsies are increasingly being used in clinical studies as prognostic, predictive, and pharmacodynamics biomarkers, as surrogates of tumor response, to detect minimal residual disease after treatment with curative intent and to inform on mechanisms of treatment resistance and treatment switching. There is also steady progress in the development of liquid biopsies for early detection of cancers. The advantages of liquid biopsies are that they are minimally invasive and readily repeatable. The liquid biopsy that has been most widely adopted is circulating tumor DNA (ctDNA) as it is readily measured without specialist equipment in most molecular biology laboratories. Circulating tumor cells (CTCs) are technically more challenging and have yet to realize their full potential as biomarkers in clinic. However, CTC technology is constantly improving, and CTCs have several exciting applications beyond ctDNA that, once optimized, will assist personalized cancer medicine, including their use as cultures for real-time therapy testing and their utility, in some cancer types, to derive mouse models. I will discuss the use of CTCs from patients with lung cancer in this presentation. CTCs in both non-small cell lung cancer (NSCLC) and SCLC enumerated by CellSearch (EpCAM and cytokerin positive, CD45 negative) hold prognostic information. However, while CellSearch CTCs are scarce in NSCLC, most likely due to epithelial-to-mesenchymal transition (EMT) that leads to downrgulation of the EpCAM surface marker used to capture them for enumeration, they are prevalent in SCLC. Single CTC copy number analysis has led to the development of classifier that, with further validation studies, could predict response to chemotherapy alongside ctDNA analysis for therapy monitoring. We have exploited SCLC CTC prevalence to derive mouse models in immune-incompetent mice (termed CDX) that allow us to explore biology and test new therapeutics. I will also update on our research using our biobank of 46 CDX models, exploring inter and intratumoral heterogeneity. The most important aspect of this approach is the ability to generate pre- and post-therapy CDX models allowing interrogation of therapy resistance mechanisms and the biology of progressing disease in a tumor type where tumor evolution is rapid and where serial tumor biopsies are rarely obtained. I will also describe our approaches to study mechanisms of tumor dissemination, including vasculogenic mimicry and new models of brain metastasis. More recently, we are developing direct CTC cultures that may allow real-time therapy testing with reporting to the clinic. I will report on our NSCLC CTC studies within the UK TRACERx consortium to study tumor evolution. We have explored the potential of CTCs found in the pulmonary draining vein of stage I-IIIa patients at surgery with curative intent to predict risk of disease recurrence and shown in a case study that a lethal subclone that gives rise to metastasis 10 months later was identified in the pulmonary vein at surgery. In summary, this presentation will exemplify utility of CTCs in lung cancer that are distinct from but complement the implementation of ctDNA. Citation Format: Caroline Dive. Multiple uses of circulating tumor cells in lung cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA19.

Abstract B42: Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis

Abstract Introduction: Breast cancer (BC) is the leading type of cancer and the second leading cause of cancer-related deaths among women in the US. Metastasis accounts for 90% of solid tumor-related mortality and is mainly mediated by hematogenous spread of circulating tumor cells (CTCs). Compared to single cells, clustered CTCs mediate metastasis at a 20-100 times higher efficiency and are associated with lower overall survival. We have recently identified a new mechanism of CTC cluster formation through cellular aggregation instead of cohesive shedding and demonstrated that CTC clusters have enhanced stemness (Cancer Discovery 2019). However, the cellular heterogeneity and molecular mechanisms underlying CTC cluster formation and polyclonal metastasis have yet to be fully elucidated. We hypothesize that molecular drivers of metastasis initiation enhance cancer stemness and CTC cluster formation and serve as a novel therapeutic target for BC metastasis. Methods: Using single-cell RNA sequencing, we compared tumor cells from the primary breast tumor site and lung metastases of breast cancer patient-derived xenograft. We identified genes with differential expression levels in the lung mets and determined their functional importance in CTC clustering, cancer stemness, and lung colonization. Upon the candidate gene modulation, we performed proteomic and transcriptome analyses to elucidate the downstream signaling pathways involved in CTC cluster formation and lung metastasis. Finally, we explored therapeutic intervention options in blocking CTC cluster formation and lung metastasis. New Results: Compared to the primary breast tumor cells, we identified a stemness gene signature enriched in a subpopulation of the CD44+ lung metastases, with 30- to 60-fold higher expression of CD34, CD36, VCAM1, ZEB1, ALDH1A1, TGFBR2, TSPAN8, and ICAM1. Patient blood analyses (N=40) revealed that CD44 and many of these new candidate proteins are enriched in CTC clusters in comparison to single CTCs. We then examined the CSC-related properties of these tumor cells, such as tumorigenesis, sphere formation, and lung metastasis. Knockdown of selected surface molecules from the signature genes significantly reduced the efficiency of lung metastasis of BC cells in vivo. A subset of these tumor cells had increased stemness and highest tumor growth upon orthotopic implantation in vivo. Knockdown of the signature genes dramatically reduced the self-renewal ability in mammosphere formation of breast tumor cells in vitro. In addition, our studies also revealed that these tumor cells cluster through CD44 and other surface protein-mediated homophilic binding between two neighboring tumor cells. Neutralizing antibodies significantly blocked tumor cluster formation and lung colonization. Conclusion: We identified new molecular mediators of CTC aggregation and lung metastasis in breast cancer. We anticipate that a specific blocking of tumor clustering could decrease cancer progression and improve survival of breast cancer patients. Citation Format: Rokana Taftaf, Xia Liu, Salendra Singh, Yuzhi Jia, David Scholten, Massimo Cristofanilli, William A. Muller, Vinay Varadan, Huiping Liu. Identification of molecular drivers in circulating tumor cell cluster formation and lung metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B42.

Abstract B14: Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images

Abstract Analysis of circulating tumor cells (CTCs) provides exclusive benefits compared with other liquid biopsy techniques due to their advanced clinical applications including single-cell multiomics (DNA, RNA, proteins, and metabolites) analysis and the ability to generate CTCs-derived xenograft models. Current CTC analysis primarily relies on labor-intensive and human-biased manual CTC enumeration or semiautomated work, hindering clinical adaptation of CTC analysis into practice. Beyond CTC enumeration, recent studies focused on detection and characterized features of circulating tumor microemboli (CTM) to improve cancer monitoring and diagnosis, but the added complexity of CTM makes CTC analysis even more challenging. Development of an automated, unbiased, and specialized computation method for CTC analysis remains a clinical unmet need. We developed a deep learning-based framework by applying computer vision to efficiently automate CTC enumeration and characterization with high throughput and accuracy. Fluorescence microscopy CTCs images (CK+/CD45-/DAPI+) have been prepared from blood samples of non-small cell lung cancer patients by the “CMx” CTC capture platform. This study applied an “active learning” model that only requires a small upfront training dataset while being able to accumulate more training data to make improvements with minimal annotation efforts. We have collected 20 images annotated by subject matter experts to train the initial model. Using an iterative process where feedback from the subject matter experts was used to influence the model, we achieved continuous improvements using adaptive annotations. After the model had been well trained and validated by 4 extra images, we used 18 new test images to benchmark the model performance. Preliminary results demonstrated that our AI model outperformed current semiautomated methods having higher sensitivity and reduced time for CTC enumeration in a lung cancer CTC sample analysis. In 18 test samples, the model predicted 34% more total CTCs than the current method (1,775 versus 1,328 counts). By applying the model to the 24 training samples, the model also recovered 45% more total CTCs absent from the original human annotation (2,507 versus 1,732 counts). In some samples, enumeration assisted by the model saved 90% of time over the current method (<20 min vs ~4 hrs.) for CTC enumeration, which would result in an enormous advantage if the datasets being analyzed included a large number of samples. Furthermore, our AI model was able to characterize features of CTM, including CTC clusters and CTC-associated immune cells, in addition to enumerating the samples. The AI model developed in this study is a promising method for CTC analysis that significantly improved throughput, accuracy, and reproducibility, leading to an approach of CTC analysis better suited for clinical adoption. Development of AI-driven CTC/CTM analysis provides additional resources and advancements to the field of liquid biopsy for the diagnosis and treatment of disease. Citation Format: Wei-Lei Yang, Chi-Bin Li, Jia-Yang Chen, Samuel Chen, Ten-Jen Liu, Ying-Chih Chang. Artificial intelligence assists automation and high performance of circulating tumor cells enumeration and circulating tumor microemboli characterization in fluorescence microscopy images [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B14.

Dynamic evaluation of mesenchymal circulating tumor cells in patients with colorectal cancer: Clinical associations and prognostic value.

Circulating tumor cells (CTCs), as the precursor of metastases, gain mesenchymal traits through the epithelial‑mesenchymal transition (EMT) process, thereby mediating tumor metastasis. However, the dynamic changes and clinical value of mesenchymal CTCs (MCTCs) in colorectal cancer (CRC) patients remain inconclusive. The aim of the present study was to explore the prognostic value of dynamic changes of MCTCs in CRC patients using our previously developed CTCBIOPSY® device with an immunocytochemistry assay. The results revealed that 74 out of 175patients were pre‑MCTCs‑positive and 41 out of 127patients were post‑MCTCs‑positive. Dynamical monitoring revealed that the status of MCTCs remained dynamically changed under the pressure of anticancer therapy, and these dynamic changes were significantly associated with lymphovascular invasion (P<0.001) and TNM stage (P=0.033). Moreover, Kaplan‑Meier survival analyses revealed that the median recurrence‑free survival (RFS) and overall survival (OS) were significantly different between four groups (pre‑MCTC‑→post‑MCTC‑; pre‑MCTC‑→post‑MCTC+; pre‑MCTC+→post‑MCTC‑; pre‑MCTC+→post‑MCTC+), and patients with pre‑MCTCs+→post‑MCTCs+ had a significant shorter RFS (P=0.001) and OS (P<0.001) than the others. Univariate and multivariate Cox regression analyses demonstrated that persistent positivity of MCTCs before and after anticancer therapy was an independent risk factor affecting the RFS (HR: 1.302, 95%CI: 1.033‑1.639, P=0.025) and OS (HR: 1.366, 95%CI: 1.070‑1.742, P=0.012) of CRC patients. Collectively, these findings provided the evidence that the dynamic change of MCTCs during anticancer therapy can be a useful prognostic tool in CRC, indicating the important value of molecular profiling of CTCs‑EMT traits in cancer management.

Required Evidence for Clinical Applications of Liquid Biopsy Using Especially CTCs in Lung Cancer

As therapies have become more and more dependent on tumor as well as patient characteristics, obtaining tumor material has become of great importance. Liquid biopsies hold much potential as shown by a large amount of evidence across several studies. Clinical applications for circulating tumor cells (CTCs) are unfortunately still lacking. In part this is due to a lack of studies comparing liquid biopsies to conventional diagnostics and response measurements as well as studies showing that liquid biopsies can be used to switch therapies leading to improved outcomes. However, liquid biopsies using ctDNA for specific markers such as EGFR, ALK, ROS1 or RET have clinical applications because specific drugs are available.

Diagnosis of leptomeningeal metastasis (LM) through identification of circulating tumor cells (CTCs) in cerebrospinal fluid (CSF).

3567 Background: Diagnosis of LM from solid tumors can be challenging. The TargetSelector (TS) CTC detection assay has demonstrated highly specific and sensitive CTC capture both for epithelial (CK+) and non-epithelial (CK-) subsets. The assay utilizes a ten-antibody (ab) capture cocktail followed by biotinylated secondary abs that bind to CTCs, enriched in a microfluidic device. TS targeted next-generation sequencing (NGS) assay detects somatic mutations in 12 breast cancer-related genes. The aim was to determine whether TS can improve sensitivity in the diagnosis of LM compared to CSF cytology by lumbar puncture (LP). Methods: CSF was collected prospectively from patients (pts) with a prior solid tumor diagnosis and suspicion of LM. CTCs were isolated from CSF using the TS platform. Cells were stained with cytokeratin (CK), CD45, streptavidin and DAPI. CTCs captured in a microchannel were classified as CK + or -. Peripheral blood samples obtained at time of LP underwent similar CTC analysis. Cell-free total nucleic acids (cfTNA) were extracted from plasma and CSF followed by NGS. Data analysis used the Ion Torrent Suite with annotation and report curation by Ion Reporter and Oncomine Knowledgebase Reporter software respectively. Results: There were 14 pts (13 women and 1 man), median age 56 years (range 32-75) with cancers of the breast (10), lung (1), colon (1), CNS lymphoma (1) or glioma (1). Pts had received a median of 2.5 lines of systemic metastatic therapy (range 0-8). CSF cytology was not sent for 1 pt and TS was not performed for 1 pt. TS and standard cytology had 89% agreement in pts with metastatic breast cancer (MBC, 8/9). Of the 6 pts for whom CTCs were detected in CSF by TS, 3 pts had + cytology (all MBC), 2 pts had - cytology and 1 pt with MBC was not tested by cytology. Of the 3 pts with + CSF by cytology (all MBC), all were detected by TS (Table). Among 5 MBC pts with CTCs present in CSF, ER status was concordant in 2 of 5 (40%). HER2 status was concordant in 3 of 4 (75%) evaluable pts and not determined in 1 pt. Analysis of cfDNA from CSF identified somatic mutations in 3 pts (TP53, PIK3CA, CCND1, respectively). In 1 of 3 pts, the mutation identified in the CSF (PIK3CA) in HR+/HER2- MBC was also identified in the blood. Conclusions: TargetSelector is a viable platform for the detection of breast cancer CTCs in the CSF. NGS performed on CSF samples can identify potentially actionable mutations. [Table: see text]

A Multiplex PCR-Based Next Generation Sequencing-Panel to Identify Mutations for Targeted Therapy in Breast Cancer Circulating Tumor Cells

Targeted therapy has become the preferred approach to treat most cancers, including metastatic breast cancer. Using liquid biopsies, which can act as a dynamic diagnostic tool, is an appealing concept to identify effective therapies. In order to identify mutations from circulating tumor cells (CTCs) on single cell level, we have developed a multiplex PCR-based next generation sequencing-panel. The CTCs were enriched using the CellSearch system and isolated by micromanipulation followed by whole genome amplification of their DNA. Afterwards, mutation hotspot regions in the PIK3CA, the ESR1, the AKT1, and the ERBB2 genes were amplified and barcoded. Sequencing was performed on a MiSeq system. The assay was validated with cells from various cell lines displaying the expected mutations. Mutations that provide the basis for potential targeted therapies were detected in 10 out of 13 patients in all analyzed genes. In four patients, mutations in more than one gene were observed—either in the same cell or in different cells, suggesting the presence of different tumor cell clones, which might be targeted with combination therapies. This assay is a time and cost effective tool to investigate the most relevant genomic positions indicative for targeted therapies in metastatic breast cancer. It can support therapy decision to improve the treatment of cancer patients.

Detection of Circulating Tumor Cells in Breast Cancer Patients by Nanopore Sensing with Aptamer-Mediated Amplification.

Circulating tumor cells (CTCs) have been utilized in the diagnosis and prognosis of tumor. However, the CTC concentration is extremely low to be detected in peripheral blood. Many existing methods suffer from either expensive labeling or complex operation. In this study, we constructed a label- and enzyme-free and sensitive method to detect the breast cancer CTCs. First of all, a probe containing a breast cancer cell-specific aptamer and a complementary single-stranded DNA (trigger DNA P1) were designed. When the target cells are present, the aptamer binds to the CTCs and releases P1 which triggers the strand displacement amplification. This process generates three-way junction structure DNA, the specific translocation signals of which are identified by nanopore assay. The detection limit of tumor cells is 5 in the current experimental setup and can be further reduced. Furthermore, the method is demonstrated in a clinical sample test with high recovery rate and accuracy. Our results suggest that this method could be applied to early diagnosis of metastatic recurrence and prognosis determination.

Nanoemulsions to support ex vivo cell culture of breast cancer circulating tumor cells

The culture and expansion of circulating tumor cells (CTCs) for ex vivo assays plays an important role in precision medicine. However, it still represents a big challenge in translational research. Generating knowledge about the characteristics of CTCs can help to shed light about the metastasis process. Furthermore, ex vivo culture of CTCs might allow performing functional analyses and testing different drugs, to guide clinical therapies. In this work, we present a new methodology based on the use of nanosystems to support ex vivo culture of CTCs. We have formulated oil-in-water (O/W) nanoemulsions (NEs) composed by lipids and fatty acids, and have demonstrated that they can help increasing cell viability on different breast cancer cell lines. Moreover, we have generated a CTC model from breast cancer mice xenografts, to prove the ability of the NEs to facilitate their culture and expansion. Additionally, we have postulated a mechanism of action based on the cell consumption of the NEs, which are acting as energy suppliers, driving proliferation. This work corroborates the potential of nanotechnology to provide valuable tools for precision oncology, and the ability of our NEs to improve proliferation of breast cancer CTCs for the establishment of CTCs culture protocols.

MP09-15 PROGRAMMED DEATH-LIGAND 1 POSITIVE AND NON-EPITHELIAL SUBTYPE IN CIRCULATING TUMOR CELLS PREDICT LOCALLY ADVANCED PROSTATE CANCER PROGRESSION AND GUIDE THE IMMUNOTHERAPY AFTER SURGERY

INTRODUCTION AND OBJECTIVE: Circulating tumor cells (CTCs) is considered as “liquid biopsy” for tumor prognosis, and the detection of programmed death-ligand 1 (PD-L1) expressing in CTCs could promote the predictive efficiency. However, there are not standard of cut-offs to discriminate PD-L1 positive/negative expression in CTCs in prostate cancer (PCa). This study aimed to evaluate the rates of PD-L1 positive and non-epithelial (NE) subset in CTCs isolated from patients with high-risk (HRPC) and locally advanced PCa (LAPC) before surgery, and explored the clinical utility of these markers. METHODS: Four PCa cell lines were applied in spiking experiments to establish the cut-offs of NE (hybrid and mesenchymal) and PD-L1+ CTCs. From June 2016 to December 2018, 166 and 79 patients with HRPC and LAPC were collected. All patients underwent radical prostatectomy. Preoperative CTCs were enumerated by Canpatrol CTC enrichment system. PD-L1 expressing in CTCs were identified by the RNA in situ hybridization. And the correlation between CTCs markers and other parameters in terms of clinical outcome, tumor tissues immunochemistry staining, and the effect of immunotherapy was analyzed. RESULTS: The lowest rates of NE and PD-L1+ CTCs were observed in 22Rv1 cell line, the cut-offs of NE and PD-L1+ CTCs were established in vitro, being 45% and 25%, respectively. In HRPC cohort, the NE CTCs positive group (≥45%) had a higher PSA level and more advanced pathological tumor stage than negative group (P=0.041 and P=0.004). Multivariate analysis revealed that NE CTCs rate, not total CTCs amount, was an independent prognostic factor for progression-free survival (PFS) (HR=4.68, P=0.013). In LAPC cohort, the PD-L1+ CTCs positive group (≥25%) displayed a higher PSA level and more frequently lymph node metastasis than negative group (P=0.027 and P=0.028), with median PFS of 15.6 mouths vs. 22.6 mouths, HR=2.70, P=0.018). PD-L1 expression in CTCs was not associated with its expression in tumor tissue staining. While a significant correlation was observed between PD-L1+ CTCs rate and ERG or PTEN expression in tissues (P=0.010 and P=0.009). Furthermore, we found that patients with PD-L1+ CTCs positive expression exhibited a better PSA response to Durvalumab treatment than those with negative expression. CONCLUSIONS: A novel cut-off value of PD-L1+ and NE CTCs could be utilized as powerful prognosis parameter for HRPC and LAPC patients after surgery, and adjuvant immunotherapy can be adopted accordingly. Source of Funding: None.

Clinical Significance of Circulating Tumor Cells in Gastrointestinal Carcinomas.

The idea of a liquid biopsy to screen, surveil and treat cancer patients is an intensively discussed and highly awaited tool in the field of oncology. Despite intensive research in this field, the clinical application has not been implemented yet and further research has to be conducted. However, one component of the liquid biopsy is circulating tumor cells (CTCs) whose potential for clinical application is evaluated in the following. CTCs can shed from primary tumors to the peripheral blood at any time point during the progress of a malignant disease. Following, one single CTC can be the origin for distant metastasis at later cancer stage. Thus, CTCs have great potential to either be used in cancer diagnostics and patient stratification or to function as a target for new therapeutic approaches to stop tumor dissemination and metastasis at the very early beginning. Due to the biological fundamental role of CTCs in tumor progression, here, we provide an overview of CTCs in gastrointestinal cancers and their potential use in the clinical setting. In particular, we discuss the usage of CTC for screening and stratifying patients’ risk. Moreover, we will discuss the potential role of CTCs for treatment specification and treatment monitoring.

Abstract PD2-01: Exploring CDK4/6 inhibitor therapy response and drug resistance development at the single cell level in metastatic breast cancer CTCs

Abstract Although the survival of patients diagnosed with breast cancer has greatly improved over the last decades, the metastatic stage of the disease still remains incurable. Blood circulating tumor cells (CTCs) are assumed to be the source for metastatic onset and the enumeration of CTCs has already proven its prognostic value for survival in metastatic breast cancer (mBC) patients. However, the biological profiles of CTCs are so far not considered in therapy selection, but hold the potential to display the disease stage in a minimally invasive procedure. Here we serially collect liquid biopsies of mBC patients throughout their course of therapy with the CDK4/6 inhibitor palbociclib to monitor treatment response and drug resistance development. To this end, we combine transcriptomic, genomic and proteomic analyses of primary CTCs on bulk but also on the single cell level. Furthermore, we generated patient-specific, in vitro expanded CTC cell lines to intensively study mechanisms of drug resistance and identify new therapeutic targets by drug sensitivity assays. By analyzing mBC CTCs of serial liquid biopsies we monitored cell cycle arrest upon palbociclib treatment and drug resistance development. Drug sensitivity and successive drug resistance can be observed in primary and in vitro expanded, patient-specific CTCs. As revealed by RNA-seq of bulk and single CTCs, acquired resistance to palbociclib in mBC CTCs is associated with altered transcriptional programs including RB transcriptional corepressor 1 (RB1) function and can be linked to a loss-of-function mutation in the RB1 gene. Interestingly, apart from altered RB1 function, we can identify a global transcriptional transition from palbociclib sensitivity to resistance. Based on the transcriptomic information, pathways that are upregulated in resistant CTCs were determined and the patient-derived CTC cell lines were used to validate and specifically target tumor cells that are resistant to CDK4/6 inhibitors. Using drug screening assays on the CTC-derived cell lines, we identified potential drugs that in combination with palbociclib could circumvent the acquired resistance and help to prolong patient survival. Our study indicates that information about the biological state of serially collected CTCs can help to earlier detect the onset of resistance and should be included in clinical disease management. This will help to spare the patient severe side effects from ineffective drug treatment. Citation Format: Andreas Trumpp, Lisa Becker, Michael Fletcher, Roberto Würth, Anand Thippeswamy, Simon Haas, Lars Velten, Laura Michel, Frederik Marme, Sarah Jauch, Mirjam Becker, Lars Steinmetz, Andreas Schneeweiss, Marc Zapatka, Peter Lichter, Martin Sprick. Exploring CDK4/6 inhibitor therapy response and drug resistance development at the single cell level in metastatic breast cancer CTCs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-01.

PD-L1 profiling of circulating tumor cells for immune checkpoint inhibitor therapy in gastroesophageal cancers.

29 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Gastroesophageal cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Gastroesophageal cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 106 patients among whom 20 were diagnosed with gastric adenocarcinomas (AD) and 86 with either esophageal AD or esophageal squamous cell carcinomas (SCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK positivity. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 103 out of 106 samples (97.2%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 103 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 19 gastric ADs, 5 (26.3%) samples were positive for PD-L1:22c3 and 2 (10.5%) were positive for PD-L1:28-8; all gastric ADs were ‘Low’ for either PD-L1 subtype. Among the 84 esophageal carcinomas (AD+SCC), 32 samples (38.1%) were positive for PD-L1:22c3 (26 Low + 5 Moderate + 1 High) and 16 (19.1%) were positive for PD-L1:28-8 (11 Low + 5 Moderate). Conclusions: CTCs in Gastroesophageal cancers can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.