Mutations In Circulating Tumor Cells Research Articles

Rapid detection of EGFR mutation in CTCs based on a double spiral microfluidic chip and the real-time RPA method.

Circulating tumor cells (CTCs) are cells shed from primary or metastatic tumors and spread into the peripheral bloodstream. Mutation detection in CTCs can reveal vital genetic information about the tumors and can be used for "liquid biopsy" to indicate cancer treatment and targeted medication. However, current methods to measure the mutations in CTCs are based on PCR or DNA sequencing which are cumbersome and time-consuming and require sophisticated equipment. These largely limited their applications especially in areas with poor healthcare infrastructure. Here we report a simple, convenient, and rapid method for mutation detection in CTCs, including an example of a deletion at exon 19 (Del19) of the epidermal growth factor receptor (EGFR). CTCs in the peripheral blood of NSCLC patients were first sorted by a double spiral microfluidic chip with high sorting efficiency and purity. The sorted cells were then lysed by proteinase K, and the E19del mutation was detected via real-time recombinase polymerase amplification (RPA). Combining the advantages of microfluidic sorting and real-time RPA, an accurate mutation determination was realized within 2h without professional operation or complex data interpretation. The method detected as few as 3 cells and 1% target variants under a strongly interfering background, thus, indicating its great potential in the non-invasive diagnosis of E19del mutation for NSCLC patients. The method can be further extended by redesigning the primers and probes to detect other deletion mutations, insertion mutations, and fusion genes. It is expected to be a universal molecular diagnostic tool for real-time assessment of relevant mutations and precise adjustments in the care of oncology patients.

Whole-Exome Sequencing on Circulating Tumor Cells Explores Platinum-Drug Resistance Mutations in Advanced Non-small Cell Lung Cancer

Circulating tumor cells (CTCs) have important applications in clinical practice on early tumor diagnosis, prognostic prediction, and treatment evaluation. Platinum-based chemotherapy is a fundamental treatment for non-small cell lung cancer (NSCLC) patients who are not suitable for targeted drug therapies. However, most patients progressed after a period of treatment. Therefore, revealing the genetic information contributing to drug resistance and tumor metastasis in CTCs is valuable for treatment adjustment. In this study, we enrolled nine NSCLC patients with platinum-based chemotherapy resistance. For each patient, 10 CTCs were isolated when progression occurred to perform single cell–level whole-exome sequencing (WES). Meanwhile the patients’ paired primary-diagnosed formalin-fixed and paraffin-embedded samples and progressive biopsy specimens were also selected to perform WES. Comparisons of distinct mutation profiles between primary and progressive specimens as well as CTCs reflected different evolutionary mechanisms between CTC and lymph node metastasis, embodied in a higher proportion of mutations in CTCs shared with paired progressive lung tumor and hydrothorax specimens (4.4–33.3%) than with progressive lymphatic node samples (0.6–11.8%). Functional annotation showed that CTCs not only harbored cancer-driver gene mutations, including frequent mutations of EGFR and TP53 shared with primary and/or progressive tumors, but also particularly harbored cell cycle–regulated or stem cell–related gene mutations, including SHKBP1, NUMA1, ZNF143, MUC16, ORC1, PON1, PELP1, etc., most of which derived from primary tumor samples and played crucial roles in chemo-drug resistance and metastasis for NSCLCs. Thus, detection of genetic information in CTCs is a feasible strategy for studying drug resistance and discovering new drug targets when progressive tumor specimens were unavailable.

Abstract 598: Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer

Abstract Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

EGFR mutation detection of lung circulating tumor cells using a multifunctional microfluidic chip

Microfluidics has become a reliable platform for circulating tumor cells (CTCs) detection because of its high integration, small size, low consumption of reagents and rapid response. Here, we developed a multifunctional microfluidic device consists of three parts, including CTCs capture area, single-layer membrane valves area, and microcavity nucleic acid detection and analysis region based on digital polymerase chain reaction (dPCR), allowing CTCs capture, lysis, and genetic characterization to be performed on a single chip. The CTCs capture chip is coupled to the nucleic acid detection chip via a control valve. CTCs were firstly trapped in the CTC capture area, and then lysed using proteinase K to release nucleic acids. Subsequently CTCs lysate was transferred into nucleic acid detection area consisting of 12800 micro-cavity chambers for nucleic acids detection. To evaluate the performance of this chip, this study detected EGFR-L858R mutation in lung cancer cell lines H1975 and A549 cells, as well as leukocytes from normal donors. The results showed that positive signals were only observed in H1975 cells, and the detected value had a high linear relationship with the expected value (R2 = 0.9897). In conclusion, this multi-functional microfluidic chip that integrates CTCs capture, lysis and nucleic acid detection can successfully detect gene mutations in CTCs, providing reference for tumor-targeted drugs and precise diagnosis and treatment.

Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA

Abstract There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications. Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [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 5363.

Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2–5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Genetic characterization of a unique neuroendocrine transdifferentiation prostate circulating tumor cell-derived eXplant model

Transformation of castration-resistant prostate cancer (CRPC) into an aggressive neuroendocrine disease (CRPC-NE) represents a major clinical challenge and experimental models are lacking. A CTC-derived eXplant (CDX) and a CDX-derived cell line are established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis from a CRPC patient resistant to enzalutamide. The CDX and the derived-cell line conserve 16% of primary tumor (PT) and 56% of CTC mutations, as well as 83% of PT copy-number aberrations including clonal TMPRSS2-ERG fusion and NKX3.1 loss. Both harbor an androgen receptor-null neuroendocrine phenotype, TP53, PTEN and RB1 loss. While PTEN and RB1 loss are acquired in CTCs, evolutionary analysis suggest that a PT subclone harboring TP53 loss is the driver of the metastatic event leading to the CDX. This CDX model provides insights on the sequential acquisition of key drivers of neuroendocrine transdifferentiation and offers a unique tool for effective drug screening in CRPC-NE management.

KRAS mutations by digital PCR in circulating tumor cells isolated from the mesenteric vein are associated with residual disease and overall survival in resected colorectal cancer patients.

In colorectal cancer (CRC), circulating tumor cells (CTCs) are released into the mesenteric veins (MV). We chose to determine whether KRAS mutations detected in CTCs from blood obtained at the time of surgery could be a marker of survival. From 52 surgically resected CRC patients who later relapsed, samples of tumor tissue, normal tissue, and blood from the peripheral vein (PV) and MV were obtained from each patient at the time of surgery. KRAS mutations were assessed by Sanger sequencing and digital PCR (DGPCR) in tissue samples and by DGPCR in CTCs. Mutant KRAS copy number was assessed in CTCs. Results were correlated with overall survival (OS). Sanger sequencing detected KRAS mutations in ten tumor samples (19.2%), while DGPCR detected mutations in 30 (58%). Mutations were detected in CTCs in 21 MV samples (40.4%) and 18 PV samples (34.6%). Patients with G13D mutations in CTCs from the MV had shorter OS than those with G12D mutations (28.1 vs 54.6months; p = 0.025). Patients with a high mutant KRAS copy number in CTCs had shorter OS than those with a low mutant KRAS copy number (MV: 20.5 vs 43.7months; p = 0.002; PV: 15.1 vs 38.2months; p = 0.027). DGPCR is more efficient than Sanger sequencing for detecting KRAS mutations. KRAS G13D mutations and high mutant KRAS copy number are associated with shorter OS. The analysis of KRAS mutations in CTCs from blood obtained at the time of surgery can identify patients with a higher risk of relapse.

Chemokine signaling and MAPK/ERK pathway for advanced prostate cancer treatment response.

TPS275 Background: Androgen deprivation therapy (ADT) is the backbone of therapy for metastatic hormone sensitive prostate cancer (mHSPC). Despite a high response rate, the majority of patients progress to metastatic castration-resistant prostate cancer (mCRPC). Both disease settings are heterogeneous with variable responses to treatment. While prostate specific antigen is an important biomarker for prognosis and disease monitoring, it has limitations. Biomarkers predictive of disease response and progression are critically needed. Circulating tumor cells (CTCs) are shed from tumors and are found in blood during advanced stages of disease. Serial characterization of CTCs serves as a real-time ‘liquid biopsy’ for molecular profiling of PC. Recent reports suggest that phenotypic & genomic heterogeneity in CTCs and cell-free DNA (cfDNA) is associated with response to AR-targeted therapy highlighting the importance of CTC as a predictive biomarker. In addition, there is a high concordance between mutations in CTCs’ DNA and CRPC tissue through whole cell exome sequencing. We showed that atypical chemokine receptor CXCR7 is up-regulated following AR-targeted therapies, activating MAPK/ERK signaling and resulting in treatment resistance (Li et al., Cancer Res. 2019). To examine the potential of CXCR7/MAPK/ERK signaling in predicting treatment response, we propose to evaluate MAPK/ERK gene signature, pERK and AR level in CRPC or mHSPC patients receiving systemic therapy. Methods: Men with new CRPC or mHSPC are eligible prior to initiating new therapy for the respective disease setting of ADT + AR targeted therapy or chemotherapy. For CRPC arm, we will collect samples at baseline, 3, 6 months & at time of progression. For mHSPC arm, samples are collected at baseline, after 7 months & at progression. We will enroll 120 patients. Collected samples are subjected to identification, isolation, and enumeration of CTCs. Our primary molecular testing will include AR and pERK staining. CfDNA exome and methylome analysis will be performed to evaluate genomic heterogeneity and epigenomic alterations. We will explore pairwise association among biomarkers of interest, stratified by subgroups, using measures of association.

Abstract 439: Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells

Abstract Background. RareCyte has developed platform technology for visual identification and single cell retrieval of rare cells in blood, including circulating tumor cells (CTCs). There is increasing interest in mutational analysis of circulating tumor cells (CTCs) as a liquid biopsy application. Because of the minuscule amount of DNA present in a single cell (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as elevated error rates that can lead to false positive mutations. Amplicon-based next generation sequencing (abNGS) is a high-throughput method which enables genetic confirmation of malignancy and discovery of de novo pathogenic mutations. Here we present a method for performance of single cell abNGS on model CTCs without prior WGA using a commercially available pan-cancer hotspot panel. Methods. A549 lung cancer cells as model CTCs (mCTCs) were spiked into whole blood, which was processed by AccuCyte® separation onto slides. After formalin fixation, multi-parameter immunofluorescence and automated imaging (CyteFinder®) were used to identify CTCs - visualized as nucleated cells expressing epithelial markers (cytokeratin or EpCAM) and not expressing white blood cell markers. mCTCs were mechanically retrieved by CytePicker® into PCR tubes and either amplified by WGA (PicoPLEX®) or lysed in a PCR-compatible lysis buffer. WGA products or cell lysates were used as template for the AmpliSeq™ Cancer HotSpot Panel v2 for Illumina® library preparation; additional PCR cycles were added during target amplification to compensate for low DNA input in the non-WGA samples. Libraries were sequenced on an Illumina MiSeq and analyzed using the BaseSpace bioinformatics suite. Results. Single mCTCs that underwent amplicon-based NGS library prep direct from cell lysate (non-WGA) displayed increased uniformity of coverage with decreased target dropout when compared to WGA cells. Median read depth increased 7-fold with the non-WGA method. On average, 8 of 15 variants present in bulk A549 genomic DNA were observed in single mCTCs sequenced after WGA, while 12 out of 15 were observed with the non-WGA method. Additionally, the false positive error frequency of non-WGA samples was < 5% of the WGA samples. The non-WGA method was applied to CTCs identified in blood from a prostate cancer patient and confirmed presence of PTEN and TP53 mutations identified by cell-free DNA analysis. Conclusions. Amplicon-based targeted single-cell sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. Citation Format: Nolan G. Ericson, Arturo B. Ramirez, Alisa C. Clein, Celestia S. Higano, Daniel E. Sabath, Eric P. Kaldjian. Targeted single cell DNA sequencing without prior whole genome amplification for mutational analysis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 439.

Abstract P5-11-05: Concordance of genomic alterations with targeted sequencing of circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) in endocrine-resistant metastatic breast cancer

Abstract Background: Liquid biopsy has emerged as a valuable strategy for analyzing resistance biomarkers in advanced breast cancer. Differences in genomic alterations between CTC and cfDNA has been reported before and may reflect differences in the cell compartments of origin. Our aim was to determine the mutational concordance between CTC and cfDNA in luminal metastatic breast cancer (MBC) with different levels of endocrine resistance. Methods: We evaluated the mutational spectrum in matched cfDNA and CTC samples of 38 patients with luminal (hormone receptors [HR] positive, HER2 negative) MBC. Using QIAmp Circulating Nucleic Acid Kit, cfDNA was isolated from plasma samples (5 mL), and from CTC samples obtained from plasma (7.5 mL). CTC were isolated by Epcam-based immune-magnetic enrichment and confirmed with AdnaTest Breast Cancer Detect (Epcam, Muc1, HER2). Sequencing of both cfDNA and CTC was performed using the Oncoming Breast cfDNA Assay (150 hotspots, 10 genes). Limit of detection range was 0.01-0.15%, with 92.1% cases equal or less than 0.1%. Concordance between mutational profile of CTC and cfDNA was analyzed and correlated with endocrine resistance and patient characteristics. Results: Forty-seven patients with HR+ HER2- MBC were included, yielding enough cfDNA for analysis in 38 cases (80%), with isolation of CTC in 8 cases (16.7%). Rate of mutation detection in CTC was 87.5% (7/8), with TP53 mutations in all cases and no cases of ESR1 mutations; two cases of ERBB3 and PIK3CA mutations were found only in CTC, without a correlate in cfDNA. In cfDNA, genetic alterations were observed in 63.4% of cases, with 70.8% of TP53 mutations, 37.5% of PI3KCA mutations and 25% ESR1 mutations (2 D538G, 3 Y537S, 2 V392I). Average number of mutations was 2.28 in CTC and 1.96 in cfDNA, with a range of 1-4 in both cases. Concordance between cfDNA and CTC was low, with only 20% of CTC mutations simultaneously detected in cfDNA. CTC were only detected in cases with primary or secondary endocrine resistance. The percentage of patients with genetic alterations in cfDNA varied according to the degree of endocrine resistance (no resistance, 42.8%; secondary resistance, 65.2%; primary resistance, 75%), although the difference was not statistically significant. An association was found between the rate of detection of mutations in cfDNA and the response status (progression disease: 90.9%; stable disease: 57.1%; objective response: 33.3%) (p=0.04). Conclusions: The low rate of CTC detection in HR positive MBC may limit its utility for guiding therapeutic decisions. However, the information provided by CTC sequencing does not completely overlap with the data provided by cfDNA, and may be complementary to cfDNA in endocrine-resistant MBC. Citation Format: Ayala de la Peña F, Navarro Manzano E, Fernández Pérez MP, García Martínez E, de la Morena Barrio P, Ivars Rubio A, González Billalabeitia E, Fernández Sánchez A, Teruel Montoya R, Marín Zafra G. Concordance of genomic alterations with targeted sequencing of circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) in endocrine-resistant metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-11-05.

Isolation of circulating tumor cells and detection of EGFR mutations in patients with non-small-cell lung cancer.

The aim of the present study was to develop a procedure for the isolation of circulating tumor cells (CTCs), and to evaluate its application in the detection of epidermal growth factor receptor (EGFR) mutations, and potential heterogeneity in patients with non-small-cell lung cancer (NSCLC). Peripheral blood samples were collected from 91 patients with lung cancer, 10 patients with benign disease and 10 healthy volunteers. CTCs were enriched by positive immunomagnetic separation, detected by immunocytochemistry, and processed for single-cell capture. Pure CTC DNA was amplified, and the EGFR gene was analyzed using the amplification refractory mutation system (ARMS) and digital polymerase chain reaction (dPCR). The CTC capture rate in patients with lung cancer was 61.5% (56/91), whereas no CTCs were detected in patients with benign lung disease or in healthy volunteers. The CTC-positive detection rates were 69.3% (52/75) and 25.0% (4/16) in patients with TNM stage III and IV disease, respectively. Markedly more CTCs were captured from patients with small-cell lung cancer compared with patients with other types of cancer. In patients who were positive for EGFR mutations, the detection rate of these mutations was low (16.67%, 2/12), at the single CTC level. The sensitivity increased as the number of CTCs per sample increased. A total of four patients displayed consistent detection of EGFR mutations at the 10-cell level, and one patient exhibited a clear, inconsistent and rare mutation (G719×) between CTCs. A simplified technique for isolating CTCs from blood was established, though multiple CTCs were required to sensitively detect mutations in these cells. The detection of EGFR mutations in CTCs and tissue specimens was generally homogeneous, and therefore, the CTC-level mutation analysis may potentially contribute to the discovery of heterogeneous mutations.

The Discordance of Gene Mutations between Circulating Tumor Cells and Primary/Metastatic Tumor Tissue

Non-invasive methods are widely recommended with precision medicine. Circulating tumor cells (CTCs) are suggested to be able to solve the many limitations present in tumor tissue. As sequencing methods are being improved, studies about mutations in CTCs are burgeoning. However, major questions remain to be answered about whether the mutations in the CTCs represent the mutations in primary tumor tissue and metastatic tumor tissue and how to apply the clinical utility of the mutations in CTCs. In the searched articles, we compared the genes mutations between CTCs and tumor tissue, and extracted data about the heterogeneity of the mutational status in CTCs and the change in mutations of CTCs before and during treatment. For mutations detected in single genes, we calculated the concordance of the mutations between the CTCs and primary tumor tissue. For mutations detected in multiple genes, we calculated the concordance of the mutations between the CTCs and primary/metastatic tumor tissue. The heterogeneity of the mutational status is clear in CTCs. For mutations detected in a single gene, the total concordance of mutations is 53.05%. For mutations detected in multiple genes, the concordance of mutations is extremely high and different. Before mutations in CTCs can find widespread clinical utility, we should clarify the following subjects: amplification and sequencing errors, the definition of mutations in sequenced single CTCs, the heterogeneity of the mutational status in single CTCs, and the discordance of mutations between CTCs and tumor tissue.Therefore, the clinical application of CTCs mutations might be realized after considerably more research. Funding: This work was supported by National Institutes of Health (R21HG006173, R43HG007621, HL117929). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This study was also funded by grants from the National Natural Science Foundation of China (81472823), the Fundamental Research Funds for Xi’an Jiaotong University (xjj2015093), the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University, China (XJTU1AHCR2014-007), and Major Basic Research Project of Natural Science of Shaanxi Provincial Science and Technology Department (2017ZDJC-11). Competing interests: The authors have declared no conflicts of interest.

The Promise of Circulating Tumor Cells in Metastatic CRPC

The Promise of Circulating Tumor Cells in Metastatic CRPC

Aneuploidy of chromosome 8 and mutation of circulating tumor cells predict pathologic complete response in the treatment of locally advanced rectal cancer.

Identifying patients who may or may not achieve pathologic complete response (pathCR) allows for treatment with alternative approaches in the preoperative setting. The aim of the current study was to investigate whether aneuploidy of chromosome 8 and mutations of circulating tumor cells (CTCs) could predict the response of patients with rectal cancer to preoperative chemoradiotherapy. A total of 33 patients with locally advanced rectal cancer (cT3-T4 and/or cN+) treated with neoadjuvant chemoradiotherapy between September 2014 and March 2015 were recruited. Blood samples were collected from 33 patients with pre-chemoradiotherapy rectal cancer. It was demonstrated that ≥5 copies of chromosome 8 was associated with pathCR (univariate logistic regression, P=0.042). Of the 6 patients whose CTCs had <5 copies of chromosome 8, 3 achieved pathCR (3/6, 50%), and of the 27 patients whose CTCs had ≥5 copies of chromosome 8 obtained 3 pathCR (3/27, 11.1%; Chi-square test, P=0.0255). Of the 33 patients with mutations assessed, 8 significant nonsynonymous mutations in CTCs were identified as associated with pathCR (Chi-square test, P-values range, 0.0004-0.0298; mutations in ARID1A, HDAC1, APC, ERBB3, TP53, AMER1 and AR). These results suggest that ≥5 copies of chromosome 8 and 8 nonsynonymous mutations in ARID1A, HDAC1, APC, ERBB3, TP53, AMER1 AR in CTCs were associated with pathCR. This conclusion should be validated further in larger prospective studies and the long-term follow-up survival data of this study will also be reported in the future.

Cancer panel analysis of circulating tumor cells in patients with breast cancer.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Abstract B06: Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study

Abstract Colorectal cancer presents high incidences (9.7%) and mortality rates (8.5%) worldwide in men and women. Although the early detection of colon cancer promotes high cure rates (30-40%), there are still patients who experience local recurrence as well as distant metastases. The main treatment is surgery-based and then, after risk evaluation, patients can undergo or not undergo adjuvant chemotherapy. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) seem to play an important role in these processes, being detached from tumor and circulating in blood vessels before surgery intervention. Our objective, therefore, was to evaluate the role of CTCs and CTM in stages I-III colon cancer patients. Here, we collected 10 mL of blood from presurgery colon cancer and filtrated the sample in ISET® (Isolation by Size of Epithelial Tumor cells) device. This study started on July 2016. CTCs counts were assessed and correlated with clinical and pathologic data. We included 15 patients. However, one patient was identified as metastatic after surgery and therefore excluded from these analyses. From 14 patients analyzed, 8 (57.1%) were male, with a median age of 65 years (53-87). The most prevalent cancer sites were right colon (n= 6;42.9%) and left colon (n=4;28.6%). CTCs detection rate was 85.7%, and the mean and median of CTCs were 5.1/mL and 2.6/mL, respectively (0-30.3/mL). We found CTM in 4/14 patients (28.5%). Histopathologic analysis showed that no patient had blood vessel invasion, 5 (35.7%) had lymphatic invasion, and 4 (28.6%) had perineural invasion. Four patients (28.4%) were classified as stage I and 10 (71.4%) were classified as stage II and III. From patients with RAS mutational analysis available (12), 9 (75%) presented tumor mutation. Although we present a limited sample, exact Fisher's test showed an association between lymphatic invasion and CTC number above the median (P= 0.03), and the presence of CTM was most frequent in this group of patients, although without statistical significance (P= 0.09). CTM absence was associated with pathologic T= 2 and 3 (P= 0.06), when compared to pathologic T= 4. Our next steps are to evaluate some proteins in CTCs that could confer an invasive phenotype, resistance to adjuvant treatment as well as verify the pattern of mutations in CTCs and CTMs. Moreover, this is the first study to show the efficacy of ISET method in detecting high rates of CTCs from stage I-III colon cancer patients. Citation Format: Emne Abdallah, Virgílio Silva, Bianca Flores, Alexcia Braun, Ana Urvanegia, Vanessa Alves, Marcello Fanelli, Samuel Aguiar, Ludmilla Chinen. Circulating tumor cell analysis in locally and advanced colon cancer: A pilot study [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B06.

Abstract A015: Intrapatient heterogeneity of multiple myeloma is evenly distributed in multiple myeloma lesions

Abstract Background: We previously reported that circulating tumor cells (CTCs) from myeloma patients and clonal plasma cells from the BM biopsy of the same patients have quite similar clonal structures (Cell Rep. 2017;19(1):218). Specifically, overall, 90% of CTC mutations were present in BM tumors and 93% of BM mutations were present in CTC samples, and upon analyzing the mutational variants by nucleotide change, we found that the percentages of each change in BM myeloma PCs and CTCs were concordant. This finding suggests the possibility that multiple lesions of a multiple myeloma patient basically share similar clonal structure. Methods: To examine this hypothesis, we performed xenograft dissemination model of multiple myeloma using DNA-barcoded cell library. Briefly, two million human multiple myeloma cell lines barcoded with 12mer-DNA tags were inoculated into the bone chips harvested from syngeneic donor mice and the tumor-bearing bone chips were implanted to recipient mice. After the implanted myeloma cells were disseminated to murine host bone marrow via hematogenous dissemination, tumor cells were harvested from multiple BM sites and peripheral blood then analyzed the clonal structure by next-generation sequencing. Results: The clonal structures of tumor cells were highly concordant between left and right femur bone marrow from same mice. In addition, the clonal structures of tumor in host murine bone marrow were also highly concordant with those of CTCs harvested from same mice as we observed in clinical samples previously. Discussion: Recent studies of massive parallel sequencing of tumor cells obtained from the BM of patients with MM have demonstrated significant intrapatient clonal heterogeneity. Most patients have both clonal and subclonal variants even in single biopsy site. In a previous study, we have shown that the variant landscapes of tumors were highly concordant between CTCs and single BM biopsy specimen in same patients. Together with these findings, the result of our xenograft model may indicate that the tumor lesions of myeloma patients basically consist of heterogeneous but highly shared tumor cell population across the multiple lesions. The concordance of clonal structures among the multiple BM lesions is thought to be due to the fact that the clonal structure of BM myeloma tumors reflects that of CTC. Together, this study supports validity of the CTC for genetic profiling. Citation Format: Yuji Mishima, Yasuhito Terui, Kiyohiko Hatake. Intrapatient heterogeneity of multiple myeloma is evenly distributed in multiple myeloma lesions [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A015.

545P - Next-generation sequencing of circulating tumor cells isolated from peripheral blood of patients with head and neck, and gastrointestinal cancer

Background: Recently, precision medicine has gained attention for the prediction of therapeutic strategies in individual patients. Because the biological behavior of tumor cells has changed over time according to the pressures from treatment, real-time monitoring of tumor biology is needed to provide suitable information for choosing the most effective therapy for each patient. Circulating tumor cells (CTCs) can reflect current tumor status from primary sites and metastases from the bloodstream without invasive testing. In the current CTCs capture platforms of flow cytometry, gradient centrifugation, filtration, and droplets have been employed. A novel label-free inertial microfluidics approach (MFA) based on biomechanical properties has been developed at National Singapore University (Hou et al, Sci Rep 2013), and is able to capture CTCs independent of EpCAM expression. To identify genomic alterations in CTCs as basic data for precision medicine, we investigated the genomic profiling of CTCs using next-generation sequencing (NGS). Methods: Participants in this prospective study comprised 31 patients with advanced head and neck cancer (HN), esophageal cancer (EC), gastric cancer (GC), or colorectal cancer (CRC). CTCs were isolated using an MFA technique from 5 ml of whole blood. NGS was performed after whole-genome amplification (WGA) of DNA extracted from CTCs. Results: The underlying pathology was HN in 11 patients, EC in 8 patients, GC in 1 patient, and CRC in 11 patients. CTCs were detected from the peripheral blood of all patients (median number of CTCs, 14.5/ml; range, 3-133/ml). The most frequently detected mutations in CTCs were to APC, EGFR, RB1 and SMAD4 (10.5%) in HN and EC, and TP53 (27.2%), MET, RB1, and SMAD4 (18.2%) in CRC. NGS was successfully performed using WGA-treated DNA from CTCs and related material. Conclusions: We were able to effectively capture CTCs from patients with HN, EC, GC and CRC, and successfully performed NGS of CTCs using a microfluidic separation system without antibodies. Another trial is now ongoing to assess correlations between emergence of gene mutations in CTCs and changes in therapeutic effect during molecular-targeted therapy. Legal entity responsible for the study: Division of Early Detection for Cancer, National Cancer Center Research Institute Funding: None Disclosure: All authors have declared no conflicts of interest.

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care.

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.