Individual Circulating Tumor Cells Research Articles

Phenotypic Heterogeneity of Circulating Tumor Cells Informs Clinical Decisions between AR Signaling Inhibitors and Taxanes in Metastatic Prostate Cancer.

The heterogeneity of an individual patient's tumor has been linked to treatment resistance, but quantitative biomarkers to rapidly and reproducibly evaluate heterogeneity in a clinical setting are currently lacking. Using established tools available in a College of American Pathologists-accredited and Clinical Laboratory Improvement Amendments-certified clinical laboratory, we quantified digital pathology features on 9,225 individual circulating tumor cells (CTC) from 179 unique metastatic castration-resistant prostate cancer (mCRPC) patients to define phenotypically distinct cell types. Heterogeneity was quantified on the basis of the diversity of cell types in individual patient samples using the Shannon index and associated with overall survival (OS) in the 145 specimens collected prior to initiation of the second or later lines of therapy. Low CTC phenotypic heterogeneity was associated with better OS in patients treated with androgen receptor signaling inhibitors (ARSI), whereas high heterogeneity was associated with better OS in patients treated with taxane chemotherapy. Overall, the results show that quantifying CTC phenotypic heterogeneity can help inform the choice between ARSI and taxanes in mCRPC patients. Cancer Res; 77(20); 5687-98. ©2017 AACR.

Three-Dimensional Scaffold Chip with Thermosensitive Coating for Capture and Reversible Release of Individual and Cluster of Circulating Tumor Cells.

Tumor metastasis is attributed to circulating tumor cells (CTC) or CTC clusters. Many strategies have hitherto been designed to isolate CTCs, but there are few methods that can capture and gently release CTC clusters as efficient as single CTCs. Herein, we developed a three-dimensional (3D) scaffold chip with thermosensitive coating for high-efficiency capture and release of individual and cluster CTCs. The 3D scaffold chip successfully combines the specific recognition and physically obstructed effect of 3D scaffold structure to significantly improve cell clusters capture efficiency. Thermosensitive gelatin hydrogel uniformly coated on the scaffold dissolves at 37 °C quickly, and the captured cells are gently released from chip with high viability. Notably, this platform was applied to isolate CTCs from cancer patients' blood samples. This allows global DNA and RNA methylation analysis of collected single CTC and CTC clusters, indicating the great potential of this platform in cancer diagnosis and downstream analysis at the molecular level.

Abstract 3781: Expression of neuroendocrine markers in circulating tumor cells from patients with prostate cancer visceral metastases

Abstract Purpose: Therapeutic resistance to Androgen Receptor (AR) targeted therapies in patients with Castration Resistant Prostate Cancer (CRPC) is complex and originates from a wide range of molecular alterations including AR point mutations, gene rearrangements and splice variant expression. An aggressive subtype of CRPC involves neuroendocrine de-differentiation that is often found in patients with visceral metastases. To evaluate neuroendocrine signatures in patients progressing on AR targeted therapies, we measured gene expression in circulating tumor cells (CTCs) as a minimally-invasive approach to examine putative resistance mechanisms to AR targeted therapies. Methods: We utilized the VERSA platform, an integrated CTC capture and analysis technology, to capture CTCs using antibodies for EpCAM or TROP2 in parallel. mRNA was extracted on chip from live cells for comparison of gene expression profiles for markers of neuroendocrine de-differentiation (SYP, CHGA, GHGB, MYCN, NCAM) and the AR signaling pathway. Fixed PBMC were stained and imaged with fluorescence microscopy and CTCs were enumerated by defining CTCs as EpCAM or TROP2 captured, intact nuclei, negative for CD45/CD11b/CD14/CD34 and cytokeratin positive. Synaptophysin or Chromogranin A protein expression was quantified in individual CTCs and compared to tumor biopsy results where available. Results: Expression of the Synaptophysin (SYP) gene was found in 39% of the patients in our initial 18 patient cohort. These patients could be split into two groups. Group 1 patients had low AR expression with minimal detection of AR target genes. Of these four patients, three had visceral metastases. Group 2 patients had high levels of AR expression including expression of the splice variants ARV7 and ARV9, and robust expression of KLK3 (PSA) and PSMA. Of these three patients, two had visceral metastases. Subsets of the patients in both groups also exhibited expression of genes identified in neuroendocrine prostate cancer including Chromogranin B, MYCN, and NCAM. A subset of the patients tested for gene expression are being screened for CTC detection of protein markers of neuroendocrine phenotypes including Chromogranin A and Synaptophysin. Conclusions: We report the first identification of neuroendocrine gene expression signatures in CTCs from patients with CRPC experiencing disease progression on advanced AR targeted therapies. These signatures may serve as predictive biomarkers of resistance to AR targeted therapies. Future studies will evaluate these signatures in prospective clinical trials. Citation Format: Jamie M. Sperger, Anupama Singh, David Niles, Charlotte N. Stahlfeld, David J. Beebe, Joshua M. Lang. Expression of neuroendocrine markers in circulating tumor cells from patients with prostate cancer visceral metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2017-3781

Abstract 5895: Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer

Abstract Background: Breast cancer metastasis is the cause of breast cancer-related mortality. The tumor microenvironment (TME) plays a critical role in governing tumor initiation, progression and metastasis. In breast cancer, cancer associated fibroblasts (CAFs) are master regulators of the TME. Cancer metastasis occurs in part by transport of circulating tumor cells (CTCs) in the circulation. CTCs in clusters, rather than individual CTCs, have a greater capacity to establish metastases. We recently demonstrated that CAFs also circulate (cCAFs), both in clusters with CTCs and individually, in the blood of the majority of breast cancer patients with metastases, some patients without overt metastases, and in no patients with no evidence of disease. Our lab has also established primary breast cancer and primary CAF cell lines from dissociated breast tumors of different molecular subtypes; these are ideal models to dissect tumor-stromal interactions, both in vitro and in vivo. We hypothesize that cCAFs originate from the primary tumor, and further, that cCAFs cluster with CTCs to facilitate metastatic seeding. Methods: We used separately labeled CAFs and primary breast cancer cells (DT28) co-injected into NSG mice and followed by a novel 2-color IVIS. We also modeled the ability of CAFs and breast cancer cells to form clusters in culture and used our microfluidic filter technology to evaluate the composition of clusters resulting from admixed CAFs and BC cells of differing metastatic capacity – metastatic DT28 and non-metastatic MCF-7. Results: Through sequential dual-color IVIS we demonstrate that we can monitor the presence of CAFs and breast cancer cells at orthotopic tumor injection sites and to sites of distant metastasis, indicating that cCAFs originate from the primary tumor. Evaluation of cCAFs and CTCs in tumor bearing mice indicates that breast cancer cells with high metastatic potential mobilize greater numbers of cCAFs. CAFs formed robust clusters with metastatic DT28 cells, while non-metastatic MCF-7 cells clustered with each other but not with CAFs suggesting that the ability of breast cancer cells to form clusters with CAFs, both in vitro and in vivo, is reflective of the metastatic capability of the breast cancer cell line. Conclusions: The intrinsic metastatic capabilities of breast cancer cells are augmented by contact and clustering with cCAFs. In turn, the ability of CAFs to mobilize into circulation is in part conferred by properties of aggressive breast cancer cells. These data corroborate observations from our pilot clinical study that indicated the presence of cCAFs is overwhelmingly associated with the presence of metastatic breast cancer. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina-Saenz, Pedro Ferrer, Svetlana Speransky, Toni Yeasky, Dorraya El-Ashry. Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5895. doi:10.1158/1538-7445.AM2017-5895

Abstract 1717: Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis

Abstract Introduction: Presence of circulating tumor cells has prognostic value in multiple malignancies, and molecular analysis of CTCs is currently ongoing in numerous clinical trials. Most CTC enrichment methods rely on standard epithelial and leukocyte markers (CK+CD45-), so recovered cells are assumed to be of epithelial origin but never shown to be bona fide tumor cells. Conversely, atypical cells lacking the characteristic marker profile may not be analyzed, even though they may represent important tumor subpopulations. Here we evaluate a rapid, non-exhaustive, and cost-effective first-pass genomic analysis of individual candidate CTCs. This approach allows efficient upfront CNA-based confirmation that a given cell is of tumor origin, while leaving abundant DNA for deeper subsequent analysis in cells of interest. Methods: Whole peripheral blood of metastatic prostate cancer patients was enriched for CTCs using the CellSearch® system (Janssen Diagnostics) under an IRB-approved protocol, and 5 samples with >5 CTCs were selected for further study. Next, the DEPArray™ v2 system (Menarini Silicon Biosystems) was used to identify and isolate single CTCs (CK+CD45-DAPI+) and paired white blood cells (WBCs; CK-CD45+DAPI+) from the enriched samples. In addition, cells negative for both cytokeratin and CD45 but with characteristic malignant morphology (large with high nuclear-cytoplasmic ratio) were isolated. Recovered single cells were whole-genome amplified with Ampli1™ WGA and quality controlled by Ampli1 QC. Ampli1 LowPass kit was then used to prepare NGS libraries for absolute CNA profiling by low-pass WGS. Results: Thirty-three single CTCs (CK+CD45-DAPI+) and 30 WBCs (CK-CD45+DAPI+), as well as 47 putative CTCs with non-conventional phenotype (CK-CD45-DAPI+) were isolated. Single-cell WGA products with high Genome-Integrity Index (QC score ≥3) were prioritized for CNA analysis. Ampli1 LowPass data demonstrated copy number gains/losses confirming tumor origin of the CK+ cells, while WBCs showed a normal profile. In addition, a portion of the cells having non-conventional phenotype also demonstrated copy number alterations consistent with tumor origin. Discussion: We demonstrate a WGA and low-pass WGS approach on single CTCs sorted from enriched peripheral blood, which offers a dual benefit: i) it allows rapid, non-exhaustive upfront identification of bona fide tumor cells for further study, and ii) it reveals genetic similarities and diversities (vis a vis copy number alteration) across CTCs of classical as well as non-conventional phenotypes, which may better represent clonal diversity. In a clinical setting, this molecular approach may be more effective for reliably identifying and characterizing heterogeneous CTCs, yielding profiles that more accurately reflect disease evolution and inform treatment strategies. Citation Format: Gareth Morrison, Valeria Sero, Yucheng Xu, Jacek Pinski, Sue Ingles, David Quinn, Claudio Forcato, Genny Buson, Chiu-Ho Webb, Kyle Horvath, Aditi Khurana, Gianni Medoro, Suman Verma, Matthew Moore, Philip Cotter, Nicolò Manaresi, Farideh Bischoff, Amir Goldkorn. Orthogonal identification of circulating tumor cells (CTCs) using single cell low pass whole-genome sequencing (WGS) and copy-number alteration (CNA) analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2017-1717

Abstract 1740: Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC)

Abstract Background: Epithelial Plasticity (EP) is a proposed mechanism of immune escape, resistance to programmed cell death, resistance to specific drugs, and promotion of metastasis. EP has been extensively explored in mathematical, cell biological, and animal models, which predict the presence of these tumor cells in circulation. Most studies on CTCs in human subjects are based on enrichment of cells expressing EpCAM prior to enumeration or molecular interrogation, which precludes analysis of cells that might be EP. The non-enrichment Epic Sciences platform was utilized to identify CTCs in metastatic castration-resistant prostate cancer patient samples phenotypically consistent with EP: CTCs that do not express cytokeratins (CK), but do express malignant or EP biomarkers: Androgen Receptor (AR), AR splice variant 7 isoform (AR-V7) or N-cadherin. Phenotypic and genotypic analyses of individual CTCs were performed to assess the malignant origin of CK- CTCs and their prevalence associated with overall patient survival. Methods: 221 pre-therapy mCRPC blood samples were collected at Memorial Sloan-Kettering Cancer Center and shipped to Epic Sciences. Patients represent a diverse number of previous therapies and tumor burdens. All nucleated cells were plated to glass slides and subjected to DAPI and immunofluorescent (IF) staining of cytokeratin (CK), CD45 and another tumor marker (AR, AR-V7, or N-cadherin), followed by CTC identification by fluorescent scanners and malignant morphology. All samples were stained for AR, with a subset of samples stained for AR-V7 or N-Cadherin as well. Additionally, 83 AR(+)/CK(-) and 9 AR-V7(+)/CK(-) CTCs were analyzed for copy number variation (CNV) by low pass whole genome single CTC sequencing. Abnormal genomes were identified by amplifications and deletions of known oncogenes and tumor suppressors. Results: 3931 AR(+) CTCs were detected, of which 2996 (76%) expressed CK and 935 (24%) did not. The AR(+)/CK(-) CTCs had a lower median AR signal, less nucleoli, and more nuclear speckles by digital pathology vs. CK(+) CTCs. AR(+)/CK(-) and AR-V7(+)/CK(-) CTCs had gross genomic alterations consistent with malignant prostate origin (AR gain, PTEN loss, RB1 loss, 8q gain, 8p loss, etc), often sharing the same genomic alterations with CK(+) CTCs in the same patients, with some having unique CNV profiles Presence of AR(+)/CK(-) CTCs was associated with worse overall survival (median 13.7 mo vs. 33.9 mo, p < 0.0001) and remained additive and independent to treatment Line, pre-therapy PSA, and therapy type in multivariate models. Conclusions: Similar to CTCs expressing epithelial markers, CK(-) CTCs have gross genomic aberrations consistent with metastatic prostate cancer and are a negative prognostic factor associated with worse overall survival in both iterative threshold and multivariable analyses. Citation Format: Ryon P. Graf, Yipeng Wang, Nicole Schreiber, Brigit McLaughlin, Stephanie Greene, Angel Rodriguez, Adam Jendrisak, Jerry Lee, Mark Landers, Ryan Dittamore, Howard I. Scher. Phenotypic, genomic, and clinical associations of Circulating Tumor Cells (CTCs) lacking epithelial biomarkers in metastatic Castration Resistant Prostate Cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2017-1740

Inflammatory breast cancer: a model for investigating cluster-based dissemination

Metastases claim more than 90% of cancer-related patient deaths and are usually seeded by a subset of circulating tumor cells shed off from the primary tumor. In circulation, circulating tumor cells are found both as single cells and as clusters of cells. The clusters of circulating tumor cells, although many fewer in number, possess much higher metastatic potential as compared to that of individual circulating tumor cells. In this review, we highlight recent insights into molecular mechanisms that can enable the formation of these clusters—(a) hybrid epithelial/mesenchymal phenotype of cells that couples their ability to migrate and adhere, and (b) intercellular communication that can spatially coordinate the cluster formation and provide survival signals to cancer cells. Building upon these molecular mechanisms, we also offer a possible mechanistic understanding of why clusters are endowed with a higher metastatic potential. Finally, we discuss the highly aggressive Inflammatory Breast Cancer as an example of a carcinoma that can metastasize via clusters and corroborates the proposed molecular mechanisms.

Detection of FAM172A expressed in circulating tumor cells is a feasible method to predict high-risk subgroups of colorectal cancer.

Previous studies used to enumerate circulating tumor cells to predict prognosis and therapeutic effect of colorectal cancer. However, increasing studies have shown that only circulating tumor cells enumeration was not enough to reflect the heterogeneous condition of tumor. In this study, we classified different metastatic-potential circulating tumor cells from colorectal cancer patients and measured FAM172A expression in circulating tumor cells to improve accuracy of clinical diagnosis and treatment of colorectal cancer. Blood samples were collected from 45 primary colorectal cancer patients. Circulating tumor cells were enriched by blood filtration using isolation by size of epithelial tumor cells, and in situ hybridization with RNA method was used to identify and discriminate subgroups of circulating tumor cells. Afterwards, FAM172A expression in individual circulating tumor cells was measured. Three circulating tumor cell subgroups (epithelial/biophenotypic/mesenchymal circulating tumor cells) were identified using epithelial-mesenchymal transition markers. In our research, mesenchymal circulating tumor cells significantly increased along with tumor progression, development of distant metastasis, and vascular invasion. Furthermore, FAM172A expression rate in mesenchymal circulating tumor cells was significantly higher than that in epithelial circulating tumor cells, which suggested that FAM172A may correlate with malignant degree of tumor. This hypothesis was further verified by FAM172A expression in mesenchymal circulating tumor cells, which was strictly related to tumor aggressiveness factors. Mesenchymal circulating tumor cells and FAM172A detection may predict highrisk stage II colorectal cancer. Our research proved that circulating tumor cells were feasible surrogate samples to detect gene expression and could serve as a predictive biomarker for tumor evaluation.

Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

KRAS mutation analysis of single circulating tumor cells from patients with metastatic colorectal cancer

BackgroundThe molecular profiles of tumors may inform the selection of appropriate targeted therapies. Circulating tumor cells (CTCs) reflect the real-time status of tumor genotypes. CTCs exhibit high genetic heterogeneity within a patient; accordingly, the analysis of individual CTCs, including their heterogeneity, may enable more precise treatments. We analyzed KRAS mutations in single CTCs from patients with metastatic colorectal cancer (mCRC) using a new single-cell picking system.MethodsBlood samples were obtained from 61 patients with mCRC. CTCs were enriched and fluorescently labeled using the CellSearch® System. They were recovered using the single-cell picking system based on the fluorescence intensity of marker dyes. Single CTCs and tumor tissue samples were examined for mutations in codons 12 and 13 of the KRAS gene.ResultsCTCs were detected in 27 of 61 patients with mCRC. We isolated at least two CTCs from 15 of 27 patients. KRAS genotype was evaluated in a total of 284 CTCs from 11 patients, and 15 cells with mutations were identified in four patients. In 10 of 11 patients, the KRAS status was the same in the primary tumor and CTCs. In one patient, the KRAS status was discordant between the primary tumor and CTCs. In two patients, different KRAS mutations were found among individual CTCs.ConclusionsWe successfully isolated single CTCs and detected KRAS mutations in individual cells from clinical samples using a novel application of single-cell isolation system. Using the system, we detected CTC heterozygosity and heterogeneity in KRAS status among CTCs within a patient and between CTCs and tumor tissues.

Profiling protein expression in circulating tumour cells using microfluidic western blotting

Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

FAM172A expression in circulating tumor cells for prediction of high-risk subgroups of colorectal cancer.

ObjectivesPrevious studies used enumerated circulating tumor cells (CTCs) to predict prognosis and therapeutic effect in several types of cancers. However, increasing evidence showed that only enumerated CTCs were not enough to reflect the heterogeneity of tumors. Therefore, we classified different metastasis potentials of CTCs from colorectal cancer (CRC) patients to improve the accuracy of prognosis by CTCs.MethodsBlood samples were collected from 45 primary CRC patients. CTCs were enriched by blood filtration, and the RNA in situ hybridization method was used to identify and discriminate subgroups of CTCs. Later, FAM172A expression in individual CTCs was measured.ResultsThree CTC subgroups (epithelial/biophenotypic/mesenchymal CTCs) were identified using epithelial–mesenchymal transition markers. In our research, mesenchymal CTCs significantly increased along with tumor progression, including developing distant metastasis and vascular invasion. Furthermore, FAM172A expression rate in mesenchymal CTCs was significantly higher than that in epithelial CTCs, which suggested that FAM172A may correlate with tumor malignancy. This hypothesis was further verified by FAM172A expression in mesenchymal CTCs strictly related to tumor aggressiveness factors. Finally, we revealed that mesenchymal CTCs and FAM172A expression may predict high-risk subgroups in stage II CRC.ConclusionOur research proved that CTCs could serve as feasible surrogate samples to detect gene expression as a predictive biomarker for tumor evaluation.

Abstract P1-01-05: Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process

Abstract Background: The enumeration of circulating tumor cells (CTCs) using the CellSearch assay is a well-established prognostic and predictive marker for metastatic breast cancer (MBC). However, additional prognostic markers are lacking in patients with ≥ 5 CTCs in 7.5 ml of blood. Tumor-associated macrophages (TAMs) are derived from circulating monocytes or tissue-resident macrophages. TAMs have a controversial role in metastasis and anti-tumor processes. Recent studies showed that circulating cancer associated macrophage-like cells (CAMLs) are specialized phagocytic myeloid cells and found in the peripheral blood of patients with solid tumors including breast cancer, but not in healthy individuals. The presence of CAMLs may indicate the activation of innate immunity in cancer patients. The function and prognostic value of CAMLs in MBC is unknown. In the current study, we measured CTCs and CAMLs on the CellSearch™ platform and investigated their prognostic values in MBC. Methods: Peripheral blood samples from 127 stages IV breast cancer patients were collected at baseline before starting first-line therapy. The detection and enumeration of CTCs and CAMLs in 7.5 ml blood sample were performed on the CellSearch™ system. CTCs were identified by cytokeratins (CK-8, 18, and 19) positive and CD45 negative staining. CAMLs were defined by positive staining for cytokeratins and CD45 (Adams et al, PNAS, 111(9):3514-9, 2014). CTCs and CAMLs enumeration in associations with the progression-free survival (PFS) and overall survival (OS) of patients were evaluated using Kaplan Meier curves and Cox proportional hazards modeling. Results: The image review of CAMLs by using CellSearch analysis showed heterogeneous morphological phenotypes. CAMLs are large cells presenting enlarged nuclei or multiple individual nuclei, and both cytokeratin and CD45 positive with diffused cytoplasmic staining. Among the 127 MBC patients, 38 (29.9%) had elevated CTCs (≥5 CTCs), and 21 (16.5%) had at least one CAML detected. Patients with CAMLs had a significantly increased PFS (p=0.0374) and OS (p=0.0042), compared to patients without CAMLs at baseline. Patients with elevated baseline CTCs and CAMLs had worse PFS with a hazard ratio (HR) of 4.04 (95% CI 2.16 -7.56, P<0.0001), compared to patients with < 5 CTCs and without CAMLs. The combined analysis of baseline CTCs enumeration and CAMLs showed similar effect on patient OS. Compared to patients with < 5 CTCs and without CAMLs, patients with < 5 CTCs and with CAMLs, patients with ≥ 5 CTCs and without CAMLs, and patients with ≥ 5 CTCs and with CAMLs, had an increasing trend of death risk, with an HR of 2.66 (95% CI 0.53-13.21), 6.14 (2.10-17.92), and 9.13 (3.05-27.37), respectively (p for trend<0.0001). Conclusion: Baseline enumerations of both individual CTCs and CAMLs are feasible and increase our ability to accurately predict outcome in MBC patients. Evaluation of CAMLs in peripheral blood may be a marker of innate immunity and provide additional prognostic values for MBC. Citation Format: Mu Z, Wang C, Ye Z, Rossi G, Austin L, Yang H, Cristofanilli M. Prognostic values of circulating tumor cells (CTC) and cancer associated macrophage-like cells (CAML) enumerations in metastatic breast cancer: The role for innate immunity in the metastatic process [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 P1-01-05.

Mutation Analysis of Cell-Free DNA and Single Circulating Tumor Cells in Metastatic Breast Cancer Patients with High Circulating Tumor Cell Counts.

The purpose of this study was to directly compare mutation profiles in multiple single circulating tumor cells (CTC) and cell-free DNA (cfDNA) isolated from the same blood samples taken from patients with metastatic breast cancer (MBC). We aimed to determine whether cfDNA would reflect the heterogeneity observed in 40 single CTCs. CTCs were enumerated by CELLSEARCH. CTC count was compared with the quantity of matched cfDNA and serum CA15-3 and alkaline phosphatase (ALP) in 112 patients with MBC. In 5 patients with ≥100 CTCs, multiple individual EpCAM-positive CTCs were isolated by DEPArray and compared with matched cfDNA and primary tumor tissue by targeted next-generation sequencing (NGS) of about 2,200 mutations in 50 cancer genes. In the whole cohort, total cfDNA levels and cell counts (≥5 CTCs) were both significantly associated with overall survival, unlike CA15-3 and ALP. NGS analysis of 40 individual EpCAM-positive CTCs from 5 patients with MBC revealed mutational heterogeneity in PIK3CA, TP53, ESR1, and KRAS genes between individual CTCs. In all 5 patients, cfDNA profiles provided an accurate reflection of mutations seen in individual CTCs. ESR1 and KRAS gene mutations were absent from primary tumor tissue and therefore likely either reflect a minor subclonal mutation or were acquired with disease progression. Our results demonstrate that cfDNA reflects persisting EpCAM-positive CTCs in patients with high CTC counts and therefore may enable monitoring of the metastatic burden for clinical decision-making. Clin Cancer Res; 23(1); 88-96. ©2016 AACR.

Molecular profiling of single circulating tumor cells from lung cancer patients

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis.

Genomic instability is a hallmark of cancer often associated with poor patient outcome and resistance to targeted therapy. Assessment of genomic instability in bulk tumor or biopsy can be complicated due to sample availability, surrounding tissue contamination, or tumor heterogeneity. The Epic Sciences circulating tumor cell (CTC) platform utilizes a non-enrichment based approach for the detection and characterization of rare tumor cells in clinical blood samples. Genomic profiling of individual CTCs could provide a portrait of cancer heterogeneity, identify clonal and sub-clonal drivers, and monitor disease progression. To that end, we developed a single cell Copy Number Variation (CNV) Assay to evaluate genomic instability and CNVs in patient CTCs. For proof of concept, prostate cancer cell lines, LNCaP, PC3 and VCaP, were spiked into healthy donor blood to create mock patient-like samples for downstream single cell genomic analysis. In addition, samples from seven metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. CTCs were enumerated and characterized using the Epic Sciences CTC Platform. Identified single CTCs were recovered, whole genome amplified, and sequenced using an Illumina NextSeq 500. CTCs were then analyzed for genome-wide copy number variations, followed by genomic instability analyses. Large-scale state transitions (LSTs) were measured as surrogates of genomic instability. Genomic instability scores were determined reproducibly for LNCaP, PC3, and VCaP, and were higher than white blood cell (WBC) controls from healthy donors. A wide range of LST scores were observed within and among the seven mCRPC patient samples. On the gene level, loss of the PTEN tumor suppressor was observed in PC3 and 5/7 (71%) patients. Amplification of the androgen receptor (AR) gene was observed in VCaP cells and 5/7 (71%) mCRPC patients. Using an in silico down-sampling approach, we determined that DNA copy number and genomic instability can be detected with as few as 350K sequencing reads. The data shown here demonstrate the feasibility of detecting genomic instabilities at the single cell level using the Epic Sciences CTC Platform. Understanding CTC heterogeneity has great potential for patient stratification prior to treatment with targeted therapies and for monitoring disease evolution during treatment.

Longitudinally collected CTCs and CTC-clusters and clinical outcomes of metastatic breast cancer.

Circulating tumor cell (CTC) is a well-established prognosis predictor for metastatic breast cancer (MBC), and CTC-cluster exhibits significantly higher metastasis-promoting capability than individual CTCs. Because measurement of CTCs and CTC-clusters at a single time point may underestimate their prognostic values, we aimed to analyze longitudinally collected CTCs and CTC-clusters in MBC prognostication. CTCs and CTC-clusters were enumerated in 370 longitudinally collected blood samples from 128 MBC patients. The associations between baseline, first follow-up, and longitudinal enumerations of CTCs and CTC-clusters with patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models. CTC and CTC-cluster counts at both baseline and first follow-up were significantly associated with patient PFS and OS. Time-dependent analysis of longitudinally collected samples confirmed the significantly unfavorable PFS and OS in patients with ≥5 CTCs, and further demonstrated the independent prognostic values by CTC-clusters compared to CTC-enumeration alone. Longitudinal analyses also identified a link between the size of CTC-clusters and patient OS: compared to the patients without any CTC, those with 2-cell CTC-clusters and ≥3-cell CTC-clusters had a hazard ratio (HR) of 7.96 [95% confidence level (CI) 2.00-31.61, P=0.003] and 14.50 (3.98-52.80, P<0.001), respectively. In this novel time-dependent analysis of longitudinally collected CTCs and CTC-clusters, we showed that CTC-clusters added additional prognostic values to CTC enumeration alone, and a larger-size CTC-cluster conferred a higher risk of death in MBC patients.

Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining.

In tissue biopsies formalin fixed paraffin embedded cancer blocks are micro-sectioned producing multiple semi-identical specimens which are analyzed and subtyped proteomically, and genomically, with numerous biomarkers. In blood based biopsies (BBBs), blood is purified for circulating tumor cells (CTCs) and clinical utility is typically limited to cell enumeration, as only 2–3 positive fluorescent markers and 1 negative marker can be used. As such, increasing the number of subtyping biomarkers on each individual CTC could dramatically enhance the clinical utility of BBBs, allowing in depth interrogation of clinically relevant CTCs. We describe a simple and inexpensive method for quenching the specific fluors of fluorescently stained CTCs followed by sequential restaining with additional biomarkers. As proof of principle a CTC panel, immunosuppression panel and stem cell panel were used to sequentially subtype individual fluorescently stained patient CTCs, suggesting a simple and universal technique to analyze multiple clinically applicable immunomarkers from BBBs.

Frequent detection of PIK3CA mutations in single circulating tumor cells of patients suffering from HER2-negative metastatic breast cancer

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch® system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.

Abstract 2394: Scalable, rapid and affordable low-pass whole genome sequencing method for single-cell copy-number profiling on LM-PCR based WGA products

Abstract Introduction: Genome-wide profiling of copy-number alterations (CNA) in single cells enables the characterization and investigation of genomic heterogeneity and evolution in tumor cell populations such as individual circulating tumor cells (CTCs), or disaggregated solid tumors. Ampli1™ WGA is a commercially available kit for whole-genome amplification from single cells suitable for array comparative genomic hybridization (aCGH), which is currently the gold standard for genetic diagnosis of CNA. However, labor and reagent costs limit aCGH applicability. Here we introduce for the first time a rapid, high-throughput low-pass Whole Genome Sequencing (WGS)-based method for CNA analysis (LPCNA) on single cells, enabling simultaneous profiling of multiple cells at lower cost and effort. Methods: Six single cells from NCI-H23, NCI-H661 and NCI-H1650 cell lines and 19 white blood cells (WBCs) were amplified using Ampli1™ WGA Kit. Only 5μl (1/10th of total WGA product) were used as sample input for LPCNA. Purified WGA products were processed for each sample in a single-tube, one-step reaction resulting in IonTorrent-compatible indexed libraries. Samples were pooled and size-selected to recover fragments with a length ranging from 300 to 400 bp. Following purification and template preparation, pools were sequenced on Ion 318™v2 Chips. Array CGH was performed for comparison, using Agilent SurePrint 4×180k arrays as previously reported. CNA analysis was performed using Control-FREEC for low-pass WGS data, or Agilent Genome Workbench for aCGH. Results: By exploiting the deterministic nature of Ampli1™ whole genome amplification, we devised a method to generate IonTorrent compatible libraries in a single reaction, drastically reducing time of bench work and cost. Method accuracy has been assessed by comparing the copy number profiles generated by our approach with about 870,000 reads/sample (average genome coverage of 0.064x, range 0.051-0.073) with those obtained by a validated aCGH method. Very good agreement in gain and loss profiles was achieved for all single tumor cells analyzed. As expected WBC analysis resulted in normal copy-number profiles with autosomes copy number = 2, with minimal false-positive rate (&amp;lt;0.6% at a resolution of 760kb with 618k reads). Highlights: This innovative low-pass sequencing approach for CNA detection on single cells enables high level multiplexing on different NGS platforms. The approach is highly scalable and flexible. From few (6-8) samples (IonTorrent PGM, 318™v2 Chip), up to 96 samples (Ion Proton, Ion PI™ Chip v3) can be processed in a single run. This approach meets the need for rapid results (&amp;lt;7h turnaround time for library preparation) and affordable cost required for massively parallel single-cell analysis such as multiple tumor cells or CTCs. This work has been carried out within IMI CANCER-ID consortium (www.cancer-id.eu). Citation Format: Genny Buson, Paola Tononi, Claudio Forcato, Francesca Fontana, Gianni Medoro, Rui Neves, Birte Möhlendick, Nikolas Stoecklein, Nicolò Manaresi. Scalable, rapid and affordable low-pass whole genome sequencing method for single-cell copy-number profiling on LM-PCR based WGA products. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2394.