Circulating Tumor Cells In Breast Cancer Research Articles

Abstract 5697: Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells

Abstract Background: There is a need for noninvasive predictive biomarkers of response to anti-PD1/PDL1 therapies. Assessment of circulating tumor cells (CTCs) is a rational approach to noninvasive sampling of tumors to understand the potential response or nonresponse to anti-PD1/PD-L1 therapies. IFN-gamma signals through the JAK/STAT cascade to induce PD-L1 via the Interferon Regulatory Factor-1 (IRF1) transcription factor, and is a potent inducer of PD-L1 expression in tumor cells. Recent studies have shown that low or absent IRF1 expression can identify melanomas that lack IFN-gamma responsiveness, and that IRF1 can have a higher predictive value of response to anti-PD1/PD-L1 therapy than PD-L1 itself. Using CTC models and the RareCyte platform, we developed a multiparameter assay that allows simultaneous PD-L1 and IRF1 assessment after CTC identification. We used this assay to better understand the relationship between IRF1 and PD-L1 expression in CTC model systems and applied this assay to blood samples from breast cancer patients. Materials and Methods: Peripheral blood from normal donors or cancer patients under an IRB-approved protocol was collected into RareCyte blood collection tubes. PD-L1(+) and PD-L1(-) CTC models were created by culturing A549 overnight with or without 10ng/mL INF-gamma. The A549 cells were spiked into normal donor blood and buffy coats isolated from 7.5mL of blood by AccuCyte® separation and spread onto slides. MDA-MB-231 cells were used as a model to represent breast cancer cells that express PD-L1 in the absence of IFN-gamma signaling. Slides were stained with a 6-marker panel that included antibodies to pan-cytokeratin (CK), EpCAM, CD45, PDL1, IRF1, and a nuclear dye on the Leica Bond Rx auto-stainer. Slides were scanned with CyteFinder® and CTCs identified by CK and/or EPCAM positivity and negative CD45 staining. Confirmed CTCs were then assessed for expression of PD-L1 and IRF1; cellular compartment was recorded for IRF1 staining. Results: Nuclear expression of IRF1 correlated with PD-L1 expression in IFN-gamma stimulated A549 cells. Unstimulated MDA-MB-231 cells expressed high levels of PD-L1, but this did not correlate with nuclear IRF1 expression. Breast cancer patient samples were identified having CTCs that expressed PD-L1. A PDL1 mean fluorescence intensity (MFI) threshold was set at the upper 95% CI of unstimulated A549 cells to define “high” and “low” PD-L1 expression in the patient samples. The population of high-PD-L1 CTCs had higher nuclear IRF1 MFI than low-PD-L1 CTCs, with mean fold-increase of 2.7 (range from 1.4 to 3.3). Conclusions: Nuclear IRF1 expression correlates with PD-L1 expression in breast cancer CTCs. Using CTCs to evaluate expression of IRF1 and PD-L1 together with assessment of CTC mutational load could be a powerful noninvasive assessment of response to anti-PD1/PD-L1 therapies. Citation Format: Lance U'Ren, Nolan Ericson, Ryan Huston, Elisabeth Mahen, VK Gadi, C. Anthony Blau, Eric Kaldjian. Simultaneous assessment of PD-L1 and IFR1 expression on breast cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5697.

Abstract 3913: Selective targeting of circulating tumor cells with agonistic EphA2 ligand

Abstract EphA2 is a tyrosine kinase receptor that is overexpressed in many cancer types like pancreatic, colon and breast cancers. Targeting cancer cells with an EphA2-targeting molecule conjugated with cytotoxic agents can spare normal cells from chemotherapy side effects. Here, we developed (123B9)2, a novel, potent ligand for the EphA2 receptor that binds to EphA2 ligand-binding domain and causes receptor activation at a sub micromolar concentration as evident in biochemical and cell-based assays. Furthermore, our molecule showed enhanced resistance to degradation, making it suitable for in vivo studies. Subsequently, we conjugated (123B9)2 with paclitaxel, a mitotic spindle inhibitor, and administered it in nude mice bearing breast cancer cells MDA-MD-231 in mammary glands. Remarkably, (123B9)2 paclitaxel-conjugate showed significant inhibition of breast cancer circulating tumor cells (CTC)s in orthotropic mice compared to paclitaxel alone. Finally, we are planning to use our molecule as a single agent to target cancer cells. Citation Format: Ahmed F. Salem, Si Wang, Sandrine Billet, Jie-Fu Chen, Parima Udompholkul, Luca Gambini, Carlo Baggio, Edwin M. Posadas, Neil A. Bhowmick, Maurizio Pellecchia. Selective targeting of circulating tumor cells with agonistic EphA2 ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3913.

Abstract 3264: Targeting bone marrow-resident dormant CTCs to overcome metastasis

Abstract Metastatic “seeding” is an early event initiated when cancer cells are shed from the primary tumor, enter into the blood stream as circulating tumor cells (CTCs), and travel to distant organs where they can remain dormant as disseminated cancer cells (DTCs) for indefinite lengths of time. The organ serving as the foremost reservoir for DTCs is the bone marrow (BM). Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. This is caused by the lack of biomarkers that can identify and isolate viable but dormant BMRCs not expressing neoplastic and/or proliferation markers. To overcome these hurdles, we developed a patient-derived CTC model of clinical dormancy. We hypothesized that a significant distinction exists between signaling pathways of BM-homing vs metastasis-competent CTCs upon transplantation in xenografts. The model was established by three sequential steps: (a) isolation of CTC-enriched cell populations from the peripheral blood of patients with metastatic breast cancer, followed by CTC implantation in NOD-SCID-gamma mice; (b) in vivo depletion of “normal” human cell lineages over 4-8 months (before onset of overt metastasis), and (c) capture and characterization of viable human CTCs from blood and BM of mice via multi-parametric flow cytometry. This strategy was implemented on a set of breast cancer patients stratified according to their primary tumor subtype (ER+/PR+, HER2+, and ER-/PR-/HER2-), and gene-signatures of putative ex vivo CTCs and BMRCs were obtained by whole genome transcriptomic arrays. First, comparative analyses of ex vivo vs de novo CTC-gene signatures identified increased mTORC2 along with decreased mTORC1 signaling activity as the most significant characteristic of human BM-resident CTCs. mTORC2/mTORC1 represent the two complementary arms of mTOR signaling - a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy. Second, heightened mTORC2 downstream targets augmented quiescent CTC populations (Ki67-/RBL2+ cells) in paired metastatic vs primary breast cancer tissues. Third, IHC analyses of breast cancer CTC xenograft tissues showed that solitary BM and tissue-resident breast cancer CTCs had high mTORC2 activity. Finally, shRNA knockdown of Rictor-the essential component of mTORC2 actions, increased Ki67/PCNA expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling pathways regulate CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in breast cancer patients having metastatic “seeding” but yet to develop overt metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio T. Scamardo, David S. Hong, Dario Marchetti. Targeting bone marrow-resident dormant CTCs to overcome metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3264.

CIRCULATING TUMOR CELLS: CLINICAL SIGNIFICANCE IN BREAST CANCER (REVIEW)

Circulating tumor cells (CTCs) constitute a heterogeneous population. Some tumor cells are cancer stem cells (CSCs), while others are in the process of the epithelial-mesenchymal transition (EMT); however, most CTCs are neither stem cells nor participants in the EMT. There is increasing interest in the study of the molecular biological characteristics of CTCs. Many researchers consider circulating tumor cells (CTC) as one of the variants of «liquid biopsy in real time». In this review, we discuss the clinical significance of CTCs in breast cancer and in particular the prognostic and predictive significance both in early stage and metastatic breast cancer, as well as the pathogenetic role of CTCs in venous thromboembolism. Evaluation of various characteristics of CTCs is promising for the study of new biomarkers and targets for targeted therapies. The clinical importance involves the determination of the heterogeneity of the CТC and in particular of the stem subpopulation of these cells, cells with signs of EMТ, with no evidence of stem cells, and with a combination of these features.

Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles

Circulating tumor cells (CTCs) are extremely rare cells found in blood of metastatic cancer patients. There is a need for inexpensive technologies for fast enrichment of CTCs from large blood volumes. Previous data showed that antibody-conjugated lipid shell immuno-microbubbles (MBs) bind and isolate cells from biological fluids by flotation. Here, blood-stable MBs targeted to several surface markers for isolation of breast tumor cells were developed. MBs coated with anti-human EpCAM antibodies showed efficient binding of EpCAM+ breast cancer cell lines SKBR-3, MCF-7, and MDA-MB-453, whereas anti-human EGFR MBs showed binding of EpCAMLOW/NEGATIVE cell lines MDA-MB-231 and BT-549. Multitargeted anti-human EpCAM/EGFR MBs bound all cell lines with over 95% efficiency. Highly concentrated MB-bound tumor cells were collected in a microliter volume via an inverted vacuum-assisted harvesting setup. Using anti-EpCAM and/or anti-EpCAM/EGFR MBs, an efficient (70–90%) recovery and fast (30min) isolation of the above-mentioned cells and cell clusters was achieved from 7.5mL of spiked human blood. Using anti-EpCAM MBs and anti-EpCAM/EGFR MBs, cytokeratin-positive, CD45-negative CTCs were detected in 62.5% (10/16) of patients with metastatic breast cancer and CTC clusters were detected in 41.7% (5/12) of CTC-positive samples. Moreover, in some samples MBs isolated cytokeratin positive, CD45 negative tumor-derived microparticles. None of these structures were detected in blood from non-epithelial malignancies. The fast and inexpensive multitargeted platform for batch isolation of CTCs can promote research and clinical applications involving primary tumors and metastases.

Molecular characterization of breast cancer CTCs associated with brain metastasis

The enumeration of EpCAM-positive circulating tumor cells (CTCs) has allowed estimation of overall metastatic burden in breast cancer patients. However, a thorough understanding of CTCs associated with breast cancer brain metastasis (BCBM) is necessary for early identification and evaluation of treatment response to BCBM. Here we report that BCBM CTCs is enriched in a distinct sub-population of cells identifiable by their biomarker expression and mutational content. Deriving from a comprehensive analysis of CTC transcriptomes, we discovered a unique “circulating tumor cell gene signature” that is distinct from primary breast cancer tissues. Further dissection of the circulating tumor cell gene signature identified signaling pathways associated with BCBM CTCs that may have roles in potentiating BCBM. This study proposes CTC biomarkers and signaling pathways implicated in BCBM that may be used either as a screening tool for brain micro-metastasis detection or for making rational treatment decisions and monitoring therapeutic response in patients with BCBM.

Challenges in enumeration of CTCs in breast cancer using techniques independent of cytokeratin expression.

IntroductionGiven the current postulated plasticity between epithelial and mesenchymal states of migratory cancer cells the detection of non-epithelial CTCs is an important scientific and clinical goal.MethodsWe used the filtration-based ISET technology to enrich circulating tumour cells (CTCs) in early breast cancer blood samples and identify them using a morphology-based immunocytochemistry (ICC) approach.ResultsWe found greater numbers of putative CTCs by this approach than by the cytokeratin-based CellSearch technology, but a high number of CTC false positives were identified in healthy volunteer samples which were not reduced in successive blood draws. Preliminary work using an oestrogen receptor (ER)-based multiplex ICC method in metastatic breast cancer ISET samples indicated a low number of ER+ CTCs even at this advanced stage.ConclusionsThis work highlights the challenges in enumerating CTCs without conventional epithelial markers.

Abstract P1-01-14: Nuclease-activated oligonucleotide probes for detection of breast cancer circulating tumor cells (CTCs): Early clinical results

Abstract Introduction: A challenge for CTC-based diagnostic tests has been the development of methods with sufficient sensitivity to detect low levels of CTCs. Expense, accuracy and complexity have also limited clinical uptake of CTCs. To overcome these limitations we explored detecting CTCs by measuring their nuclease activity with nuclease-activated probes. We present the development of a rapid and highly-sensitive CTC detection assay based on probes that are selectively digested (activated) by target nucleases expressed in breast cancer cells. Methods: Nuclease activity in samples from women with Stage IV breast cancer and healthy donors was determined and correlated with clinical data. Patients seen at University of Iowa Clincis were eligible for this IRB-approved study. Blood samples were processed using microfilter (ScreenCell) units for CTC enrichment and converted into cell lysates that were examined by means of three different chemically-optimized oligonucleotide probes. CTC-derived nuclease activity was quantified using a fluorometer. The presence of CTCs was confirmed using established CTC detection methods (e.g. RT-PCR, immunohistostaining). Results: Sensitivity of the probe assay was 5 cancer cells in buffer solution and ~200 cancer cells in 1 mL of healthy donor blood. The final study cohort included 28 breast cancer patients and 10 healthy donors. The averaged signal intensities from patient samples were significantly higher compared to the healthy donor control group, presumably arising from CTCs in the blood. Statistical analysis further reveald short incubations in the assay (<20 min) to be optimal. From an ROC analysis we obtained AUC values of 0.8821, 0.8103 and 0.9356 for the three different probes. The oligonucleotide probe being the best predictor of disease yielded 100% sensitivity in the patient samples with a specificity of 70%. The dsDNA 20 minute probe was correlated negatively with tumors being ER+/PR+ (p=0.03). The 2'f-RNA 0 minute probe correlated significantly with HER2- tumors (p=0.04). In this smaller series other trends were also suggested. Conclusion: We describe a novel diagnostic for the detection of CTCs that could overcome limitations of CTC detection assays and could provide a robust diagnostic tool for breast cancer. Future clinical assays derived from this technology could require minimal training and infrastructure and might be developed into a point-of-care testing format. Citation Format: Giangrande PH, Kruspe S, Dickey DD, Kamboj S, Clark KC, Urak K, Burghardt E, Smith B, Thomas A, McNamara JO. Nuclease-activated oligonucleotide probes for detection of breast cancer circulating tumor cells (CTCs): Early clinical results [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 P1-01-14.

Abstract P1-01-07: Maintenance of genomic integrity in dormant circulating tumor cells

Abstract More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) as well as shRNA mediated RIF1 knockdown in HCC1954 and ZR-75-1 cell lines attenuated resistance of the dormant subset to UV and bleomycin treatment. Finally, RIF1 knockdown activated both p38 and pERK pathways albeit to varying degrees in multiple cell lines resulting in metastatic inefficiency in xenograft and syngeneic mouse models. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs and be a potential biomarker of breast cancer CTC survival while in circulation. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Marchetti D, Hong DS, Scamardo A. Maintenance of genomic integrity in dormant circulating tumor cells [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 P1-01-07.

Abstract P1-01-08: The evolution of breast cancer circulating tumor cells mediating brain metastasis

Abstract Multiple studies concur that CTCs - the “seeds” of fatal metastasis - intravasate into the bloodstream throughout the early stages of cancer promoting generation of micro-metastatic reservoirs, some of which ultimately evolve to metastatic tumors. However, while it may take decades for CTCs to progress from cells in circulation to clinically detectable metastases, once detected, the tumor usually grows at an exponential rate. This difference in growth dynamics at two ends of the metastatic spectrum should also be reflected in the behavior of CTCs shed from these organ sites. Having a focus towards breast cancer brain-colonizing CTCs and their properties, we hypothesized that a gradual evolution of dormant CTCs into mitotically active CTCs with brain-metastatic potency is central to the progression of primary breast cancer to brain metastasis. We isolated CD44+/CD24- (stem-like) and Pan-Cytokeratin+ (epithelial) breast cancer CTCs from peripheral blood of two groups of breast cancer patients – five with brain metastases (BCBM) (MRI-detectable) and five without brain metastases (No BCBM). CTCs were isolated by multi-parametric flow cytometry and the expression of CTC-specific antigen was confirmed by immuno-cytochemistry and DEPArrayTM platform. Intra-cellular flow cytometry showed that No BCBM patients had 3-fold higher mitotically dormant (Ki67-) CTCs compared to BCBM patients. Whole genome expression arrays demonstrated low generalized transcriptional activity in CTCs suggestive of mitotic/metabolic dormancy. Comparison of CTC transcriptomes with ER+/PR+, HER2+ and triple negative breast cancers identified a unique CTC gene signature valid not only across the three molecular subtypes but also independent of the molecular subtype patients were initially diagnosed as. Pathway analyses predicted increased activation of pluripotency-related pathways along with decreased protein translational machinery and proliferative pathways in CTCs. Further, to dissect mechanisms of metastatic reactivation in the brain microenvironment, we compared the CTC transcriptome in BCBM vs No BCBM patients and identified a 126 gene signature potentiated in BCBM CTCs. Subsequent pathway analyses revealed upregulation of known CTC pathways like Notch along with novel hematopoietic and immune evasion networks. Cellular and functional annotations of cell migration and chemotaxis were significantly activated in BCBM along with pro-inflammatory (TNF, IL1β, NF-kB), immunomodulatory chemokines (CXCL8, CXCR4, CD86) and mitogenic growth factors (PDGF-BB). Lastly, lack of cell surface expression of Urokinase Plasminogen Activator Receptor (uPAR) and integrin beta-1 (int-β1) were used to identify CTCs in a state of metastatic dormancy. Whole genome sequencing showed significantly higher incidence of genomic mutations in proliferating CTCs compared to their dormant counterparts. We provide first time evidence of signaling pathways keenly implicated in breast cancer CTC biology based on comprehensive analyses of CTC transcriptomes. We discovered the existence of CTCs in a state of metabolic/mitotic dormancy and identified genomic instability as a switch for CTCs to revert to their proliferative phenotype. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Hong DS, Scamardo AT, Marchetti D. The evolution of breast cancer circulating tumor cells mediating brain metastasis [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 P1-01-08.

Detection of Circulating Tumor Cells in Breast Cancer Patients Using Cytokeratin-19 Real-Time RT-PCR.

PurposeThe roles of circulating tumor cells (CTCs) as predictive and prognostic factors, as well as key mediators in the metastatic cascade, have been investigated. This study aimed to validate a method to quantify CTCs in peripheral blood using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for cytokeratin (CK)-19 and to evaluate the utility of this assay in detecting CTCs in breast cancer patients.Materials and MethodsReal-time monitoring PCR of fluorescently labeled specific hybridization probes for CK-19 mRNA was established. Peripheral blood samples from 30 healthy donors, 69 patients with early breast cancer, 47 patients with locally advanced breast cancer, and 126 patients with metastatic breast cancer were prospectively obtained and analyzed for CTC detection.ResultsCK-19 mRNA was not detectable in healthy subjects using the real-time RT-PCR method. The detection rates of CK-19 mRNA in breast cancer patients were 47.8% for early breast cancer (33/69), 46.8% for locally advanced breast cancer (22/47), and 61.1% for metastatic breast cancer (77/129). The detection rate of CK-19-positive CTCs in metastatic disease was slightly higher than early or locally advanced breast cancer; however, the detection rate according to disease burden was not statistically different (p=0.097). The detection rate was higher in patients with pleural metastasis (p=0.045). CTC detection was associated with poor survival (p=0.014).ConclusionA highly specific and sensitive CK-19 mRNA-based method to detect CTCs in peripheral blood in breast cancer patients can be used in further prospective studies to evaluate the predictive and prognostic importance of CTCs.

A FISH-based method for assessment of HER-2 amplification status in breast cancer circulating tumor cells following CellSearch isolation.

IntroductionAmplification of the HER-2/neu (HER-2) proto-oncogene occurs in 10%–15% of primary breast cancer, leading to an activated HER-2 receptor, augmenting growth of cancer cells. Tumor classification is determined in primary tumor tissue and metastatic biopsies. However, malignant cells tend to alter their phenotype during disease progression. Circulating tumor cell (CTC) analysis may serve as an alternative to repeated biopsies. The Food and Drug Administration-approved CellSearch system allows determination of the HER-2 protein, but not of the HER-2 gene. The aim of this study was to optimize a fluorescence in situ hybridization (FISH)-based method to quantitatively determine HER-2 amplification in breast cancer CTCs following CellSearch-based isolation and verify the method in patient samples.MethodsUsing healthy donor blood spiked with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer cell lines, SKBr-3 and BT-474, and a corresponding negative control (the HER-2-negative MCF-7 cell line), an in vitro CTC model system was designed. Following isolation in the CellSearch system, CTC samples were further enriched and fixed on microscope slides. Immunocytochemical staining with cytokeratin and 4′,6-diamidino-2′-phenylindole dihydrochloride identified CTCs under a fluorescence microscope. A FISH-based procedure was optimized by applying the HER2 IQFISH pharmDx assay for assessment of HER-2 amplification status in breast cancer CTCs.ResultsA method for defining the presence of HER-2 amplification in single breast cancer CTCs after CellSearch isolation was established using cell lines as positive and negative controls. The method was validated in blood from breast cancer patients showing that one out of six patients acquired CTC HER-2 amplification during treatment against metastatic disease.ConclusionHER-2 amplification status of CTCs can be determined following CellSearch isolation and further enrichment. FISH is superior to protein assessment of HER-2 status in predicting response to HER-2-targeted immunotherapy in breast cancer patients. This assay has the potential of identifying patients with a shift in HER-2 status who may benefit from treatment adjustments.

The role of the liquid biopsy as a clinical tool for early prediction in prostate cancer

The evolution of the liquid biopsy from a novel biomarker discovery platform to a clinical (molecular diagnostic) assay represents a true inflection point in the practice of medical (oncologic) pathology. Initially associated with the quantitation of breast cancer circulating tumor cells (CTCs) found in blood as a measure of tumor burden, the field quickly has expanded to include the isolation and capture of cell free/tumor DNA, exosomal RNA species and peptide-protein analytes in all body fluids including CSF and urine (1-4). The improvements in specimen handling, isolation techniques and the robust identification of low abundance nucleic acids have continued to advance the field; however, challenges persist as investigators attempt to understand the importance of rare variants in a complex setting of tumoral heterogeneity, drug resistance pathways and host-immune response. Recent success including the development of the first Food and Drug Administration (FDA) approved blood-based (liquid biopsy) companion diagnostic for the drug Tarceva (erlotinib) in patients with non-small cell lung cancer have further realized the potential (5). A simple, non-invasive, liquid biopsy approach for men with a suspicion of prostate cancer that offers insight into early detection of clinically significant disease while not over-diagnosing low-risk prostate cancer would have a critical impact on reducing the number of prostate needle biopsies and most importantly limiting over-treatment (6,7).

Abstract 496: Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Abstract Background: Androgen receptor (AR) is a nuclear transcription factor and a member of the steroid hormone receptor superfamily that can be detected by immunohistochemical staining in all three subtypes of invasive breast cancer with frequency of >70% of estrogen receptor-positive tumors (ER+), >60% of HER2-positive tumors (HER2+), and 40-50% of triple-negative breast cancers (TNBC) respectively. AR-target therapies are showing early activity in AR+ breast cancer. Given early evidence of activity with agents targeting AR, it is desirable to improve our understanding of the AR biology in breast cancer. Although circulating tumor cells (CTCs) have been proposed as a liquid biopsy for biomarker detection in multiple cancer types, the rate and heterogeneity of AR expression in CTCs in breast cancer is still unclear. In this study, we determine the rate and heterogeneity of AR expression in CTCs of patients with metastatic breast cancer. Methods: Blood samples from patients with metastatic breast cancer were smeared onto glass slides and subjected to nuclear staining with DAPI and reacted with fluorescent-labeled antibodies to detect CD45 on leukocytes and cytokeratin and AR on epithelial cells; the slides were scanned on the Epic Sciences CTC platform and the data analyzed using established algorithms. Results: Among the 59 patients analyzed, 25 (42.4%) had ER+/HER2-, 12 (20.3%) had ER+/HER2+, 6 (10.2%) had ER-/HER2+, and 16 (27.1%) had TNBC based on the most recent staining. CTCs were detected in 50 of 59 (84.7%) patients. Characterization of CTCs identified CK+ CTCs (45 of 59, 76.3%), CK-negative CTCs (16 of 59, 27.1%), CK+ CTC clusters (16 of 59, 27.1%), and apoptotic CTCs (45 of 59, 76.3%), respectively. Number of CTCs per patient ranged from 0 to 282.1 per mL (median = 4.4). Six of the 50 patients (12%) with CTCs had AR+ CTCs. The number of AR+ CTCs per patient ranged from 0.3 to 52.6 per mL (median = 1.15). Among the patients with AR+ CTCs, the percentage of CTCs that were AR+ ranged from 5% to 50% (median = 21%). Among the six patients with AR+ CTCs, five patients had ER+ breast cancer and one had TNBC. Conclusions: We demonstrated that the Epic Sciences non-enriching or selecting platform can detect AR expression in CTCs in breast cancer. These preliminary results suggest the need for clinical validation of CTC AR expression as a potential test to stratify and identify patients who might benefit from AR therapy. The heterogeneity of intra-patient CTC AR expression leads us to another hypothesis that patients with AR+ CTCs might have heterogeneous disease with multiple drivers. Further studies are warranted to allow serial monitoring of changes in AR and to investigate the clinical applicability of AR+ CTCs and their heterogeneity. Citation Format: Takeo Fujii, Yipeng Wang, James M. Reuben, Rachel Krupa, Ryon Graf, Lyndsey Dugan, Jessica Kouw, Dena Marrinucci, Bora Lim, Carlos H. Barcenas, Angela N. Marx, Debu Tripathy, Ryan Dittamore, Naoto T. Ueno. Androgen receptor expression on circulating tumor cells in metastatic breast cancer. [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 496.

Abstract 3143: Deciphering mechanisms of circulating tumor cells in breast cancer dormancy

Abstract More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized EpCAM-negative breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient in repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) attenuated resistance of the dormant subset to UV and bleomycin treatment. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs. Further investigations are being pursued to assess RIF1 contributions to retain CTC LTI potential leading to CTC-driven metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Deciphering mechanisms of circulating tumor cells in breast cancer dormancy. [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 3143.

Abstract 1530: Dissecting breast cancer dormant CTC phenotypes

Abstract Tumor relapse is a clinically relevant problem in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant for periods longer than 20 years and are undetectable by current clinical tools. Uncovering phenotypes of circulating tumor cells (CTCs) - the “seeds” of intractable metastasis-offers the promise to dissect CTC heterogeneity in relation to metastatic competence, to predict biomarker assessment, and to significantly improve monitoring and treatment of cancer. However, little is known about CTC biology and how CTCs differ in their capacity to circulate while maintaining a metastatic potential. We hypothesized that EpCAM-negative breast cancer CTC subsets exist, and avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties. We collected peripheral blood of clinically diagnosed breast cancer patients with or without brain metastasis, and performed multiparametric flow cytometry to isolate EpCAM-negative CTC subsets with stem-cell properties (CD44+/CD24-), along with combinatorial expression of two neoplastic markers: urokinase plasminogen activator receptor (uPAR) and integrin beta1 (int β1). EpCAM-negative CTCs were further interrogated at a single-cell level employing DEPArray platform. Second, we were able to culture FACS-sorted CTC subsets, selected for six cell-surface expression markers (CD45-/EpCAM-negative/CD44+/CD24-/uPAR+/-/int β1+/-), as long-term in-vitro 3D CTC tumorspheres. Third, CTC subsets were interrogated for biomarker profiling and biological characteristics. We identified adhesive, proliferative and invasive properties of 3D CTC tumorspheres which were distinct per uPAR/int β1 combinatorial expression. Lastly, we performed next-generation whole-genome sequencing and mutation analyses to discover unique genomic signatures of uPAR/int β1 CTC subsets and verified as putative CTCs originally disseminated from primary breast tumor. Additional investigations are being pursued assessing the molecular and genomic characterization of uPAR/int β1 CTC subsets comprehensively. Clinical relevance of this research includes that this may enhance abilities to prospectively identify patients who may be at high-risk of developing breast cancer brain metastasis. Citation Format: Monika Vishnoi, Sirisha Peddibhotla, Wei Yin, Zhong Xue, Antonio T. Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Dissecting breast cancer dormant CTC phenotypes. [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 1530.

263. Nuclease-Activated Oligonucleotide Probes for the Rapid and Robust Detection of Breast Cancer Circulating Tumor Cells (CTCs)

Metastatic breast cancer is the second leading cause of female cancer deaths in the United States. Despite substantial progress in its treatment, metastatic breast cancer remains incurable. Early identification of breast cancer patients at greatest risk of developing metastatic disease is thus an important goal that would enable oncologists to aggressively treat these patients while the cancer is still vulnerable. In addition, this would spare patients who do not need or would not benefit from further treatments from having to endure the harmful side-effects of chemotherapeutic drug regimens. Circulating tumor cells (CTCs) are rare cancer cells found in the blood circulation of cancer patients that provide a non-invasively accessible cancer cell specimen (liquid biopsy) from patients. The number of circulating tumor cells (CTCs) in cancer patients has recently been shown to be a valuable diagnostic indicator of the state of metastatic breast cancer. In particular, patients with few or no CTCs were found to have a better overall prognosis compared to patients with high numbers of CTCs. Despite the implications of CTCs as diagnostics for advanced breast cancer treatment, a critical challenge for adopting CTC-based diagnostic tests has been the development of methods with sufficient sensitivity to reliably detect the small number of CTCs that are present in the circulation. Furthermore, current tests for CTC detection are expensive, have high false positives and negatives, have high background noise, are time consuming and require a significant level of expertise to conduct. To overcome the limitations of current CTC detection assays and develop more sensitive, rapid and cost effective CTC detection methods, we explored the potential of detecting CTCs by measuring their nuclease activity with nuclease-activated probes1. We present data towards the development of a rapid and highly-sensitive CTC detection assay based on nuclease-activated oligonucleotide probes that are selectively digested (activated) by target nucleases expressed in breast cancer cells. We confirm that these probes are not activated by serum nucleases or nucleases from a lymphoblastic cell line (e.g. K-562). Furthermore, we present extensive data towards the optimization of activity and sensitivity of these probes in cell lysates from various breast cancer cell lines. Data is also presented confirming the detection of CTCs in blood from patients with stage IV breast cancer as well as healthy controls with spiked cancer cells. Future studies will focus on developing similar probes for detection of CTCs in patients with pancreatic and ovarian cancer where early detection would greatly improve survival outcomes. In conclusion, this work describes a robust assay for detection of breast cancer CTCs that will be straightforward to implement in most clinical diagnostic labs.1. Hernandez FJ, Huang L, Olson ME, Powers KM, Hernandez LI, Meyerholz DK, Thedens DR, Behlke MA, Horswill AR, McNamara JO, 2nd. Noninvasive imaging of staphylococcus aureus infections with a nuclease-activated probe. Nat Med. 2014;20:301-306

Circulating Tumor Cells and Tumor Stem Cells Detection in the Peripheral Blood Mononuclear Cells of Breast Cancer.

Our aim was to retrospectively analyze the relationships between circulating tumor cells (CTCs) and the development of breast cancer, for elucidating the role of CTCs in breast cancer. A total of 107 female patients with primary breast cancer and 48 matched healthy female volunteers were recruited. After blood collection, isolation of peripheral blood mononuclear cells (PBMC) was performed followed by the detection of cytokeratin 19 positive (CK19(+) ) and CD44(+) /CD24(-/low) cells, as well as estrogen receptor (ER), progesterone, and CerbB2. Data were analyzed with the SPSS 20.0 software. None of the 48 volunteers were detected with CK19(+) cells in their PBMC, while in 77 patients, 72% of 107 female patients with primary breast cancer, the CK19(+) cells were detected. CK19(+) could also be detected among patients in each grouping by different clinical staging and lymph node metastasis, with statistical differences (all P < 0.05). Further, among the 83 CK19(+) specimens, 32 were also detected with CD44(+) /CD24(-/low) cells. Comparisons of CK19(+) and CD44(+) /CD24(-/low) cells in patients with different clinical features (ER positive vs. ER negative, C-erbB2 positive vs. C-erbB2 negative) and molecular subtypes (triple-negative breast cancer, ER positive, and C-erbB2 positive) showed no obvious difference (all P > 0.05). Both CTCs and tumor stem cells (TSCs) could be detected in the PBMC of breast cancer patients; besides, positive expression rate of CTCs might be obviously associated with the clinical stage and metastasis. Positive relationship of TSCs and the clinical stage of breast cancer was also proved in this study.

Abstract 363: Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy

Abstract Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was &amp;gt;30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (&amp;gt;50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2015-363

Abstract 1997: Label-free concentration of viable breast cancer CTCs for single cell Western blotting

Abstract The isolation of circulating tumor cells (CTCs) in blood can aid in cancer prognosis, characterize genetic mutations for targeted therapies, and elucidate the biological mechanisms of metastasis. While CTCs provide clinical utility, there are still several technical difficulties with CTC isolation and analysis. CTCs are very rare (1-10 cells/mL of blood) and current isolation methods rely on immunocapture, require long sample preparation, and fail to deliver viable cells. Further, recent advances in single cell analysis of CTCs have been limited to genomic studies, in which DNA/mRNA does not provide a complete picture of CTCs at the functional level, as mRNA expression may only account for a portion of protein expression. The ability to study and measure the protein expression of CTCs could provide insight into the mechanisms driving cancer progression and signaling dysregulation. Unfortunately, conventional proteomic tools (e.g. immunocytochemistry and Western blot) either do not provide sufficient specificity or sensitivity toward single cell levels. Thus, there is a lack of tools that can provide quantitative protein analysis for rare CTCs. To address this gap, we enable rare cell Western blotting by combining isolation and proteomic technologies in a unique workflow: (i) Label-free Vortex enrichment of rare larger cells from blood samples. Larger cancer cells are trapped within an array of microscale fluid vortices that form in simple rectangular reservoirs, while smaller blood cells pass through. The cancer cells are stably maintained in the vortices, allowing for solution exchange, followed by release upon lowering the flow rate. With this device, we were able to release a small volume (∼500 μL) of concentrated, viable cancer cells (MCF7) from healthy blood, at high throughput, capture efficiency (35%), purity (&amp;gt;80%), and ∼10,000 fold enrichment. CTCs were also successfully extracted and enumerated from the blood of patients with breast cancer (N = 18, from 0.75 to 23.25 CTCs/mL). (ii) High-specificity and multiplexed protein analysis of rare cancer cells with single cell Western blotting (scWB). Enriched CTCs are placed into microwells patterned into a polyacrylamide gel for the scWB assay (in-situ chemical lysis in the microwells, electrophoresis into bulk gel layer, photo-immobilization, and subsequent antibody-probing of immobilized proteins). Here, we demonstrate scWB compatibility with rare cells by spiking MCF7s into normal blood and performing protein separations. The scWB is multiplex and can assay several target proteins simultaneously. Cells stained for surface markers (anti-EpCAM) are also compatible, and the scWB can resolve complete immunocomplexes without disrupting the location of the intracellular targets (GAPDH, ERK). With the integration of these technologies, we push the limits of quantitative protein analysis of CTCs, an essential step to better understand these rare populations. Citation Format: Elly Sinkala, Elodie Sollier, Corinne Renier, James Che, Stefanie S. Jeffrey, Amy E. Herr. Label-free concentration of viable breast cancer CTCs for single cell Western blotting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1997. doi:10.1158/1538-7445.AM2015-1997