Abstract B067: The Melanoma CTC Brain-Liver Metastasis Axis

<div><p>Melanoma brain metastasis (MBM) is linked to poor prognosis and low overall survival. We hypothesized that melanoma circulating tumor cells (CTC) possess a gene signature significantly expressed and associated with MBM. Employing a multipronged approach, we provide first-time evidence identifying a common CTC gene signature for ribosomal protein large/small subunits (RPL/RPS) which associate with MBM onset and progression. Experimental strategies involved capturing, transcriptional profiling, and interrogating CTCs, either directly isolated from blood of patients with melanoma at distinct stages of MBM progression or from CTC-driven MBM in experimental animals. Second, we developed the first MRI CTC-derived MBM xenograft model (MRI-MBM CDX) to discriminate MBM spatial and temporal growth, recreating MBM clinical presentation and progression. Third, we performed the comprehensive transcriptional profiling of MRI-MBM CDXs, along with longitudinal monitoring of CTCs from CDXs possessing and/or not possessing MBM. Our findings suggest that enhanced ribosomal protein content/ribogenesis may contribute to MBM onset. Because ribosome modifications drive tumor progression and metastatic development by remodeling CTC translational events, overexpression of the CTC RPL/RPS gene signature could be implicated in MBM development. Collectively, this study provides important insights for relevance of the CTC RPL/RPS gene signature in MBM, and identify potential targets for therapeutic intervention to improve patient care for patients with melanoma diagnosed with or at high risk of developing MBM.</p>Significance:<p>This study reports first evidence of RPL/RPS gene signature driving melanoma brain metastasis. Complex multilevel approach was performed to identify MBM signature and confirm its relevance to clinical settings. Novel MRI CTC-derived MBM mouse xenograft was established to monitor MBM spatial and temporal development and progression.</p></div>

Due to the advance in technologies for rare cell isolation, circulating tumor cells (CTC) have recently received vast interest. During tumor progression tumor cells invade the primary tumor microenvironment and intravasate into blood vessels, where they are referred as CTCs. These CTCs disseminate to other organs and a subset of these cells will be able to form metastasis. The growing interest for CTC is confronted with the difficulty associated with their isolation and characterization. To address this challenge, our lab has recently optimized the ex vivo culture condition and was able to establish CTC lines from breast cancer patients. These CTC lines constitute a unique cell population in the metastatic process and gave us a rare opportunity to investigate the signal pathways involved in each step of the metastatic cascade. To assess the metastatic potential of breast cancer patient-derived CTC lines we utilized an experimental mouse model for metastasis by injecting CTCs directly into the left ventricle of the heart in female immuodeficient NSG mice. The ability of 4 CTC lines to arrest and colonize in organ was monitored by bioluminescence imaging once every 2 weeks for 5 months. CTC lines were capable of generating brain, lung, bone and ovary metastases. Most of those organs are common sites of metastases in breast cancer patients. Two patient-derived CTC lines have a high metastatic potential (over 80% of mice had metastases after 3 months) with generation of simultaneous metastases in multiple organs such as bone, lung and Ovary. These mice remained brain-metastases free for up to 8 months. However, two other patient-derived CTC lines demonstrated the brain as preferential site of metastasis despite their overall low metastatic potential. Interestingly, of 4 breast cancer patients where CTC lines were generated, one developed a metastatic brain tumor and her CTC line has the highest risk of brain metastases in our mouse model. We further investigated genetic and epigenetic determinants that regulate the organotropism of CTCs. We isolated metastatic variants corresponding to a subpopulation of CTCs with a preferential tropism for the brain, lung and bone. Gene expression analysis (RNA-seq) of these variants identified potential gene signatures of breast cancer metastasis. The pathways with the highest enrichment scores were glioblastoma multiform, ceramide biosynthesis and PCP pathway for the brain metastatic variants and were interferon and mTOR signaling for the lung metastatic variants. Next we used a method for assaying chromatin accessibility (ATAC-seq) and identified potential regulatory regions mediating the organ tropism in breast cancer. Together our data provide the evidence of a promising role of CTC as an early prognostic factor in metastasis. Additionally, we expect to develop novel organ tropism associated markers, which can be considered for potential therapeutic targets in breast cancers. Citation Format: Remi Klotz, Thomas Amal, Alan Wang, Matthew Mackay, Kathleen Heller, Lin Li, Maxwell Serowoky, Grace Lee, Jane Han, Andrew Smith, Min Yu. Understanding breast cancer metastasis through circulating tumor cells [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 1045. doi:10.1158/1538-7445.AM2017-1045

Introduction: Somatic mutations, including those in TP53, PIK3CA, and estrogen receptor alpha (ESR1), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch© (CXC kit). CTC and WBC were then purified using DEPArrayTM. DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1TM WGA). Genetic analysis was performed on individual CTC, pooled CTC and pooled WBC DNA by multiplexed PCR based targeted next generation sequencing (NGS) using the Oncomine Comprehensive Panel (targeting ∼130 onco- and tumor suppressor genes) and the Ion Torrent Proton. All patients had exome sequencing performed on research biopsies of metastases using an Illumina HiSeq 2500 platform. Results: This pilot study was conducted using high quality DNA from two patients assessed to date. Both patients had lobular carcinoma and as expected harbored somatic, deleterious CDH1 (E-cadherin) mutations (frameshift and non-sense) in both research biopsy and CTC-DNA. These data supported our approach. Patient #1 was TP53 wild type in her research biopsy, but multiple CTC harbored somatic TP53 frame-shift mutations (Table). Patient #2 harbored an ESR1 Y537S mutation in her research biopsy. However, only 4 of 7 CTC also harbored this somatic, heterozygous mutation. Prioritized mutations in CTCPt#Cell Type (CTC vs WBC), numberGeneMutationVariant fraction (expected 1=homozygous; 0.5=heterozygous)Found in research biopsy?1CTC_A2CDH1p.I584fs1YES CTC_A4 1 CTC_A7 0.54 CTC_pool* 0.74 WBC_pool 0 CTC_A2TP53p.152_156del1NO CTC_A4 1 CTC_A7 0.51 CTC_pool* 0.88 WBC_pool 0 2CTC_A9ESR1p.Y537S0.52YES CTC_D1 0.34 CTC_D2 0.46 CTC_D6 0.65 CTC_pool* 0.35 WBC_pool 0 CTC_A12 0 CTC_D3 0 CTC_D7 0 CTC_A12CDH1p.Q641X1YES CTC_A9 1 CTC_D1 1 CTC_D3 1 CTC_D6 1 CTC_pool* 1 WBC_pool 0 * pool of all CTC Conclusions: We demonstrate the ability to purify CTC, isolate, and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Both known and novel alterations were identified in comparison to research biopsy specimens. This approach allows single cell analysis demonstrating heterogeneity of mutational status in different single cells. Studies of CTC-ESR1 and other genetic abnormalities in patients with known tissue mutations who participated in Mi CTC-ONCOSEQ are now underway. Citation Format: Paoletti C, Cani AK, Aung K, Darga EP, Cannell EM, Hovelson DH, Yazdani M, Blevins AR, Tokudome N, Larios JM, Thomas DG, Brown ME, Gersch C, Schott AF, Robinson DR, Chinnaiyan AM, Bischoff F, Hayes DF, Rae JM, Tomlins SA. Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-19.

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 >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 (>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

Background: Presence of EpCAM+ cytokeratin+ nucleated circulating tumor cells (CTC) in metastatic carcinoma patients is associated with poor survival and may be used to guide treatment. Assessment of treatment targets on these CTC holds the promise of a liquid biopsy. However the proportion of patients in which a sufficient number of CTC are detected in 7.5 mL of blood is not sufficient. In this study we estimate the number of CTC in blood of patients with metastatic disease, explore the relationship with survival and determine whether assay modifications and/or increases in sample volume are needed to achieve the required increase in number of CTC detected. Methods: EpCAM+CK+DNA+CD45- CTC enumeration was performed with the CellSearch® system in 7.5 mL of blood of 836 patients with metastatic breast, colorectal and prostate cancer patients (Cristofanilli, NEJM 2004, Cohen, JCO 2008, De Bono, CCR 2008). EpCAM+, CD45-, Nucleic acid+ CTC were enumerated in 100ul of NH4CL lysed blood from 140 metatatic cancer patients and compared to CTC as enumerated with the CellSearch system. Image analysis for automated CTC enumeration and modeling of CTC frequencies was performed in Matlab. Results: The median number of CTC in 7.5mL of blood metastatic breast, colorectal and prostate is 5 and in 39% of patients no CTC are detected. The best fit of the CTC frequency distribution was used to extrapolate the sample volume to 5 liters of blood and predicted that 99% (95% confidence interval, CI, 95-99.8%) of patients had at least 1 CTC before initiation of therapy, which decreased to 97% (95% CI 87-99.5%) after the first cycles of therapy. One hundred CTC per liter of blood are present in ∼80% of patients, 1 CTC per ml of blood in ∼40% of patients and 10 CTC per ml of blood in ∼20% of patients. The median survival of patients with CTC is reduced by 6.6 months for each tenfold CTC increase. CTC definitions that do not include a requirement for presence of DNA, EpCAM, cytokeratin 8,18,19 (CK) or absence of CD45 are less predictive of survival, than the strictest definition requiring a CTC to be EpCAM+CK+DNA+CD45-. Comparison of CTC detected by flow cytometry to the CellSearch CTC method on 140 patients shows that improvements in EpCAM recovery may increase the average number of detected CTC by 6.5 fold (95% CI 5.6-7.3), yet the fits predict that this will only reduce the number of patients with 0 CTC from 39% to 21% (95% CI 13-31%). Conclusions: EpCAM+CK+DNA+CD45- CTC are present in blood of all patients with metastatic breast, colorectal and prostate cancer. The predicted strong relation with survival suggests the importance of this phenotype for metastasis. To use CTC as a liquid biopsy for the majority of patients, the CTC yield needs to be improved 100-1000 fold. This requires a dramatic increase in sample volume which may be achieved by in vivo flow cytometry, or through processing an apheresis product. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2391. doi:1538-7445.AM2012-2391

Introduction: CTCs can overexpress HER2 discordant from tumor HER2 expression. We aimed to describe characteristics of a CTC-defined group of pts with metastatic breast cancer (MBC) that is tumor HER2- and CTC HER2+ (HER2 tumor- CTC+). Methods: We retrospectively analyzed data from pts treated at Northwestern University who had serial evaluation of CTCs and circulating tumor DNA (ctDNA). We included pts with pathologically confirmed HER2- MBC and HER2+ CTCs. CTCs were enumerated with the CellSearch immunomagnetic kit (Menarini Silicon Biosystems), HER2 expression on CTCs was determined using the CellSearch CXC Kit in 7.5 cc whole blood, and ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health). Results: Among 98 pts with HER2- MBC and CTC analysis, 46 (47%) had at least 1 HER2+ CTC. In this cohort the median age was 53. At initial BC diagnosis, 80% had early stage or locally advanced BC and 20% had de-novo metastatic disease. Baseline histology was 65% ductal, 20% lobular, 2% mixed ductal and lobular, and 13% unknown. Pathology of metastatic tumor was hormone receptor positive (HR+)/HER2- in 78% and triple negative in 22%. Detailed HER2 immunohistochemistry (IHC) and FISH results from metastases were available from 63% of pts of whom 72% had an IHC score of 0 or 1 and 28% had an IHC score of 2 with negative FISH testing. The median time from the most recent pathologic metastatic tumor assessment to the detection of a HER2+ CTC was 6.5 mo. Twenty-two pts had simultaneous (within 8 weeks) HER2- tumor confirmation and HER2+ CTC detection. The median lines of endocrine therapy (ET) for MBC prior to detection of HER2+ CTCs was 1 (range 0-5, 41% no ET, 17% 1 line, 41% >2 lines). Pts received a median of 2 (range 0-10) prior systemic therapies for MBC prior to detection of HER2+ CTCs, (20% 0 lines, 41% 1-3 lines, and 39% >4 lines). Among these 46 pts, CTCs were analyzed longitudinally in 104 samples, with HER2+ CTCs detected in 77 samples. Number of HER2+ CTCs at initial detection ranged from <5 in 24%, 5-50 in 43%, and >50 in 33%, with a median of 11.5 HER2+ CTCs. CTC clusters were noted in 37% of pts. At initial detection the proportion of CTCs that were HER2+ was 0-25% in 13% of pts, 26-50% in 46% of pts, and 51-100% in 41% of pts. Seven pts had ERBB2 aberrations in ctDNA. Of 12 pts with tumor sequencing, 2 had ERBB2 mutations, 1 had ERBB3 amplification, and 1 had overexpression of ERBB3 RNA. After detection of HER2+ CTCs, 18 pts received HER2 directed therapy (with chemotherapy in 13 pts, with endocrine therapy in 4 pts, and as monotherapy in 1 pt). Imaging demonstrated a partial response or stable disease in 9 pts (clinical benefit rate 50%), including in 1 pt with trastuzumab monotherapy, progressive disease in 8 pts, and not evaluated in 1 pt. Conclusions: HER2+ CTCs are frequently detected simultaneously or soon after HER2- tumor assessment in MBC. Within this newly defined subgroup, the several responses seen with HER2 targeted therapy serve as a proof of concept that HER2 tumor- CTC+ patients can benefit from HER2 targeted therapy. Future studies are needed to determine a clinically relevant threshold for HER2+ CTCs to guide further study of HER2 therapy combinations in HER2 tumor- CTC+ pts. Citation Format: Shah AN, Gerratana L, Zhang Q, Davis AA, Zhang Y, Flaum L, Behdad A, Platanias L, Gradishar WJ, Cristofanilli M. HER2-negative metastatic breast cancer with HER2-positive circulating tumor cells (CTCs): A new CTC-defined HER2-positive subgroup [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-08.

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.

Today, circulating tumor cells (CTCs) can provide important prognostic information about a patient. However, to one day help guide decisions for precision medicine, more and increasingly sophisticated information will have to be extracted from each patient blood sample. We describe a new microfluidic CTC isolation technology, the Harpoon CTC Isolator and Chip (Hrp Isolator). From a single sample the Hrp Isolator can enable the analysis of plasma components such as ctDNA, as well as the ability to enrich all circulating tumor cell types, regardless of their antigenic expression. To enrich the CTCs from 7.5 mL of whole blood, the Hrp Isolator removes the red blood cells and platelets based on their small size and the white blood cells by negative depletion using magnetic particles. The time required to process 7.5 mLs of blood is approximately 90 minutes. In contrast, the CELLSEARCH® CTC Test (CS), the only US FDA-cleared test, uses antigens expressed by CTCs to target the CTCs and positively select them out of blood. To enumerate CTCs from patient samples, the output from the Hrp Isolator was then loaded into and run on the CELLSEARCH® System using the CS test (Hybrid method). CTCs were enumerated from 37 breast and prostate cancer patients by CS and by the Hybrid method. At least 1 CTC was found in 23 (62%) patient samples by the Hybrid method versus 17 (46%) by CS. The product of the Hrp Isolator enrichment contained on average the same numbers of CTCs as were found by CS (mean = 13.3 ± 18 by CS versus 12.0 ± 16 by Hybrid) in patient samples having <75 CTCs. For samples with high CTC counts (>75), the Hybrid method may have underestimated the total Hrp Isolator CTC count. An important feature of the Hrp Isolator is its ability to enrich CTCs that are negative or low expressers of epithelial cell adhesion molecule (EpCAM) or cytokeratin (CK). These cells could have been missed by positive-selection technologies such as CS and may not have been counted using the Hybrid method. CTC spiked samples were run on the Hrp Isolator and the CTC enriched product was spun onto microscope slides, stained, and examined using a Vectra Multispectral Imaging System (Hrp+Vec method). Using the Hrp+Vec method, 79% to 100% of spiked CTCs were recovered depending on the slide fixation method used. Also, using Hrp+Vec we confirmed the ability of the Harpoon Isolator to enrich EpCAM-/CK+ and EpCAM+/CK- CTCs from patient samples. This establishes the existence of “non-canonical” tumor cell types in circulation and adds to the biological diversity of the CTC population in circulation. These results emphasize the need to further characterize all tumor cell populations and to determine what, if any, clinical significance may be assigned to these diverse CTC types. Citation Format: David Chianese, Mark Connelly, Carrie Morano, Thai Bui, Steven Gross, Renouard Sanders, Tom Barber, Ravi Kapur, Shyamala Maheswaran, Mehmet Toner, Daniel Haber. Isolation of canonical and non-canonical circulating tumor cells (CTCs) by negative depletion using the Harpoon CTC Isolator and Chip. [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 2422.

Introduction: Current CTC detection platforms require the processing of patient blood within 48 hrs after collection to preserve CTC integrity and biomarker expression. Here we demonstrate the recovery of CTCs from banked patient blood samples that have been cryopreserved for up to 7 years. Our results show that cryopreserved CTCs have equivalent morphology and protein expression patterns as CTCs in freshly processed blood and are suitable for genomic analyses. This process enables retrospective studies of banked patient blood samples inclusive of CTC enumeration, biomarker quantification, FISH and genomic analysis via the Epic Platform. Methods: 30 cryopreserved mCRPC patient blood samples were sent to Epic Sciences alongside 10 matched fresh blood draws (<48 h after draw). All nucleated cells from mCRPC patient samples or cell line-spiked healthy donor (HD) controls were plated onto glass slides and subjected to IF staining, CTC identification by fluorescent scanners and algorithmic analysis. Traditional CTCs (CK+, CD45-, intact DAPI nuclei and morphologically distinct) and CK- CTCs (CK-, CD45-, intact and distinct) were identified. Androgen receptor (AR) expression was assessed by IF staining and PTEN status determined by FISH. Relative CTC enumeration, morphology, protein expression and genomic status were compared between freshly processed and cryopreserved samples. Results: Initial testing in VCaP (AR+) and PC3 (AR-) cell line-spiked HD blood demonstrated preservation of cell morphology and biomarker expression. In VCaP, CK signal decreased 20% and AR decreased 6% after freezing, but CK and AR expression were 90- and 8-fold higher than negative controls, respectively. In patients, CTCs were detected in 7/10 freshly processed samples and in equal relative abundance in 6/10 matched frozen samples. Cryopreserved CTCs retain CK and AR expression as compared to freshly processed samples: there were no significant differences in mean CK or AR expression among all matched samples compared, and% AR+ CTCs correlated strongly between fresh and frozen samples (r2 = 0.96). Furthermore, PTEN FISH on cryopreserved CTCs resulted in a near-equal percentage of PTEN variant cells as compared to matched fresh draws. After concordance studies, an additional 20 frozen mCRPC patient samples were processed: CTCs were detected in 16/20 samples, with 11/20 harboring AR+ CTCs. Conclusion: Recovery of CTCs in cryopreserved patient blood samples is feasible using the Epic Platform. These data demonstrate concordance of CTC recovery, protein characterization and genomic analysis in an initial cohort of cryopreserved mCRPC samples. Further studies characterizing cryopreserved CTCs in mCRPC and in cancers of other tissues are warranted, with the goal of enabling retrospective protein and genomic characterization of banked patient samples. Citation Format: David Lu, Melissa Harvey, Ravi Madan, Christopher Heery, Jennifer Marte, Sharon Beasley, Mark Landers, Rachel Krupa, Jessica Louw, Justin Wahl, Natalee Bales, Dena Marrinucci, Jeffrey Schlom, James Gulley, Ryan Dittamore. Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples. [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 1586. doi:10.1158/1538-7445.AM2015-1586

Introduction: Monitoring CTCs from a blood draw could enable a minimally invasive, longitudinal observation of an evolving disease. Antibody drug conjugates (ADC) are a promising approach to deliver highly potent chemotherapeutics to cells expressing target proteins. An ADC that targets 5T4, an oncofetal antigen expressed on tumor initiating cells, is currently in clinical development. Patients with tumors or CTCs expressing 5T4 may benefit from this therapeutic. We sought to develop a 5T4 CTC test and to evaluate the incidence of 5T4 expressing CTCs in breast, ovarian, and lung cancer patients. Experimental Procedures: Blood samples from breast (n = 10), ovarian (n = 10) and NSCLC (n = 25) patients on active therapy were collected and shipped to Epic Sciences. Upon receipt, nucleated blood cells were plated onto glass microscope slides and stored at -80C. Two slides per patient were thawed and stained for cytokeratins (CK), CD45, 5T4, and DAPI. CTCs were detected using a combination of CD45 exclusion, CK and 5T4 expression, and morphology parameters. CTC enumeration and 5T4 expression were analyzed alongside pathology, staging and treatment information. Results: Assay Development: A novel CTC assay utilizing an antibody against 5T4 was developed. Dilutions of high, medium and low/negative 5T4 expressing cell lines were spiked into normal whole blood to assess the linearity (R2 = 0.99) of CTC detection. These cell lines as well as 5T4 high and 5T4 low patient samples were used to assess intra- and inter-assay reproducibility of% 5T4(+) CTCs. All patient and calibration control samples met acceptance criteria of ≤ 30% CV and ≤ 20% CV, respectively. Breast Cancer: 7 of 10 patients had traditional CTCs (median: 6/mL range: 0-23/mL). 9 of 10 patients had additional subpopulations of CK(-) CTCs, apoptotic CTCs, and CTC clusters. Among non-apoptotic CTCs, 5T4 positivity was detected in 6 of 10 patient samples, with the highest% positivity seen in patients with corresponding ER(+) primary tumors. Ovarian Cancer: 3 of 10 patients had traditional CTCs (median: 0/mL, range: 0-41/mL). Additional subpopulations were identified in the same 3 of 10 patients. NSCLC: 14 of 25 patients had traditional CTCs (median: 1/mL, range: 0-45/mL). 20 of 25 patients had additional subpopulations. Conclusions: We have developed a robust, sensitive and specific assay to quantify 5T4 expression on CTCs and tested this assay on small cohorts of breast, ovarian and lung cancer patients on active therapy. CTCs expressing 5T4 were detected in a subset of patients from each indication. Citation Format: Steven R. Pirie-Shepherd, Iman Jilani, Eric Tucker, David Valenta, Ryon Graf, Amanda Anderson, Dena Marrinucci, Puja Sapra, Eric L. Powell. Circulating tumor cell (CTC) detection and 5T4 characterization in breast, ovarian, and lung cancer patients on active therapy. [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 565. doi:10.1158/1538-7445.AM2015-565

Introduction: Breast cancers (BCa) in younger women are more likely to be fast-growing, higher grade and hormone receptor-negative. As such these tumors tend to be associated with a worse prognosis even after systemic treatments with surgery, chemotherapy and radiation. The mechanisms that contribute to these tumors' aggressiveness remain largely unknown. Surveillance of circulating tumor cells (CTCs) may help to understand BCa prognosis and predict treatment benefit, especially for women with metastatic disease. Herein, we report a new finding of the correlation between age and the changes of CTC after the systemic therapies. Methods: 160 whole blood samples (7.5ml/each) were collected from 100 patients with stage III/IV BCa at baseline, before and after systemic therapy. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search). CTCs were confirmed using Anti-CK-PE, CD25, and EpCaM antibody and DAPI nuclear stain. Anti-CD45-APC antibodies were used to identify leukocytes. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD25-. Database of CTCs was generated and linked with clinical database. Student test was used for statistics. Results: Among 100 subjects who met inclusion criteria, 54 subjects (68%) at least 1 detectable CTCs in the baseline blood drawn prior to treatment. Of those, 37 patients had ≥ 5 CTCs, 17 patients had 1-5 CTCs, and 46 patients had no CTCs. There was no significant difference on the average age between these CTCs groups, which are 54.89, 53.52 and 53.30 respectively (P>0.05). Among the initial cohort, 42 subjects had at least 2 blood draws before and after systemic therapies respectively. Among these 42 patients, 11 had more CTCs after systemic therapy [Group 1 (26%), the increased range of CTC is from 1 to 756] and 17 patients had no CTCs before or after therapy [Group 2 (40%)]. There were 14 patients who had significantly fewer CTCs after systemic therapy [Group 3 (34%), the decreased range of CTC is from -1 to -23]. There was a significant difference in the average ages of each group (48.45, 56.00 and 57.28 years respectively; P=0.02). The results indicated that younger age (less than 50 years old) is associated with persistent of higher CTCs after systemic therapy likely reflecting decreased benefit from systemic therapy. Conclusion: Our original findings suggest age-related differences in CTCs response after systemic therapies for advanced BCa. These findings may indicate that younger patients may be at higher risk of developing more distant metastases as suggested by the increased numbers of CTCs after the systemic therapies. We propose that a combination of baseline CTCs detection and age be considered as a potential new criteria for selecting the systemic therapies for BCa patients. Citation Format: Qiang Zhang, Lorenzo Gerratana, Lisa Flaum, Youbin Zhang, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) after systemic therapy is associated with younger age in stage III/IV breast cancer patients [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 1597.

Background: Circulating tumor cells (CTCs) are associated with prognosis in metastatic breast cancer (BC). We evaluated the association of inflammatory/tumor markers and CTCs in women with progressing metastatic breast cancer prior to commencing a new line of systemic therapy. Methods: From February 2013 to April 2015, 96 patients with metastatic BC about to start a new treatment (due to progression), without current diabetes or use of anti-inflammatory agents, were recruited from four Ontario cancer hospitals. Women provided fasting blood for inflammatory and tumor markers and CTC measurement; CTCs were assayed within 72 hours of collection using CellSearch. Blood was frozen at -80C until assays were performed in a single batch (C-reactive protein (CRP), IL-6, PAI-1, Ca15-3, Ca125, VEGF, TNFa). Associations of CTCs with blood factors were evaluated using Pearson correlation coefficients after transforming the variables to normality. For CTCs the transformation log(x+0.5) was used. Associations with categorical variables were tested using one-way analysis of variance. P values <0.05 were significant. Results: Median age of patients was 60.5 years, 87 (90.6%) were post-menopausal, 83 (86.5%) had hormone receptor positive BC, 16 (16.7%) HER2 positive BC, 10 (10.4%) triple negative; 75 (78.1%) grade II/III. At the time of CTC measurement, bone, lung, liver and brain metastases were present in 79%, 44%, 40% and 6% of patients respectively, with 54%, 37%, 35% and 3% having progression at these sites respectively. PAI-1 and CA15-3 exceeded the limit of the assay in 11 and 5 cases respectively (the upper limit of the assay was used in the analysis). 33.4% of patients were starting first line therapy, 25% second line and 16.7% third line. CTC counts (per 7.5cc) ranged from 0 to 1238 (median 2, geometric mean 3.63); none were detected in 29 (30.2%) patients, 1 to 4 in 25 (26%) and 5 or more in 42 (43.8%) patients. CTCs were not associated with age, estrogen receptor, progesterone receptor, HER2, line of treatment, lymph-vascular invasion or tumor grade. Compared to metastatic disease at other sites, CTCs were higher in the presence of bone (p=0.027) and liver metastases (p=0.002) and with progressing bone (p=0.018) and liver (p=0.012) metastases. CTCs were significantly associated with CRP (R =0.25, p=0.014), IL-6 (R=0.31, p=0.002), PAI-1 (R=0.31, p=0.002), Ca15-3 (R=0.44, p=<0.0001) and Ca 125 (R=0.21, p=0.04) but not with VEGF and TNFa (R = 0.11, p= 0.29 and R = 0.16, p=0.11, respectively). Conclusion: CTCs were associated with bone and liver metastases and with higher levels of inflammatory and tumor markers, potentially reflecting tumor burden. Additional inflammatory marker assays are underway. Future studies are warranted to confirm these findings. Citation Format: Lohmann AE, Chang M, Dowling RJO, Ennis M, Amir E, Elser C, Brezden-Masley C, Vandenberg T, Lee E, Fazae K, Stambolic V, Goodwin PJ. Association of inflammatory and tumor markers with circulating tumor cells in metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-12.

Background: Thousands of circulating tumor cells (CTCs) may be released into the blood stream of a cancer patient each day. However, only viable CTCs can cause metastasis. These viable CTCs hold the promise of deciphering the tumor heterogeneity, uncovering the mechanism of resistances to therapies, and enabling personalized treatments. Despite the continuously mounting interest, isolation of these cells remains challenging due to the multitude of factors, including rarity of the CTCs in blood, their constant exposure to therapeutic agents, and the harsh environment in the circulation. To address this, we have developed a novel microfluidic biochip capable of CTC isolation from unprocessed whole blood. Herein, we compare isolation of CTCs from the central and peripheral blood samples of metastatic breast cancer (MBC) patients using our biochip to assess impact of blood collection location on CTC count and viability. Methods: We included 19 patients with MBC, of which 26% are white, 63% are African American, and 11% are Latino. The median age of the cohort is 58 (34-75y). All patients are undergoing therapy. A total of 36 samples (7.5 mL each) were collected from either an antecubital vein (peripheral vein) or the subcutaneous port catheter (central vein) at different time points. All samples were processed within 5 hours of blood drawn in our novel microfluidic biochip (only 40 minutes per sample, without pretreatment), followed by in situ immunostaining. CK+/EpCAM+, DAPI+ and CD45- cells were counted as CTCs. In situ live/dead assay using Calcein AM and propidium iodide was performed for some samples right after CTC isolation. Results: CTCs were detected in all the samples (36/36), with a median of 15 CTCs per sample. A >5 CTCs count was found in 86% of the samples (31/36). No correlation was found between CTC count and race/ethnicity. The CTC count in central venous samples was in the range of 6-105 (median 27), while the range was 1-45 (median 10) in the peripheral samples. The difference was found to be statistically significant (p<0.001 using ANOVA). While no correlation between CTC count and patient age was found for the overall samples, we noted a significantly higher CTC count in central venous blood of younger patients (<60y) than of senior patients (p<0.05). Live/dead assay immediately following the microfluidic isolation found live CTCs in patient samples. Conclusions: Viable CTCs were rapidly isolated and detected in MBC samples using our novel microfluidic biochip. Our results show that CTC count in blood drawn from central vein yields significantly higher numbers than from the peripheral vein, suggesting that central venous samples might be preferred for prognosis and monitoring to drug response in MBC patients. Viable CTCs may provide an opportunity to evaluate the mechanism of resistance to therapies. More studies with larger cohorts are needed to further confirm these findings. Citation Format: Jian Zhou, Qiyue Luan, Celine Macaraniag, Arielle Guzman, Maria Mantice, Oana Danciu, Kent Hoskins, Ian Papautsky. Isolation of viable circulating tumor cells from peripheral and central venous blood using a rapid microfluidic biochip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5124.

Background : The detection of circulating tumor cells (CTCs) in peripheral blood is an independent predictor of the efficacy of systemic therapy and a prognostic marker for patients with metastatic breast cancer. One of the leading techniques to detect CTCs uses immune-magnetic separation followed by immunocytochemistry. A microdevice can capture and enumerate CTCs using distinctive physiological difference (size and deformability) between cancer cells and blood cells. This microdevice thus obtains a larger CTC yield than that of affinity based separation which enriches the samples from a particular subgroup of cells based on biomarker (EpCAM) used. In this study, we investigated CTCs in peripheral blood from metastatic breast cancer patients using this microdevice. Patients and methods: We examined blood samples of 9 patients with heavily treated locally recurrent or metastatic breast cancer. Informed consent from these patients was obtained before blood extraction. Blood samples were taken into sodium EDTA tubes after discarding the first 1ml of blood samples. Two ml whole blood were subjected to the microdevice (Clear cell system), and CTCs were trapped in the microwells: Trapped cells were analyzed by immunocytochemistry with monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (CK8/18), along with 4,2-diamidino-2-phenylndole dihydrochloride (DAPI) for nuclei. CK8/18- positive, DAPI-positive and CD45-negative cells were defined as CTCs. Three patients were examined using both this microdevice and affinity-based separation with EpCAM, to compare the yield of CTCs. Results: Of the 9 patients: 7 had ER-positive primary tumors, and 6 had PgR-positive ones, HER2 overexpression was detected in 2 primary tumors. CTCs were detected in 8 patients. The single patient in whom CTCs were not detected suffered from local recurrence (axillary lymph node metastasis) only, with no distant metastases. We were also unable to detect CTCs using EpCAM affinity method for this patient. The number of detected CTCs in the other patients ranged from 19/2ml to 156/2ml (mean 90/2ml), and the sizes of CTCs varied from 5 to 16μm. CK8/18-negative and DAPI positive were detected in most patients, and these cells tended to be larger than CK8/18-positive cells, suggesting that epithelial–mesenchymal transition (EMT) might occur in CTCs. The total number of CTCs detected by the microdevice from 2 patients was larger than that of CTCs detected by EpCAM affinity method (107/2ml vs 1/7.5ml, and 19/2ml vs 39/7.5ml). Conclusion: CTCs detected by this microdevice varied in regard to the size of trapped cells and characteristics examined by immunochemistry, suggesting the heterogeneity of CTCs. Further research on this heterogeneity is vital in order to develop personalized treatment for patients with metastatic breast cancer. Citation Format: Tozuka K, Nagai SE, Inoue K, Komatsu K, Matsumoto H, Hayashi Y, Kurozumi S, Suganuma M. Enumeration of heterogeneous circulating tumor cells (CTCs) in metastatic breast cancer patients based on size and deformability. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-20.

Introduction: The prognostic relevance of Circulating Tumor Cells (CTCs) in patients with metastatic breast cancer (MBC) has been shown in multiple clinical trials. Beside their quantification it is of particular interest to analyze the predictive value of these cells and investigate if therapeutic decisions can be based on the phenotype of CTCs. The DETECT study concept evaluates this possible implication of CTCs. The DETECT V study compares endocrine therapy vs. chemotherapy, both combined with Trastuzumab and Pertuzumab in MBC patients with a HER2-positive (Human Epidermal Growth Factor Receptor 2), hormone receptor-positive tumor. Translational projects to identify potential predictive markers are associated to the study program. The expression of predictive markers HER2 and estrogen receptor (ER) on CTCs is analyzed to establish and validate an “Endocrine Responsiveness Score” (ERS). The ERS is aiming to estimate the potential benefit of endocrine therapy. Methods: Quantification and characterization of CTCs was performed using Cell Search® CXC Kit. The staining protocol was validated using cell lines with known expression of ER and HER2. CTCs were quantified at randomization, after six weeks and at the end of treatment in two samples. Staining intensities of both markers were specified. Results: Staining was successfully established. ER staining intensity was specified as negative or positive; HER2 staining intensity as negative, weak, moderate or strong. Detection and characterization of CTCs was analyzed for the first 30 patients. At time of randomization 17 of 26 samples were CTC positive with ≥1 CTC in one sample (HER2 and ER), 6/25 in both samples. After six weeks 8 of 26 patients contained ≥1 CTC in one sample and 5 of 26 in both samples. 11 of 15 CTC positive patients at time of randomization were CTC negative in the subsequent sample after six weeks. The cumulative frequency of ER- and HER2-positivity regarding CTCs detected in all samples was analyzed. 13% of all CTCs detected were ER positive (14/115). The percentage of HER2 3+ CTCs decreased from 27% at time of randomization (25/92) to 0% after six weeks (0/87). Analyzing the distribution of the expression of ER and HER2 in all CTCs of one sample, 10 of 19 samples containing ≥2 CTC showed heterogenic intensity of HER2-specific immunofluorescence. 8 of 14 samples containing ≥2 CTC showed heterogenic intensity of ER-specific immunofluorescence, respectively. Conclusion: The implementation of “Endocrine Responsiveness Score” ERS aims to predict the potential benefit of endocrine therapy in patients with a HER2-positive, hormone receptor-positive tumor. Descriptive analysis of first patient samples has shown to detect heterogenic expression of HER2 and ER in CTCs of patients participating in the DETECT V study. Citation Format: Fehm T, Meier-Stiegen F, Riethdorf S, Rack B, Taran F-A, Pantel K, Müller V, Janni W, Huober J. DETECT V – Expression analysis of human epidermal growth factor receptor 2 and estrogen receptor on circulating tumor cells of metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-04.