Brad Foulk

Development of a method to isolate circulating tumor cells using mesenchymal-based capture

Epithelial tumor cells can become mesenchymal cells and vice versa via phenotypic transitions, a process known as epithelial plasticity. We postulate that during the process of metastasis, circulating tumor cells (CTCs) lose their epithelial phenotype and acquire a mesenchymal phenotype that may not be sufficiently captured by existing epithelial-based CTC technologies. We report here on the development of a novel CTC capture method, based on the biology of epithelial plasticity, which isolates cells based on OB-cadherin cell surface expression. Using this mesenchymal-based assay, OB-cadherin cellular events are detectable in men with metastatic prostate cancer and are less common in healthy volunteers. This method may complement existing epithelial-based methods and may be particularly useful in patients with bone metastases.

Development of a Novel c-MET–Based CTC Detection Platform

Amplification of the MET oncogene is associated with poor prognosis, metastatic dissemination, and drug resistance in many malignancies. We developed a method to capture and characterize circulating tumor cells (CTC) expressing c-MET using a ferromagnetic antibody. Immunofluorescence was used to characterize cells for c-MET, DAPI, and pan-CK, excluding CD45+ leukocytes. The assay was validated using appropriate cell line controls spiked into peripheral blood collected from healthy volunteers (HV). In addition, peripheral blood was analyzed from patients with metastatic gastric, pancreatic, colorectal, bladder, renal, or prostate cancers. CTCs captured by c-MET were enumerated, and DNA FISH for MET amplification was performed. The approach was highly sensitive (80%) for MET-amplified cells, sensitive (40%–80%) for c-MET–overexpressed cells, and specific (100%) for both c-MET–negative cells and in 20 HVs. Of 52 patients with metastatic carcinomas tested, c-MET CTCs were captured in replicate samples from 3 patients [gastric, colorectal, and renal cell carcinoma (RCC)] with 6% prevalence. CTC FISH demonstrated that MET amplification in both gastric and colorectal cancer patients and trisomy 7 with gain of MET gene copies in the RCC patient. The c-MET CTC assay is a rapid, noninvasive, sensitive, and specific method for detecting MET-amplified tumor cells. CTCs with MET amplification can be detected in patients with gastric, colorectal, and renal cancers. Implications: This study developed a novel c-MET CTC assay for detecting c-MET CTCs in patients with MET amplification and warrants further investigation to determine its clinical applicability. Mol Cancer Res; 14(6); 539–47. ©2016 AACR.

Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders

We have developed an automated assay to enumerate and characterize circulating multiple myeloma cells (CMMC) from peripheral blood of patients with plasma cell disorders. CMMC show expression of genes characteristic of myeloma and fluorescence in situ hybridisation results on CMMC correlated well with bone marrow results. We enumerated CMMC from over 1000 patient samples including separate cohorts of newly diagnosed multiple myeloma and high/intermediate risk smouldering multiple myeloma (SMM) with clinical follow‐up data. In newly diagnosed myeloma patient samples, CMMC counts correlated with other clinical measures of disease burden, including the percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (P < 0·001). Patients with CMMC counts ≥100 at remission showed reduced survival relative to patients with CMMC counts <100. Patients with undetectable CMMC in remission showed further overall survival benefits. In the SMM cohort, there was a trend toward higher CMMC in patients with higher‐risk myeloma precursor states. Significantly higher CMMC counts were observed between intermediate/high risk SMM patients that progressed versus those without progression (P = 0·031). CMMC allow a non‐invasive means of monitoring tumour biology and may have use as a prognostic test for patients with plasma cell disorders.

Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC)

Two androgen receptor (AR)‐targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration‐resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide.

Abstract 3163: Peripheral blood circulating multiple myeloma cells (CMMCs) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma

There is an increasing interest in the ability to dynamically track disease burden and perform molecular subtyping of patients with plasma cell disorders without invasive bone marrow sampling. Circulating multiple myeloma cells (CMMC) have been detected in elevated numbers in the peripheral blood of patients with plasma cell disorders using flow cytometry or circulating cell enrichment platforms. We developed an automated CELLSEARCH® assay to enrich, enumerate, and perform a triplex FISH assay for t(4;14), t(14;16), and del 17p on CMMC (CD138+CD38+, CD45-CD19-) isolated from a 4 mL peripheral blood sample (Gross, et.al. Blood 2011; 118(21):1825). Here we present the enumeration and cytogenetic profiling of CMMC from separate cohorts of patients across the spectrum of plasma cell disorders. The first cohort consisted of newly diagnosed multiple myeloma patients enrolled in the CoMMpass study (ClinicalTrials.gov Identifier: NCT01454297). One or more CMMC per four ml blood were detected in 684/698 (98%) of newly diagnosed myeloma patients with median CMMC count of 413 per 4 mL of blood. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (p 100 at remission. CMMC FISH results (n = 57) showed overall agreement of 85%, 91% and 80% with bone marrow FISH results and 81%, 91%, and 95% agreement with bone marrow CNV/RNAseq results for the t(4;14), t(14;16), and del 17p assays, respectively. The second cohort of patients consisted of intermediate/high risk smoldering myeloma patients enrolled in a Phase 2 study of Siltuximab (ClinicalTrials.gov Identifier: NCT01484275). One or more CMMC per 4 mL blood was detected at baseline in 74/79 (94%) of intermediate/high risk smoldering myeloma patients with median CMMC count of 100 per 4 mL of blood. Significantly higher CMMC counts were observed between patients in the placebo arm that progressed versus those without progression (n = 34, p = 0.031). This is in contrast to standard metrics of percentage of bone marrow plasma cells and serum M protein levels where statistically significant differences were not seen between progressors and non-progressors in the placebo arm (p = 0.068 and p = 0.070, respectively). CMMCs were collected from a third cohort of 35 patients across the plasma cell disease spectrum with an emphasis on MGUS and SMM. CMMC counts were associated with the disease burden of patients within this cohort. CMMC may be a useful non-invasive tool for disease monitoring and characterization across the plasma cell disorder spectrum. In myeloma, CMMC may be a useful prognostic marker at remission to delineate those patients at risk for relapse. In SMM, CMMC may be useful for predictive patients at risk of progression to MM. Citation Format: Brad Foulk, Mike Schaffer, Steve Gross, Chandra Rao, Denis Smirnov, Shalini Chaturvedi, Manjula Reddy, Madeline Repollet, Claudia Rojas, Daniel Auclair, Mary DeRome, The MMRF CoMMpass Network, Brendan Weiss, A. Kate Sasser. Peripheral blood circulating multiple myeloma cells (CMMCs) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma. [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 3163.

Abstract 375: Phenotypic and molecular characterization of circulating tumor cells (CTCs) in patients with castration resistant prostate cancer (CRPC) undergoing treatment with abiraterone acetate or enzalutamide

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Despite the introduction of a number of new treatment options (such as abiraterone acetate and enzalutamide) for castration resistant prostate cancer (CRPC), patients with this disease eventually progress. In order to understand how to help such patients, it is paramount to understand the biological characteristics of the resistant disease that emerges in response to new lines of therapy. Unfortunately, repeat biopsy samples are difficult to attain and may not reflect the full complexity of the disease. Circulating tumor cells (CTCs) provide a powerful alternative to biopsies for determining the molecular characteristics of cells that emerge following development of resistance. The presence of ≥ 5 CTCs measured with the CellSearch system is associated with poor survival in patients with CRPC and increases in the number of CTCs following treatment with abiraterone acetate or enzalutamide appears to be indicative of a lack of clinical response. Such association with clinical outcome suggests that CTCs play an important role in metastatic process. Moreover, detailed molecular characterization of CTCs at various points of the disease course may be useful in understanding of evolution of tumors and aid in selection of optimal therapeutic regimens. To better characterize resistant phenotypes that emerge following treatment with abiraterone acetate or enzalutamide, we collected blood from CRPC patients that prior to starting abiraterone acetate or enzalutamide, or upon a switch between the two therapies. We also collected blood at 4, 8, 12 weeks and at progression. CTCs were enumerated using CellSearch and also stained for synaptophysin (SYP) expression. Blood was also collected for analysis of myeloid derived suppressor cells (MDSCs) that are often associated with cancer progression. Like most other CTC isolation instruments, CellSearch is a CTC-enrichment device that efficiently captures CTCs while also retaining a few thousand non-specifically captured white blood cells. To obtain molecular profiles of CTCs, we isolated single tumor cells and performed RNA sequencing of pools of a small number of cells, which enabled us to track changes of molecular characteristics of CRPC upon progression. Initial observations suggest that CRPC is an evolving heterogeneous disease that utilizes several escape mechanisms upon application of specific treatments. Citation Format: Jaymala Patel, Vipul Bhargava, Miaoling He, Brad Foulk, Denis A. Smirnov, Przemyslaw Twardowski, Marcin Kortylewski, Sumanta Kumar Pal, Jeremy O. Jones. Phenotypic and molecular characterization of circulating tumor cells (CTCs) in patients with castration resistant prostate cancer (CRPC) undergoing treatment with abiraterone acetate or enzalutamide. [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 375. doi:10.1158/1538-7445.AM2015-375

Abstract 1597: A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients

Multiple Myeloma is bone marrow tumor that frequently results in the aberrant release of neoplastic plasma cells into circulation. The CELLSEARCH® system (Janssen Diagnostics, Raritan, NJ) has been used to capture, enumerate, and perform FISH analysis on circulating multiple myeloma cells (CMMC) from patients with active multiple myeloma, smoldering myeloma, and MGUS (Gross, et.al. Poster #1825, ASH 2011). This method utilizes a blood fixative and subsequently permeabilizes the cells, which confounds the ability to do mRNA analysis on these samples. Here we present a novel method to isolate pure circulating myeloma cells that is compatible with downstream genetic analysis. Blood was obtained through a commercial vendor (Conversant Bio) from multiple myeloma patients. Samples were processed in parallel using the existing method for enumeration and a novel method for genetic profiling. For CMMC enumeration, blood was collected in CellSave tubes (Janssen Diagnostics) and CMMC were enriched using paramagnetic ferrofluid recognizing CD138 and CS1 antigens. Cells were permeabilized and stained on the CELLSEARCH® AUTOPREP with CD38 (PE), CD45 (APC), CD19 (APC) and DAPI. CMMC were enumerated using the CELLTRACKS ANALYZER II® scanning platform. CMMC were defined as DAPI +, CD138+, CD38+, CD45-, CD19-. For genetic profiling, 4 ml EDTA blood was enriched using CD138 and CS1 ferrofluids on the AUTOPREP with no subsequent permeabilization. CMMC were stained offline using CD38 (Texas Red), CD45 (FITC), CD19 (FITC), CD3 (FITC) and NucBlue. Samples were transferred to glass a bottomed petri dish and observed through an inverted microscope using a Texas Red/FITC dual filter, individual FITC and Texas red filters, and a DAPI filter. CMMC were defined as NucBlue+, CD138+, CD38+, CD45-, CD19-, and CD3-. Pools of individual CMMC were picked from the sample using an Eppendorf Transferman micromanipulator. For comparison, contaminating leukocytes were also collected for analysis. RNA from picked cells was amplified using the SMARTer Ultra Low Input Kit (Clontech) and qPCR was performed for myeloma and leukocyte specific markers. Detection of CMMC using the novel method for genetic profiling was found to be concordant with the existing method for enumeration. We found this approach to provide a deep and unbiased characterization of gene pathways activated in CMMC by RT qPCR and RNASeq. This method will have applications in longitudinal studies of active myeloma patients as a way to gather information about relapse and minimal residual disease between bone marrow draws. Citation Format: Greg Brittingham, Chandra Rao, Peter Vulfson, Vipul Bhargava, Denis Smirnov, Brad Foulk. A novel method for isolation and genetic analysis of pure populations of circulating plasma cells from multiple myeloma patients. [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 1597. doi:10.1158/1538-7445.AM2015-1597

Abstract 3063: Molecular characterization of individual circulating tumor cells by RNA sequencing

Background: Numbers of circulating tumor cells (CTC) measured with the CellSearch system in patients with metastatic carcinomas are associated with poor survival. Such association with clinical outcome suggests that CTCs play an important role in metastatic process. Molecular characterization of CTCs can provide important insights into phenotypic characteristics of cells necessary to participate in this process. Moreover, detailed molecular characterization of CTCs at various points of the disease course may be useful in understanding of evolution of tumors and aid in selection of optimal therapeutic regimens. Like most other CTC isolation instruments CellSearch is a CTC-enrichment device that efficiently captures CTCs while also retaining few thousands non-specifically captured white blood cells. To obtain deep molecular profiles of CTCs isolation of single cells is required and development of specialized next-generation sequencing protocols is necessary. Method/Results: We developed a workflow to enable isolation of single CTCs or pools of few CTCs following enrichment with the FDA approved CellSearch platform. Experiments with cancer cell lines spiked into donor blood showed 80% cellular recovery from blood using CellSearch and subsequent 40-50% capture of individual cancer cells using Fluidigm C1 Single Cell system. Following analysis of gene expression via RNAseq expression of ∼5000 genes can be detected in isolated tumor cells. We will discuss reagents and molecular approaches utilized to ensure efficient isolation and quality assessment of nucleic acids recovered from isolated tumor cells. We will discuss impact of sample preservation has on transcriptome profiling. We will share insights into prostate and lung cancers that one can gain from such analysis. Conclusion: Characterization of individual CTCs by RNA sequencing is a promising approach to characterize metastatic processes and tumor evolution. Citation Format: Jaymala Patel, Brad Foulk, Vipul Bhargava, Denis A. Smirnov. Molecular characterization of individual circulating tumor cells by RNA sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2014-3063