Mark Landers

High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.

Analytical Validation and Capabilities of the Epic CTC Platform: Enrichment-Free Circulating Tumour Cell Detection and Characterization

The Epic Platform was developed for the unbiased detection and molecular characterization of circulating tumour cells (CTCs). Here, we report assay performance data, including accuracy, linearity, specificity and intra/inter-assay precision of CTC enumeration in healthy donor (HD) blood samples spiked with varying concentrations of cancer cell line controls (CLCs). Additionally, we demonstrate clinical feasibility for CTC detection in a small cohort of metastatic castrate-resistant prostate cancer (mCRPC) patients. The Epic Platform demonstrated accuracy, linearity and sensitivity for the enumeration of all CLC concentrations tested. Furthermore, we established the precision between multiple operators and slide staining batches and assay specificity showing zero CTCs detected in 18 healthy donor samples. In a clinical feasibility study, at least one traditional CTC/mL (CK+, CD45-, and intact nuclei) was detected in 89 % of 44 mCRPC samples, whereas 100 % of samples had CTCs enumerated if additional CTC subpopulations (CK-/CD45- and CK+ apoptotic CTCs) were included in the analysis. In addition to presenting Epic Platforms performance with respect to CTC enumeration, we provide examples of its integrated downstream capabilities, including protein biomarker expression and downstream genomic analyses at single cell resolution.

Phenotypic diversity of circulating tumour cells in patients with metastatic castration-resistant prostate cancer

To use a non‐biased assay for circulating tumour cells (CTCs) in patients with prostate cancer (PCa) in order to identify non‐traditional CTC phenotypes potentially excluded by conventional detection methods that are reliant on antigen‐ and/or size‐based enrichment.

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

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

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

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

Correlation of FCGRT genomic structure with serum immunoglobulin, albumin and farletuzumab pharmacokinetics in patients with first relapsed ovarian cancer

Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that binds to folate receptor alpha. A Ph3 trial in ovarian cancer patients treated with carboplatin/taxane plus FAR or placebo did not meet the primary statistical endpoint. Subgroup analysis demonstrated that subjects with high FAR exposure levels (Cmin>57.6μg/mL) showed statistically significant improvements in PFS and OS. The neonatal Fc receptor (fcgrt) plays a central role in albumin/IgG stasis and mAb pharmacokinetics (PK). Here we evaluated fcgrt sequence and association of its promoter variable number tandem repeats (VNTR) and coding single nucleotide variants (SNV) with albumin/IgG levels and FAR PK in the Ph3 patients. A statistical correlation existed between high FAR Cmin and AUC in patients with the highest quartile of albumin and lowest quartile of IgG1. Analysis of fcgrt identified 5 different VNTRs in the promoter region and 9 SNVs within the coding region, 4 which are novel.

Detection and Characterization of Circulating Tumour Cells from Frozen Peripheral Blood Mononuclear Cells

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via Epic CTC Platform or from “frozen” PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples.

Abstract 1588: Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) patients

Background: Androgen signaling directed therapies, including Abiraterone (A) and Enzalutamide (E), prolong survival in patient (pts) with mCRPC and are FDA approved. Resistance to A and E is associated with the presence of the ARv7 splice variant which, along with other AR ligand binding domain (LBD) alterations, may constitutively activate AR. Previous work showed marked heterogeneity in cell types and protein expression in CTCs of mCRPC pts mandating single cell analysis to assess the AR LBD to understand passenger vs. driving clonal subtypes. We have developed and begun clinical validation for AR N terminal (AR N), AR C terminal (AR C), ARv7 protein and ARv7 mRNA enabling single CTC analysis. Methods: Cell lines (VCaP, LnCaP, LnCaP-95, 22RV1, & PC3) expressing varying levels of AR LBD alterations were spiked into healthy donor blood. 47 pt samples were collected for CTC analysis utilizing the Epic Sciences platform which 24 pts (6 treatment naive, 10 post A or E, and 8 post taxane) were selected for AR N/C/v7 analysis due to CTC prevalence across clinical decision point where change in therapy was needed. Epic analysis included ID of traditional CTCs, CK- CTCs, small CTCs, and CTC clusters. Cell lines & pts were analyzed for AR N, AR C and ARv7 IF, and a subset of patients CTC for ARv7 mRNA by RNA FISH. Results: Immunofluorescent protein expression of AR N, AR C and ARv7 in cell lines were consistent with published profiles. AR C & ARv7 were detected only in AR N+ CTCs indicating assay specificity for both targets. ARv7 expression was observed in traditional, CK-, small CTCs and CTC clusters, but no association was seen between ARv7+ and any CTC subtype. AR v7 RNA FISH was specific to AR v7 IF+ pts. Conclusions: AR C loss is more sensitive in detecting AR LBD alterations than ARv7. Variable ARv7/AR N ratios in different cell types suggest intrapatient AR heterogeneity. The low prevalence of ARv7+ CTCs of total and AR N+ CTCs suggests many pts may have other driving resistance mechanisms. Citation Format: James Kelvin, David Lu, Davin Packer, Richard Bambury, Dana Rathkopf, Nicole Schreiber, Zaina Arslan, Natalie Prigozhina, David Brown, Rachel Krupa, Edward Swangren, Mark Landers, Florence Lee, Dena Marrinucci, Ryan Dittamore, Howard I. Scher. Single cell analysis of AR N terminal, AR C terminal and the ARv7 splice variant in the CTCs of metastatic castration resistant prostate cancer (mCRPC) 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 1588. doi:10.1158/1538-7445.AM2015-1588

Androgen receptor expression on circulating tumor cells in metastatic breast cancer

Purpose Androgen receptor (AR) is frequently detected in breast cancers, and AR-targeted therapies are showing activity in AR-positive (AR+) breast cancer. However, the role of AR in breast cancers is still not fully elucidated and the biology of AR in breast cancer remains incompletely understood. Circulating tumor cells (CTCs) can serve as prognostic and diagnostic tools, prompting us to measure AR protein expression and conduct genomic analyses on CTCs in patients with metastatic breast cancer. Methods Blood samples from patients with metastatic breast cancer were deposited on glass slides, subjected to nuclear staining with DAPI, and reacted with fluorescent-labeled antibodies to detect CD45, cytokeratin (CK), and biomarkers of interest (AR, estrogen receptor [ER], and HER2) on all nucleated cells. The stained slides were scanned and enumerated by non-enrichment-based non-biased approach independent of cell surface epithelial cell adhesion molecule (EpCAM) using the Epic Sciences CTC platform. Data were analyzed using established digital pathology algorithms. Results Of 68 patients, 51 (75%) had at least 1 CTC, and 49 of these 51 (96%) had hormone-receptor-positive (HR+)/HER2-negative primary tumors. AR was expressed in CK+ CTCs in 10 patients. Of these 10 patients, 3 also had ER expression in CK+ CTCs. Single cell genomic analysis of 78 CTCs from 1 of these 3 patients identified three distinct copy number patterns. AR+ cells had a lower frequency of chromosomal changes than ER+ and HER2+ cells. Conclusions CTC enumeration and analysis using no enrichment or selection provides a non-biased approach to detect AR expression and chromosomal aberrations in CTCs in patients with metastatic breast cancer. The heterogeneity of intrapatient AR expression in CTCs leads to the new hypothesis that patients with AR+ CTCs have heterogeneous disease with multiple drivers. Further studies are warranted to investigate the clinical applicability of AR+ CTCs and their heterogeneity.

Abstract A061: Tumor mutation burden (TMB), microsatellite instability (MSI), and chromosomal instability (CIN) analysis using low pass whole genome sequencing of single circulating tumor cell (CTC)

Background: Genomic instability (GI) is a hallmark of cancer often associated with poor patient outcome. TMB, MSI, and CIN represent the majority of GI in metastatic patients. Recent studies show that TMB and MSI are emerging immune checkpoint inhibitor drug sensitivity biomarkers and CIN is a sensitivity marker for PARP inhibitors. Assessment of genomic instability in bulk tumor samples is well explored, but it is limited by sample availability and tumor heterogeneity. Analysis of ctDNA is feasible for TMB and MSI analysis but not CIN, and it also suffers in sensitivity and specificity in patients who harbor subclonal GI, limiting the clinical utility of these assays to detect early clonal alterations. The Epic Sciences CTC platform is a non-enrichment-based approach for the detection and characterization of rare tumor cells in clinical blood samples, and could provide insight into subclonal heterogeneity. Here we present downstream single cell GI assay(s) for the detection of TMB, MSI and CIN from individual CTCs using next-generation sequencing and PCR. Methods: Contrived samples were prepared by spiking three prostate cancer cell lines, LNCaP, PC3, and VCaP, into healthy donor blood. Red blood cells were lysed, all nucleated cells deposited onto slides, slides immunofluorescence stained (DAPI, CK, CD45, and Androgen Receptor), and identified cancer cells individually picked up from the slides. Each recovered cell was lysed, whole genome amplified (WGA), shotgun library prepared, and low pass whole genome sequenced using Illumina NextSeq 500. Data were analyzed for TMB scores (# of INDELs per Mbp) and large-scale transitions (LSTs, a surrogate of CIN). MSI was measured using Qiagen Type-It microsatellite PCR kit for four sites (BAT26, BAT25, D2S123, and D5S346). Samples from metastatic castration resistant prostate cancer (mCRPC) patients were included to evaluate clinical feasibility. Results: TMB scores for LNCaP (average 652, coefficient of variation 15%) were significantly higher than PC3 (558, 0.9%), VCaP (548, 1.1%), and WBC from healthy donor (540, 7.6%) with p Citation Format: Angel Rodriguez, Jerry Lee, Ramsay Sutton, Rhett Jiles, Yipeng Wang, Mark Landers, Ryan Dittamore. Tumor mutation burden (TMB), microsatellite instability (MSI), and chromosomal instability (CIN) analysis using low pass whole genome sequencing of single circulating tumor cell (CTC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A061.