Denis Smirnov

Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

BACKGROUND Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

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 2755: Comparison of blood based liquid biopsy methodologies for improved risk assessment of prostate cancer (PCa)

Approximately 28,000 men die from prostate cancer in the US each year. Predictive biomarkers can provide patient risk assessment to enable therapeutic decision making. Compared to tumor, blood based liquid biopsies offer greater flexibility for noninvasive sample collection and allows for continuous monitoring of treatment response. While various liquid biopsy sample types and methodologies are currently available, it is critical to identify which method offers the highest sensitivity and specificity for clinical decision making. In this study we systematically compared specificity and sensitivity of prostate cancer detection using a pre-selected panel of PCa specific mRNA transcripts using four different methodologies. In this prospective laboratory study, we collected five blood tubes from a cohort of 40 metastatic castrate resistant prostate cancer (mCRPC) patients and 20 age matched healthy volunteers (HV). Circulating tumor cells (CTCs) were enumerated and blood samples enriched for CTCs were isolated for mRNA evaluation. Two tubes of plasma samples were utilized to isolate exosomes using ExoRNAeasy columns and CellSearch EpCAM capture respectively, while RNA was extracted directly from whole blood collected in PAXgene tubes. RNA isolated from blood samples enriched for CTCs, two exosome preps and whole blood samples were tested using qPCR (Fluidigm) for a panel of 48 prostate specific mRNA transcripts and three endogenous controls previously shown to be detected in whole blood. Receiver operator analysis (ROC) was performed to compare expression of mRNA transcripts across all four methods. Results showed PCa specific genes were detected in whole blood, Exo-52 exosome prep and samples enriched for CTCs, while exosomes isolated using CellSearch EpCAM capture failed to detect any PCa transcripts. ROC showed ExoRNAeasy exosome prep detected PCa specific mRNA transcripts with significantly higher sensitivity and specificity compared to three other methodologies. Area under curve (AUC) for ExoRNA prep 0.65 to 0.75 (average of z score at 3.27 p= 0.0023), compared to AUC for PAXgene is 0.55 to 0.83, (average of z score at 2.64 and p=0.05) and CTC samples is 0.52 to 0.67, (average of z score at 1.63 and p= 0.19). For CellSearch exosome method, the AUC is 0.47 to 0.52, average of z score at 0.66 and p= 0.32). These early results support the utility of exosome derived mRNA assay as a viable alternative to whole blood and CTC based assays. Further testing for expression of markers associated with prognosis and treatment response is necessary to warrant its prognostic or predictive clinical utility. Citation Format: Yashoda Rajpurohit, Shibu Thomas, Jaymala Patel, Dong Shen, Michael Gormley, Denis Smirnov, Deborah Ricci. Comparison of blood based liquid biopsy methodologies for improved risk assessment of prostate cancer (PCa) [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 2755. doi:10.1158/1538-7445.AM2017-2755

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, PAnnDespite 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.nnCitation 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