Chandra Rao

Characterization of Circulating Endothelial Cells in Acute Myocardial Infarction

Features of endothelial cells in blood samples may eventually permit prediction of atherosclerotic plaque rupture events. Tell-Tale Cells In Edgar Allan Poe’s short story The Tell-Tale Heart, the narrator murders an old man and hides the body under the floorboards. The guilty murderer imagines that he hears the beating of the dead man’s heart emanating from the corpse underfoot—unwanted evidence of his guilt. But what if the body could leak evidence of a fragile condition before suffering a heart attack? The tale told could be frightening, yes, but the information may allow intervention at a crucial time in the pathophysiological process of heart disease. Now, Damani et al. take crucial first steps toward defining a clinical measure that could predict a thus-far unpredictable, myocardial infarction (MI)–associated event: acute atherosclerotic plaque rupture. Many people tell a personal story of a friend or relative who had a normal stress test just weeks before suffering a heart attack as a result of plaque rupture. Indeed, diagnosis of stable coronary artery disease (CAD) is now possible using stress tests and coronary artery imaging. In contrast, there are no clinically useful tests that warn of impending cardiovascular maladies caused by atherosclerotic plaque rupture. Physicians thus require a noninvasive, clinically feasible assay for a macromolecule or cell in blood that can identify people at risk for this condition, which is increasing in incidence as the population ages and widens. Endothelial cells (ECs) are normally found lining the blood vessels, and leakage into the circulation is evidence of ongoing injury to arteries that occurs on the way to potentially lethal plaque rupture. Elevated amounts of circulating endothelial cells (CECs) were previously linked to acute arterial catastrophes, but these measures have not yet made it into the clinic. Using automated, clinically feasible, three-channel fluorescence microscopy technology that can detect and permit isolation of rare cells, the authors measured and characterized CECs in healthy subjects and in patients who had experienced a type of heart attack known to manifest after acute arterial plaque rupture. CECs were elevated significantly in patients, relative to controls, and this elevation was not correlated with other measures of heart tissue death. Damani et al. also found that acute MI patients specifically displayed multicellular, multinuclear EC clusters and ECs with larger cellular and nuclear areas, relative to age-matched controls and patients with peripheral vascular disease (narrowing of arteries in the legs and feet). Although the study must be conducted in more patients and validated in an independent cohort, the new work suggests that tell-tale CECs may be useful in the clinic as evidence of ongoing plaque rupture and as a warning of possible heart attack in the near future. Acute myocardial infarction (MI), which involves the rupture of existing atheromatous plaque, remains highly unpredictable despite recent advances in the diagnosis and treatment of coronary artery disease. Accordingly, a clinical measurement that can predict an impending MI is desperately needed. Here, we characterize circulating endothelial cells (CECs) using an automated and clinically feasible CEC three-channel fluorescence microscopy assay in 50 consecutive patients with ST-segment elevation MI and 44 consecutive healthy controls. CEC counts were significantly elevated in MI cases versus controls, with median numbers of 19 and 4 cells/ml, respectively (P = 1.1 × 10−10). A receiver-operating characteristic (ROC) curve analysis demonstrated an area under the ROC curve of 0.95, suggesting near-dichotomization of MI cases versus controls. We observed no correlation between CECs and typical markers of myocardial necrosis (ρ = 0.02, creatine kinase–myocardial band; ρ = −0.03, troponin). Morphological analysis of the microscopy images of CECs revealed a 2.5-fold increase (P < 0.0001) in cellular area and a twofold increase (P < 0.0001) in nuclear area of MI CECs versus healthy controls, age-matched CECs, as well as CECs obtained from patients with preexisting peripheral vascular disease. The distribution of CEC images that contained from 2 to 10 nuclei demonstrates that MI patients were the only subject group to contain more than 3 nuclei per image, indicating that multicellular and multinuclear clusters are specific for acute MI. These data indicate that CEC counts may serve as a promising clinical measure for the prediction of atherosclerotic plaque rupture events.

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.

Circulating tumor cells as a predictive biomarker in patients with small cell lung cancer undergoing chemotherapy

BACKGROUND There are no biomarkers for assessment of disease burden or activity of therapy in SCLC. PATIENTS AND METHODS We conducted a prospective study enumerating serial CTCs in patients with newly diagnosed limited disease (LD) and extensive stage (ED) SCLC. CTCs demonstrating DNA damage and apoptosis based on γH2AX and M30 staining were also assessed. We correlated CTC number with disease stage, survival outcomes and tumor burden by RECIST. RESULTS Between 03/2011-10/2013, 50 evaluable patients were enrolled (20 LD). Baseline CTC number was higher for ED (median CTC 71 vs. 1.5 for LD; p 0.0004). Patients with <5 CTC had longer PFS but not OS (11 vs. 6.7 months, p 0.0259 and 15.5 vs. 12.9 months, p 0.4357). A higher cutoff (CTC<50 or CTC≥50) was significantly correlated with both OS (20.2 vs. 11.8 months, p 0.0116) and PFS (10 vs. 4.8 months, p 0.0002). Patients with <5 CTC on day 1 of cycle 2 had longer PFS (10 vs. 3.17 months, p<0.001) and OS (18 vs. 9 months, p 0.0001). Patients with an increase in γ2HAX-positive CTCs after chemotherapy had longer OS compared to patients without an increase (25.3 vs. 9 months, p 0.15). CONCLUSIONS This study demonstrates that CTCs at baseline and Cycle 2 of chemotherapy correlate with disease stage and survival in patients with SCLC, suggesting that CTCs may be used as a surrogate biomarker for clinical response. Confirmatory prospective clinical trials are needed before we can incorporate routine evaluation of CTCs into clinical practice.

A Whole Blood Molecular Signature for Acute Myocardial Infarction

Chest pain is a leading reason patients seek medical evaluation. While assays to detect myocyte death are used to diagnose a heart attack (acute myocardial infarction, AMI), there is no biomarker to indicate an impending cardiac event. Transcriptional patterns present in circulating endothelial cells (CEC) may provide a window into the plaque rupture process and identify a proximal biomarker for AMI. Thus, we aimed to identify a transcriptomic signature of AMI present in whole blood, but derived from CECs. Candidate genes indicative of AMI were nominated from microarray of enriched CEC samples, and then verified for detectability and predictive potential via qPCR in whole blood. This signature was validated in an independent cohort. Our findings suggest that a whole blood CEC-derived molecular signature identifies patients with AMI and sets the framework to potentially identify the earlier stages of an impending cardiac event when used in concert with clinical history and other diagnostics where conventional biomarkers indicative of myonecrosis remain undetected.

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 4972: Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin

Prostate specific membrane antigen (PSMA), a transmembrane glycoprotein, is a clinically validated marker of prostate cancer. PSMA is overexpressed in primary and metastatic prostate cancer when compared to normal prostate tissue which makes it an attractive target for immunotherapy. Several antibodies are being considered to target PSMA for the development of an anticancer drug. The expression levels of PSMA on tumor cells may play a role in response to treatment with anti PSMA drugs. Therefore, it is important to monitor PSMA expression on tumor cells before and during treatment. We used CellSearch® CTC assay to determine the presence of PSMA on circulating tumor cells (CTCs) using anti PSMA Centyrin as a biomarker. Centyrins are a class of alternative scaffold molecules which are proteins of small size (∼ 10 kDa). They are engineered to bind to target molecules with high specificity and sensitivity. The anti PSMA Centyrin was tested for specificity and sensitivity using tissue cultured tumor cell lines spiked into normal healthy blood and then compared to anti PSMA antibody. The anti PSMA Centyrin showed 100% positivity with the high PSMA expressing cell line (LNCap), 25% positivity with the low PSMA expressing cell line (22Rv1) and 0% positivity with the PSMA negative cell line (PC3-9). The anti PSMA Centyrin was further tested with prostate patient blood samples to determine the expression levels of PSMA on CTCs. The patient samples which contain CTCs are positive for anti PSMA Centyrin and the percentage of CTCs positive for PSMA ranged from 25-100%. Thus, PSMA expression on CTCs can be determined using CellSearch® system and anti PSMA Centyrin as a biomarker in a clinical trial. Citation Format: Bhavesh Vaidya, Peter Vulfson, Renouard Sanders, Donna Klein, Shalom Goldberg, Steve Jacobs, Chandra Rao. Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin. [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 4972.

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