Emma E. van der Toom

Technical challenges in the isolation and analysis of circulating tumor cells

Increasing evidence suggests that cancer cells display dynamic molecular changes in response to systemic therapy. Circulating tumor cells (CTCs) in the peripheral blood represent a readily available source of cancer cells with which to measure this dynamic process. To date, a large number of strategies to isolate and characterize CTCs have been described. These techniques, however, each have unique limitations in their ability to sensitively and specifically detect these rare cells. In this review we focus on the technical limitations and pitfalls of the most common CTC isolation and detection strategies. Additionally, we emphasize the difficulties in correctly classifying rare cells as CTCs using common biomarkers. As for assays developed in the future, the first step must be a uniform and clear definition of the criteria for assigning an object as a CTC based on disease-specific biomarkers.

Circulating tumour cells as biomarkers of prostate, bladder, and kidney cancer

Circulating tumour cells (CTCs) have been studied as biomarkers of a number of solid malignancies. Potential clinical applications for CTC analysis include early cancer detection, disease staging, monitoring for recurrence, prognostication, and to aid in the selection of therapy. In the field of urologic oncology, CTCs have been most widely studied as prognostic biomarkers of castration-resistant prostate cancer. Additionally, emerging data support a role for CTCs to help identify which patients are most likely to respond to novel androgen-pathway targeted therapies, such as abiraterone and enzalutamide. CTCs have also been studied as predictive biomarkers of bladder cancer, in particular as a means to identify patients whose disease has been clinically understaged. Less is known regarding CTCs in kidney cancer; this has been attributed to the fact that a minority of renal tumours express EpCAM, the epithelial cell surface protein commonly used by CTC assays for positive cell selection. However, alternative approaches using markers specific for kidney cancer are being explored.

Disseminated tumor cells and dormancy in prostate cancer metastasis.

It has been reported that disseminated tumor cells (DTCs) can be found in the majority of prostate cancer (PCa) patients, even at the time of primary treatment with no clinical evidence of metastatic disease. This suggests that these cells escaped the primary tumor early in the disease and exist in a dormant state in distant organs until they develop in some patients as overt metastases. Understanding the mechanisms by which cancer cells exit the primary tumor, survive the circulation, settle in a distant organ, and exist in a quiescent state is critical to understanding tumorigenesis, developing new prognostic assays, and designing new therapeutic modalities to prevent and treat clinical metastases.

Nucleolin staining may aid in the identification of circulating prostate cancer cells

Micro‐Abstract In advanced prostate cancer, there is a need for biomarkers to monitor response to therapy and determine prognosis. Current tests for circulating tumor cells (CTCs) rely on epithelial markers with limited sensitivity and specificity. We showed that the staining pattern of nucleolin, a protein associated with proliferative cells, aids in the classification of prostate cancer CTCs. Introduction: Circulating tumor cells (CTCs) have great potential as circulating biomarkers for solid malignancies. Currently available assays for CTC detection rely on epithelial markers with somewhat limited sensitivity and specificity. We found that the staining pattern of nucleolin, a common nucleolar protein in proliferative cells, separates CTCs from white blood cells (WBCs) in men with metastatic prostate cancer. Patients and Methods: Whole peripheral blood from 3 men with metastatic prostate cancer was processed with the AccuCyte CTC system (RareCyte, Seattle, WA). Slides were immunostained with 4′,6‐diamidino‐2‐phenylindole (DAPI), anti–pan‐cytokeratin, anti‐CD45/CD66b/CD11b/CD14/CD34, and anti‐nucleolin antibodies and detected using the CyteFinder system. DAPI nucleolin colocalization and staining pattern wavelet entropy were measured with novel image analysis software. Results: A total of 33,718 DAPI‐positive cells were analyzed with the novel imaging software, of which 45 (0.13%) were known CTCs based on the established AccuCyte system criteria. Nucleolin staining pattern for segmentable CTCs demonstrated greater wavelet entropy than that of WBCs (median wavelet entropy, 6.86 × 107 and 3.03 × 106, respectively; P = 2.92 × 10−22; approximated z statistic = 9.63). Additionally, the total nucleolin staining of CTCs was greater than that of WBCs (median total pixel intensity, 1.20 × 105 and 2.55 × 104 integrated pixel units, respectively; P = 2.40 × 10−21; approximated z statistic = 9.41). Conclusion: Prostate cancer CTCs displayed unique nucleolin expression and localization compared to WBCs. This finding has the potential to serve as the basis for a sensitive and specific CTC detection method.

A simple selection-free method for detecting disseminated tumor cells (DTCs) in murine bone marrow

Bone metastasis is a lethal and incurable disease. It is the result of the dissemination of cancer cells to the bone marrow. Due to the difficulty in sampling and detection, few techniques exist to efficiently and consistently detect and quantify disseminated tumor cells (DTCs) in the bone marrow of cancer patients. Because mouse models represent a crucial tool with which to study cancer metastasis, we developed a novel method for the simple selection-free detection and quantification of bone marrow DTCs in mice. We have used this protocol to detect human and murine DTCs in xenograft, syngeneic, and genetically engineered mouse models. We are able to detect and quantify bone marrow DTCs in mice that do not have overt bone metastasis. This protocol is amenable not only for detection and quantification purposes but also to study the expression of markers of numerous biological processes or tissue-specificity.

Characterization of Urothelial Cancer Circulating Tumor Cells with a Novel Selection-Free Method

OBJECTIVE To investigate circulating tumor cells (CTCs) as biomarkers of urothelial carcinoma (UC). To date, the majority of work on this topic has utilized the CellSearch test, which has limited sensitivity due to reliance on positive selection for the cell surface protein epithelial cell adhesion molecule (EpCAM). We used a novel selection-free method to enumerate and characterize CTCs across a range of UC stages. MATERIALS AND METHODS Blood samples from 38 patients (9 controls, 8 nonmuscle invasive bladder cancer [NMIBC], 12 muscle-invasive bladder cancer [MIBC], and 9 metastatic UC) were processed with the AccuCyte-CyteFinder system. Slides were stained for the white blood cell markers CD45 and CD66b and the epithelial markers EpCAM and pancytokeratin. CTCs were defined as any cytokeratin postive and white blood cell marker negative cell. Separately, the more restrictive CellSearch definition was applied, with the additional requirement of EpCAM positivity. The Kruskal-Wallis ANOVA test compared CTC counts by stage. RESULTS Greater than or equal to 1 CTC was detected in 2 of 8 (25%) patients with NMIBC, 7 of 12 (58%) with MIBC, and 6of 9 (67%) with metastatic disease. No control had CTCs. Comparing CTC counts between groups, the only statistically significant comparison was between controls and patients with metastatic UC (P = .009). With EpCAM positivity as a CTC requirement, no CTCs were detected in any patient with NMIBC, and only 2 (17%) patients with MIBC had CTCs. CTCs tended to be larger in metastatic patients. CONCLUSION CTCs were detected at all UC stages and exhibited phenotypic diversity of cell size and EpCAM expression. EpCAM negative CTCs that would be missed with the CellSearch test were detected in patients with NMIBC and patients with MIBC.

Analogous detection of circulating tumor cells using the AccuCyte®-CyteFinder® system and ISET system in patients with locally advanced and metastatic prostate cancer

Circulating tumor cells (CTCs) can provide important information on patients prognosis and treatment efficacy. Currently, a plethora of methods is available for the detection of these rare cells. We compared the outcomes of two of those methods to enumerate and characterize CTCs in patients with locally advanced and metastatic prostate cancer (PCa). First, the selection‐free AccuCyte® − CyteFinder® system (RareCyte®, Inc., Seattle, WA) and second, the ISET system (Rarecells Diagnostics, France), a CTC detection method based on cell size‐exclusion.

MP20-20 IDENTIFICATION OF DISSEMINATED TUMOR CELLS IN THE BONE MARROW WITH DISEASE-SPECIFIC MARKERS AT RADICAL PROSTATECTOMY

with a PSADT <3 months had a median 9 months to metastases, 16 months to PCSM, and 15 months to ACM. In contrast, men with a PSADT 15 months, had a median time to metastases of 50 months, 67 months to PCSM, and 46 months to ACM. CONCLUSIONS: We found PSADT was a strong predictor of metastases, ACM, and PCSM in patients with M0 CRPC. As with patients at earlier disease stages, <3, 3-8.9, 9-14.9, and 15 are reasonable PSADT cut-points for risk stratification in men with M0 CRPC. These cut-points can be used for selecting high-risk men for clinical trials.

PD65-04 NUCLEOLIN STAINING MAY AID IN THE IDENTIFICATION OF CIRCULATING PROSTATE CANCER CELLS

used to predict risk of biochemical recurrence and guide adjuvant therapy after prostatectomy, but it is not clear to what extent these features correlate with genetic markers of disease aggressiveness. In this study, we sought to determine patterns of biomarker expression in African Americans, who are at higher risk of developing aggressive prostate cancer, and whether these biomarkers correlate with adverse pathologic features at prostatectomy. METHODS: We ran the Decipher test retrospectively on radical prostatectomy specimens obtained between December 2008 and April 2016 from a cohort of 72 African American men at a single institution. Data obtained included Decipher GRID expression levels of additional RNA biomarkers as well as the pathologic features at prostatectomy of each specimen. Fisher’s Exact Test analysis was used to determine correlations between biomarkers and the following pathologic features: perineural invasion (PNI), extraprostatic extension (EPE), seminal vesical invasion (SVI), lymphovascular invasion (LVI), and margin positivity. RESULTS: The most common biomarkers expressed were SPINK1 (37.5%, n1⁄427), ERG (18.1%, n1⁄413), NKX3 (13.95, n1⁄410), and PCA3 (11.1%, n1⁄48). The triple negative genotype (ERG-/ETS-/SPINK-) was 38.9% (n1⁄428). There were no statistically significant relationships between any biomarker expression levels and pathologic features associated with aggressive prostate cancer. PCA3 did show a nonsignificant association with LVI (p 1⁄4 0.09). CONCLUSIONS: Our study employed one of the largest African American cohorts to date and failed to show a relationship between adverse pathologic features and the RNA expression patterns of markers implicated in prostate cancer. Further studies are necessary to determine the impact of these abnormal expression patterns on clinical outcomes and whether they are better predictors of disease recurrence than traditional clinicopathologic variables.

Bladder Cancer: Epidemiology & Evaluation IIIPD57-02 CHARACTERIZATION OF UROTHELIAL CANCER CIRCULATING TUMOR CELLS WITH A NOVEL SELECTION-FREE METHOD

regression was used to build a predictive risk model and create a hematuria risk index. RESULTS: Within a cohort of 6417 patients with microscopic hematuria, a total of 177 (2.8%) were diagnosed with a neoplasm. On multivariate analysis, age between 50-59 (OR1⁄41.96, 2 points), age over 60 (OR1⁄45.21, 4 points), history of gross hematuria (OR1⁄43.15, 3 points), current or past smoking history (OR1⁄41.51, 1 point), male gender (OR1⁄42.57, 2 points), >25 red blood cell per high power field (OR1⁄42.94, 2 points), Non-Hispanic Black (OR1⁄41.73, 1 point), and Non-Hispanic White (OR1⁄42.31, 2 points) were all significant predictors of malignancy. A modified Hematuria Risk Index (0 to 14 points) was developed from these factors, which demonstrated an improved area under the receiver operating characteristic curve of 0.841 compared to our previous model at 0.807. We observed natural breaks in the scores that grouped the patients into low (0-4 points, 41.7%), moderate (5-9 points, 49.0%), and high-risk of cancer (10-14 points, 9.2%). Malignancy was found in 0.4%, 2.5% and 15.0% of patients from the low, moderate and high-risk groups, respectively. CONCLUSIONS: Advance age, history of gross hematuria, current or past smoking history, male gender, >25 red blood cell per high power field, and certain ethnic groups are significant predictors for malignancy in the setting of microscopic hematuria. Classification of patients into low, moderate and high-risk groups will improve patient counseling and will hopefully reduce the need for invasive endoscopy and ionizing radiation exposure for patients within the low-risk category.