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 metastase

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 biomarker

Nucleolin Staining May Aid in the Identification of Circulating Prostate Cancer Cells

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. 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. 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 × 10(7) and 3.03 × 10(6), 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 × 10(5) and 2.55 × 10(4) integrated pixel units, respectively; P = 2.40 × 10(-21); approximated z statistic = 9.41). 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 metho

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-specificit

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

To investigate circulating tumor cells (CTCs) as biomarkers of urothelial carcinoma (UC). The majority of this work to date has utilized the CellSearch test, which has limited sensitivity due to reliance on positive selection for the cell surface protein EpCAM. We used a novel selection-free method to enumerate and characterize CTCs across a range of UC stages. Blood samples from 38 patients (9 controls, 8 non-muscle 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 (WBC) markers CD45 and CD66b and the epithelial markers EpCAM and pancytokeratin (CK). CTCs were defined as any CK+/WBC- 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. ≥1 CTC was detected in 2/8(25%) patients with NMIBC, 7/12(58%) with MIBC, and 6/9(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=0.009). With EpCAM positivity as a CTC requirement, no CTCs were detected in any NMIBC patient, and only 2(17%) MIBC patients had CTCs. CTCs tended to be larger in metastatic patients. 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 NMIBC and MIBC patient

Prostate-specific markers to identify rare prostate cancer cells in liquid biopsies

WOS: 000455051200006PubMed ID: 30479377Despite improvements in early detection and advances in treatment, patients with prostate cancer continue to die from their disease. Minimal residual disease after primary definitive treatment can lead to relapse and distant metastases, and increasing evidence suggests that circulating tumour cells (CTCs) and bone marrow-derived disseminated tumour cells (BM-DTCs) can offer clinically relevant biological insights into prostate cancer dissemination and metastasis. Using epithelial markers to accurately detect CTCs and BM-DTCs is associated with difficulties, and prostate-specific markers are needed for the detection of these cells using rare cell assays. Putative prostate-specific markers have been identified, and an optimized strategy for staining rare cancer cells from liquid biopsies using these markers is required. The ideal prostate-specific marker will be expressed on every CTC or BM-DTC throughout disease progression (giving high sensitivity) and will not be expressed on non-prostate-cancer cells in the sample (giving high specificity). Some markers might not be specific enough to the prostate to be used as individual markers of prostate cancer cells, whereas others could be truly prostate-specific and would make ideal markers for use in rare cell assays. The goal of future studies is to use sensitive and specific prostate markers to consistently and reliably identify rare cancer cells.NCI [F32CA206394, U54CA143803, CA163124, CA093900, CA143055]; Prostate Cancer Foundation; Patrick C. Walsh Fund; Cure for Cancer FoundationThis work is supported by NCI grants U54CA143803, CA163124, CA093900, and CA143055 as well as the Prostate Cancer Foundation, the Patrick C. Walsh Fund and a gift from the Stutt family. E.E.v.d.T. is supported by the Cure for Cancer Foundation. K.C.V. is supported by NCI grant F32CA206394

A systematic review on mutation markers for bladder cancer diagnosis in urine

OBJECTIVES: To systematically summarise the available evidence on urinary bladder cancer (BC) mutation markers. Gene mutations are expected to provide novel biomarkers for urinary BC diagnosis. To date, evidence on urinary BC mutation markers has not proven sufficient to be adopted by clinical guidelines. In the present systematic review, diagnostic accuracy of urinary mutation analysis is separately assessed for primary BC diagnosis (BC detection) and for follow-up of BC patients (BC surveillance). METHODS: A literature search (PubMed, Embase.com and Wiley/Cochrane Library) and systematic review was performed up to 31 October 2019. As studies were too heterogeneous, no quantitative analysis could be performed. RESULTS: In total, 25 studies were summarised by qualitative analysis. For BC detection, diagnostic accuracy differed considerably for single mutation markers (sensitivity 1-85%, specificity 84-100%), and for marker panels (sensitivity 50-94%, specificity 43-97%). Similarly, for BC surveillance, diagnostic accuracy was highly variable for single mutation markers (sensitivity 0-85%, specificity 66-100%), and for marker panels (sensitivity 51-84%, specificity 66-96%). CONCLUSION: Urinary mutation analysis showed to be a promising diagnostic tool for non-invasive BC diagnosis. Nonetheless, we observed substantial differences in diagnostic accuracy of urinary BC mutation markers among publications. To translate the data summarised in the present review to future clinical practice, heterogeneity in research design, BC population, mutation analysis technique and urinary DNA should be considered. Eventual clinical implementation of urinary BC mutation markers can only be achieved by collecting more and stronger evidence. Combining different molecular assays might overcome current shortcomings of urinary mutation analysis

A surface tension magnetophoretic device for rare cell isolation and characterization

The cancer community continues to search for an efficient and cost-effective technique to isolate and characterize circulating cells (CTCs) as a 'real-time liquid biopsy'. Existing methods to isolate and analyze CTCs require various transfer, wash, and staining steps that can be time consuming, expensive, and led to the loss of rare cells. To overcome the limitations of existing CTC isolation strategies, we have developed an inexpensive 'lab on a chip' device for the enrichment, staining, and analysis of rare cell populations. This device utilizes immunomagnetic positive selection of antibody-bound cells, isolation of cells through an immiscible interface, and filtration. The isolated cells can then be stained utilizing immunofluorescence or used for other downstream detection methods. We describe the construction and initial preclinical testing of the device. Initial tests suggest that the device may be well suited for the isolation of CTCs and could allow the monitoring of cancer progression and the response to therapy over tim