Characterization of circulating tumor cell aggregates identified in patients with epithelial tumors.

Circulating tumor cells (CTCs) have been implicated as a population of cells that may seed metastasis and venous thromboembolism (VTE), two major causes of mortality in cancer patients. Thus far, existing CTC detection technologies have been unable to reproducibly detect CTC aggregates in order to address what contribution CTC aggregates may make to metastasis or VTE. We report here an enrichment-free immunofluorescence detection method that can reproducibly detect and enumerate homotypic CTC aggregates in patient samples. We identified CTC aggregates in 43% of 86 patient samples. The fraction of CTC aggregation was investigated in blood draws from 24 breast, 14 non-small cell lung, 18 pancreatic, 15 prostate stage IV cancer patients and 15 normal blood donors. Both single CTCs and CTC aggregates were measured to determine whether differences exist in the physical characteristics of these two populations. Cells contained in CTC aggregates had less area and length, on average, than single CTCs. Nuclear to cytoplasmic ratios between single CTCs and CTC aggregates were similar. This detection method may assist future studies in determining which population of cells is more physically likely to contribute to metastasis and VTE.

High-definition imaging of circulating tumor cells and associated cellular events in non-small cell lung cancer patients: a longitudinal analysis.

Sampling circulating tumor cells (CTCs) from peripheral blood is ideally accomplished using assays that detect high numbers of cells and preserve them for downstream characterization. We sought to evaluate a method using enrichment free fluorescent labeling of CTCs followed by automated digital microscopy in patients with non-small cell lung cancer. Twenty-eight patients with non-small cell lung cancer and hematogenously seeded metastasis were analyzed with multiple blood draws. We detected CTCs in 68% of analyzed samples and found a propensity for increased CTC detection as the disease progressed in individual patients. CTCs were present at a median concentration of 1.6 CTCs ml⁻¹ of analyzed blood in the patient population. Higher numbers of detected CTCs were associated with an unfavorable prognosis.

Fluid biopsy for circulating tumor cell identification in patients with early-and late-stage non-small cell lung cancer: a glimpse into lung cancer biology.

Circulating tumor cell (CTC) counts are an established prognostic marker in metastatic prostate, breast and colorectal cancer, and recent data suggest a similar role in late stage non-small cell lung cancer (NSCLC). However, due to sensitivity constraints in current enrichment-based CTC detection technologies, there are few published data about CTC prevalence rates and morphologic heterogeneity in early-stage NSCLC, or the correlation of CTCs with disease progression and their usability for clinical staging. We investigated CTC counts, morphology and aggregation in early stage, locally advanced and metastatic NSCLC patients by using a fluid-phase biopsy approach that identifies CTCs without relying on surface-receptor-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. HD-CTCs were analyzed in blood samples from 78 chemotherapy-naïve NSCLC patients. 73% of the total population had a positive HD-CTC count (>0 CTC in 1 mL of blood) with a median of 4.4 HD-CTCs mL⁻¹ (range 0-515.6) and a mean of 44.7 (±95.2) HD-CTCs mL⁻¹. No significant difference in the medians of HD-CTC counts was detected between stage IV (n = 31, range 0-178.2), stage III (n = 34, range 0-515.6) and stages I/II (n = 13, range 0-442.3). Furthermore, HD-CTCs exhibited a uniformity in terms of molecular and physical characteristics such as fluorescent cytokeratin intensity, nuclear size, frequency of apoptosis and aggregate formation across the spectrum of staging. Our results demonstrate that despite stringent morphologic inclusion criteria for the definition of HD-CTCs, the HD-CTC assay shows high sensitivity in the detection and characterization of both early- and late-stage lung cancer CTCs. Extensive studies are warranted to investigate the prognostic value of CTC profiling in early-stage lung cancer. This finding has implications for the design of extensive studies examining screening, therapy and surveillance in lung cancer patients.

Digital Dementia Registry Bavaria-digiDEM Bayern: study protocol for a multicentre, prospective, longitudinal register study.

INTRODUCTION: Dementia is one of the most relevant widespread diseases, with a prevalence of currently 50 million people with dementia worldwide. The care of people with dementia will be one of the major challenges for healthcare systems worldwide. Digitalisation offers new possibilities to improve both dementia healthcare and health outcomes research as a fundament for national healthcare planning. The 'Digital Dementia Registry Bavaria-digiDEM Bayern' aims to improve the understanding of the complexity and long-term progression of dementia and the current care situation in Bavaria. Moreover, by offering digital services, digiDEM will actively contribute to improving the care situation in Bavaria. METHODS AND ANALYSIS: digiDEM will recruit people with dementia and their family caregivers in all administrative regions of Bavaria. All participants will undergo dementia screening prior to study inclusion in order to identify people with mild cognitive impairment and mild-to-moderate dementia. Participants will be followed up over a period of three years. Sociodemographic data, type of dementia, symptoms, diagnosis, cognitive trajectories, activities of daily living, behavioural and psychological symptoms, falls, resource utilisation, caregiver burden, quality of life, needs of people with dementia and their caregivers, mobility, use of media and sources of information will be assessed. The project will implement a digital web-based platform for data collection. Data will be collected by means of standardised online or face-to-face interviews. ETHICS AND DISSEMINATION: The study obtained ethical approval from the Ethics Committee of the Medical Faculty of Friedrich-Alexander-University Erlangen-Nürnberg (FAU) (application number: 253_20 B). Findings will be used for evidence-based decision-making for health decision-makers in order to optimise dementia healthcare in the state of Bavaria. Specific analyses will be conducted for the participating research partners. Results of the study will be published in peer-reviewed journals.

Regulatory T cells control macrophage accumulation and activation in lymphoma.

Strategies of manipulating immunosuppressive regulatory T cells (Treg) in cancer patients are currently evaluated in clinical trials. Treg suppress immune responses of tumor-specific T cells; yet, relatively little is known about the impact of Treg on innate immune cells in tumor models in vivo. Many tumors lose expression of MHC class I. Therefore, our study aimed at defining strategies to strengthen immune responses against a high tumor burden of the MHC class I-deficient mouse lymphoma RMA-S. We demonstrate that Treg depletion in mice led to tumor rejection that was dependent on T cells, NK cells and IFN-gamma. In the absence of Treg elevated levels of IFN-gamma were produced by tumor-infiltrating T cells and NK cells. Tumor rejection observed in the absence of Treg correlated with a substantial IFN-gamma-dependent increase in the numbers of tumor-infiltrating leukocytes. The most abundant cell population in the tumors was macrophages. Tumor-infiltrating macrophages from Treg-depleted mice expressed increased amounts of MHC class II, produced highly enhanced levels of pro-inflammatory cytokines and inhibited tumor cell proliferation. It was reported that tumor-infiltrating macrophages have multi-faceted functions promoting or counteracting tumor growth. In our study, high numbers of macrophages infiltrating RMA-S tumors in the absence of Treg correlated with tumor rejection suggesting that macrophages are additional targets for Treg-mediated immune suppression in cancer.

Natural killer cell accumulation in tumors is dependent on IFN-gamma and CXCR3 ligands.

Several studies have correlated high numbers of tumor-infiltrating natural killer (NK) cells with a good prognosis for cancer patients. Our study aimed at identifying factors controlling intratumoral NK cell accumulation in s.c. injected NK cell sensitive tumor models and at studying their effect on survival time of recipient mice. We observed that fewer NK cells infiltrated the tumors in IFN-gamma receptor knockout (IFN-gammaR(-/-)) mice compared with wild-type controls that correlated with decreased survival rate. Exogenous application of IFN-gamma in the tumor augmented levels of ligands of the chemokine receptor CXCR3, increased NK cell accumulation, and prolonged survival. Furthermore, our data show that CD27(high) NK cells, which under steady-state conditions express CXCR3, preferentially accumulated in the tumor tissue. Accordingly, significantly lower numbers of tumor-infiltrating NK cells were detected in CXCR3(-/-) mice, and the capacity of adoptively transferred CXCR3(-/-) NK cells to accumulate in the tumor was severely impaired. Finally, exogenous application of the CXCR3 ligand CXCL10 in the tumor or ectopic expression of CXCL10 by tumor cells increased the numbers of NK cells in the tumors and prolonged NK cell-dependent survival. Our results identify IFN-gamma and the expression of CXCR3 on NK cells as prerequisites for NK cell infiltration into tumors. Exploiting strategies to augment NK cell accumulation in the tumor might lead to the development of effective antitumor therapies.