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Background Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. Methods We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. Results CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. Conclusions Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Background Detection of asbestos-associated diseases like asbestosis or mesothelioma is still challenging. We sought to improve the diagnosis of benign asbestos-associated disease (BAAD) by detection of the protein cysteine-rich angiogenic inducer 61 (Cyr61) in human plasma. Methods Plasma Cyr61 was quantified using an enzyme-linked immunosorbent assay. Plasma samples from males diagnosed with BAAD, but without a malignant disease (n = 101), and malignant mesothelioma (n = 21; 15 males, 6 females), as well as nonasbestos-exposed healthy control participants (n = 150; 58 males, 92 females) were analyzed. Clinical sensitivity and specificity of Cyr61 were determined by receiver operating characteristic analysis. Results The median plasma Cyr61 concentration for healthy control participants was 0.27 ng/mL. Cytoplasmic Cyr61 in peripheral blood mononuclear cells from healthy control participants was evenly distributed, as detected by immunofluorescent staining. The increase in plasma Cyr61 concentrations in the BAAD study group was statistically significant compared to the healthy control participants (P Conclusions In our study, plasma Cyr61 protein concentrations showed to be a new biomarker for asbestos-associated diseases like BAAD and mesothelioma in men, which deserves further investigation in large-scale cohort studies.

Treatment with axicabtagene ciloleucel (Axi-cel) CD19-CAR-T (chimeric antigen receptor T) cells has been approved for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). Because treatment success as well as side effects might depend on CAR-T cell expansion in vivo, we aimed at developing digital PCR (dPCR) assays for detection and quantification of CAR-T cells. To this end, we cloned and sequenced the complete cDNA of the CAR construct. We designed different combinations of primers and dual-labeled hydrolysis probes located in various CAR regions. Three combinations were successfully tested on CAR-positive and -negative cells in duplex reactions with a reference gene (REF) to concomitantly assess cell numbers. All assays demonstrated excellent specificity and reproducibility with neglectable inter-assay variations. For all three assays, almost perfect correlation between the two dPCRs (Axi-cel versus REF) was observed, and the limit of detection was one single CAR-transduced cell corresponding to a sensitivity of 0.01% for 100 ng genomic DNA. After cross-validation, we used one assay to monitor Axi-cel CAR-T numbers in patients. CAR-T expansion and contraction followed the expected kinetics with median peak value of 11.2 Axi-cel CAR-T cells/μL at 11.3 days (median). Clinically, we observed only two partial responses (PRs) in the five patients with CAR-T cell peak numbers below median, whereas four of the five patients with comparatively good expansion showed clinical responses (two complete responses [CRs] and two PRs) on day 30. In conclusion, we established a novel dPCR assay for the sensitive detection of transgenic CAR-T cells, which should be very useful in the context of Axi-cel treatment., Graphical Abstract

Data on the association between chimeric antigen receptor (CAR)-T-cell kinetics and patient outcome in the nontrial setting are missing, mainly due to the lack of broadly available CAR-T-cell diagnostic quantification tools. We performed prospective quantification of axicabtagene ciloleucel (axi-cel) in 21 patients treated for aggressive B-cell lymphoma at our clinic. Median peak CAR-T-cell count was 16.14 CAR-T cells/µL. Patients with 16.14/μL or higher peak CAR-T cells (strong expanders) had more day-30 objective responses (91% vs 40%, P = .02). In univariate analysis, peak CAR-T cell ≥ 16.14 (P < .001), normal platelet counts at start of lymphodepletion (P < .001), no prior stem cell transplant (P = .04), and peak CAR-T cells as continuous variable (P = .03) were associated with better progression-free survival (PFS). After adjusting for platelet counts and prior stem cell transplantation, peak CAR-T cells below median was still associated with shorter PFS (relative risk, 0.15, 95% confidence interval, 0.04-0.59, P = .007). Low platelet counts also maintained significant impact on PFS. Our data demonstrate association of axi-cel levels and outcome in a nontrial setting and for the first time use a cutoff to segregate weak and strong expanders with respective outcomes.

Simple Summary Metastasis is the leading cause of death in breast cancer, and it can be predicted by the detection of circulating tumor cells in the blood and disseminated tumor cells in the bone marrow, which are usually detected by epithelial marker proteins. However, tumor cells with mesenchymal attributes down-regulate the expression of epithelial marker proteins, and are therefore difficult to detect. Here, we found that the protein-cysteine–rich angiogenetic inducer 61 (Cyr61) is strongly expressed in tumor cells with mesenchymal attributes. Cyr61 expression was undetectable in normal blood cells, suggesting that Cyr61 might represent a tumor-associated protein. Functional experiments showed that the loss of Cyr61 reduces the viability of breast tumor cells. Thus, Cyr61 might represent an interesting anti-metastatic target that needs to be explored in future studies. Abstract (1) Background: the early detection of cancer cells in the blood or bone marrow of breast cancer patients improves the understanding of metastasis. Disseminating tumor cells in the bone marrow with a pronounced manifestation of mesenchymal markers (mDTC) are difficult to detect by epithelial markers, but they are relevant in the initiation of metastasis. (2) Methods: the breast cancer mDTC cell line BC-M1 was analyzed by mass spectrometry, which revealed high levels of the protein-cysteine–rich angiogenic inducer 61 (Cyr61). The function of Cyr61 was investigated using shRNA and hypoxia. Peripheral blood samples from 35 breast cancer patients were investigated for CTCs defined as cytokeratin-positive/CD45-negative cells. (3) Results: the Cyr61 levels are elevated in mDTC lines from breast, lung, and prostate cancer patients. The loss of Cyr61 resulted in the diminished expression of hypoxia-inducible factor 1-alpha, and increased apoptosis. Cyr61 was present in 47 (43%) of the 109 detected circulating tumor cells (CTCs), while the blood and bone marrow cells from healthy controls were Cyr61-negative. (4) Conclusions: Cyr61 is expressed in mDTC lines, supports the viability of cancer cells, and classifies a new subset of cytokeratin-positive CTCs, which deserves further investigation.

Background: Owing to late diagnosis in advanced disease stages, prognosis of patients with epithelial ovarian cancer (EOC) is poor. The quantification of deregulated levels of microRNAs could facilitate earlier diagnosis and improve prognosis of EOC. Methods: Seven microRNAs (miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429) were quantified in the serum of 180 EOC patients and 66 healthy women by TaqMan PCR microRNA assays. Median follow-up time was 21 months. The effects of miR-7 and miR-429 on apoptosis, cell proliferation, migration and invasion were investigated in two (EOC) cell lines. Results: Serum levels of miR-25 (P=0.0001) and miR-93 (P=0.0001) were downregulated, whereas those of miR-7 (P=0.001) and miR-429 (P=0.0001) were upregulated in EOC patients compared with healthy women. The four microRNAs discriminated EOC patients from healthy women with a sensitivity of 93% and a specificity of 92%. The levels of miR-429 positively correlated with CA125 values (P=0.0001) and differed between FIGO I–II and III–IV stages (P=0.001). MiR-429 was an independent predictor of overall survival (P=0.011). Overexpressed miR-429 in SKOV3 cells led to suppression of cell migration (P=0.037) and invasion (P=0.011). Increased levels of miR-7 were associated with lymph node metastases (P=0.0001) and FIGO stages III–IV (P=0.0001). Overexpressed miR-7 in SKOV3 cells resulted in increased cell migration (P=0.001) and invasion (P=0.011). Additionally, the increased levels of miR-376a correlated with FIGO stages III–IV (P=0.02). Conclusions: Our data indicate the diagnostic potential of miR-7, miR-25, miR-93 and miR-429 in EOC and the prognostic potential of miR-429. This microRNA panel may be promising molecules to be targeted in the treatment of EOC.

Background and Objectives Atherosclerotic changes of arteries are the leading cause for deaths in cardiovascular disease and greatly impair patient’s quality of life. Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that regulates potentially pro-as well as anti-atherogenic processes. Here, we investigate whether serum-S1P concentrations are associated with peripheral artery disease (PAD) and carotid stenosis (CS). Methods and Results Serum was sampled from blood donors (controls, N = 174) and from atherosclerotic patients (N = 132) who presented to the hospital with either clinically relevant PAD (N = 102) or CS (N = 30). From all subjects, serum-S1P was measured by mass spectrometry and blood parameters were determined by routine laboratory assays. When compared to controls, atherosclerotic patients before invasive treatment to restore blood flow showed significantly lower serum-S1P levels. This difference cannot be explained by risk factors for atherosclerosis (old age, male gender, hypertension, hypercholesteremia, obesity, diabetes or smoking) or comorbidities (Chronic obstructive pulmonary disease, kidney insufficiency or arrhythmia). Receiver operating characteristic curves suggest that S1P has more power to indicate atherosclerosis (PAD and CS) than high density lipoprotein-cholesterol (HDL-C). In 35 patients, serum-S1P was measured again between one and six months after treatment. In this group, serum-S1P concentrations rose after treatment independent of whether patients had PAD or CS, or whether they underwent open or endovascular surgery. Post-treatment S1P levels were highly associated to platelet numbers measured pre-treatment. Conclusions Our study shows that PAD and CS in humans is associated with decreased serum-S1P concentrations and that S1P may possess higher accuracy to indicate these diseases than HDL-C.

Introduction Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates pathophysiological processes involved in sepsis progression, including endothelial permeability, cytokine release, and vascular tone. The aim of this study was to investigate whether serum-S1P concentrations are associated with disease severity in patients with sepsis. Methods This single-center prospective-observational study includes 100 patients with systemic inflammatory response syndrome (SIRS) plus infection (n = 40), severe sepsis (n = 30), or septic shock (n = 30) and 214 healthy blood donors as controls. Serum-S1P was measured by mass spectrometry. Blood parameters, including C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), lactate, and white blood cells (WBCs), were determined by routine assays. The Sequential Organ Failure Assessment (SOFA) score was generated and used to evaluate disease severity. Results Serum-S1P concentrations were lower in patients than in controls (P P P P Conclusions In patients with sepsis, serum-S1P levels are dramatically decreased and are inversely associated with disease severity. Since S1P is a potent regulator of endothelial integrity, low S1P levels may contribute to capillary leakage, impaired tissue perfusion, and organ failure in sepsis.