Cross-Species Comparison of the Pan-RAF Inhibitor LY3009120's Anti-Tumor Effects in Equine, Canine, and Human Malignant Melanoma Cell Lines.

Malignant melanomas (MMs) are the abnormal proliferation of melanocytes and are one of the lethal skin cancers in humans, equines, and canines. Accordingly, MMs in companion animals can serve as naturally occurring animal models, completing conventional cancer models. The common constitutive activation of the MAPK and PI3K pathways in MMs has been described in all three species. Targeting the related pathways is considered a potential option in comparative oncologic approaches. Herein, we present a cross-species comparative analysis exposing a set of ten melanoma cell lines (one human, three equine, and six canine) derived from primary tumors or metastasis to a pan-RAF and RAF dimer inhibitor (LY3009120). Cellular response (proliferation, biomass, metabolism, early and late apoptosis/necrosis, and morphology) and the presence of pathogenic single-nucleotide variants (SNVs) within the mutational hotspot genes BRAF exon 11 and 15, NRAS exon 2 and 3, KRAS exon 2, and KIT exon 11 were analyzed. This study showed that equine malignant melanoma (EMM) cells (MelDuWi) harbor the KRAS p.Q61H mutation, while canine malignant melanoma (CMM) cells (cRGO1 and cRGO1.2) carry NRAS p.G13R. Except for EMM metastasis cells eRGO6 (wild type of the above-mentioned hotspot genes), all melanoma cell lines exhibited a decrease in dose dependence after 48 and 72 h of exposure to LY3009120, independent of the mutation hotspot landscape. Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in all melanoma cell lines except for eRGO6. The anti-tumor effects of LY3009120 were observed in nine melanoma cell lines, indicating the potential feasibility of experimental trials with LY3009120. The present study reveals that the irradiation-resistant canine metastasis cells (cRGO1.2) harboring the NRAS p.G13R mutation are significantly LY3009120-sensitive, while the equine metastases-derived eRGO6 cells show significant resistance to LY3009120, which make them both valuable tools for studying resistance mechanisms in comparative oncology.

Exploring Thiazolopyridine AV25R: Unraveling of Biological Activities, Selective Anti-Cancer Properties and In Silico Target and Binding Prediction in Hematological Neoplasms.

Thiazolopyridines are a highly relevant class of small molecules, which have previously shown a wide range of biological activities. Besides their anti-tubercular, anti-microbial and anti-viral activities, they also show anti-cancerogenic properties, and play a role as inhibitors of cancer-related proteins. Herein, the biological effects of the thiazolopyridine AV25R, a novel small molecule with unknown biological effects, were characterized. Screening of a set of lymphoma (SUP-T1, SU-DHL-4) and B- acute leukemia cell lines (RS4;11, SEM) revealed highly selective effects of AV25R. The selective anti-proliferative and metabolism-modulating effects were observed in vitro for the B-ALL cell line RS4;11. Further, we were able to detect severe morphological changes and the induction of apoptosis. Gene expression analysis identified a large number of differentially expressed genes after AV25R exposure and significant differentially regulated cancer-related signaling pathways, such as VEGFA-VEGFR2 signaling and the EGF/EGFR pathway. Structure-based pharmacophore screening approaches using in silico modeling identified potential biological AV25R targets. Our results indicate that AV25R binds with several proteins known to regulate cell proliferation and tumor progression, such as FECH, MAP11, EGFR, TGFBR1 and MDM2. The molecular docking analyses indicates that AV25R has a higher binding affinity compared to many of the experimentally validated small molecule inhibitors of these targets. Thus, here we present in vitro and in silico analyses which characterize, for the first time, the molecular acting mechanism of AV25R, including cellular and molecular biologic effects. Additionally, this predicted the target binding of the molecule, revealing a high affinity to cancer-related proteins and, thus, classified AVR25 for targeted intervention approaches.

Circulating endothelial cells and microparticles as diagnostic and prognostic biomarkers in small-cell lung cancer.

OBJECTIVES: It has been proposed that circulating endothelial cells (CECs) and microparticles (MPs) may be useful for the assessment of patients with non-small-cell lung cancer (NSCLC). However, little is known about the potential clinical relevance of these biomarkers in small-cell lung cancer (SCLC). Therefore, we investigated the utility of baseline levels of CECs and MPs in SCLC patients. MATERIALS AND METHODS: An immunomagnetic separation (IMS) technique was used to isolate and quantify CECs in the peripheral blood, while plasma samples were analyzed using flow cytometry for the measurement of circulating MPs. RESULTS: We prospectively collected data from 56 patients and 41 healthy individuals. Forty-three patients presented at initial diagnosis and 13 patients presented at relapse. Baseline levels of CECs and MPs were significantly higher in SCLC patients either at initial diagnosis or at relapse than in healthy subjects (p < 0.0002 and p < 0.007, respectively). However, estimated tumor volume (ETV) was significantly correlated with basal MP values (p < 0.0001) but not with pretreatment CECs (p = 0.57). The amount of baseline CECs and MPs was significantly lower in patients with an objective response (OR, n = 23) than in those with progressive disease (PD, n = 15) after treatment (p = 0.016 and 0.05, respectively). With cut-off values of 110 cells/mL for CECs and 1257 events/µL for MPs according to receiver operating characteristics (ROC) analysis, baseline levels of these biomarkers were not significantly correlated with either progression-free survival (PFS) or overall survival (OS). However, patients with 6-month PFS displayed significantly decreased pretreatment CEC counts (p = 0.042), whereas basal MP values significantly increased in 1-year survivors compared with those in non-survivors (p = 0.05). CONCLUSION: Our results suggest that baseline CECs and MPs may be predictive biomarkers of tumor response and long-term survival in SCLC patients.

The impact of baseline body mass index on clinical outcomes in metastatic breast cancer: a prospective study.

OBJECTIVE: The prognostic value of body mass index (BMI) in metastatic breast cancer (MBC) has not been fully elucidated. In a prospective study to investigate the chemo-sensitizing effect of statins on clinical outcomes in MBC patients who were scheduled to receive palliative chemotherapy (Carboplatin and Vinorelbine), we sought to investigate the relationship between baseline BMI and clinical outcomes; response, overall survival (OS) and progression free survival (PFS), over a median follow-up of 40-months. RESULTS: Eighty-Two MBC patients were enrolled and categorized using baseline BMI as underweight (BMI, < 18.5 kg/m2, n = 1), normal-weight (BMI, 18.5-24.9 kg/m2, n = 20), overweight (BMI, 25-29.9 kg/m2, n = 34), and obese (BMI, ≥ 30 kg/m2, n = 27). Median OS was 10 months in normal/underweight, 19 months in overweight, and 16 months in obese (P = 0.083). Univariate Cox model revealed that overweight patients were significantly less likely to die of MBC as normal BMI patients (hazard ratio [HR] = 0.54, 95% confidence interval [CI], (0.29-0.98), P = 0.044). Similarly, multivariate Cox model, after adjusting for age, number of metastatic sites, chemotherapy line's grade, HER2 and hormone receptors status, confirmed longer survivorship of overweight in comparison with normal BMI patients (HR = 0.51, 95% CI (0.26-0.99), P = 0.047). Our data suggest that being overweight could improve OS in MBC patients.

Circulating endothelial cells for evaluation of tumor response in non-small cell lung cancer patients receiving first-line chemotherapy.

BACKGROUND: Circulating endothelial cells (CECs) reflect the neovascularization in the tumor mass. We therefore investigated the potential role of CEC kinetics after first-line chemotherapy in advanced non-small cell lung cancer (NSCLC) patients. METHODS: Peripheral blood samples were obtained from 45 healthy subjects and 51 naïve patients with advanced NSCLC. Quantification of CD146+ CECs was performed using immunomagnetic separation (IMS). RESULTS: Pretreatment and posttreatment CEC levels in NSCLC patients were significantly higher than in healthy subjects (p<0.0001). An objective response was achieved after chemotherapy with partial response (PR) or stable disease (SD) in 26 patients, whereas the remaining 25 patients had progressive disease (PD). Baseline CEC levels were significantly higher in PR/SD patients than in PD patients (p = 0.039). After chemotherapy, CEC count significantly decreased in PR/SD patients (p = 0.014) and increased in patients with PD (p = 0.019). Moreover, there was a significant difference in the percentage change of CEC counts between the 2 groups (p = 0.0016). No significant difference in the median progression-free survival and overall survival (OS) was observed between patients with high baseline CEC counts and those with low baseline CEC levels. However, patients with high percentage change in CEC count had longer OS than those with low percentage change after chemotherapy (p = 0.05). CONCLUSIONS: Changes in CEC counts after chemotherapy reflect tumor response in advanced NSCLC patients. Moreover, high percentage changes in CEC counts after chemotherapy may predict longer OS in advanced NSCLC. High baseline CEC levels might be an indicator of tumor response in advanced NSCLC patients after first-line chemotherapy.

Predictive and prognostic value of circulating endothelial cells in non-small cell lung cancer patients treated with standard chemotherapy.

PURPOSE: Monitoring circulating endothelial cells (CECs) count reflects the tumor vasculature in cancer patients and might be a predictor of response to chemotherapy. We therefore investigated the clinical significance of changes in CECs count after three cycles of platinum-based chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). METHODS: Peripheral blood samples were collected from 89 naive NSCLC patients at diagnosis and after chemotherapy. The CECs were quantified by an immuno-magnetic technique and fluorescent microscopy. After chemotherapy, patients were assessed according to the response evaluation criteria in solid tumors as partial response (PR), stable disease (SD) or progression disease (PD). RESULTS: Baseline CECs levels were significantly higher in PR patients (n = 62) than those in patients with SD/PD (n = 27) (p = 0.0007). Although there was no significant correlation between baseline CECs levels and progression-free survival (PFS) (p = 0.287), patients with high percentage change in CECs count after chemotherapy had significantly longer PFS than those with low percentage change (p = 0.048). Regarding treatment efficacy, CECs count significantly decreased after chemotherapy in comparison with CECs count at baseline in patients with PR (p < 0.0001). By contrast, CECs levels after chemotherapy were significantly higher than those at diagnosis in patients with PD (p = 0.002). Moreover, there was no significant change between pre- and post-treatment CECs amount in patients with SD (p = 0.681). CONCLUSIONS: Baseline CECs levels might be an early predictive biomarker for treatment efficacy in advanced NSCLC patients. Our results suggest the change in CECs count after chemotherapy as a prognostic factor for tumor response and PFS in NSCLC.

Circulating endothelial cells as a biomarker in non-small cell lung cancer patients: correlation with clinical outcome.

BACKGROUND: Circulating endothelial cells (CECs) have been proposed as a biomarker for the assessment of patients with solid tumors. However, few data are available in non-small cell lung cancer (NSCLC). We therefore analyzed the clinical significance of CECs in newly diagnosed NSCLC patients. In addition, we tried to determine the prognostic value of CECs in NSCLC. METHODS: In this prospective study, 151 newly diagnosed NSCLC patients and 25 healthy volunteers were included. Furthermore, 25 patients with a partial response (n=15) or stable disease (n=10) after treatment were evaluated at recurrence with a mean follow-up of 117 days (range: 47-364 days). CECs were counted using magnetic beads coupled to a specific antibody against CD146. RESULTS: The pre-treatment CEC count was significantly higher in patients with all histological subtypes of NSCLC than in healthy volunteers (p<0.0001). High baseline CEC counts were significantly correlated with advanced clinical stages (p=0.026), weight loss (p=0.03), and poorly differentiated NSCLC (p=0.02). The amount of CECs increased significantly at recurrence compared with their amount after treatment in 20/21 assessable patients (p=0.0001). Nevertheless, there was no significant correlation between baseline CEC count and median duration of progression-free survival (p=0.402). CONCLUSIONS: Increased CEC counts were present in patients with newly diagnosed NSCLC compared with healthy subjects. Elevated levels of baseline CECs correlated with high-risk factors in NSCLC. In addition, increased CEC count during follow-up seems to be correlated with recurrence in NSCLC patients.