Serotoninergic receptor ligands improve Tamoxifen effectiveness on breast cancer cells

Background: Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. Methods: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. Results: We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 μM - 99.2 μM) compared to SKBR3 (IC50 range 43.3 μM - 260 μM) and MDA-MB231 BC cells (IC50 range 91.3 μM - 306 μM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. Conclusions: These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients

The Framingham cardiovascular risk score in multiple sclerosis

Background and purpose: Cardiovascular risk factors can increase the risk of multiple sclerosis (MS) and modify its course. However, such factors possibly interact, determining a global cardiovascular risk. Our aim was to compare the global cardiovascular risk of subjects with and without MS with the simplified 10-year Framingham General Cardiovascular Disease Risk Score (FR) and to evaluate its importance on MS-related outcomes. Methods: Age, gender, smoking status, body mass index, systolic blood pressure, type II diabetes and use of antihypertensive medications were recorded in subjects with and without MS to estimate the FR, an individualized percentage risk score estimating the 10-year likelihood of cardiovascular events. Results: In total, 265 MS subjects were identified with 530 matched controls. A t test showed similar FR in cases and controls (P = 0.212). Secondary progressive MS presented significantly higher FR compared to relapsing-remitting MS (P < 0.001). Linear regression analysis showed a direct relationship between FR and Expanded Disability Status Scale (P < 0.001) and MS Severity Scale (P < 0.001). Conclusion: The FR, evaluating the global cardiovascular health by the interaction amongst different risk factors, relates to MS disability, severity and course

A Multicenter, Phase 2 Study of Vascular Endothelial Growth Factor Trap (Aflibercept) in Platinum- and Erlotinib-Resistant Adenocarcinoma of the Lung

IntroductionAflibercept (vascular endothelial growth factor [VEGF] trap), a recombinant fusion protein, blocks the activity of VEGF-A and placental growth factor and has demonstrated activity in pretreated patients with lung cancer in a phase I trial. This study evaluated the efficacy and safety of intravenous aflibercept in patients with platinum- and erlotinib-resistant lung adenocarcinoma.MethodsAn open-label, single arm, multicenter trial was conducted, with the primary end point of response rate (modified RECIST). Additional endpoints included safety, duration of response, progression-free survival, and overall survival. Patients with platinum- and erlotinib-resistant lung adenocarcinoma were eligible. Aflibercept 4.0 mg/kg intravenous every 2 weeks was administered until progression of disease or intolerable toxicity.ResultsNinety-eight patients were enrolled; 89 were evaluable for response. Median age was 60 years, 41% were men with Eastern Cooperative Oncology Group performance status 0/1/2 in 35/55/9% of patients. The overall response rate was 2.0%, (95% confidence interval, 0.2-7.2%). Median progression-free survival was 2.7 months, and overall was survival 6.2 months. Six- and 12-month survival rates were 54 and 29%, respectively. A median of four cycles was administered (range 1-22). Common grade 3/4 toxicities included dyspnea (21%), hypertension (23%), and proteinuria (10%). Two cases of grade 5 hemoptysis were reported, and one case each of tracheoesophageal fistula, decreased cardiac ejection fraction, cerebral ischemia, and reversible posterior leukoencephalopathy.ConclusionsAflibercept has minor single agent activity in heavily pretreated lung adenocarcinoma, and is well tolerated, with no unexpected toxicities. Further studies evaluating aflibercept in lung cancer, in combination with chemotherapy and other targeted therapies, are ongoing

A Perspective on Challenges and Issues in Biomarker Development and Drug and Biomarker Codevelopment

A workshop sponsored by the National Cancer Institute and the US Food and Drug Administration addressed past lessons learned and ongoing challenges faced in biomarker development and drug and biomarker codevelopment. Participants agreed that critical decision points in the product life cycle depend on the level of understanding of the biology of the target and its interaction with the drug, the preanalytical and analytical factors affecting biomarker assay performance, and the clinical disease process. The more known about the biology and the greater the strength of association between an analytical signal and clinical result, the more efficient and less risky the development process will be. Rapid entry into clinical practice will only be achieved by using a rigorous scientific approach, including careful specimen collection and standardized and quality-controlled data collection. Early interaction with appropriate regulatory bodies will ensure studies are appropriately designed and biomarker test performance is well characterized

COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (<it>EGFR</it>) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect <it>KRAS </it>mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for <it>KRAS </it>mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.</p> <p>Methods</p> <p>We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in <it>KRAS </it>codons 12 and 13 using the Roche LightCycler^®instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (<it>T_c</it>) of 81°C increased the sensitivity of the assay >10-fold for the majority of <it>KRAS </it>mutations.</p> <p>Results</p> <p>We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant <it>KRAS </it>mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.</p> <p>Conclusions</p> <p>We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for <it>KRAS </it>mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.</p

ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein

P-glycoprotein (P-gp) pumps multiple types of drugs out of the cell, using energy generated from ATP, and confers multidrug resistance (MDR) on cancer cells. ZD6474 is an orally active, selective inhibitor of the vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection tyrosine kinases. This study was designed to examine whether ZD6474 reverses P-gp-mediated MDR in cancer cells. Here, we show that clinically achievable levels of ZD6474 reverse P-gp-mediated MDR of the P-gp-overexpressing cell lines derived from breast cancer, MCF-7/adriamycin (ADR), and human oral epidermoid carcinoma, KBV200 to ADR, docetaxel, and vinorelbine. This ability to reverse the P-gp-mediated resistance is comparable to that of another frequently used reversal agent known as verapamil. ZD6474 itself moderately inhibits the proliferation of both MCF-7 and MCF-7/ADR cells with almost equal activity, but its inhibitory effect is not altered by co-incubation with verapamil, suggesting that ZD6474 may not be a substrate of P-gp. In addition, ZD6474 increases the intracellular accumulation of the P-gp substrate, rhodamine-123, and ADR, by enhancing the uptake and/or decreasing the efflux of these compounds in resistant cells. Further studies show that ZD6474 stimulates ATPase activity in a dose-dependent manner, which is required for the proper function of P-gp. In contrast, ZD6474 does not inhibit the expression level of P-gp. Our results suggest that ZD6474 is capable of reversing MDR in cancer cells by directly inhibiting the function of P-gp, a finding that may have clinical implications for ZD6474

Evaluation of Somatic Mutations in Solid Metastatic Pan-Cancer Patients

Metastasis continues to be the primary cause of all cancer-related deaths despite the recent advancements in cancer treatments. To evaluate the role of mutations in overall survival (OS) and treatment outcomes, we analyzed 957 metastatic patients with seven major cancer types who had available molecular testing results with a FoundationOne CDx® panel. The most prevalent genes with somatic mutations were TP53, KRAS, APC, and LRP1B. In this analysis, these genes had mutation frequencies higher than in publicly available datasets. We identified that the somatic mutations were seven mutually exclusive gene pairs and an additional fifty-two co-occurring gene pairs. Mutations in the mutually exclusive gene pair APC and CDKN2A showed an opposite effect on the overall survival. However, patients with CDKN2A mutations showed significantly shorter OS (HR: 1.72, 95% CI: 1.34–2.21, p \u3c 0.001) after adjusting for cancer type, age at diagnosis, and sex. Five-year post metastatic diagnosis survival analysis showed a significant improvement in OS (median survival 28 and 43 months in pre-2015 and post-2015 metastatic diagnosis, respectively, p = 0.00021) based on the year of metastatic diagnosis. Although the use of targeted therapies after metastatic diagnosis prolonged OS, the benefit was not statistically significant. However, longer five-year progression-free survival (PFS) was significantly associated with targeted therapy use (median 10.9 months (CI: 9.7–11.9 months) compared to 9.1 months (CI: 8.1–10.1 months) for non-targeted therapy, respectively, p = 0.0029). Our results provide a clinically relevant overview of the complex molecular landscape and survival mechanisms in metastatic solid cancers

Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization