Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine.

Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor.

Mutation-Enrichment Next-Generation Sequencing for Quantitative Detection of Mutations in Urine Cell-Free DNA from Patients with Advanced Cancers.

Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of A quantitative, mutation-enrichment next-generation sequencing test for detecting mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer. With 90 to 110 mL of urine, the cfDNA test had an analytical sensitivity of 0.002% to 0.

A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma.

Introduction: In approximately 60% of patients with NSCLC who are receiving EGFR tyrosine kinase inhibitors, resistance develops through the acquisition of EGFR T790M mutation. We aimed to demonstrate that a highly sensitive and quantitative next-generation sequencing analysis of EGFR mutations from urine and plasma specimens is feasible.

Methods: Short footprint mutation enrichment next-generation sequencing assays were used to interrogate EGFR activating mutations and the T790M resistance mutation in urine or plasma specimens from patients enrolled in TIGER-X (NCT01526928), a phase 1/2 clinical study of rociletinib in previously treated patients with EGFR mutant-positive advanced NSCLC.

ApoStream(™), a new dielectrophoretic device for antibody independent isolation and recovery of viable cancer cells from blood.

Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream(™) that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber.

Expression of Id-1 is regulated by MCAM/MUC18: a missing link in melanoma progression.

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis.

Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype.

The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines.

CREB inhibits AP-2alpha expression to regulate the malignant phenotype of melanoma.

Background: The loss of AP-2alpha and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2alpha during melanoma progression remains unknown.

Methodology/principal Findings: Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2alpha protein expression in two metastatic melanoma cell lines.

Crosstalk between protease-activated receptor 1 and platelet-activating factor receptor regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and melanoma metastasis.

The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter.

Overexpression of protease-activated receptor-1 contributes to melanoma metastasis via regulation of connexin 43.

Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level.

Silencing cAMP-response element-binding protein (CREB) identifies CYR61 as a tumor suppressor gene in melanoma.

Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis.

Inflammation and melanoma metastasis.

Metastatic melanoma is extremely refractory to existing chemotherapeutic drugs and bioimmune adjuvant therapies, and the life span of patients with metastatic melanoma is often measured in months. Understanding the mechanisms responsible for the development of tumor metastasis is critical for finding successful curative measures. An expending amount of data reveal the importance of inflammatory microenvironment and stroma in cancer initiation and progression, which brings new directions and approaches to cancer treatment.

Emerging roles of PAR-1 and PAFR in melanoma metastasis.

Melanoma growth, angiogenesis and metastatic progression are strongly promoted by the inflammatory tumor microenvironment due to high levels of cytokine and chemokine secretion by the recruited inflammatory and stromal cells. In addition, platelets and molecular components of procoagulant pathways have been recently emerging as critical players of tumor growth and metastasis. In particular, thrombin, through the activity of its receptor protease-activated receptor-1 (PAR-1), regulates tumor cell adhesion to platelets and endothelial cells, stimulates tumor angiogenesis, and promotes tumor growth and metastasis.

Expression profiling of Galectin-3-depleted melanoma cells reveals its major role in melanoma cell plasticity and vasculogenic mimicry.

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells.

Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA.

The thrombin receptor [protease-activated receptor-1 (PAR-1)] is overexpressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and up-regulation of genes involved in adhesion, invasion, and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral short hairpin RNA and found significant decreases in both tumor growth (P < 0.

Tumor immunotherapy in melanoma: strategies for overcoming mechanisms of resistance and escape.

The incidence of melanoma has been steadily increasing over the last 3 decades. Currently, there are several approved treatments for metastatic melanoma, including chemotherapy and biologic therapy as both single treatments and in combination, but none is associated with a significant increase in survival. The chemotherapeutic agent dacarbazine is the standard treatment for metastatic melanoma, with a response rate of 15-20%, although most responses are not sustained.

Transcriptional control of the melanoma malignant phenotype.

Malignant progression and tumor metastasis is a complex process enabled by the intrinsic genomic instability of the tumor cells and supported by a plethora of recruited cell types within the tumor microenvironment. Propelled by a selective pressure to overcome tissue homeostatic mechanisms, a metastatic "super-cell" emerges whose phenotype is associated with the cellular capacity for uncontrolled growth, resistance to apoptosis, high invasive potential, and effective neoangiogenesis. While genetic alterations arise early in the course of melanoma development, the progression toward metastatic disease is accompanied by deregulated expression of a number of transcription factors.

P53 protein and pathogenesis of melanoma and nonmelanoma skin cancer.

The p53 tumor suppressor gene and gene product are among the most diverse and complex been shown to have a direct correlation with cancer development and have been shown to occur in nearly 50% of all cancers. p53 mutations are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific 'signature' mutations that can result in oncogenic transformation. There are certain 'hot-spots' in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site.

Models and mechanisms in malignant melanoma.

Human melanoma represents the fastest growing malignancy in the US. The etiology of melanoma is highly debated as is the role of ultraviolet (UV) radiation in the initiation and progression of melanoma. This article discusses data from UV exposure and its relationship to the development of melanoma from various models of melanoma as well as various genetic alterations seen in oncogenic transformation of melanocytes.

ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis.

Melanoma is a very aggressive and highly angiogenic tumor in which standard treatments have had only limited success. Patients with advanced disease have a 5-year survival rate of 5%. In search for alternatives, we identified a natural product extracted from the fungus Aspergillus niger, termed ACTIBIND, that inhibits tumor growth and metastasis of melanoma in vivo.

Bioimmunotherapy for melanoma using fully human antibodies targeting MCAM/MUC18 and IL-8.

Metastatic melanoma is associated with high rate of patients' mortality and represents a great challenge for cancer therapies because of its notorious resistance to chemotherapeutic drugs. Considerable efforts have been made over the last 2 decades in pursuit of new treatment modalities and identification of molecular events associated with melanoma progression and development of metastases. The acquisition of the metastatic phenotype is associated with overexpression of the adhesion molecule MCAM/MUC18 and the angiogenic factor IL-8.

Platelet-activating factor mediates MMP-2 expression and activation via phosphorylation of cAMP-response element-binding protein and contributes to melanoma metastasis.

Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines.

Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development.

Chronic exposure to ultraviolet (UV) radiation causes skin cancer in humans and mice. We have previously shown that in hairless SKH-hr1 mice, UVB-induced p53 mutations arise very early, well before tumor development. In this study, we investigated whether discontinuation of UVB exposure before the onset of skin tumors results in the disappearance of p53 mutations in the skin of hairless SKH-hr1 mice.

Cellular and molecular events leading to the development of skin cancer.

The transition from a normal cell to a neoplastic cell is a complex process and involves both genetic and epigenetic changes. The process of carcinogenesis begins when the DNA is damaged, which then leads to a cascade of events leading to the development of a tumor. Ultraviolet (UV) radiation causes DNA damage, inflammation, erythema, sunburn, immunosuppression, photoaging, gene mutations, and skin cancer.

Genomic alterations in spontaneous and carcinogen-induced murine melanoma cell lines.

We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups.