Whole genome analysis reveals the genomic complexity in metastatic cutaneous squamous cell carcinoma.

Metastatic cutaneous squamous cell carcinoma (CSCC) is a highly morbid disease requiring radical surgery and adjuvant therapy, which is associated with a poor prognosis. Yet, compared to other advanced malignancies, relatively little is known of the genomic landscape of metastatic CSCC. We have previously reported the mutational signatures and mutational patterns of CCCTC-binding factor (CTCF) regions in metastatic CSCC. However, many other genomic components (indel signatures, non-coding drivers, and structural variants) of metastatic CSCC have not been reported. To this end, we performed whole genome sequencing on lymph node metastases and blood DNA from 25 CSCC patients with regional metastases of the head and neck. We designed a multifaceted computational analysis at the whole genome level to provide a more comprehensive perspective of the genomic landscape of metastatic CSCC. In the non-coding genome, 3' untranslated region (3'UTR) regions of EVC (48% of specimens), PPP1R1A (48% of specimens), and ABCA4 (20% of specimens) along with the tumor-suppressing long non-coding RNA (lncRNA) LINC01003 (64% of specimens) were significantly functionally altered (Q-value < 0.05) and represent potential non-coding biomarkers of CSCC. Recurrent copy number loss in the tumor suppressor gene PTPRD was observed. Gene amplification was much less frequent, and few genes were recurrently amplified. Single nucleotide variants driver analyses from three tools confirmed TP53 and CDKN2A as recurrently mutated genes but also identified C9 as a potential novel driver in this disease. Furthermore, indel signature analysis highlighted the dominance of ID signature 13 (ID13) followed by ID8 and ID9. ID9 has previously been shown to have no association with skin melanoma, unlike ID13 and ID8, suggesting a novel pattern of indel variation in metastatic CSCC. The enrichment analysis of various genetically altered candidates shows enrichment of "TGF-beta regulation of extracellular matrix" and "cell cycle G1 to S check points." These enriched terms are associated with genetic instability, cell proliferation, and migration as mechanisms of genomic drivers of metastatic CSCC.

Cancer Progression Gene Expression Profiling Identifies the Urokinase Plasminogen Activator Receptor as a Biomarker of Metastasis in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) of the head and neck region is the second most prevalent skin cancer, with metastases to regional lymph nodes occurring in 2%-5% of cases. To further our understanding of the molecular events characterizing cSCC invasion and metastasis, we conducted targeted cancer progression gene expression and pathway analysis in non-metastasizing (PRI-) and metastasizing primary (PRI+) cSCC tumors of the head and neck region, cognate lymph node metastases (MET), and matched sun-exposed skin (SES). The highest differentially expressed genes in metastatic (MET and PRI+) versus non-metastatic tumors (PRI-) and SES included PLAU, PLAUR, MMP1, MMP10, MMP13, ITGA5, VEGFA, and various inflammatory cytokine genes. Pathway enrichment analyses implicated these genes in cellular pathways and functions promoting matrix remodeling, cell survival and migration, and epithelial to mesenchymal transition, which were all significantly activated in metastatic compared to non-metastatic tumors (PRI-) and SES. We validated the overexpression of urokinase plasminogen activator receptor (uPAR, encoded by PLAUR) in an extended patient cohort by demonstrating higher uPAR staining intensity in metastasizing tumors. As pathway analyses identified epidermal growth factor (EGF) as a potential upstream regulator of PLAUR, the effect of EGF on uPAR expression levels and cell motility was functionally validated in human metastatic cSCC cells. In conclusion, we propose that uPAR is an important driver of metastasis in cSCC and represents a potential therapeutic target in this disease.

Gene expression profiling of perineural invasion in head and neck cutaneous squamous cell carcinoma.

Perineural invasion (PNI) is frequently associated with aggressive clinical behaviour in head and neck cutaneous squamous cell carcinoma (HNcSCC) leading to local recurrence and treatment failure. This study evaluates the gene expression profiles of HNcSCC with PNI using a differential expression analysis approach and constructs a tailored gene panel for sensitivity and specificity analysis. 45 cases of HNcSCC were stratified into three groups (Extensive, Focal and Non PNI) based on predefined clinicopathological criteria. Here we show HNcSCC with extensive PNI demonstrates significant up- and down-regulation of 144 genes associated with extracellular matrix interactions, epithelial to mesenchymal transition, cell adhesion, cellular motility, angiogenesis, and cellular differentiation. Gene expression of focal and non PNI cohorts were indistinguishable and were combined for further analyses. There is clinicopathological correlation between gene expression analysis findings and disease behaviour and a tailored panel of 10 genes was able to identify extensive PNI with 96% sensitivity and 95% specificity.

Comprehensive Mutational and Phenotypic Characterization of New Metastatic Cutaneous Squamous Cell Carcinoma Cell Lines Reveal Novel Drug Susceptibilities.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2-5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.

Thulium oxide nanoparticles as radioenhancers for the treatment of metastatic cutaneous squamous cell carcinoma.

Metastases from cutaneous squamous cell carcinoma (cSCC) occur in 2%-5% of cases. Surgery is the standard treatment, often combined with adjuvant radiotherapy. Concurrent carboplatin treatment with post-operative radiotherapy may be prescribed, although it has not shown benefit in recent clinical trials in high-risk cSCC patients. The novel high-Z nanoparticle thulium (III) oxide has been shown to enhance radiation dose delivery to brain tumors by specific uptake of these nanoparticles into the cancerous tissue. As the dose-enhancement capacity of thulium oxide nanoparticles following radiotherapy against metastatic cSCC cells is unknown, its efficacy as a radiosensitizer was evaluated, with and without carboplatin. Novel and validated human patient-derived cell lines of metastatic cSCC were used. The sensitivity of the cells to radiation was investigated using short-term proliferation assays as well as clonogenic survival as the radiobiological endpoint. Briefly, cells were irradiated with 125 kVp orthovoltage x-rays (0-6 Gy) with and without thulium oxide nanoparticles (99.9% trace metals basis; 50 µg ml-1) or low dose carboplatin pre-sensitization. Cellular uptake of the nanoparticles was first confirmed by microscopy and found to have no impact on short-term cell survival for the cSCC cells, highlighting the biocompatibility of thulium oxide nanoparticles. Clonogenic cell survival assays confirmed radio-sensitization when exposed to thulium nanoparticles, with the cell sensitivity increasing by a factor of 1.24 (calculated at the 10% survival fraction) for the irradiated cSCC cells. The combination of carboplatin with thulium oxide nanoparticles with irradiation did not result in significant further reductions in survival compared to nanoparticles alone. This is the first study to provide in vitro data demonstrating the independent radiosensitization effect of high-Z nanoparticles against metastatic cSCC with or without carboplatin. Further preclinical investigations with radiotherapy plus high-Z nanoparticles for the management of metastatic cSCC are warranted.

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer.

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM-, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets.

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease.

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer.

Metastasis is the cause of death in the majority (∼90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5 -N, N-(hexamethylene)amiloride (HMA) reportedly show robust antitumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases, and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and antikaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model, and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.

Cryopreservation for delayed circulating tumor cell isolation is a valid strategy for prognostic association of circulating tumor cells in gastroesophageal cancer.

AIM: To demonstrate the feasibility of cryopreservation of peripheral blood mononuclear cells (PBMCs) for prognostic circulating tumor cell (CTC) detection in gastroesophageal cancer. METHODS: Using 7.5 mL blood samples collected in EDTA tubes from patients with gastroesopheagal adenocarcinoma, CTCs were isolated by epithelial cell adhesion molecule based immunomagnetic capture using the IsoFlux platform. Paired specimens taken during the same blood draw (n = 15) were used to compare number of CTCs isolated from fresh and cryopreserved PBMCs. Blood samples were processed within 24 h to recover the PBMC fraction, with PBMCs used for fresh analysis immediately processed for CTC isolation. Cryopreservation of PBMCs lasted from 2 wk to 25.2 mo (median 14.6 mo). CTCs isolated from pre-treatment cryopreserved PBMCs (n = 43) were examined for associations with clinicopathological variables and survival outcomes. RESULTS: While there was a significant trend to a decrease in CTC numbers associated with cryopreserved specimens (mean number of CTCs 34.4 vs 51.5, P = 0.04), this was predominately in samples with a total CTC count of > 50, with low CTC count samples less affected (P = 0.06). There was no significant association between the duration of cryopreservation and number of CTCs. In cryopreserved PBMCs from patient samples prior to treatment, a high CTC count (> 17) was associated with poorer overall survival (OS) (n = 43, HR = 4.4, 95%CI: 1.7-11.7, P = 0.0013). In multivariate analysis, after controlling for sex, age, stage, ECOG performance status, and primary tumor location, a high CTC count remained significantly associated with a poorer OS (HR = 3.7, 95%CI: 1.2-12.4, P = 0.03). CONCLUSION: PBMC cryopreservation for delayed CTC isolation is a valid strategy to assist with sample collection, transporting and processing.

N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines.

The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.