Analytical Validation of a Novel 6-Gene Signature for Prediction of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative, Early-Stage Breast Cancer.

BACKGROUND: OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study. METHODS: Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays. RESULTS: The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue. CONCLUSION: The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.

Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. METHODS: We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. RESULTS: We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. CONCLUSIONS: Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility.

Development of a 3D pigmented skin model to evaluate RNAi-induced depigmentation.

Because current skin whitening agents often have insufficient efficacy and side effects, we aim to develop effective and safe therapeutics using RNA interference (RNAi). We established a pigmented human-reconstructed skin model as a first step in the development of novel siRNA-based depigmenting agents. Histological characterization revealed that our model had a similar morphology as normal human skin, expressed keratinocyte differentiation as well as basement membrane markers, and showed a high degree of pigmentation. The utility of the model to study RNAi-induced depigmentation was validated by incorporation of melanocytes transfected with siRNA against tyrosinase, a key enzyme in skin pigmentation. This resulted in a strong reduction in pigmentation and inhibition of melanin transfer proving that siRNA-mediated gene silencing in melanocytes worked successfully in our model. Therefore, this self-made 3D skin model will be a useful and easy tool to validate the whitening potential of candidate genes with a presumed function in melanin synthesis or transfer.

Prognostic value of the 6-gene OncoMasTR test in hormone receptor-positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors.

AIM: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. METHODS: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. RESULTS: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes = 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P ≤ 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. DISCUSSION: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.

Validation of the OncoMasTR Risk Score in Estrogen Receptor-Positive/HER2-Negative Patients: A TransATAC study.

PURPOSE: To test the validity of OncoMasTR Molecular Score (OMm), OMclin1, and OncoMasTR Risk Score (OMclin2) prognostic scores for prediction of distant recurrence (DR) in estrogen receptor (ER)-positive/HER2-negative breast cancer treated with 5 years' endocrine therapy only and compare their performance with the Oncotype DX Recurrence Score (RS). EXPERIMENTAL DESIGN: OMm incorporates three master transcription regulator genes. OMclin1 combines OMm, tumor size, grade, and nodal status; OMclin2 incorporates OMm, tumor size, and nodal status. OMclin1 and OMclin2 were evaluated for 646 postmenopausal patients with ER-positive/HER2-negative primary breast cancer with 0-3 involved lymph nodes in TransATAC. Patients were randomized to 5 years' anastrozole or tamoxifen without chemotherapy. RS was available in all cases. We used likelihood ratio-χ 2, C-index, and Kaplan-Meier analyses to assess prognostic information. RESULTS: OMm, OMclin1, and OMclin2 were highly prognostic for prediction of DR in years 0-10 among all patients [likelihood ratio (LR)-χ 2 = 25.4, 48.7, and 45.0, respectively, all P < 0.001; C-index = 0.67, 0.71, and 0.71, respectively], compared with RS (LR-χ 2 = 18.8; P < 0.001; C-index = 0.63). All three scores provided significant additional prognostic value beyond clinical treatment score, Nottingham Prognostic Index, and Ki67. OMclin1 and OMclin2 categorized 190 and 267 node-negative patients as low risk (DR rates: 2.9% and 4.9%, respectively). In comparison, RS categorized 296 node-negative patients as low-risk and 128 patients as intermediate-risk (DR rate: 6.6% and 17.3%, respectively). CONCLUSIONS: OMm, OMclin1, and OMclin2 were highly prognostic for early and late DR in women with early-stage ER-positive breast cancer receiving 5 years' endocrine therapy. In TransATAC, OMclin1 and the OncoMasTR Risk Score (OMclin2) were superior to RS in identifying patients at increased risk of DR.

Low expression of pro-apoptotic proteins Bax, Bak and Smac indicates prolonged progression-free survival in chemotherapy-treated metastatic melanoma.

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

Apelin: A putative novel predictive biomarker for bevacizumab response in colorectal cancer.

Bevacizumab (bvz) is currently employed as an anti-angiogenic therapy across several cancer indications. Bvz response heterogeneity has been well documented, with only 10-15% of colorectal cancer (CRC) patients benefitting in general. For other patients, clinical efficacy is limited and side effects are significant. This reinforces the need for a robust predictive biomarker of response. To identify such a biomarker, we performed a DNA microarray-based transcriptional profiling screen with primary endothelial cells (ECs) isolated from normal and tumour colon tissues. Thirteen separate populations of tumour-associated ECs and 10 of normal ECs were isolated using fluorescence-activated cell sorting. We hypothesised that VEGF-induced genes were overexpressed in tumour ECs; these genes could relate to bvz response and serve as potential predictive biomarkers. Transcriptional profiling revealed a total of 2,610 differentially expressed genes when tumour and normal ECs were compared. To explore their relation to bvz response, the mRNA expression levels of top-ranked genes were examined using quantitative PCR in 30 independent tumour tissues from CRC patients that received bvz in the adjuvant setting. These analyses revealed that the expression of MMP12 and APLN mRNA was significantly higher in bvz non-responders compared to responders. At the protein level, high APLN expression was correlated with poor progression-free survival in bvz-treated patients. Thus, high APLN expression may represent a novel predictive biomarker for bvz unresponsiveness.

Prognostic and predictive biomarkers in melanoma: an update.

Malignant melanoma is one of the most aggressive cancers. Several new therapeutic strategies that focus on immuno- and/or targeted therapy have been developed, which have entered clinical trials or already been approved. This review provides an update on prognostic and predictive biomarkers in melanoma that may be used to improve the clinical management of patients. Prognostic markers include conventional histopathological characteristics, chromosomal aberrations, gene expression patterns and miRNA profiles. There is a trend towards multi-marker assays and whole-genome molecular screening methods to determine the prognosis of individual patients. Predictive biomarkers, including targeted components of signal transduction, developmental or transcriptional pathways, can be used to determine patient response towards a particular treatment or combination thereof. The rapid evolution of sequencing technologies and multi-marker screening will change the spectrum of patients who become candidates for therapeutic agents, and in addition create new ethical and regulatory challenges.

miR-145 overexpression suppresses the migration and invasion of metastatic melanoma cells.

MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression which play important roles in tumorigenesis and cancer metastasis. Since they are often highly deregulated in various types of cancer, miRNAs may be effective treatment targets. miRNA profiling studies of melanoma have led to the identification of several tumor suppressor miRNAs. One of these include miR-145, although functional data proving its specific function are limited. Therefore, in this study, we examined the expression levels of miR-145 in three melanoma cell lines (BLM, FM3P and WM793). Additional gain-of-function experiments revealed that miR-145 exerts an anti-proliferative effect in the primary, non-invasive melanoma cell line, WM793, whereas cell migration and the invasive potential of metastatic melanoma cells was suppressed following transfection with miR-145 mimics. In order to investigate the mechanisms by which miR-145 exerts its invasion suppressor function, we examined the expression level of target genes [fascin homolog 1 (FSCN1), myosin­Va (MYO5A and SOX9] and that of an indirect target (RAB27A) following the overexpression of miR-145. The results showed that SOX9, MYO5A and RAB27A were not involved in the biological effects caused by miR-145 mimics. Surprisingly, we discovered that miR-145 in melanoma, in contrast to many other tumor types, does not necessarily act via the target, FSCN1, since the downregulation of FSCN1 did not inhibit cell proliferation or migration but, on the contrary, increased cell invasion in two out of the three melanoma cell lines examined. Our in vitro data is in accordance with previously reported in vivo data describing the low expression of FSCN1 in malignant melanomas when compared to dysplastic nevi, suggesting that the expression of FSCN1 decreases as the formation and progression stage of melanoma advances. In conclusion, our data provide evidence that miR-145 is an invasion suppressor in metastatic melanoma cells. Despite the fact that it remains unclear which genes or pathways are regulated by miR-145 in melanoma, miR-145 may serve as a useful therapeutic agent in melanoma when re-expressed in situ.

Identification of miR-145 as a key regulator of the pigmentary process.

The current treatments for hyperpigmentation are often associated with a lack of efficacy and adverse side effects. We hypothesized that microRNA (miRNA)-based treatments may offer an attractive alternative by specifically targeting key genes in melanogenesis. The aim of this study was to identify miRNAs interfering with the pigmentary process and to assess their functional role. miRNA profiling was performed on mouse melanocytes after three consecutive treatments involving forskolin and solar-simulated UV (ssUV) irradiation. Sixteen miRNAs were identified as differentially expressed in treated melan-a cells versus untreated cells. Remarkably, a 15-fold downregulation of miR-145 was detected. Overexpression or downregulation of miR-145 in melan-a cells revealed reduced or increased expression of Sox9, Mitf, Tyr, Trp1, Myo5a, Rab27a, and Fscn1, respectively. Moreover, a luciferase reporter assay demonstrated direct targeting of Myo5a by miR-145 in mouse and human melanocytes. Immunofluorescence tagging of melanosomes in miR-145-transfected human melanocytes displayed perinuclear accumulation of melanosomes with additional hypopigmentation of harvested cell pellets. In conclusion, this study has established an miRNA signature associated with forskolin and ssUV treatment. The significant down- or upregulation of major pigmentation genes, after modulating miR-145 expression, suggests a key role for miR-145 in regulating melanogenesis.

Lipid-mediated gene delivery to the skin.

Cutaneous gene delivery methods have been developed over the past decades as therapeutic strategies for the treatment of a variety of skin disorders. Both viral and non-viral techniques have been frequently described. Mainly due to safety concerns, the application of viral methods is being questioned and non-viral alternatives are gaining major interest. Lipid-based vesicles for the delivery of plasmid DNA by topical application onto the skin hold great potential and have been investigated thoroughly. Here, we give an overview of the different lipid vesicles that have been described in literature. Next to the conventional phospholipid liposomes, new generation liposomes like niosomes and Transfersomes® have been developed for enhanced (trans)dermal delivery. In addition, we draw attention to other lipid-based delivery systems, that could not be classified into one of these categories. Clearly, lipid-based delivery vehicles demonstrate very promising results for DNA delivery into and through the skin, especially for cutaneous vaccination purposes. Apart from simple topical application onto the skin, liposomes have also been described in combination with delivery enhancing techniques. Here we describe this combined approach for some specific skin disorders.

Griscelli syndrome: a model system to study vesicular trafficking.

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutations in either the myosin VA (GS1), RAB27A (GS2) or melanophilin (GS3) genes. The three GS subtypes are commonly characterized by pigment dilution of the skin and hair, due to defects involving melanosome transport in melanocytes. Here, we review how detailed studies concerning GS have contributed to a better understanding of the molecular mechanisms involved in vesicle transport and membrane trafficking processes. Additionally, we demonstrate that the identification and biological analysis of novel disease-causing mutations highlighted the functional importance of the RAB27A-MLPH-MYO5A tripartite complex in intracellular melanosome transport. As the small GTPase Rab27a is able to interact with multiple effectors, including Slp2-a and Myrip, we report on their presumed role in melanosome transport. Furthermore, we summarize data suggesting that RAB27B and RAB27A are functionally redundant and hereby provide further insight into the pathogenesis of GS2. Finally, we discuss how the gathered knowledge about the RAB27A-MLPH-MYO5A tripartite complex can be translated into a possible therapeutic application to reduce (hyper)pigmentation of the skin.