Genomic and proteomic findings in early melanoma and opportunities for early diagnosis.

Overdiagnosis of early melanoma is a significant problem. Due to subtle unique and overlapping clinical and histological criteria between pigmented lesions and the risk of mortality from melanoma, some benign pigmented lesions are diagnosed as melanoma. Although histopathology is the gold standard to diagnose melanoma, there is a demand to find alternatives that are more accurate and cost-effective. In the current "omics" era, there is gaining interest in biomarkers to help diagnose melanoma early and to further understand the mechanisms driving tumor progression. Genomic investigations have attempted to differentiate malignant melanoma from benign pigmented lesions. However, genetic biomarkers of early melanoma diagnosis have not yet proven their value in the clinical setting. Protein biomarkers may be more promising since they directly influence tissue phenotype, a result of by-products of genomic mutations, posttranslational modifications and environmental factors. Uncovering relevant protein biomarkers could increase confidence in their use as diagnostic signatures. Currently, proteomic investigations of melanoma progression from pigmented lesions are limited. Studies have previously characterised the melanoma proteome from cultured cell lines and clinical samples such as serum and tissue. This has been useful in understanding how melanoma progresses into metastasis and development of resistance to adjuvant therapies. Currently, most studies focus on metastatic melanoma to find potential drug therapy targets, prognostic factors and markers of resistance. This paper reviews recent advancements in the genomics and proteomic fields and reports potential avenues, which could help identify and differentiate melanoma from benign pigmented lesions and prevent the progression of melanoma.

Proteomics: An emerging approach for the diagnosis and classification of cutaneous squamous cell carcinoma and its precursors.

Cutaneous squamous cell carcinoma (cSCC) and its precursors, actinic keratosis (AK) and Bowen's disease (BD), are the most common types of keratinocytic skin lesions (KSL) which account for the majority of non-melanoma skin cancer lethality. Currently, clinical and histopathological criteria are used for the diagnosis, classification and therapeutic intervention of KSLs, however discrepancies exist between the clinical presentations and histologic analyses of these lesions, making the diagnosis difficult. The identification of biomarkers as companion diagnostics for accurately stratifying KSL types is required to support the paradigm shift in current cancer care to personalised, precision medicine and ameliorate the negative impact of misdiagnoses or delayed diagnoses on patient outcome. Also, it is essential to elaborate on the poorly defined molecular modifications required for the initiation, development and progression of KSL from normal keratinocytes. By harnessing recent technological advances in molecular profiling techniques, it is anticipated that greater insight into the various combinations of proteomic events or alternative pathways underlying carcinogenesis will be gained. This review will explore recent genomic studies in KSL followed by assessing the feasibility and significance of mass spectrometry-based proteomics profiling as a promising approach to a better understanding of the oncogenic pathways underpinning the formation and progression of KSL lesions and in aiding the identification of novel biomarkers and new therapeutic targets. The development of non-invasive tools such as tape-stripping coupled with proteomic analysis alone or in conjunction with imaging and genomic technologies will complement existing clinical and histopathological parameters, leading to an improvement in patient outcomes.

Data Independent Acquisition Proteomic Analysis Can Discriminate between Actinic Keratosis, Bowen's Disease, and Cutaneous Squamous Cell Carcinoma.

Actinic keratosis, Bowen's disease and cutaneous squamous cell carcinoma (cSCC) are heterogeneous keratinocytic skin lesions. Biomarkers that can accurately stratify these lesion types are needed to support a new paradigm of personalized and precise management of skin neoplasia. In this paper, we used a data independent acquisition proteomics workflow, sequential window acquisition of all theoretical mass spectra, to analyze formalin-fixed paraffin-embedded samples of normal skin and keratinocytic skin lesions, including well-differentiated, moderately differentiated and poorly differentiated cSCC lesions. We quantified 3,574 proteins across the 93 samples studied. Differential abundance analysis identified 19, 5, and 6 protein markers exclusive to actinic keratosis, Bowen's disease and cSCC lesions, respectively. Among cSCC lesions of various levels of tumor differentiation, 118, 230, and 17 proteins showed a potential as biomarkers of well-differentiated, moderately differentiated and poorly differentiated cSCC lesions, respectively. Bioinformatics analysis revealed that actinic keratosis and cSCC lesions were associated with decreased apoptosis, and Bowen's disease lesions with over-representation of the DNA damage repair pathway. Differential expression of alternatively spliced FGFR2, Rho guanosine triphosphatase signaling, and RNA metabolism proteins were associated with the level of cSCC tumor differentiation. Proteome profiles also separated keratinocytic skin lesion subtypes on principal components analysis. Overall, protein markers have excellent potential to discriminate keratinocytic skin lesion subtypes and facilitate new diagnostic and therapeutic strategies.

Tape Stripped Stratum Corneum Samples Prove to be Suitable for Comprehensive Proteomic Investigation of Actinic Keratosis.

PURPOSE: Actinic keratoses (AK) are premalignant tumors that can be clinically difficult to differentiate from skin cancer. An easy, quick, and reliable noninvasive alternative to biopsy is needed to definitively confirm the clinical diagnoses. This study evaluates Tape Stripping (TS) of stratum corneum (SC) for noninvasive biomarker analysis of AK. METHOD: Lesional and nonlesional human SC samples are obtained by application of stripping tapes on the skin of five AK patients. Following sample preparation, protein digests are analyzed by LC-MS/MS. Bioinformatics analyses are performed using Funrich, Ingenuity Pathway Analysis (IPA), and Oncomine bioinformatics and analytical tools. RESULTS: Of the total 613 unique proteins identified, 477 overlap with proteins identified in the proteomic analysis of formalin-fixed and paraffin-embedded (FFPE) AK samples. Additionally, 32 proteins are significantly increased and four proteins decreased in AK samples compared to the normal skin (p < 0.05). In line with proteomic analysis of FFPE samples, IPA and Funrich analysis show that differentially abundant proteins in the TS AK samples are implicated in PI3K/AKT and EGF signaling pathways. These findings are confirmed at the transcript level. CONCLUSION: Tape stripped AK sample is suitable for biomarker analysis. The application of this technique further could revolutionize management of keratinocytic skin tumors by reducing the need for traditional invasive biopsy.

Differential proteomic analysis of actinic keratosis, Bowen's disease and cutaneous squamous cell carcinoma by label-free LC-MS/MS.

BACKGROUND: The boundaries between actinic keratosis (AK), Bowen's disease (BD), and cutaneous squamous cell carcinoma (cSCC) are sometimes not clear. Large-scale proteomic profiling studies of these lesions are also non-existent. OBJECTIVE: To evaluate proteomic changes between normal epidermis, AK, BD and cSCC that could support a molecular classification and improve our understanding of disease progression. METHODS: Microdissected formalin-fixed paraffin embedded samples of normal epidermis (n = 4, pooled), AK (n = 10), BD (n = 10) and cSCC (n = 10) were analyzed by mass spectrometry. Following normalization and multiple testing adjustments, differential abundance analysis was performed using Linear Models for Microarray data. Proteins were filtered for significance (adjusted p-value ≤ 0.05) and fold change of at least ±1.5. Comparative bioinformatics analysis was performed using Ingenuity Pathway Analysis (IPA) software. Proteomic findings were subsequently substantiated using immunohistochemistry. RESULTS: 2073 unique proteins were identified. cSCC had the highest number of differentially abundant proteins (63 proteins) followed by BD (58 proteins) and AK (46 proteins). Six proteins (APOA1, ALB, SERPINA1, HLA-B, HP and TXNDC5) were differentially abundant in cSCC compared to AK. Immunohistochemical analysis corroborated changes in MIF, RPL37A and TXNDC5. IPA analysis predicted that cell proliferation, angiogenesis and inflammatory reactions were significantly activated in cSCC compared to BD and AK. Cell death and DNA damage were predicted to be inhibited in BD. CONCLUSION: Our study supports the concept that AK and BD are precursors of cSCC. The identification of proteome changes indicates disruption of repair, pro-apoptotic, and tumor promoting pathways. Our findings will help select targets for classification and treatment.

Endocannabinoids in arthritis: current views and perspective.

Preclinical and clinical studies using cannabis-based therapy have been shown to provide both analgesia and anti-inflammatory effects, with an overall alleviation of clinical symptoms in animal models of arthritis, highlighting its promising therapeutic application for humans. Despite this, the development of cannabis-based therapeutics remains in its infancy, with further investigation into its efficacy and safety profile in patients still required. This synopsis reviews the various components of the endocannabinoid system in health and disease and their potential as therapeutic targets.

Evaluation of rapid GeneXpert MTB/RIF method using DNA tissue specimens of vertebral bones in patients with suspected spondylitis TB.

PURPOSE: To detect Mycobacterium tuberculosis DNA and rifampicin resistance in vertebral bone tissue specimens from spondylitis TB suspects. METHODS AND RESULTS: The rapid GeneXpert MTB/RIF and MGIT 960 liquid culture methods have been used in the specimens. Results from 70 suspects with spondylitis TB shown that 31.42% identified positive for spondylitis TB using culture method, while 88.57% shown positive results using rapid GeneXpert MTB/RIF method. The validity of GeneXpert MTB/RIF shown sensitivity value of 100%, specificity value of 16.6%, PPV of 35.48%, and NPV of 100%. CONCLUSION: GeneXpert has a high sensitivity but low specificity value in this study.

In Silico Analysis Validates Proteomic Findings of Formalin-fixed Paraffin Embedded Cutaneous Squamous Cell Carcinoma Tissue.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer but there are no comprehensive proteomic studies on this entity. MATERIALS AND METHODS: We employed liquid chromatography coupled with tandem mass spectrometry (MS/MS) using formalin-fixed paraffin-embedded (FFPE) cSCC material to study the tumor and normal skin tissue proteomes. Ingenuity Pathway Analysis (IPA) was used to interpret the role of altered proteins in cSCC pathophysiology. Results were validated using the Human Protein Atlas and Oncomine database in silico. RESULTS: Of 1,310 unique proteins identified, expression of an average of 144 and 88 proteins were significantly (p<0.05) increased and decreased, respectively, in the tumor samples compared to their normal counterparts. IPA analysis revealed disruptions in proteins associated with cell proliferation, apoptosis, and migration. In silico analysis confirmed that proteins corresponding to 12 antibodies, and genes corresponding to 18 proteins were differentially expressed between the two categories, validating our proteomic measurements. CONCLUSION: Label-free MS-based proteomics is useful for analyzing FFPE cSCC tissues.

Evaluating disease activity in patients with ankylosing spondylitis and rheumatoid arthritis using 99mtc-glucosamine.

OBJECTIVE: To evaluate the clinical utility of a novel radiotracer, 99mTc-glucosamine, in assessing disease activity of both rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Material and Methods: Twenty-five patients with RA (nine males and 16 females) and 12 patients with AS (all male) at various stages of disease were recruited for the study. A clinical history and examination was performed, followed by the measurement of hematological, biochemical, and autoimmune serological parameters to assess disease activity. 99mTc-glucosamine was intravenously administered and scans were compared with other imaging modalities, including plain X-ray, magnetic resonance imaging (MRI), and bone scans. RESULTS: In patients with AS, 99mTc-glucosamine scans were more capable of identifying active disease and differentiating between inflammatory and non-inflammatory causes. In patients with RA, 99mTc-glucosamine accumulated at all known sites of disease involvement. Uptake was most pronounced in patients with active untreated disease. The relative tracer activity in the involved joints increased with time compared with that in the adjoining soft tissue, liver, and cardiac blood pool. Using Spearman's correlation coefficient, there was a positive correlation among glucosamine scan scores, C-reactive protein (p=0.048), and clinical assessment (p=0.003), which was not noted with bone scans. CONCLUSION: The radiotracer was well tolerated by all patients, with no adverse reactions. 99mTc-glucosamine imaging could detect spinal inflammation in AS. With respect to RA, 99mTc-glucosamine was a viable alternative to 99mTc-labeled methylene diphosphonate nuclear bone scans for imaging inflamed joints and had the added advantage of demonstrating a significant clinical correlation between disease activity and scan findings.

Cyclization enhances function of linear anti-arthritic peptides.

This study describes the biophysical and immunomodulatory features of a cyclic peptide termed C1 which consists of alternating d-, l-amino acids and is capable of inhibiting IL-2 production in vitro and reducing the induction and extent of T-cell mediated inflammation in animal models. Solid-state nuclear magnetic resonance demonstrates that the peptide orders the lipid bilayer, suggesting a transmembrane orientation, and this is supported by surface plasmon resonance indicating strong binding affinity of C1 to model membranes. In vitro cell viability and proliferation assays show that C1 does not disrupt the integrity of cell surface membranes. Permeation studies of C1 and analogs across human epidermis cells show that the stability and skin permeability are enhanced by cyclization. Treatment with C1 in an asthma and in an arthritis animal model resulted in a suppressed immune response. Cyclization may be a useful means of enhancing biological linear peptide activity and improving delivery.

Alanine scan of an immunosuppressive peptide (CP): analysis of structure-function relationships.

Core peptide is a hydrophobic peptide, the sequence of which is derived from the T-cell antigen receptor alpha-chain transmembrane region. Previous studies have shown that core peptide can inhibit T-cell-mediated immune responses both in vitro and in vivo. Here, we report the role each constituent amino acid plays within core peptide using an alanine scan and the amino acid effect on function using a biological antigen presentation assay. The biophysical behaviour of these analogues in model membranes was analysed using surface plasmon resonance studies and then binding correlated with T-cell function. Removal of any single hydrophobic amino acid between the two charged amino acids in core peptide (R, K) resulted in lower binding. Changing the overall net charge of core peptide, by removing either of the positively charged residues (R or K), had varying effects on peptide binding and IL-2 production. There was a direct correlation (ρ = 0.718) between peptide binding to model membranes and peptide ability to inhibit IL-2. Except for IL-2 inhibition, production of other T-cell cytokines such as GM-CSF, IFN-γ, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and T-cell antigen receptor alpha-chain was not detected using a fluorescent bead immunoassay. This study provides important structure-function relationships essential for further drug design.

Anti-arthritic effects of immunomodulatory peptide injected in joints.

OBJECTIVE: To examine the clinical and histological outcomes following intra-articular injection of an immunomodulatory peptide (CP) during an acute attack of adjuvant induced arthritis in rats. METHODS: Arthritis in rats was induced by injecting 0.1 mL of Mycobacterium Tuberculosis (MTB; 10mg/ml) intra-dermally at the base of the tail. After the onset of arthritis, affected ankle joints were injected with either 20 µl of water, dexamethasone (4 mg/ml), cyclosporine (0.38 mg; equivalent to 3mg/kg in humans), CP (60 mg/ml; 12.5 mg/ml; 6.25 mg/ml; 3.125 mg/ml) or a CP analogue (CPD; 6.25mg/ml). There were a minimum of 5 rats/group and intra-articular injection was given on two separate occasions following the onset of arthritis. Rat weight, paw thickness, paw-width, ankle width and arthritic joint count were measured. Affected joints were X-rayed and joint tissue taken for histology. RESULTS: Rats given dexamethasone and cyclosporine had a significant improvement in clinical outcomes compared to the control group. CP at the highest dose was significantly better compared to the water group and the clinical effects were dose dependent. Histology of the joints showed severe inflammation and destruction in the water treated group with minimal inflammatory effects in the dexamethasone and cyclosporine group. Joints injected with cyclosporine were characterized by extensive fibrosis. The joints from CP treated rats showed mild to moderate inflammation with histological features somewhere between the cyclosporine and water injected groups. CONCLUSION: This study demonstrates that CP given intra-articularly has antiarthritic effects and raises new possibilities for treatment.

99mTc-technetium labeling of antiarthritic peptides to evaluate homing and biodistribution at inflamed joints.

PURPOSE: The aim of this study was to investigate the biodistribution and localization of an anti-inflammatory nonapeptide coupled to synovial targeting peptide (HAP-1) in rat adjuvant-induced arthritis. PROCEDURE: N(ε)-functionalized histidine derivative was coupled to the N-terminus of core peptide (CP) and HAP-1 to allow coupling of (99m)Tc-tricarbonyl linker (Isolink). Synovial homing peptide HAP-1 was linked to CP through a peptide bond prior to labeling with (99m)Tc. Peptides were purified by high-performance liquid chromatography, characterized by mass spectrometry, radiolabeled and injected into normal and arthritic rats to determine biodistribution and localization. RESULTS: Gamma scintigraphy imaging showed that the biodistribution of all (99m)Tc-labeled peptides were higher in the arthritic joints compared with the normal nonarthritic joints, at all three time points (10 min, 1 h, 3 h postinjection) and attributed to increased blood flow to inflammatory sites. HAP-1 and CP-HAP-1 showed a greater uptake and localization to arthritic joints compared with controls. There was no difference in the physiological biodistribution of these agents in the heart, kidneys and the bladder. CONCLUSIONS: This study highlights the versatility of using the His derivative linker for (99m)Tc tagging of a variety of peptides. It also demonstrates greater peptide localization and thereby bioavailability of therapeutic peptides to inflamed joints following specific conjugation to homing peptides. The ability to localize peptide/drugs to inflamed synovium has important therapeutic implications.

NMR study of the structure and self-association of Core peptide in aqueous solution and DPC micelles.

Core peptide is a hydrophobic peptide derived from the T-cell antigen receptor-alpha chain (TCR-alpha) transmembrane region with therapeutic potential. The mechanism by which the peptide inserts into the membrane, including any requirements to change conformational or association states during the insertion, is unclear. Here, the self-association and secondary structure of Core peptide in aqueous solution and in dodecylphosphocholine (DPC) micelles were examined using various nuclear magnetic resonance (NMR) techniques. NMR diffusion measurements were performed on 0.05, 1, and 5 mM Core peptide in D2O. These samples had pH values varying from 3 to 4. A constant measured diffusion coefficient of 2 X 10(-10) m2 s(-1) was observed in these samples indicating that Core peptide was monomeric. Multidimensional NMR experiments (i.e., TOCSY and NOESY) revealed the formation of beta-strands in water at low pH and random coil in DPC micelles. The results of this study reveal that at relatively low pH, the insertion mechanism must involve Core peptide in the monomeric state but it undergoes a conformational transition during membrane insertion.

T-cell antigen receptor (TCR) transmembrane peptides: A new paradigm for the treatment of autoimmune diseases.

Cell surface membranes are generally considered as inert and hydrophobic providing a stable physical barrier that anchor proteins and maintain cellular homeostasis between the intra- and the extra-cellular environment. The integral proteins that transverse membranes do so once or multiple times and can function alone or as part of a larger complex. Far from being inert, there is a multiplicity of biophysical factors that drive protein-protein and protein-lipid interactions within membranes that are being increasingly recognised as very important for cellular function. Unravelling these "hot-spots" on the contact surface of transmembrane (TM) proteins and targeting peptides to these sites to interrupt the cohesive interaction between the proteins provides both an enormous challenge and a huge therapeutic potential that as yet remains unrecognized. Indeed, with biopharmaceutical research on the rise, TM peptides may prove a useful innovation. Using the T-cell antigen receptor (TCR) as a model system of multi-subunits interacting at the TM via electrostatic charges the potential for peptides as therapeutic agents to interfere with normal immune responses is discussed. The principles of such can be extended to other similar receptor systems including those involved in cancer or infection.

Therapeutic application of transmembrane T and natural killer cell receptor peptides.

Autoimmune diseases primarily mediated by T-cells effect a significant proportion of the population and include common and distressing conditions such as diabetes, multiple clerosis, inflammatory bowel disease, skin diseases and arthritis. Current treatments are restrictive in terms of range of options and side-effect profiles and new drugs and new approaches are always eagerly sought. With the T-cell antigen receptor (TCR) as a model system we have identified a new approach to inhibit T-cell activation. By means of peptides derived from the transmembrane TCR-alpha chain region we have shown that T-cells, the major effector cells of disease, can be inhibited in vitro and the immune responses leading to disease ameliorated in animal models. The exact molecular mechanism of peptide action is still uncertain and assumed to involve a disturbance in transmembrane protein-protein interactions mediated by amino acid charges that disrupt normal signaling pathways. This chapter summarizes the results to date ofTCR core peptide (CP); the most effective peptide noted so far, in terms of function, behavior in membranes and future development and application as a therapeutic agent. The lessons learned from this model can be applied to other multi-subunit receptors that serve critical cellular functions and open new doors for drug design, development and application.

Kinetic and conformational properties of a novel T-cell antigen receptor transmembrane peptide in model membranes.

Core peptide (CP; GLRILLLKV) is a 9-amino acid peptide derived from the transmembrane sequence of the T-cell antigen receptor (TCR) alpha-subunit. CP inhibits T-cell activation both in vitro and in vivo by disruption of the TCR at the membrane level. To elucidate CP interactions with lipids, surface plasmon resonance (SPR) and circular dichroism (CD) were used to examine CP binding and secondary structure in the presence of either the anionic dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), or the zwitterionic dimyristoyl-L-alpha-phoshatidyl choline (DMPC). Using lipid monolayers and bilayers, SPR experiments demonstrated that irreversible peptide-lipid binding required the hydrophobic interior provided by a membrane bilayer. The importance of electrostatic interactions between CP and phospholipids was highlighted on lipid monolayers as CP bound reversibly to anionic DMPG monolayers, with no detectable binding observed on neutral DMPC monolayers.CD revealed a dose-dependent conformational change of CP from a dominantly random coil structure to that of beta-structure as the concentration of lipid increased relative to CP. This occurred only in the presence of the anionic DMPG at a lipid : peptide molar ratio of 1.6:1 as no conformational change was observed when the zwitterionic DMPC was tested up to a lipid : peptide ratio of 8.4 : 1.

T-cell antigen receptor assembly and cell surface expression is not affected by treatment with T-cell antigen receptor-alpha chain transmembrane Peptide.

A synthetic peptide termed core peptide (CP), which corresponds to a specific sequence of the TCR-alpha chain transmembrane domain, is known to inhibit IL-2 production in antigen stimulated T-cells. The molecular mechanism of the TCR inhibition is not known. This study examined the effects of CP on TCR subunit assembly and TCR cell surface expression in vitro. Co-transfection experiments between TCR-alpha and CD3-delta using COS-7 cells, and the interaction between TCR-alpha and the CD3 proteins in a T-cell line (2B4) were analysed after incubation with CP or its conjugates. Results indicate that CP co-precipitates with CD3-delta and CD3-epsilon in vitro, without any effect on TCR-alpha/CD3-delta dimerisation or TCR multisubunit assembly and cell surface expression.