Skin Punch Biopsy Samples Research Articles

Establishment of Primary Adult Skin Fibroblast Cell Lines from African Savanna Elephants (Loxodonta africana).

Following population declines of the African savanna elephant (Loxodonta africana) across the African continent, the establishment of primary cell lines of endangered wildlife species is paramount for the preservation of their genetic resources. In addition, it allows molecular and functional studies on the cancer suppression mechanisms of elephants, which have previously been linked to a redundancy of tumor suppressor gene TP53. This methodology describes the establishment of primary elephant dermal fibroblast (EDF) cell lines from skin punch biopsy samples (diameter: ±4 mm) of African savanna elephants (n = 4, 14-35 years). The applied tissue collection technique is minimally invasive and paves the way for future remote biopsy darting. On average, the first explant outgrowth was observed after 15.75 ± 6.30 days. The average doubling time (Td) was 93.02 ± 16.94 h and 52.39 ± 0.46 h at passage 1 and 4, respectively. Metaphase spreads confirmed the diploid number of 56 chromosomes. The successful establishment of EDF cell lines allows for future elephant cell characterization studies and for research on the cancer resistance mechanisms of elephants, which can be harnessed for human cancer prevention and treatment and contributes to the conservation of their genetic material.

The cannabinoid receptors system in horses: Tissue distribution and cellular identification in skin.

BackgroundThe endocannabinoid system (ECS) is composed of cannabinoid receptors type 1 (CBR1) and type 2 (CBR2), cannabinoid‐based ligands (endogenous chemically synthesized phytocannabinoids), and endogenous enzymes controlling their concentrations. Cannabinoid receptors (CBRs) have been identified in invertebrates and in almost all vertebrate species in the central and peripheral nervous system as well as in immune cells, where they control neuroimmune homeostasis. In humans, rodents, dogs, and cats, CBRs expression has been confirmed in the skin, and their expression and tissue distribution become disordered in pathological conditions. Cannabinoid receptors may be a possible therapeutic target in skin diseases.ObjectivesTo characterize the distribution and cellular expression of CBRs in the skin of horses under normal conditions.AnimalsFifteen healthy horses.MethodsUsing full‐thickness skin punch biopsy samples, skin‐derived primary epidermal keratinocytes and dermal‐derived cells, we performed analysis of Cnr1 and Cnr2 genes using real‐time PCR and CBR1 and CBR2 protein expression by confocal microscopy and Western blotting.ResultsNormal equine skin, including equine epidermal keratinocytes and dermal fibroblast‐like cells, all exhibited constant gene and protein expression of CBRs.Conclusions and Clinical ImportanceOur results represent a starting point for developing and translating new veterinary medicine‐based pharmacotherapies using ECS as a possible target.

Determination of Filaggrin Gene Single Nucleotides Polymorphisms in Patients with Atopic Dermatitis

Objectives: The protein, filaggrin, is important in barrier function and epidermal differentiation facilitation. Filaggrin gene (FLG) mutations have been identified as the cause of ichthyosis vulgaris (IV), and certain mutations have been associated with atopic dermatitis (AD). We aimed to investigate genetic polymorphism of FLG in Mongolian AD patients. Methods: FLG mutations were determined using sequence analysis in 46 AD patients and 12 IV patients. Severity of AD was assessed using the Scoring Atopic Dermatitis (SCORAD) index. Allergen specific IgE were determined from serum. Filaggrin expression in skin punch biopsy samples of AD patients was investigated using immunohistochemistry (IHC). Results: Several single nucleotides polymorphisms (SNPs) (1150C>T, 1741A>T, 1791C>T, 2181C>G, 2191A>G, and 2263G>A) were demonstrated in AD patients using sequence analysis. Total IgE levels were significantly associated with age (p=0.03) and duration of disease (p=0.02). Presence of SNPs and mixed allergen specific IgE was significantly correlated (p=0.02); 2 SNPs were significantly associated with food allergen specific IgE levels (p=0.009). 2263G>A SNP was significantly correlated with food allergen specific IgE (p=0.003) and a history of atopic diseases (p=0.03). Conclusion: New mutations or genetic polymorphisms with ethnic characteristics may be detected among Mongolians.

A phase 1 dose-escalation and expansion study of binimetinib (MEK162), a potent and selective oral MEK1/2 inhibitor

Background:Binimetinib (MEK162; ARRY-438162) is a potent and selective oral MEK 1/2 inhibitor. This phase 1 study determined the maximum tolerated dose (MTD), safety, pharmacokinetic and pharmacodynamic profiles, and preliminary anti-tumour activity of binimetinib in patients with advanced solid tumours, with expansion cohorts of patients with biliary cancer or KRAS- or BRAF-mutant colorectal cancer.Methods:Binimetinib was administered twice daily. Expansion cohorts were enroled after MTD determination following a 3+3 dose-escalation design. Pharmacokinetic properties were determined from plasma samples. Tumour samples were assessed for mutations in RAS, RAF, and other relevant genes. Pharmacodynamic properties were evaluated in serum and skin punch biopsy samples.Results:Ninety-three patients received binimetinib (dose-escalation phase, 19; expansion, 74). The MTD was 60 mg twice daily, with dose-limiting adverse events (AEs) of dermatitis acneiform and chorioretinopathy. The dose for expansion patients was subsequently decreased to 45 mg twice daily because of the frequency of treatment-related ocular toxicity at the MTD. Common AEs across all dose levels included rash (81%), nausea (56%), vomiting (52%), diarrhoea (51%), peripheral oedema (46%), and fatigue (43%); most were grade 1/2. Dose-proportional increases in binimetinib exposure were observed and target inhibition was demonstrated in serum and skin punch biopsy samples. Three patients with biliary cancer had objective responses (one complete and two partial).Conclusions:Binimetinib demonstrated a manageable safety profile, target inhibition, and dose-proportional exposure. The 45 mg twice daily dose was identified as the recommended phase 2 dose. The three objective responses in biliary cancer patients are encouraging and support further evaluation in this population.

Three years of retrospective evaluation of skin biopsy results in childhood.

OBJECTIVE:In our study, we aimed to evaluate retrospectively histopathological diagnoses of children based on their skin biopsies, and determine the prevalence of the disease in question.METHODS:Among patients who applied to Medeniyet University Goztepe Training and Research Hospital between January 2011 and February 2014, we retrospectively evaluated demographic data and histopathological diagnoses of patients aged between 0-17 years whose skin punch biopsy samples were obtained.RESULTS:The study population (n=566) with skin biopsy results consisted of 287 (50.7%) male, and 279 (49.2%) female patients with a mean age of 10.04±4.84 years. Biopsy materials were obtained from the various age groups as follows: 0-2 years, n=31 (5.4%); 3-5 years, n=67 (11.8%) 6-11 years, n=165 (29.1%), and 12-17 years, n=303 (53.5%). Among all age groups, we took biopsies mostly from patients with noninfectious erythematous squamous (24%) and vascular (21.2%) diseases. The determined histopathological diagnoses were leukocytoclasis vasculitis (18.9%), psoriasis (7.4%), melanocytic nevus (5.4%), and contact dermatitis (5.1%) respectively.CONCLUSION:We determined that skin punch biopsy examinations were done most frequently during adolescence and are mostly necessary for diagnosis of erythematous squamous and vascular diseases. If clinical evidence-based prevalence studies are supported with histopathological data, more significant results can be obtained.

Genomic Profiling of Pre-Clinical Models of Inflammatory Breast Cancer Identifies a Signature of Epithelial Plasticity and Suppression of TGFβ Signaling

Abstract Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

P4-09-08: Secretory Leukocyte Protease Inhibitor as a Differential Diagnostic Biomarker of Tumor Emboli in Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of locally advanced breast cancer. Pathologically, IBC is characterized by the presence of nests of cells, defined as tumor emboli, that undergo lymphovascular invasion into the skin and chest wall and represent the metastatic lesion of IBC. Due to the distinct presentation of IBC, it is often misdiagnosed due to a lack of defined biomarkers that are specific for tumor emboli in biopsy samples of IBC patients. To date, the only validated marker that has shown to consistently identify IBC tumor emboli is the surface glycoprotein E-cadherin. Materials and Methods: With the goal of identifying genes specifically expressed by IBC tumor emboli, the present studies performed whole transcriptome analysis of tumor emboli from skin punch biopsy samples from IBC patients and tumor emboli from the Mary-X pre-clinical model of IBC which were isolated using laser capture microdissection. Results: Secretory leukocyte peptidase inhibitor (SLPI) was a gene identified as being highly expressed by tumor emboli. This gene was previously identified as a metastasis related gene, has been reported to be associated with poor prognosis in serous ovarian carcinoma and is implicated in mediating resistance to paclitaxel. Immunofluorescence staining and confocal microscopy revealed that SLPI is a robust marker of tumor emboli in skin punch biopsies from IBC patient and in Mary-X tumor emboli. SLPI differentially delineates between IBC tumor emboli and hair follicles present in the skin, which also stain with E-cadherin. An additional marker, Cytokeratin 15 (CK15), can be used in combination with SLPI to differentially identify the components of the skin that are CK15 positive, and IBC tumor emboli that reside within the skin, which are CK15 negative. Use of podoplanin antibodies, which stain lymphovascular endothelium, demonstrate that IBC tumor emboli that express SLPI protein, are completely encircled within the dermal lymphovasculature. Conclusions: These studies are among the first to identify SLPI as a potential biomarker of tumor emboli in skin punch biopsy samples from IBC patients and in pre-clinical models of IBC. Studies are ongoing to establish the molecular function and role of SLPI in mediating the survival and invasion of IBC tumor emboli. Collectively, these studies are the first to identify SLPI as a potential biomarker of IBC tumor emboli in skin punch biopsy samples. In addition to serving as a potential biomarker of tumor emboli, SLPI may also serve as a therapeutic target for development of approaches to eradicate IBC tumor emboli. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-08.

Abstract PR-4: Gene amplification of anaplastic lymphoma kinase in inflammatory breast cancer.

Abstract Inflammatory breast cancer (IBC) is the most lethal form of breast cancer associated with a 40% 5-year survival in spite of appropriate multidisciplinary care, suggesting resistance to current therapies. IBC serves as a paradigm for tumors that have a program of accelerated metastasis and exhibit characteristics of an mesenchymal-epithelial transition, with robust expression of E-cadherin, and a lack of expression of ZEB1 and TFG beta. Although a few genes such as RHO-C GTPase have been shown to be expressed in IBC, to date, there has been no primary genetic alteration linked to the metastasis observed in IBC patients. Methods: Using reverse phase proteomics, whole transcriptome analysis, fluorescence in situ hybridization, real time PCR and western blotting, we designed a discovery strategy to identify the signaling pathways that are activated in IBC, using tissue samples from primary IBC patients (core and skin punch biopsy samples), established IBC cell lines and newly developed new IBC cell lines and xenograft models, designated as FC-IBC-01, and FC-IBC-02, that were established from tumor cells isolated from IBC patients who had developed pleural effusion under an IRB-approved protocol. Results: Activation of phospho-ALK-tyrosine 1586 as a common occurrence in IBC cell lines at a level similar to that found in non-small cell lung cancer (NSCLC) patients with ALK genetic abnormalities. Our studies revealed first time evidence for amplification (3–7 fold) of the receptor tyrosine kinase ALK in 13/15 IBC patient tumor specimens thus far examined by fluorescence in situ hybridization (FISH) and in 66% of IBC cell lines, including Mary-X, and the newly developed FC-IBC01, and FC-IBC02 xenograft models. Interestingly, ALK amplification is not detected in SUM149, SUM190 and KPL-4 cell lines which are the most commonly studied IBC cell lines. Tumor cells isolated from IBC patients with pleural effusion whose tumor was ALK amplified were very sensitive to the anti-proliferative effects of the small molecule cMet/ALK inhibitor crizotinib [PF-02341066] at submicromolar levels (IC50=0.88 nM) but were completely resistant to paclitaxel. Thus far we have not found EML4-ALK or NPM-ALK translocation in IBC cell lines nor have we identified ALK mutations at sites reported to be present large cell anaplastic lymphoma, NSCLC, or neuroblastoma. Analysis of signaling pathway maps reveal that ALK is a central regulator of tumor growth, survival and metastatic progression through its association to multiple downstream effectors including PI3K/AKT, mTOR, and JAK/STAT3. In contrast, cMet is not differentially expressed or phosphorylated in pre-clinical models of IBC. We are currently assessing the activities of the combination of PI3K/mTOR inhibitors and small molecule ALK inhibitors to determine synergistic effects. Discussion: These studies are the first to suggest that ALK may be a primary molecular driver in the rapid metastasis exhibited in IBC and also suggests that proteomics based approaches are critically important to incorporate into studies to identify targets for development of effective treatments for IBC patients. Furthermore, these studies provide a critical path to clinical evaluation of ALK inhibitors in IBC patients with disease progression for whom there are few therapeutic options. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-4.

Skin Concentrations and Pharmacokinetics of Posaconazole after Oral Administration

A randomized, single-center, open-label study of posaconazole (POS) was performed to determine the concentration of POS in the skin of 30 healthy adult human subjects receiving 400 mg POS oral suspension twice daily for 8 days with a high-fat meal. Blood samples for plasma POS level determination were collected at prespecified times on day 1 and day 8. From each subject, two 4-mm skin punch biopsy samples were obtained, one immediately before or after both the first and last doses of POS. A MIC(90) value of 250 ng/ml, which encompasses the majority of common dermatophytes, was used to calculate the time above the MIC(90) in plasma and skin. On days 1 and 8, POS attained peak plasma concentrations at median times of 8 and 5 h, respectively. On days 1 and 8, POS peak skin concentrations were attained at 12 and 3 h, respectively; peak skin concentrations were produced from a single composite profile. On day 8, POS concentrations in skin and plasma for the entire dosing interval were severalfold higher than the MIC(90). POS dosed at 400 mg twice daily per os was well tolerated in healthy subjects. Two subjects reported increased alanine aminotransferase (ALT) levels. The findings of this study demonstrate adequate skin penetration and have certain implications for the treatment of dermatophytic skin and nail infections.

Reduced Innervation of Sweat Glands in Diabetic Neuropathy

The aim of this study was to evaluate a novel method for quantification of sweat gland innervation and to investigate whether the values obtained correlate with severity of diabetic neuropathy. Investigators obtained skin-punch biopsy samples from the thigh and lower leg of 30 patients with diabetes and mild neuropathy and 64 healthy subjects. After immunostaining the samples for sweat glands and for nerve fibers, …

PCR-Based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-Day postinfection period.

Borrelia burgdorferi infection in beagle dogs was studied quantitatively with skin punch biopsy samples and blood samples collected at 4- and 2-week intervals, respectively, over a 500-day period. Thereafter, 25 tissue samples of each dog were collected for further analysis. Starting at day 120 after tick challenge, 12 dogs were treated with antibiotics (azithromycin, ceftriaxone, or doxycycline) for 30 consecutive days. Four dogs received no antibiotic therapy. Quantification of B. burgdorferi DNA was done with an ABI Prism 7700 Sequence Detection System with oligonucleotide primers and a fluorescence-labeled probe designed to specifically amplify a fragment of the ospA gene of B. burgdorferi strain N40. All 16 dogs became infected with B. burgdorferi after tick challenge. In skin biopsy samples, spirochete numbers peaked at day 60 postinfection (<1.5 x 10(6) organisms per 100 microgram of extracted DNA), at the same time when clinical signs of arthritis developed in 11 of 16 dogs, and decreased to almost undetectable levels during the following 6 months. The number of B. burgdorferi organisms detected in skin biopsy samples was inversely correlated with the antibody levels measured by enzyme-linked immunosorbent assay. Antibiotic treatment reduced the amount of detectable spirochete DNA in skin tissue by a factor of 1,000 or more. At the end of the experiment, B. burgdorferi DNA was detectable at low levels (10(2) to 10(4) organisms per 100 microgram of extracted DNA) in multiple tissue samples regardless of treatment. However, more tissue samples of untreated dogs than of antibiotic-treated dogs were positive, and tissue samples of untreated dogs also were positive by culture. Only 1.6% of 576 blood samples of all dogs were positive for B. burgdorferi by PCR.

In Vitro Induction of Human Skin Ornithine Decarboxylase by the Tumor Promoter 12-&lt;italic&gt;O&lt;/italic&gt;-Tetradecanoylphorbol-13-Acetate&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

A method was developed for the in vitro induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) of ornithine decarboxylase (ODC) activity in human skin punch biopsy samples. Addition of TPA to 1 ml serum-free minimum essential medium containing a single 3-mm human skin punch biopsy sample obtained from a surgical specimen resulted in an induction of ODC activity with a peak activity at 5 hours after TPA addition. In vitro induction of human epidermal ODC activity was dependent on the TPA concentration in the medium; about a twofold increase in ODC activity was observed 6 hours after the addition of 0.1 microM TPA, and about a fivefold increase in ODC activity was observed with 1 microM TPA. TPA also caused about a fivefold to sixfold increase in ODC activity in 3-mm skin punch biopsy samples from healthy volunteers. Human skin punch biopsy samples remained responsive to TPA induction of ODC activity even when stored in serum-free medium at 4 degrees C for 24 hours. A similar degree of induction of ODC activity by TPA was observed whether whole unfractionated human epidermis or a soluble epidermal extract was used for ODC assays. Increased ODC activity was the result of the increase in enzymatically active ODC protein, quantitated by a [3H]difluoromethylornithine-binding assay. Thus human skin, like mouse skin, is responsive to TPA for ODC induction.

Erythromelalgia Caused by Platelet-Mediated Arteriolar Inflammation and Thrombosis in Thrombocythemia

Erythromelalgia was the presenting symptom in 26 of 40 patients with thrombocythemia in its primary form or when associated with polycythemia vera. The localized painful burning, redness, and warm congestion in the extremities could be accurately documented with thermography. Skin punch biopsy samples taken from the affected areas showed typical arteriolar inflammation, fibromuscular intima proliferation, and thrombotic occlusions. Erythromelalgia often progressed to ischemic acrocyanosis or necrosis in toes or fingers. Complete relief of pain and restoration of microvascular circulation disturbances was obtained with the cyclo-oxygenase inhibitors aspirin and indomethacin, but not with sodium-salicylate or the platelet inhibitors dipyridamole, sulfinpyrazone, ticlopidine, and dazoxiben. The erythromelalgia was alleviated during busulfan-induced remissions of thrombocythemia and its recurrence coincided with relapsing thrombocythemia. These observations suggest a causal relationship between erythromelalgia and thrombocythemia, in which platelet-mediated inflammatory and occlusive arteriolar changes play a part in the etiology of erythromelalgia.