Comparative microarray analysis of microRNA expression profiles in primary cutaneous malignant melanoma, cutaneous malignant melanoma metastases, and benign melanocytic nevi

Perturbations in microRNA (miRNA) expression profiles have been reported for cutaneous malignant melanoma (CMM) predominantly when examined in cell lines. Despite the rapidly growing number of newly discovered human miRNA sequences, the availability of up-to-date miRNA expression profiles for clinical samples of primary cutaneous malignant melanoma (PCMM), cutaneous malignant melanoma metastases (CMMM), and benign melanocytic nevi (BMN) is limited. Specimens excised from the center of tumors (lesional) from patients with PCMM (n=9), CMMM (n=4), or BMN (n=8) were obtained during surgery. An exploratory microarray analysis was performed by miRNA expression profiling based on Agilent platform screening for 1205 human miRNAs. The results from the microarray analysis were validated by TaqMan quantitative real-time polymerase chain reaction. In addition to several miRNAs previously known to be associated with CMM, 19 unidentified miRNA candidates were found to be dysregulated in CMM patient samples. Among the 19 novel miRNA candidates, the genes hsa-miR-22, hsa-miR-130b, hsa-miR-146b-5p, hsa-miR-223, hsa-miR-301a, hsa-miR-484, hsa-miR-663, hsa-miR-720, hsa-miR-1260, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-3663-3p, hsa-miR-4281, and hsa-miR-4286 were upregulated, and the genes hsa-miR-24-1*, hsa-miR-26a, hsa-miR-4291, hsa-miR-4317, and hsa-miR-4324 were downregulated. The results of this study partially confirm previous CMM miRNA profiling studies identifying miRNAs that are dysregulated in CMM. However, we report several novel miRNA candidates in CMM tumors; these miRNA sequences require further validation and functional analysis to evaluate whether they play a role in the pathogenesis of CMM.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

Comparative analysis of volatile metabolomics signals from melanoma and benign skin: a pilot study

The analysis of volatile organic compounds (VOC) as biomarkers of cancer is both promising and challenging. In this pilot study, we used an untargeted approach to compare volatile metabolomic signatures of melanoma and matched control non-neoplastic skin from the same patient. VOC from fresh (non-fixed) biopsied tissue were collected using the headspace solid phase micro extraction method (HS SPME) and analyzed by gas chromatography and mass spectrometry (GCMS). We applied the XCMS analysis platform and MetaboAnalyst software to reveal many differentially expressed metabolic features. Our analysis revealed increased levels of lauric acid (C12:0) and palmitic acid (C16:0) in melanoma. The identity of these compounds was confirmed by comparison with chemical standards. Increased levels of these fatty acids are likely to be a consequence of up-regulated de novo lipid synthesis, a known characteristic of cancer. Increased oxidative stress is likely to cause an additional increase in lauric acid. Implementation of this study design on larger number of cases will be necessary for the future metabolomics biomarker discovery applications.     

Circulating and local nuclear expression of survivin and fibulin-3 genes in discriminating benign from malignant respiratory diseases: correlation analysis

Survivin is an inhibitor of apoptosis as well as a promoter of cell proliferation. Fibulin-3 is a matrix glycoprotein that displays potential for tumor suppression or propagation. The present study aimed to validate the expression levels of survivin and fibulin-3 in benign and malignant respiratory diseases. This case–control study included 219 patients categorized into five groups. Group A included 63 patients with lung cancer, group B included 63 patients with various benign lung diseases, group D included 45 patients with malignant pleural mesothelioma (MPM), and group E included 48 patients with various benign pleural diseases. Group C included 60 healthy individuals (control group). Serum survivin and fibulin-3 levels were measured by ELISA, whereas their nuclear expressions in the lung and pleura were assessed via Western blot analysis. The results showed significantly higher survivin serum levels and significantly lower fibulin-3 levels in group A compared with in group B and controls (P<0.001). There were significantly higher serum levels of survivin and fibulin-3 in group D compared with in group E and controls (P<0.001), consistent with observed nuclear survivin and fibulin-3 expression levels. Fibulin-3 was determined to have higher value than survivin in discriminating lung cancer from MPM (P<0.05). Survivin and fibulin-3 could be useful diagnostic markers for lung and pleural cancers, and fibulin-3 expression was particularly useful in differentiating lung cancer from MPM.     

Estimating a multivariate familial correlation using joint models for canonical correlations: application to memory score analysis from familial Hispanic Alzheimer's disease study

Summary .  Analysis of multiple traits can provide additional information beyond analysis of a single trait, allowing better understanding of the underlying genetic mechanism of a common disease. To accommodate multiple traits in familial correlation analysis adjusting for confounders, we develop a regression model for canonical correlation parameters and propose joint modeling along with mean and scale parameters. The proposed method is more powerful than the regression method modeling pairwise correlations because it captures familial aggregation manifested in multiple traits through maximum canonical correlation.     

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.     

Biochemical correlation of Raman spectra of normal,benign and malignant breast tissues: A spectral deconvolution study

The aim of this study was to understand and correlate spectral features and biochemical changes in normal, fibroadenoma and infiltrating ductal carcinoma of breast tissues using Raman spectra that were part of the spectroscopic models developed and evaluated by us earlier. Spectra were subjected to curve fitting and intensities plots of resultant curve resolved bands were computed. This study has revealed that fat (1301 and 1440 cm^?1), collagen (1246, 1271, and 1671 cm^?1) and DNA (1340 and 1480 cm^?1) bands have strong presence in normal, benign and malignant breast tissues, respectively. Intensity plots of various combinations of curved resolved bands were also explored to classify tissue types. Combinations of fat (1301 cm^?1) and collagen (1246, 1271, and 1671 cm^?1)/amide I; DNA (1340 cm^?1) and fat (1301 cm^?1); collagen (1271 cm^?1) and DNA (1480 cm^?1) are found to be good discriminating parameters. These results are in tune with findings of earlier studies carried out on western population as well as our molecular biological understanding of normal tissues and neoplastic processes. Thus the finding of this study further demonstrates the efficacy Raman spectroscopic approaches in diagnostic applications as well as in understanding molecular phenomenon in breast cancers. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 539–546, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Atmospheric conditions, lunar phases, and childbirth: a multivariate analysis

Our objective was to assess extrinsic influences upon childbirth. In a cohort of 1,826 days containing 17,417 childbirths among them 13,252 spontaneous labor admissions, we studied the influence of environment upon the high incidence of labor (defined by 75th percentile or higher), analyzed by logistic regression. The predictors of high labor admission included increases in outdoor temperature (odds ratio: 1.742, P = 0.045, 95%CI: 1.011 to 3.001), and decreases in atmospheric pressure (odds ratio: 1.269, P = 0.029, 95%CI: 1.055 to 1.483). In contrast, increases in tidal range were associated with a lower probability of high admission (odds ratio: 0.762, P = 0.030, 95%CI: 0.515 to 0.999). Lunar phase was not a predictor of high labor admission (P = 0.339). Using multivariate analysis, increases in temperature and decreases in atmospheric pressure predicted high labor admission, and increases of tidal range, as a measurement of the lunar gravitational force, predicted a lower probability of high admission.     

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.     

Endemicity analysis,parsimony and biotic elements: a formal comparison using hypothetical distributions

There is as yet no general agreement regarding the most appropriate solution to the problem of identifying areas of endemism, not even in particular cases. In this study, we compared Endemicity Analysis (EA), Parsimony Analysis of Endemicity (PAE), and Biotic Elements Analysis (BE) based on their ability to identify hypothetical predefined patterns that represent nested, overlapping, and disjoint areas of endemism supported by species with different degrees of sympatry. We found that PAE performs poorly when applied to patterns that either overlap with each other or are supported by species with imperfect sympatry. BE exhibits a counterintuitive sensitivity to the degree of congruence among the distributions of endemic species, being unable to recognize areas of endemism supported by perfectly sympatric species. In contrast, in all cases examined we found that EA results in a high proportion of correctly identified distributional patterns. In addition to highlighting the strengths and limitations of these approaches, our results show how different methods can lead to seemingly conflicting conclusions and caution about the possibility of identifying distributional patterns that are merely methodological artefacts. © The Willi Hennig Society 2012.     

Rapid generation of region-specific genomic clones by chromosome microdissection: isolation of DNA from a region frequently deleted in malignant melanoma.

Malignant melanoma is frequently characterized by the deletion of the long arm of chromosome 6 (usually encompassing 6q16-q21). In an effort to saturate this region with DNA markers, microdissection and molecular cloning of DNA from banded human metaphases recent development of a novel chromosome microdissection scheme that omits microchemical manipulation of DNA. Microdissection was targeted on band 6q21. Direct PCR amplification of dissected DNA was first used as a probe in chromosomal in situ hybridization of normal metaphases to confirm the specificity of material excised for cloning. A genomic library of 20,000 clones, which is highly enriched for sequences encompassing 6q21, was then constructed. Clones from this library have been mapped against a human-rodent somatic cell hybrid mapping panel that divides chromosome 6 into seven regions, confirming the localization of probes within the target region. Direct PCR amplification of DNA excised by microdissection greatly simplifies and facilitates this chromosome band-specific cloning strategy. The isolation of microclones from this region of chromosome 6 should assist in establishing a physical map of the melanoma deletion region.     

Short-window spectral analysis using AMVAR and multitaper methods: a comparison

We compare two popular methods for estimating the power spectrum from short data windows, namely the adaptive multivariate autoregressive (AMVAR) method and the multitaper method. By analyzing a simulated signal (embedded in a background Ornstein–Uhlenbeck noise process) we demonstrate that the AMVAR method performs better at detecting short bursts of oscillations compared to the multitaper method. However, both methods are immune to jitter in the temporal location of the signal. We also show that coherence can still be detected in noisy bivariate time series data by the AMVAR method even if the individual power spectra fail to show any peaks. Finally, using data from two monkeys performing a visuomotor pattern discrimination task, we demonstrate that the AMVAR method is better able to determine the termination of the beta oscillations when compared to the multitaper method.     

Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors.

We have used a double-labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti-keratin antibodies were tested: KL1 recognizing Mr 55,000-57,000 keratins, and "anti-glandular epithelia," LE41, RGE-53, and LP2K specific for CK n. 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA-CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA-keratin-positive population allowed accurate DNA analysis without interference due to debris background and non-epithelial cells. Moreover, double-labelling using the CK19 antibody gave a better identification of near-diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P less than 0.001), KL1 (P less than 0.01), CK 8 (P less than 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.