BackgroundScleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.Methodology and FindingsWe analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of ‘intrinsic’ genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.Conclusions and SignificanceGenome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

The terminology for human papillomavirus (HPV)–associated squamous lesions of the lower anogenital tract has a long history marked by disparate diagnostic terms derived from multiple specialties. It often does not reflect current knowledge of HPV biology and pathogenesis. A consensus process was convened to recommend terminology unified across lower anogenital sites. The goal was to create a histopathologic nomenclature system that reflects current knowledge of HPV biology, optimally uses available biomarkers, and facilitates clear communication across different medical specialties. The Lower Anogenital Squamous Terminology (LAST) Project was cosponsored by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology and included 5 working groups; 3 work groups performed comprehensive literature reviews and developed draft recommendations. Another work group provided the historical background and the fifth will continue to foster implementation of the LAST recommendations. After an open comment period, the draft recommendations were presented at a consensus conference attended by LAST work group members, advisors, and representatives from 35 stakeholder organizations including professional societies and government agencies. Recommendations were finalized and voted on at the consensus meeting. The final, approved recommendations standardize biologically relevant histopathologic terminology for HPV-associated squamous intraepithelial lesions and superficially invasive squamous carcinomas across all lower anogenital tract sites and detail the appropriate use of specific biomarkers to clarify histologic interpretations and enhance diagnostic accuracy. A plan for disseminating and monitoring recommendation implementation in the practicing community was also developed. The implemented recommendations will facilitate communication between pathologists and their clinical colleagues and improve accuracy of histologic diagnosis with the ultimate goal of providing optimal patient care.

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20 ؉ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.

Activating mutations in the neuroblastoma rat sarcoma viral oncogene homolog (NRAS) gene are common genetic events in malignant melanoma being found in 15-25% of cases. NRAS is thought to activate both mitogen activated protein kinase (MAPK) and PI3K signaling in melanoma cells. We studied the influence of different components on the MAP/extracellular signal-regulated (ERK) kinase (MEK) and PI3K/mammalian target of rapamycin (mTOR)-signaling cascade in NRAS mutant melanoma cells. In general, these cells were more sensitive to MEK inhibition compared with inhibition in the PI3K/ mTOR cascade. Combined targeting of MEK and PI3K was superior to MEK and mTOR 1,2 inhibition in all NRAS mutant melanoma cell lines tested, suggesting that PI3K signaling is more important for cell survival in NRAS mutant melanoma when MEK is inhibited. However, targeting of PI3K/mTOR 1,2 in combination with MEK inhibitors is necessary to effectively abolish growth of NRAS mutant melanoma cells in vitro and regress xenografted NRAS mutant melanoma. Furthermore, we showed that MEK and PI3K/mTOR 1,2 inhibition is synergistic. Expression analysis confirms that combined MEK and PI3K/mTOR 1,2 inhibition predominantly influences genes in the rat sarcoma (RAS) pathway and growth factor receptor pathways, which signal through MEK/ERK and PI3K/mTOR, respectively. Our results suggest that combined targeting of the MEK/ERK and PI3K/mTOR pathways has antitumor activity and might serve as a therapeutic option in the treatment of NRAS mutant melanoma, for which there are currently no effective therapies.O ncogenic mutations in codons 12, 13, or 61 of the rat sarcoma (RAS) family of small GTPases, Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma RAS viral oncogene homolog (NRAS) occur in approximately one-third of all human cancers with NRAS mutations found in about 15-20% of melanomas (1-7). Mutated RAS proteins activate signaling pathways that promote the cell division cycle and cell growth and suppress apoptosis. Small interfering RNA (siRNA)-mediated depletion of NRAS in melanoma cell lines inhibits proliferation and renders cells sensitive to chemotherapy, making mutant NRAS and its signaling effectors relevant targets for melanoma therapy (8, 9). Efforts at developing therapeutics that inhibit mutant RAS directly have so far not been successful. The high affinity of RAS for GTP and the high concentrations of GTP intracellularly has meant that the identification of small molecules, which selectively prevent accumulation of RAS-GTP, has not been possible (10). Targeting mutant NRAS with siRNA is still limited to preclinical models because of the significant challenge in delivering antisense oligonucleotides in vivo. The response of NRAS mutant melanoma and other melanomas to various chemotherapeutic regiments has been very scarce with only 6% of patients responding (11). Alternatively, farnesyltransferase inhibitors (FTIs) were thought to inhibit RAS activation by blocking farnesy...

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