Isaac M. Neuhaus

Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ∼75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

A high-resolution association mapping panel for the dissection of complex traits in mice.

Systems genetics relies on common genetic variants to elucidate biologic networks contributing to complex disease-related phenotypes. Mice are ideal model organisms for such approaches, but linkage analysis has been only modestly successful due to low mapping resolution. Association analysis in mice has the potential of much better resolution, but it is confounded by population structure and inadequate power to map traits that explain less than 10% of the variance, typical of mouse quantitative trait loci (QTL). We report a novel strategy for association mapping that combines classic inbred strains for mapping resolution and recombinant inbred strains for mapping power. Using a mixed model algorithm to correct for population structure, we validate the approach by mapping over 2500 cis-expression QTL with a resolution an order of magnitude narrower than traditional QTL analysis. We also report the fine mapping of metabolic traits such as plasma lipids. This resource, termed the Hybrid Mouse Diversity Panel, makes possible the integration of multiple data sets and should prove useful for systems-based approaches to complex traits and studies of gene-by-environment interactions.

Temporal Dissection of Tumorigenesis in Primary Cancers

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

Memory regulatory T cells reside in human skin.

Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, are a dynamic and heterogeneous population of cells that control immune responses and prevent autoimmunity. We recently identified a subset of Tregs in murine skin with properties typical of memory cells and defined this population as memory Tregs (mTregs). Due to the importance of these cells in regulating tissue inflammation in mice, we analyzed this cell population in humans and found that almost all Tregs in normal skin had an activated memory phenotype. Compared with mTregs in peripheral blood, cutaneous mTregs had unique cell surface marker expression and cytokine production. In normal human skin, mTregs preferentially localized to hair follicles and were more abundant in skin with high hair density. Sequence comparison of TCRs from conventional memory T helper cells and mTregs isolated from skin revealed little homology between the two cell populations, suggesting that they recognize different antigens. Under steady-state conditions, mTregs were nonmigratory and relatively unresponsive; however, in inflamed skin from psoriasis patients, mTregs expanded, were highly proliferative, and produced low levels of IL-17. Taken together, these results identify a subset of Tregs that stably resides in human skin and suggest that these cells are qualitatively defective in inflammatory skin disease.

Comparative Efficacy of Nonpurpuragenic Pulsed Dye Laser and Intense Pulsed Light for Erythematotelangiectatic Rosacea

BACKGROUND Erythematotelangiectatic (ET) rosacea is commonly treated with a variety of laser and light‐based systems. Although many have been used successfully, there are a limited number of comparative efficacy studies. OBJECTIVE To compare nonpurpuragenic pulsed dye laser (PDL) with intense pulsed light (IPL) treatment in the ability to reduce erythema, telangiectasia, and symptoms in patients with moderate facial ET rosacea. METHODS Twenty‐nine patients were enrolled in a randomized, controlled, single‐blind, split‐face trial with nonpurpuragenic treatment with PDL and IPL and untreated control. Three monthly treatment sessions were performed with initial PDL settings of 10‐mm spot size, 7 J/cm2, 6‐ms pulse duration and cryogen cooling, and initial IPL settings of 560‐nm filter, a pulse train of 2.4 and 6.0 ms in duration separated by a 15‐ms delay, and a starting fluence of 25 J/cm2. Evaluation measures included spectrophotometric erythema scores, blinded investigator grading, and patient assessment of severity and associated symptoms. RESULTS PDL and IPL resulted in significant reduction in cutaneous erythema, telangiectasia, and patient‐reported associated symptoms. No significant difference was noted between PDL and IPL treatment. CONCLUSION A series of nonpurpuragenic PDL and IPL treatments in ET rosacea was performed with similar efficacy and safety, and both modalities seem to be reasonable choices for the treatment of ET rosacea.

HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy

Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.

Dermatofibrosarcoma Protuberans: A Review of the Literature

Background Dermatofibrosarcoma protuberans (DFSP) is a rare soft tissue tumor that is locally aggressive and has traditionally had a high rate of recurrence after surgical intervention. Objective To review the current literature on DFSP and present its epidemiology, clinical features, histology, immunohistochemisty, tumor biology, prognosis, and treatment options. Materials and Methods Literature review using PubMed search for articles related to DFSP. Results DFSP is a slow‐growing tumor that often presents as a skin‐colored plaque on the trunk, although it may arise anywhere on the body. DFSP has a distinctive histologic appearance but can mimic other diseases, and immunhistochemical studies can be helpful in making the diagnosis of DFSP. Most tumors have a translocation between chromosomes 17 and 22. Surgery is the treatment of choice, in the form of Mohs micrographic surgery (MMS) or wide local excision (WLE), although imatinib has emerged as a promising treatment option for advanced disease. Conclusion For DFSP, MMS is associated with a lower rate of recurrence than WLE and is particularly useful for tumors on the head and neck. Imatinib is used in advanced disease and may take on a larger role in the treatment of DFSP in the future.

ASDS guidelines of care: injectable fillers.

S 1 1 5 Injectable soft tissue augmentation materials available for use in the United States have proliferated during the past 5 years. In addition to bovine collagen and autologous fat, physicians can now choose between numerous other options, including hyaluronic acid derivatives, poly-L-lactic acid, injectable calcium hydroxylapatite, injectable liquid silicone, polymethylmethacrylate microspheres, and human collagen. While these materials are generally safe and versatile, each has specific features that inform its best use. The guidelines presented herein are not intended to delineate the standard of care but rather to present how most injectors commonly use these materials in patients. These guidelines are a consensus document produced by the ASDS Guidelines of Care Task Force in conjunction with recognized experts in the field who have volunteered their time. While efforts have been made to maintain accuracy and timeliness at the point of submission, these guidelines will no doubt evolve over time and should be considered in the context of the patients’ individual needs. In the 1970s, research into collagen production led to a bovine formulation that could be placed in a syringe and injected. Because of bovine collagen’s temporary persistence, and requirement for skin testing, other fillers were developed and tested in Europe, Asia, and North America in the 1990s. Today, there are a number of fillers to choose from, each with its own strengths and drawbacks.

Genetic Architecture of Insulin Resistance in the Mouse

Insulin resistance (IR) is a complex trait with multiple genetic and environmental components. Confounded by large differences between the sexes, environment, and disease pathology, the genetic basis of IR has been difficult to dissect. Here we examine IR and related traits in a diverse population of more than 100 unique male and female inbred mouse strains after feeding a diet rich in fat and refined carbohydrates. Our results show dramatic variation in IR among strains of mice and widespread differences between sexes that are dependent on genotype. We uncover more than 15 genome-wide significant loci and validate a gene, Agpat5, associated with IR. We also integrate plasma metabolite levels and global gene expression from liver and adipose tissue to identify metabolite quantitative trait loci (mQTL) and expression QTL (eQTL), respectively. Our results provide a resource for analysis of interactions between diet, sex, and genetic background in IR.

Transcriptomic Responses to Sodium Chloride-Induced Osmotic Stress: A Study of Industrial Fed-Batch CHO Cell Cultures

The rapidly expanding market for monoclonal antibody and Fc‐fusion‐protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc‐fusion‐protein to hyperosmotic stress, a well‐known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132–145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine‐hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant‐protein productivity.