Bonnie E. Gould Rothberg

Activated Wnt/ß-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model

This study demonstrates that in malignant melanoma, elevated levels of nuclear ß-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of ß-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/ß-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear ß-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/ß-catenin signaling.

Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Tissue Biomarkers for Prognosis in Cutaneous Melanoma: A Systematic Review and Meta-analysis

In the clinical management of early-stage cutaneous melanoma, it is critical to determine which patients are cured by surgery alone and which should be treated with adjuvant therapy. To assist in this decision, many groups have made an effort to use molecular information. However, although there are hundreds of studies that have sought to assess the potential prognostic value of molecular markers in predicting the course of cutaneous melanoma, at this time, no molecular method to improve risk stratification is part of recommended clinical practice. To help understand this disconnect, we conducted a systematic review and meta-analysis of the published literature that reported immunohistochemistry-based protein biomarkers of melanoma outcome. Three parallel search strategies were applied to the PubMed database through January 15, 2008, to identify cohort studies that reported associations between immunohistochemical expression and survival outcomes in melanoma that conformed to the REMARK criteria. Of the 102 cohort studies, we identified only 37 manuscripts, collectively describing 87 assays on 62 distinct proteins, which met all inclusion criteria. Promising markers that emerged included melanoma cell adhesion molecule (MCAM)/MUC18 (all-cause mortality [ACM] hazard ratio [HR] = 16.34; 95% confidence interval [CI] = 3.80 to 70.28), matrix metalloproteinase-2 (melanoma-specific mortality [MSM] HR = 2.6; 95% CI = 1.32 to 5.07), Ki-67 (combined ACM HR = 2.66; 95% CI = 1.41 to 5.01), proliferating cell nuclear antigen (ACM HR = 2.27; 95% CI = 1.56 to 3.31), and p16/INK4A (ACM HR = 0.29; 95% CI = 0.10 to 0.83, MSM HR = 0.4; 95% CI = 0.24 to 0.67). We further noted incomplete adherence to the REMARK guidelines: 14 of 27 cohort studies that failed to adequately report their methods and nine studies that failed to either perform multivariable analyses or report their risk estimates were published since 2005.

Gestational weight gain and subsequent postpartum weight loss among young, low-income, ethnic minority women

OBJECTIVE Document weight change trajectories that lead to gestational weight gain or postpartum weight loss outside clinical recommendations established by the Institute of Medicine. STUDY DESIGN Women aged 14-25 receiving prenatal care and delivering singleton infants at term (n = 427). Medical record review and 4 structured interviews conducted: second and third trimester, 6- and 12-months postpartum. Longitudinal mixed modeling to evaluate weight change trajectories. RESULTS Only 22% of participants gained gestational weight within Institute of Medicine guidelines. There were 62% that exceeded maximum recommendations-more common among those overweight/obese (body mass index ≥25.0; P < .0001). 52% retained ≥10 lb 1-year postpartum. Increased weight gain and retention documented among smokers and women with pregnancy-induced hypertension; breastfeeding promoted postpartum weight loss (all P < .02). Body mass index by race interaction suggested healthier outcomes for Latinas (P = .02). CONCLUSION Excessive pregnancy weight gain and inadequate postpartum weight loss are highly prevalent among young low-income ethnic minority women. Pregnancy and postpartum are critical junctures for weight management interventions.

Melanoma Prognostic Model Using Tissue Microarrays and Genetic Algorithms

PURPOSE As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. METHODS Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. RESULTS Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than -0.052, p21(WAF1) nuclear compartment AQUA score of more than 12.98, p16(INK4A) ln(non-nuclear/nuclear AQUA score ratio) of < or = -0.083, beta-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of < or = 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. CONCLUSION This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy.

Biomarkers: The Useful and the Not So Useful—An Assessment of Molecular Prognostic Markers for Cutaneous Melanoma

Among individuals with localized (Stage I-II) melanoma, stratifying patients by a number of phenotypic variables (e.g., depth of invasion, ulceration) yields a wide range of 10-year melanoma-specific survival rates. With the possible exception of Ki-67, no molecular assessment is routinely used. However, there have been a tremendous number of studies assessing protein expression by immunohistochemistry toward the goal of better prediction of recurrence. In a previous systematic review, which required publication of multivariable prognostic models as a strict inclusion criterion, we identified 37 manuscripts that collectively reported on 62 proteins. Data for 324 proteins extracted from 418 manuscripts did not meet our inclusion criteria for that study, but are revisited here, emphasizing trends of protein expression across either melanocytic lesion progression or gradations of tumor thickness. These identified 101 additional proteins that stratify melanoma, organized according to the Hanahan and Weinberg functional capabilities of cancer.

The semaphorin 7A receptor Plexin C1 is lost during melanoma metastasis.

The transformation of normal melanocytes, or melanocyte stem cells, to melanoma, is a complex process involving multiple mechanisms. Loss of tumor suppressor proteins, which function as brakes on cell growth, migration, or cell survival, was recognized early on as an important mechanism for initiation and progression of melanoma. Semaphorins and their cognate receptors, Plexins and neuropilins, are involved in neuronal pathfinding, immune function, and tumor progression through effects on blood vessel growth and cell migration. Semaphorin 7A (Sema7A) is a membrane-linked semaphorin that is expressed by human keratinocytes, and we have shown that Sema7A binds to human melanocytes through β1-integrins and the Plexin C1 receptor. Functional studies showed that Sema7A stimulates cytoskeletal reorganization in human melanocytes, resulting in adhesion and dendrite formation. Downstream targets of Plexin C1 signaling in human melanocytes include cofilin and LIM kinase II, both of which are critical mediators of cell adhesion and migration. In this report, we analyzed the expression of Plexin C1 using immunohistochemistry on sections of primary and matched metastatic lesions from 19 subjects and in a large melanoma tumor microarray. Our data show a significant loss of Plexin C1 in metastatic melanoma compared with primary melanoma, suggesting the possibility that the Plexin C1 receptor is a tumor suppressor protein for melanoma.

Prognostic Significance of Cadherin-Based Adhesion Molecules in Cutaneous Malignant Melanoma

Background: The need for novel molecular prognostic markers that can supplement validated clinicopathologic correlates for cutaneous malignant melanoma is well recognized. Proteins that mediate the epithelial-mesenchymal transition, the process by which a cancer cell disengages from its parent tumor, are important candidates. Methods: The prognostic relevance of E-cadherin, N-cadherin, and P-cadherin, calcium-dependent transmembrane glycoproteins that regulate cell-cell adhesion, and their adaptors, α-catenin, β-catenin, and p120-catenin, was evaluated on a cohort of 201 primary and 274 metastatic melanoma tumors using fluorescence-based immunohistochemical methods and Automated Quantitative Analysis of protein expression on digitally captured photomicrographs. Results: Increasing levels of N-cadherin expression improved overall survival (log-rank = 7.31; P = 0.03) but did not retain significance following adjustment for established clinicopathologic correlates (P = 0.50). Higher levels of E-cadherin approached significance for favorable prognosis on both univariate (P = 0.13) and multivariable (P = 0.10) analyses. Hierarchical clustering of the composite profiles for all six markers identified four unique clusters that yielded differential overall survival (log-rank = 10.54; P = 0.01). Cluster 4, expressing high E-cadherin and N-cadherin levels, possessed the most favorable outcome and cluster 2, featuring low E-cadherin and α-catenin but modest N-cadherin, showed least favorable outcomes. Cluster 2 remained significant on multivariable analysis (hazard ratio, 3.29; 95% confidence interval, 1.50-7.19; P = 0.003). Conclusions: Although none of the cadherin-based adhesion molecules were independently prognostic, multimarker profiles were significant. Similar to epithelial-derived tumors, loss of E-cadherin correlates with poor outcome. In contrast, for neural crest–derived cutaneous malignant melanoma, N-cadherin overexpression can be associated with either a successful epithelial-mesenchymal transition or a favorably differentiated tumor. Additional cadherin profiles are needed to discriminate these distinctive phenotypes. (Cancer Epidemiol Biomarkers Prev 2008;17(4):949–58)

Identification and comparative analysis of human colonocyte short-chain fatty acid response genes☆

Short-chain fatty acids (SCFAs) butyrate, propionate, and acetate produced during fiber fermentation promote colonic differentiation and can reverse or suppress neoplastic progression. We sought to identify candidate genes responsible for SCFA activity on colonocytes and to compare the relative activities of independent SCFAs. cDNA was generated from polyA+ mRNA isolated from control Caco-2 cells and cells treated with equimolar butyrate, propionate, and acetate. GeneCalling, a restriction-based differential mRNA expression platform linked to a DNA sequence database lookup, was applied. A total of 30,000 individual genetic sequences were analzyed for differential expression among the three SCFAs. Differentially expressed peaks corresponding to cancer-related genes were isolated, sequenced, and cross-referenced to the GenBank human database. Gene identities were independently confirmed by oligonucleotide poisoning. More than 1000 gene fragments were identified as being substantially modulated in expression by butyrate. Butyrate tended to have the most pronounced effects and acetate the least. Five fragments selected for further study were fully sequenced and proved 100% homologous with human sequences for clusterin, amyloid precursor—like protein 2, and caudal homeobox 2 protein, not previously known to be modulated by SCFAs. In each case, a similar order of potency for the three SCFAs studied was observed. The common SCFAs appear to exert different effects. This study suggests the diversity of the SCFA response at the molecular level and facilitates identifying genes important in the biologic activity of dietary fiber.

The characterization of PPARα ligand drug action in an in vivo model by comprehensive differential gene expression profiling

Abstract. Expression pharmacogenomics includes differential gene expression (DGE) profiling of drug responses in model systems to generate a set of differentially modulated drug-responsive genes which can serve as a surrogate measure for drug action. In this manner, expression pharmacogenomics bridges the fields of genomics and medicinal chemistry. Additionally, modulated genes can be organized into metabolic and signaling pathways that highlight the mechanism of drug activity in a selected tissue. Here, we describe the application of expression pharmacogenomics to characterize a drug response in the clinically relevant in vivo model, the Sprague-Dawley rat. Following oral dosing of rats with GW9578, a novel synthetic peroxisome proliferator activated receptor alpha (PPARα) ligand indicated for lipid disorders, we applied GeneCalling, a differential mRNA transcript profiling technique, to rat liver cDNA. Following GW9578 treatment, 2.4% of the rat liver genes were differentially expressed. We confirmed the sequence identity of 50 distinctly modulated genes. DGE was observed among genes representative of at least six discrete metabolic pathways. Furthermore, we observed up-regulation of 20 genes involved in mitochondrial, peroxisomal and microsomal fatty acid oxidation, consistent with molecular biological and clinical data indicating PPARα ligand principal efficacy to be through increasing fatty acid metabolism. Those pathways regulated in our study that are potentially contributory to target effect, non-target adverse effects, or of unknown consequence include xenobiotic detoxification and steroid modification. Finally, comprehensive drug response profiling can lead to the serendipitous discovery of novel disease indications. In this case, these results suggest a potential novel indication for GW9578 in the treatment of X-linked adrenoleukodystrophy. We have shown, therefore, that the organization of DGE results into metabolic and signaling pathways can elucidate mechanisms of pharmacologically desired (i.e., efficacious) and, where appropriate, undesired (i.e., potentially deleterious) effects.