Jason E. Hawkes

Association of Inflammation-Related and microRNA Gene Expression with Cancer-Specific Mortality of Colon Adenocarcinoma

Purpose: Inflammatory genes and microRNAs have roles in colon carcinogenesis; therefore, they may provide useful biomarkers for colon cancer. This study examines the potential clinical utility of an inflammatory gene expression signature as a prognostic biomarker for colon cancer in addition to previously examined miR-21 expression. Experimental Design: Quantitative reverse transcriptase-PCR. was used to measure the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent noncancerous tissues from 196 patients. These data were used to develop models for cancer-specific mortality on a training cohort (n = 57), and this model was tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data for miR-21 were available for these patients and were compared and combined with inflammatory gene expression. Results: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor tissues were associated with poor prognosis based on Cox regression (|Z-score| >1.5) and were used to generate the inflammatory risk score (IRS). IRS was associated with cancer-specific mortality in the training, test (P = 0.01), and validation (P = 0.02) cohorts. This association was strong for stage II cases (P = 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a, and NOS2a, providing evidence that the function of this microRNA and these inflammatory genes are linked. Both IRS and miR-21 expression were independently associated with cancer-specific mortality, including stage II patients alone. Conclusion: IRS and miR-21 expression are independent predictors of colon cancer prognosis and may provide a clinically useful tool to identify high-risk patients. (Clin Cancer Res 2009;15(18):5878–87)

Inflammatory and MicroRNA Gene Expression as Prognostic Classifier of Barrett's-Associated Esophageal Adenocarcinoma

Purpose: Esophageal cancer is one of the most aggressive and deadly forms of cancer; highlighting the need to identify biomarkers for early detection and prognostic classification. Our recent studies have identified inflammatory gene and microRNA signatures derived from tumor and nontumor tissues as prognostic biomarkers of hepatocellular, lung, and colorectal adenocarcinoma. Here, we examine the relationship between expression of these inflammatory genes and micro RNA (miRNA) expression in esophageal adenocarcinoma and patient survival. Experimental Design: We measured the expression of 23 inflammation-associated genes in tumors and adjacent normal tissues from 93 patients (58 Barretts and 35 Sporadic adenocarcinomas) by quantitative reverse transcription-polymerase chain reaction. These data were used to build an inflammatory risk model, based on multivariate Cox regression, to predict survival in a training cohort (n = 47). We then determined whether this model could predict survival in a cohort of 46 patients. Expression data for miRNA-375 were available for these patients and was combined with inflammatory gene expression. Results: IFN-γ, IL-1α, IL-8, IL-21, IL-23, and proteoglycan expression in tumor and nontumor samples were each associated with poor prognosis based on Cox regression [(Z-score)>1.5] and therefore were used to generate an inflammatory risk score (IRS). Patients with a high IRS had poor prognosis compared with those with a low IRS in the training (P = 0.002) and test (P = 0.012) cohorts. This association was stronger in the group with Barretts history. When combining with miRNA-375, the combined IRS/miR signature was an improved prognostic classifier than either one alone. Conclusion: Transcriptional profiling of inflammation-associated genes and miRNA expression in resected esophageal Barretts-associated adenocarcinoma tissues may have clinical utility as predictors of prognosis. Clin Cancer Res; 16(23); 5824–34. ©2010 AACR.

High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation calling

Identifying mutations in the TP53 gene is important for cancer prognosis, predicting response to therapy, and determining genetic risk. We have developed a high‐throughput scanning assay with automatic calling to detect TP53 mutations in DNA from fresh frozen (FF) and formalin‐fixed paraffin‐embedded (FFPE) tissues. The coding region of the TP53 gene (exons 2–11) was PCR‐amplified from breast cancer samples and scanned by high‐resolution fluorescent melting curve analyses using a 384‐well format in the LightCycler 480 instrument. Mutations were confirmed by direct sequencing. Sensitivity and specificity of scanning and automatic mutation calling was determined for FF tissue (whole genome amplified [WGA] and non‐WGA) and FFPE tissue. Thresholds for automatic mutation calling were established for each preparation type. Overall, we confirmed 27 TP53 mutations in 68 primary breast cancers analyzed by high‐resolution melting curve scanning and direct sequencing. Using scanning and automatic calling, there was high specificity (>95%) across all DNA preparation methods. Sensitivities ranged from 100% in non‐WGA DNA from fresh tissue to 86% in WGA DNA and DNA from formalin‐fixed, paraffin‐embedded tissue. Scanning could detect mutated DNA at a dilution of 1:200 in a background of wild‐type DNA. Mutation scanning by high‐resolution fluorescent melting curve analyses can be done in a high‐throughput and automated fashion. The TP53 scanning assay can be performed from a variety of specimen types with high sensitivity/specificity and could be used for clinical and research purposes. Hum Mutat 29(5), 757–764, 2008.

microRNAs in Psoriasis

Psoriasis is a chronic inflammatory skin condition resulting from a complex interplay among the immune system, keratinocytes, susceptibility genes, and environmental factors. However, the pathogenesis of psoriasis is not completely elucidated. microRNAs represent a promising class of small, noncoding RNA molecules that function to regulate gene expression. Although microRNA research in psoriasis and dermatology is still relatively new, evidence is rapidly accumulating for the role of microRNAs in the pathogenesis of psoriasis and other chronic inflammatory conditions. In this article, we present a comprehensive review of what is known about microRNAs and their role in the pathogenesis of psoriasis.

Isolation of circulating microRNAs from microvesicles found in human plasma

Intact miRNAs can be isolated from the circulation in significant quantities despite the presence of extremely high levels of RNase activity. The remarkable stability of circulating miRNAs makes them excellent candidates for biomarkers in diagnostic applications as well as therapeutic targets in a variety of disease states including melanoma. Circulating RNA molecules are resistant to degradation by RNases because they are encapsulated in membrane-bound microvesicles. We describe a convenient method for the use of ExoQuick, a proprietary resin developed by Systems Biosciences (Mountain View, CA), whereby microvesicles can be purified under gentle conditions using readily available laboratory equipment. This protocol allows for isolation all microvesicles, regardless of their origin, and provides a convenient method for identifying potential cancer-specific biomarkers from biological fluids including serum and plasma.

The Snowballing Literature on Imiquimod-Induced Skin Inflammation in Mice: A Critical Appraisal.

Since 2009, the imiquimod- or Aldara-induced (3M Pharmaceuticals, St. Paul, MN) model of acute skin inflammation has become the most widely used mouse model in preclinical psoriasis studies. Although this model offers researchers numerous benefits, there are important limitations and possible confounding variables to consider. The imiquimod model requires careful consideration and warrants scrutiny of the data generated by its use. In this perspective, we provide an overview of the advantages and disadvantages of this mouse model and offer suggestions for its use in psoriasis research.

Increased MicroRNA-34b and -34c Predominantly Expressed in Stromal Tissues Is Associated with Poor Prognosis in Human Colon Cancer

The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. To elucidate the roles of miR-34 family in colon carcinogenesis, miR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR, and we examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status and Affymetrix microarray data. All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression that is consistent with the proposed regulation of miR-34a/b/c by TP53. To examine where the miR-34 family is expressed, the expression of miR-34 family was compared between epitheliums and stromal tissues using laser microdissection technique. The expression of miR-34b/c was increased significantly in stromal tissues, especially in cancer stroma, compared with epithelial tissue. In conclusion, increased miR-34b/c predominantly expressed in stromal tissues is associated with poor prognosis in colon cancer. MiR-34 may contribute to cancer-stromal interaction associated with colon cancer progression.

Skin cancer screening: recommendations for data-driven screening guidelines and a review of the US Preventive Services Task Force controversy

Melanoma is usually apparent on the skin and readily detected by trained medical providers using a routine total body skin examination, yet this malignancy is responsible for the majority of skin cancer-related deaths. Currently, there is no national consensus on skin cancer screening in the USA, but dermatologists and primary care providers are routinely confronted with making the decision about when to recommend total body skin examinations and at what interval. The objectives of this paper are: to propose rational, risk-based, data-driven guidelines commensurate with the US Preventive Services Task Force screening guidelines for other disorders; to compare our proposed guidelines to recommendations made by other national and international organizations; and to review the US Preventive Services Task Forces 2016 Draft Recommendation Statement on skin cancer screening.

Report of a novel OCA2 gene mutation and an investigation of OCA2 variants on melanoma risk in a familial melanoma pedigree

BACKGROUND Oculocutaneous albinism type 2 (OCA2) is caused by mutations of the OCA2 gene. Individuals affected by OCA2 as well as other types of albinism are at a significantly increased risk for sun-induced skin-cancers, including malignant melanoma (MM). OBJECTIVE To identify the molecular etiology of oculocutaneous albinism in a previously uncharacterized melanoma pedigree and to investigate the relationship between two OCA2 variants and melanoma predisposition in this pedigree. METHODS DNA and RNA were isolated from the peripheral blood of seven patients in a familial melanoma pedigree. Electron microscopy was performed on the individual with clinical oculocutaneous albinism. OCA2, TYRP1, MC1R, CDKN2A/p16, CDKN2A/p19ARF, and CDK4 genes were sequenced in affected individuals. The relationship between OCA2 variants and melanoma was assessed using a pedigree likelihood-based method. RESULTS The proband was determined to be an OCA2 compound heterozygous mutation carrier with a previously reported conservative missense mutation (V443I) and a novel non-conservative missense mutation (L734R). The pedigree contained individuals diagnosed with both cutaneous and iris melanoma. Based on co-segregation analysis, the odds of these OCA2 variants being high penetrance loci for melanoma was: 1.3-to-1 if we include the iris melanoma as affected and 6.5-to-1 if we only consider cutaneous melanoma as affected. CONCLUSION The discovery of this novel OCA2 variant adds to the body of evidence on the detrimental effects of OCA2 gene mutations on pigmentation, supports existing GWAS data on the relevance of the OCA2 gene in melanoma predisposition, and may ultimately assist in the development of targeted molecular therapies in the treatment of OCA and melanoma.

Lack of GNAQ and GNA11 Germ-Line Mutations in Familial Melanoma Pedigrees with Uveal Melanoma or Blue Nevi

Approximately 10% of melanoma cases are familial, but only 25–40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A – the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s) in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), Guanine nucleotide-binding protein G(q) subunit alpha-11 (GNA11), and BRCA1-associated protein-1 (BAP1). Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.