Nancy E. Thomas

Expression of p16INK4a in peripheral blood T-cells is a biomarker of human aging

Expression of the p16INK4a tumor suppressor sharply increases with age in most mammalian tissues, and contributes to an age‐induced functional decline of certain self‐renewing compartments. These observations have suggested that p16INK4a expression could be a biomarker of mammalian aging. To translate this notion to human use, we determined p16INK4a expression in cellular fractions of human whole blood, and found highest expression in peripheral blood T‐lymphocytes (PBTL). We then measured INK4/ARF transcript expression in PBTL from two independent cohorts of healthy humans (170 donors total), and analyzed their relationship with donor characteristics. Expression of p16INK4a, but not other INK4/ARF transcripts, appeared to exponentially increase with donor chronologic age. Importantly, p16INK4a expression did not independently correlate with gender or body‐mass index, but was significantly associated with tobacco use and physical inactivity. In addition, p16INK4a expression was associated with plasma interleukin‐6 concentration, a marker of human frailty. These data suggest that p16INK4a expression in PBTL is an easily measured, peripheral blood biomarker of molecular age.

A method for normalizing histology slides for quantitative analysis

Inconsistencies in the preparation of histology slides make it difficult to perform quantitative analysis on their results. In this paper we provide two mechanisms for overcoming many of the known inconsistencies in the staining process, thereby bringing slides that were processed or stored under very different conditions into a common, normalized space to enable improved quantitative analysis.

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

PURPOSE To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. MATERIALS AND METHODS A large collection of melanoma cell lines (n=37) were treated with 0-8Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. RESULTS Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G(1) arrest. CONCLUSIONS Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

Tumor-Infiltrating Lymphocyte Grade in Primary Melanomas Is Independently Associated With Melanoma-Specific Survival in the Population-Based Genes, Environment and Melanoma Study

PURPOSE Although most hospital-based studies suggest more favorable survival with tumor-infiltrating lymphocytes (TILs) present in primary melanomas, it is uncertain whether TILs provide prognostic information beyond existing melanoma staging definitions. We addressed the issue in an international population-based study of patients with single and multiple primary melanomas. PATIENTS AND METHODS On the basis of the Genes, Environment and Melanoma (GEM) study, we conducted follow-up of 2,845 patients diagnosed from 1998 to 2003 with 3,330 invasive primary melanomas centrally reviewed for TIL grade (absent, nonbrisk, or brisk). The odds of TIL grades associated with clinicopathologic features and survival by TIL grade were examined. RESULTS Independent predictors (P < .05) for nonbrisk TIL grade were site, histologic subtype, and Breslow thickness, and for brisk TIL grade, they were age, site, Breslow thickness, and radial growth phase. Nonbrisk and brisk TIL grades were each associated with lower American Joint Committee on Cancer (AJCC) tumor stage compared with TIL absence (P(trend) < .001). Death as a result of melanoma was 30% less with nonbrisk TIL grade (hazard ratio [HR], 0.7; 95% CI, 0.5 to 1.0) and 50% less with brisk TIL grade (HR, 0.5; 95% CI, 0.3 to 0.9) relative to TIL absence, adjusted for age, sex, site, and AJCC tumor stage. CONCLUSION At the population level, higher TIL grade of primary melanoma is associated with a lower risk of death as a result of melanoma independently of tumor characteristics currently used for AJCC tumor stage. We conclude that TIL grade deserves further prospective investigation to determine whether it should be included in future AJCC staging revisions.

DNA‐methylation profiling distinguishes malignant melanomas from benign nevi

DNA methylation, an epigenetic alteration typically occurring early in cancer development, could aid in the molecular diagnosis of melanoma. We determined technical feasibility for high‐throughput DNA‐methylation array‐based profiling using formalin‐fixed paraffin‐embedded tissues for selection of candidate DNA‐methylation differences between melanomas and nevi. Promoter methylation was evaluated in 27 common benign nevi and 22 primary invasive melanomas using a 1505 CpG site microarray. Unsupervised hierarchical clustering distinguished melanomas from nevi; 26 CpG sites in 22 genes were identified with significantly different methylation levels between melanomas and nevi after adjustment for age, sex, and multiple comparisons and with β‐value differences of ≥0.2. Prediction analysis for microarrays identified 12 CpG loci that were highly predictive of melanoma, with area under the receiver operating characteristic curves of >0.95. Of our panel of 22 genes, 14 were statistically significant in an independent sample set of 29 nevi (including dysplastic nevi) and 25 primary invasive melanomas after adjustment for age, sex, and multiple comparisons. This first report of a DNA‐methylation signature discriminating melanomas from nevi indicates that DNA methylation appears promising as an additional tool for enhancing melanoma diagnosis.

Indications for lymphatic mapping and sentinel lymphadenectomy in patients with thin melanoma (Breslow thickness ≤1.0 mm)

AbstractBackground: Patients with thin (Breslow thickness ≤1.0 mm) melanoma have a good prognosis (5-year survival >90%). Consequently, the added benefit of lymphatic mapping and sentinel lymphadenectomy (LM/SL) in these patients is controversial. We hypothesize that LM/SL with a focused examination of the sentinel node (SN) will detect a significant number of SN metastases in patients with thin melanoma and that certain clinical or histopathologic factors may serve as predictors of SN tumor involvement. Methods: Over 6 years, 349 patients with melanoma underwent LM/SL and were prospectively entered into an institutional review board (IRB)-approved database. LM/SL was performed with a combined radiotracer and blue dye technique. SNs were serially sectioned, and each section was examined by a dermatopathologist at multiple levels with hematoxylin and eosin as well as immunohistochemical stains. Results: One hundred forty-six patients (42%) had a melanoma with Breslow thickness ≤1.0 mm; six (4%) of these 146 patients had a tumor-involved SN. On multivariate analysis, none of the clinical or histopathologic factors examined were significantly associated with SN tumor involvement in patients with thin melanoma. Completion lymphadenectomy was performed on all patients with a tumor-involved SN. None of the patients had non-SN tumor involvement. Conclusions: The incidence of SN tumor involvement in patients with thin melanoma is considerable. Although we were unable to identify predictors of SN tumor involvement in patients with thin melanoma, efforts to identify predictors of SN tumor involvement should continue. Until better predictors are identified, we continue to advocate offering LM/SL to patients with thin melanomas who demonstrate clinical or histopathologic characteristics that have historically been associated with an increased risk of recurrence and mortality.

Vitamin D receptor polymorphisms in patients with cutaneous melanoma

The vitamin D receptor (VDR) gene has been associated with cancer risk, but only a few polymorphisms have been studied in relation to melanoma risk and the results have been inconsistent. We examined 38 VDR gene single nucleotide polymorphisms (SNPs) in a large international multicenter population‐based case‐control study of melanoma. Buccal DNAs were obtained from 1,207 people with incident multiple primary melanoma and 2,469 with incident single primary melanoma. SNPs with known or suspected impact on VDR activity, haplotype tagging SNPs with ≥10% minor allele frequency in Caucasians, and SNPs reported as significant in other association studies were examined. Logistic regression was used to calculate the relative risks conferred by the individual SNP. Eight of 38 SNPs in the promoter, coding, and 3′ gene regions were individually significantly associated with multiple primary melanoma after adjusting for covariates. The estimated increase in risk for individuals who were homozygous for the minor allele ranged from 25 to 33% for six polymorphisms: rs10875712 (odds ratios [OR] 1.28; 95% confidence interval (CI), 1.01–1.62), rs4760674 (OR 1.33; 95% CI, 1.06–1.67), rs7139166 (OR 1.26; 95%CI, 1.02–1.56), rs4516035 (OR 1.25; 95%CI, 1.01–1.55), rs11168287 (OR 1.27; 95%CI, 1.03–1.57) and rs1544410 (OR 1.30; 95%CI, 1.04–1.63); for two polymorphisms, homozygous carriers had a decreased risk: rs7305032 (OR 0.81; 95%CI 0.65–1.02) and rs7965281 (OR, 0.78; 95%CI, 0.62–0.99). We recognize the potential false positive findings because of multiple comparisons; however, the eight significant SNPs in our study outnumbered the two significant tests expected to occur by chance. The VDR may play a role in melanomagenesis.

Clinicopathologic Features of Incident and Subsequent Tumors in Patients with Multiple Primary Cutaneous Melanomas

Background0.6–12.7% of patients with primary cutaneous melanoma will develop additional melanomas. Pathologic features of tumors in patients with multiple primary cutaneous melanomas have not been well described. In this large, international, multicenter, case–control study, we compared the clinicopathologic features of a subsequent melanoma with the preceding (usually the first) melanoma in patients with multiple primary cutaneous melanomas, and with those of melanomas in patients with single primary cutaneous melanomas.MethodsMultiple primary melanoma (cases) and single primary invasive melanoma (controls) patients from the Genes, Environment and Melanoma (GEM) study were included if their tumors were available for pathologic review and confirmed as melanoma. Clinicopathologic characteristics of invasive subsequent and first melanomas in cases and invasive single melanomas in controls were compared.ResultsA total of 473 pairs comprising a subsequent and a first melanoma and 1,989 single melanomas were reviewed. Forward stepwise regression modeling in 395 pairs with complete data showed that, compared with first melanomas, subsequent melanomas were more commonly contiguous with a dysplastic nevus, more prevalent on the head/neck and legs than other sites, and thinner. Compared with single primary melanomas, subsequent melanomas were more likely to be associated with a contiguous dysplastic nevus, more prevalent on the head/neck and legs, and thinner. The same differences were observed when subsequent melanomas were compared with single melanomas. First melanomas were more likely than single melanomas to have associated solar elastosis and no observed mitoses.ConclusionsThinner subsequent than first melanomas suggest earlier diagnosis, perhaps due to closer clinical scrutiny. The association of subsequent melanomas with dysplastic nevi is consistent with the latter being risk factors or risk markers for melanoma.

Validation of the VE1 immunostain for the BRAF V600E mutation in melanoma

BRAF mutation status, and therefore eligibility for BRAF inhibitors, is currently determined by sequencing methods. We assessed the validity of VE1, a monoclonal antibody against the BRAF V600E mutant protein, in the detection of mutant BRAF V600E melanomas as classified by DNA pyrosequencing.