Hanne E. Puntervoll

Importance of P-Cadherin, β-Catenin, and Wnt5a/Frizzled for Progression of Melanocytic Tumors and Prognosis in Cutaneous Melanoma

Purpose: It has been proposed that melanoma cells shift from E-cadherin to N-cadherin expression during tumor development, and recent gene profiling has shown increased expression of Wnt5a/Frizzled in aggressive melanomas possibly by interactions with β-catenin. We therefore wanted to investigate the role of cadherin subtypes, β-catenin, and Wnt5a/Frizzled in melanocytic tumors, with focus on prognosis in nodular melanomas. Experimental Design: The immunohistochemical expression of E-cadherin, N-cadherin, P-cadherin, β-catenin, and Wnt5a/Frizzled was examined using tissue microarrays of 312 melanocytic tumors. Results: Cytoplasmic expression of P-cadherin was associated with increasing tumor thickness (P = 0.005) and level of invasion (P = 0.019), whereas membranous staining was associated with thinner (P = 0.012) and more superficial (P = 0.018) tumors. Increased cytoplasmic P-cadherin was associated with reduced survival (P = 0.047). Lack of nuclear β-catenin expression was related to increased tumor thickness (P = 0.002) and poor patient survival in univariate (P = 0.0072) and multivariate (P = 0.004) analyses. Membranous expression of N-cadherin was significantly increased from primary tumors to metastatic lesions, whereas E-cadherin staining tended to be decreased. Wnt5a and its receptor Frizzled were highly coexpressed, and nuclear expression of both markers was significantly reduced from benign nevi to melanomas, with a shift from nuclear to cytoplasmic expression in malignant tumors. In addition, Wnt5a expression was significantly associated with nuclear β-catenin expression. Conclusions: Alterations in the expression and subcellular localization of cell adhesion markers are important in the development and progression of melanocytic tumors, and strong cytoplasmic P-cadherin expression and loss of nuclear β-catenin staining were associated with aggressive melanoma behavior and reduced patient survival.

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants

Background CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. Methods All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. Results Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001). MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010). Conclusion Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

Mutation analysis of the EGFR-NRAS-BRAF pathway in melanomas from black Africans and other subgroups of cutaneous melanoma.

Earlier studies have shown frequent mutations in the BRAF and NRAS genes in cutaneous melanoma, but these alterations have not been examined in the rare category of melanoma from black Africans. Moreover, the frequency of epidermal growth factor receptor (EGFR) mutations in melanocytic tumors is not known. We therefore examined 165 benign and malignant melanocytic lesions (including 118 invasive melanomas and 18 metastases collected as consecutive cases from various time periods and from two different pathology departments; the 51 nodular melanomas were randomly selected from a larger, consecutive, population-based series of nodular melanomas) with respect to alterations in the EGFR, BRAF and NRAS genes. Mutations in EGFR (exons 18–21) were not detected. EGFR protein expression was observed in a subgroup of melanomas, but without associations with clinicopathologic phenotype or prognosis. Cytoplasmic EGFR expression was, however, significantly increased from benign nevi to melanomas. Mutations in BRAF and NRAS were detected in superficial melanoma (25 and 29%, respectively), nodular melanoma (29 and 28%, respectively) and lentigo maligna melanoma (15 and 16%, respectively). In a series of melanomas from black Africans (n=26), only two BRAF mutations (8%) were found, both being different from the common T1799A substitution. Moreover, melanomas from black Africans exhibited mutations in NRAS exon 1 only (12%), whereas NRAS exon 2 mutations were predominant in melanomas from Caucasians. Thus, the frequencies of BRAF and NRAS mutations were particularly low in melanomas from black Africans, supporting a different pathogenesis of these tumors.

Loss of BMI-1 expression is associated with clinical progress of malignant melanoma

BMI-1 is a member of the Polycomb group of genes (PcGs) and is involved in embryonic gene regulation and maintenance of adult stem cells. It has been suggested that BMI-1 protein is important in cell cycle regulation, since both p16/INK4a and p14/ARF are downstream BMI-1 targets. BMI-1 has been implicated in the development and progression of several malignancies, but its role in melanocytic tumors of the skin is unknown. In the present study, using immunohistochemistry on 178 benign and malignant melanocytic lesions and two different antibodies, BMI-1 expression was reduced in melanomas compared with benign nevi. In established melanomas, loss of BMI-1 expression was associated with features of aggressive tumors, such as increased tumor cell proliferation, presence of necrosis and increased expression of both N-cadherin and β3-integrin, indicating a more invasive and mesenchymal phenotype. Low BMI-1 expression was associated with low p14 and CDK4 but not with p16 expression. Low levels of BMI-1 expression were also significantly associated with decreased patient survival.

Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

Purpose: The human SIM2 gene is located within the Downs syndrome critical region of chromosome 21 and encodes transcription factors involved in brain development and neuronal differentiation. SIM2 has been assigned a possible role in the pathogenesis of solid tumors, and the SIM2-short isoform (SIM2-s) was recently proposed as a molecular target for cancer therapy. We previously reported SIM2 among the highly up-regulated genes in 29 prostate cancers, and the purpose of our present study was to examine the expression status of SIM2 at the transcriptional and protein level as related to outcome in prostate cancer. Experimental Design: By quantitative PCR, mRNA in situ hybridization, and immunohistochemistry, we evaluated the expression and significance of SIM2 isoforms in 39 patients with clinically localized prostate cancer and validated the expression of SIM2-s protein in an independent cohort of 103 radical prostatectomies from patients with long and complete follow-up. Results: The SIM2 isoforms (SIM2-s and SIM2-l) were significantly coexpressed and increased in prostate cancer. Tumor cell expression of SIM2-s protein was associated with adverse clinicopathologic factors like increased preoperative serum prostate-specific antigen, high histologic grade, invasive tumor growth with extra-prostatic extension, and increased tumor cell proliferation by Ki-67 expression. SIM2-s protein expression was significantly associated with reduced cancer-specific survival in multivariate analyses. Conclusions: These novel findings indicate for the first time that SIM2 expression might be important for clinical progress of human cancer and support the recent proposal of SIM2-s as a candidate for targeted therapy in prostate cancer.

Clinical Efficacy and Safety of Bevacizumab Monotherapy in Patients with Metastatic Melanoma: Predictive Importance of Induced Early Hypertension

Background VEGF driven angiogenesis plays a key role in cancer progression. We determined the clinical efficacy of bevacizumab monotherapy in patients with metastatic melanoma. Methods and Findings Thirty-five patients with metastatic melanoma in progression were enrolled in this phase II, single arm clinical trial. Each patient received bevacizumab monotherapy 10 mg/kg q14 d until intolerable toxicity or disease progression occurred. Clinical efficacy was evaluated as objective response, disease control (DC), and survival. We observed one complete (3%) and 5 partial (14%) responses. In addition, 5 patients experienced stable disease >6 months (14%) while 24 patients had progressive disease (PD, 69%), corresponding to a total DC at 6 months in 11 out of 35 patients (31%). Median progression free survival (PFS) was 2.14 months and median overall survival (OS) was 9 months (1.12–49). Seven of the 11 patients experiencing DC developed early hypertension (<2 months) compared to 3/24 of patients with PD (P = 0.001), and hypertension was associated with PFS (P = 0.005) and OS (P = 0.013). Conclusion Bevacizumab monotherapy demonstrated promising clinical efficacy in patients with metastatic melanoma with disease control in 31% of the patients. Induced early hypertension was a marker for clinical efficacy of bevacizumab. Trial Registration ClinicalTrials.gov NCT00139360.

BRAF-V600E expression in primary nodular melanoma is associated with aggressive tumour features and reduced survival

Background:Around 50% of primary melanomas harbour BRAF mutations, but their prognostic impact has not been clear. Recently, a BRAF-V600E mutation-specific antibody has become available for immunohistochemistry. Here, we investigated for the first time the prognostic impact of BRAF-V600E protein expression in primary melanoma.Methods:In a patient series of 248 nodular melanomas, BRAF-V600E and total BRAF expression were assessed by immunohistochemistry using tissue microarray sections of paraffin-embedded archival tissue. Mutation status was assessed by real-time PCR in cases with sufficient tumour tissue (n=191).Results:Positive BRAF-V600E expression was present in 86 (35%) of the cases, and was significantly associated with increased tumour thickness, presence of tumour ulceration and reduced survival. Further, BRAF-V600E expression was an independent prognostic factor by multivariate analysis, whereas BRAF mutation status was not significant. There was 88% concordance between BRAF-V600E expression and mutation status.Conclusions:Our findings indicate that BRAF-V600E expression is a novel prognostic marker in primary melanoma.

Abstract 135: Associations between VEGF polymorphisms and clinical outcome in breast cancer

Introduction Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis and is involved in the development and progression of solid tumors. Recent advances in sequencing technologies have allowed the identification of genetic variants associated with development and progression of different diseases including breast cancer. Despite evidence suggesting that these genetic variants can be used for improved risk stratification, results are not conclusive. In this work we have evaluated the relationship between 8 VEGF SNPs (single nucleotide polymorphisms) and survival as well as clinico-pathologic characteristics in breast cancer. Material and Methods In this study 8 VEGF SNPs (rs3025039, rs69947, rs833070, rs2010963, rs3025010, rs25468, rs3025035 and rs833068) were selected. DNA was extracted from non-tumor FFPE-samples from a series of 367 breast cancer patients from a Norwegian population. Associations between SNPs and categorical variables were analyzed by chi-square and Fisher exact tests. We used logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). Survival was calculated by the Kaplan-Meier method (log-rank test). Results Our results showed that for a subset of patients, rs3025035 CT and TT were associated with relapse (p = 0.044; OR 3.07, 95%CI 1.08-8.76). Accordingly the rs3025035 CC genotype was related to longer time to recurrence after surgery (p = 0.10). Interestingly, in patients with histologic grade 3, a trend between CC and improved survival was also found in rs3025035 (p = 0.10), whereas the CC genotype in rs3025039 was associated with worse prognosis in this group (p = 0.037). Finally, the haplotype rs3025035T-rs3025039C was associated with relapse (p = 0.015; OR 3.72, 95% CI 1.31-10.62). Conclusions We report for the first time an association of the rs3025035 CC genotype with disease relapse and suggest that it could be a prognostic marker in breast cancer. Independent studies are needed to confirm our result. Citation Format: Jessica Furriol, Hanne E. Puntervoll, Goril Knutsvik, Monica Mannelqvist, Sura Aziz, Elisabeth Wik, Lars A. Akslen. Associations between VEGF polymorphisms and clinical outcome in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 135. doi:10.1158/1538-7445.AM2015-135

Frequencies of KIT and GNAQ mutations in acral melanoma

To the Editor , Cutaneous melanoma is a heterogeneous disease involving multiple molecular pathways, with specific patterns of somatic alterations associated with different melanoma subtypes and body sites. Acral lentiginous melanomas occur on palms and soles and account for approximately 2–3% of melanomas in white-skinned persons but are much more common in Asian, Hispanic and black populations. Acral melanomas have been shown to harbor gain of function mutations in the KIT gene (MIM# 164920),1 with most mutations occurring in exon 9, 11, 13, 17 and 18. In previous studies, KIT mutation status in acral melanomas of Caucasian and Asian populations are described, whereas the molecular data on such tumors from black Africans are very limited. Hence, we wanted to explore the mutation patterns in a series of acral melanomas from a black African population. We examined somatic mutation status in KIT exons 9, 11, 13, 17 and 18, as well as mutations in GNAQ (MIM# 600998) exon 5, another oncogene, known to be frequently mutated in uveal melanomas. We included 24 African acral melanomas previously examined for mutations in BRAF exons 11 and 15, NRAS exons 1 and 2 and EGFR exons 18, 19, 20 and 21,2 in addition to 12 Caucasian acral melanomas. The latter were also screened for mutations in BRAF (exons 11 and 15) and NRAS (exons 1 and 2), as these tumors were not included in our prior study.2 To confirm the low GNAQ mutation rates in cutaneous melanoma, we further included 68 melanocytic lesions: 9 conventional melanocytic nevi, 9 Spitz nevi, 16 superficial spreading melanoma (SSM), 3 SSM in situ, 11 cases of lentigo maligna melanoma (LMM), 10 nodular melanomas and 10 metastases from nodular melanoma. The Caucasian material was collected at the Department of Pathology, The Gade Institute, Haukeland University Hospital, Bergen, Norway. The African acral melanomas were collected at the Department of Pathology, Muhimbili University College of Health Sciences, Dar Es Salaam, Tanzania. Tumor tissue was manually dissected from formalin-fixed paraffin embedded (FFPE) sections, followed by DNA extraction using the E.Z.N.A tissue DNA Kit (Omega Biotec Inc., Norcross, GA, USA). Polymerase chain reaction (PCR) amplification was performed using the Qiagen Multiplex PCR Master Mix kit (Qiagen, GmbH, D-40724 Hilden, Germany). Sanger sequencing was performed using the Big Dye terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA), before analyzing on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). When comparing mutation frequencies between the African and Caucasian acral melanomas, the Fisher exact test was performed using the IBM Statistical Package for Social Sciences, version 19 (SPSS Inc, USA). The Norwegian Data Inspectorate and the Regional Committee for Ethics in Research (Health Region III; 136.04) approved the study, which was performed in accordance with the Helsinki Declaration. The KIT analysis of African acral melanomas revealed two mutations (8%), one D579del (1735_1737delGAT) deletion and one S864L (TCT>TTT) mutation (Table 1). D579del is located within the juxta-membrane domain (exon 11) and has previously been observed in acral melanomas. The S864L (exon 18) has to our knowledge not been previously reported before, although a S864F mutation has been described in a case of primary childhood

Abstract 101: Analysis of GNAQ mutations in subtypes of malignant melanoma

Background: The MAP kinase pathway has been implicated in the pathogenesis of cutaneous melanoma, and mutations in BRAF and NRAS are commonly found in subtypes of melanomas as well as in benign nevi. Recently, several publications have reported frequent mutations in codon 209 of GNAQ in blue nevi and uveal melanoma, revealing an alternative path to MAP kinase activation. Aim of study: We wanted to investigate the prevalence of GNAQ mutations in codon 209 in a broad spectre of cutaneous melanoma, previously screened for mutations in BRAF, NRAS and EGFR. Methods: 75 benign and malignant tumors of different subgroups were collected at the Department of Pathology, the Gade Institute, Haukeland University Hospital, including: Spitz nevi, superficial spreading melanoma, superficial spreading melanoma in situ, lentigo malignant melanoma, nodular melanoma, metastases from nodular melanoma and uveal melanoma. Additionally, 14 acral African melanomas were collected at the Department of Pathology, Muhimbili University college of Health Sciences, Dar Es Salaam, Tanzania. Tumor tissue was manually dissected from formalin fixated paraffin-embedded sections, before GNAQ exon 5 was screened for mutations in codon 209 by direct Sanger sequencing. Further sample collection and analysis are ongoing. Results: The sequence analysis detected mutations in codon 209 in all five uveal melanomas tested (four harbored a Q209L mutation, and one had a Q209P mutation). However no mutations were found in any of the other subtypes of malignant melanoma. Conclusion: Our study shows that there appears to be a general lack of GNAQ mutations in cutaneous melanoma. This confirms that there may be different routes to MAP kinase activation in malignant melanoma as suggested by other studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 101. doi:1538-7445.AM2012-101