Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-1027

Complete Loss of PTEN Protein Expression Correlates with Shorter Time to Brain Metastasis and Survival in Stage IIIB/C Melanoma Patients with BRAFV600 Mutations Amanda D. Bucheit, Guo Chen, Alan Siroy, et al. Clin Cancer Res 2014;20:5527-5536. Published OnlineFirst August 27, 2014.

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Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-1027

Clinical Cancer Research

Biology of Human Tumors

Complete Loss of PTEN Protein Expression Correlates with Shorter Time to Brain Metastasis and Survival in Stage IIIB/C Melanoma Patients with BRAFV600 Mutations Amanda D. Bucheit1, Guo Chen1, Alan Siroy2, Michael Tetzlaff2, Russell Broaddus2, Denai Milton3, Patricia Fox3, Roland Bassett3, Patrick Hwu1, Jeffrey E. Gershenwald4,5, Alexander J. Lazar2, and Michael A. Davies1,6

Abstract Purpose: Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAFV600 mutations. The prevalence, pathologic features, and clinical outcomes associated with PTEN loss in patients with stage IIIB/C melanoma were interrogated to improve our understanding of the clinical significance of this molecular event. Experimental Design: Archival tissue from lymphadenectomy specimens among patients (n ¼ 136) with stage IIIB or IIIC melanoma was assessed by DNA sequencing for activating BRAF and NRAS mutations, and by immunohistochemistry for the expression of PTEN protein. Associations of these molecular aberrations with demographics, tumor characteristics, and clinical outcomes were determined. Results: The prevalence of BRAFV600 mutations (40% overall), NRAS mutations (10%), and PTEN loss (25%) did not vary by pathologic substage. BRAF/NRAS mutation status did not correlate with distant disease-free survival (DDFS) or overall survival (OS). Complete loss of PTEN expression correlated with shorter OS but not DDFS. When stratified by specific sites of distant metastasis, PTEN loss was associated with significantly shorter time to melanoma brain metastasis (MBM), but not to liver, lung, or bone metastasis. Analysis of PTEN in mutationally defined subsets showed that PTEN loss was significantly associated with OS and time to MBM in patients with BRAFV600 mutations. Conclusions: Loss of PTEN protein expression correlates significantly with decreased OS and time to MBM in stage IIIB/C melanoma patients with BRAFV600 mutations. The findings add to evidence supporting a significant role for PTEN loss and the PI3K–AKT pathway in melanoma. Clin Cancer Res; 20(21); 5527–36. 2014 AACR.

Introduction The clinical management of melanoma is evolving rapidly due to improved understanding of the molecular drivers of this disease. Substitutions affecting the V600 residue of the BRAF serine-threonine kinase are the most common activat-

1 Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas. 2Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas. 3Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas. 4Department of Surgical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas. 5Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, Texas. 6 Department of Systems Biology, University of Texas MD Anderson Cancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Michael A. Davies, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, 77030. Phone: 713-792-3454; Fax: 713-563-3454; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-14-1027 2014 American Association for Cancer Research.

ing mutation detected in patients with cutaneous melanoma (40%–45%; refs. 1, 2). BRAFV600 mutations increase the kinase activity of BRAF and result in constitutive activation of the RAS–RAF–MAPK pathway. Recurrent point mutations affecting residues G12, G13, and Q61 of NRAS are the second most common activating event in melanoma (20%; ref. 2). Previous studies have demonstrated that NRAS mutations are essentially mutually exclusive with BRAFV600 mutations, likely due to the fact that they also activate signaling through RAS-RAF-MAPK (3, 4). In addition to the high prevalence of oncogenic mutations, the importance of this pathway in melanoma is supported by the clinical results observed with inhibitors targeting it. Mutation-selective BRAF inhibitors (vemurafenib and dabrafenib) and MEK inhibitors (trametinib) have been approved by the FDA for the treatment of patients with metastatic melanoma with BRAFV600 mutations (5–7). MEK inhibitors have also demonstrated clinical activity in patients with activating NRAS mutations, and clinical trials of MEK inhibitors alone and in combination with other agents are planned and/or ongoing (8). Mutant RAS proteins utilize multiple signaling pathways to mediate their oncogenic effects. One such pathway is the

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Translational Relevance Multiple clinical trials with new agents are now underway or planned for patients with regional metastases from melanoma. The appropriate design and interpretation of these and future investigations will be strengthened by developing an improved understanding of the clinical characteristics and outcomes associated with molecular aberrations in this disease. This integrated analysis of BRAF and NRAS mutations, PTEN loss, and clinical outcomes in patients with stage IIIB/C disease adds to growing evidence that melanomas with loss of PTEN represent a clinically significant subpopulation. PTEN loss was associated with shorter time to brain metastasis and overall survival specifically in melanomas with concurrent BRAFV600 mutations, thus supporting functional interactions observed preclinically between these oncogenic events. The novel association of PTEN loss with shorter time to brain metastasis also supports the rationale behind further examination of the role of the PI3K–AKT pathway in this common and devastating complication of advanced melanoma.

PI3K–AKT pathway. In addition to NRAS mutations, this pathway can be activated in melanoma by loss of function of the phosphatase PTEN (9). Genetic analyses of melanoma cell lines and clinical specimens have demonstrated that inactivating mutations and deletions of the PTEN gene are relatively rare (10%), and are largely mutually exclusive with NRAS mutations in melanoma, but frequently overlap with BRAFV600 mutations (3, 4). Loss of PTEN mRNA and protein expression has been detected more frequently (20%–30%), potentially due to epigenetic mechanisms (10–12). Our previous quantitative proteomic analyses of cell lines and frozen tumor samples in multiple tumor types have shown that loss of PTEN protein expression correlates with greater activation of the AKT and other PI3K pathway effectors than a number of other oncogenic events, including PIK3CA and NRAS mutations (9, 13). Multiple inhibitors against targets in the PI3K–AKT pathway are in various phases of clinical testing in melanoma and other malignancies (14). In addition to serving as therapeutic targets, molecular aberrations can impact patient management in cancer by improving risk models of disease progression and mortality. Several previous studies have examined the clinical associations and prognostic significance of BRAFV600 and NRAS mutations in primary melanomas (15–19). We and others have also reported their prevalence and prognostic significance in patients with stage IV melanoma (20, 21). However, currently there is very little information about the clinical significance of BRAF, NRAS, or PTEN in patients with stage III melanoma. Patients with stage III melanoma have highly variable outcomes, and currently there are no validated molecular markers that add to existing prognostic models utilizing clinical and pathologic features (22). The

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identification of markers that could improve risk stratification in patients with stage III melanoma is important, as the only currently approved adjuvant therapies for these patients (IFNa-2B and pegylated-IFNa-2b) are given for prolonged periods of time and have significant associated toxicities. Furthermore, evaluating the prognostic significance of such markers in patients with stage III melanoma will also strengthen the design and interpretation of new clinical trials that have been designed for this population based on observed activity in patients with stage IV disease. To test the hypothesis that molecular markers can improve existing prognostic models, and to improve our understanding of their potential role in the pathogenesis of this disease, we determined the mutational status of BRAF and NRAS, and the protein expression of PTEN, in lymphadenectomy specimens from patients who underwent standard-of-care surgical treatment for stage IIIB/C melanoma. This study includes analysis of PTEN expression (by IHC) in a cohort of tumors that previously underwent quantitative proteomic analysis. This analysis demonstrates that only complete loss of PTEN protein expression correlates with increased expression of activation-specific markers in the PI3K–AKT pathway, thus providing a molecular rationale to guide the evaluation of PTEN expression in melanoma clinical samples. Our subsequent analysis of BRAF, NRAS, and PTEN status in a cohort of clinically annotated lymphadenectomy specimens from patients with stage IIIB/C melanoma identifies a significant association for loss of expression of PTEN with shorter overall survival (OS) and shorter time to melanoma brain metastasis (MBM).

Materials and Methods Patients Under an Institution Review Board-approved protocol, patients who underwent standard-of-care lymphadenectomy for stage IIIB/C melanoma, who, in the case of survival, had clinical follow-up of at least 5 years, and for whom archival material was available were identified. The patients underwent lymphadenectomy surgery between 1988 and 2009. Patient data and primary tumor characteristics were collected for all patients. Patient records were reviewed for clinical events, including development of local recurrence, development and patterns of distant metastases, and OS. DNA mutation detection DNA was extracted from unstained formalin-fixed paraffin-embedded (FFPE) slides that had undergone hematoxylin and eosin-guided macrodissection to isolate portions with at least 70% viable tumor cells. DNA was extracted by the MD Anderson Biospecimen Extraction Core Facility. Samples were screened for hotspot mutations in BRAF and NRAS using the Sequenom MassArray Platform by the MD Anderson Characterized Cell Line Core facility, as previously described (9). PTEN immunohistochemistry PTEN protein expression in FFPE samples was assessed by IHC (6H2.1 antibody, Cascade Bioscience) as previously

Clinical Cancer Research

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Published OnlineFirst August 27, 2014; DOI: 10.1158/1078-0432.CCR-14-1027

PTEN Loss in Stage IIIB/C Melanoma

Figure 1. Evaluation of PTEN expression in melanoma by IHC. A, representative image of a melanoma with complete loss of PTEN protein expression in the tumor cells. Endothelial cells (labeled "EC") serve as an internal positive control and standard for comparison. B, comparison of PTEN protein expression measurement by RPPA (on frozen tumor tissue) and by IHC (FFPE from the same surgical accessions). PTEN expression in the melanomas was categorized (x-axis) based on the observed results of IHC analysis. The average PTEN protein expression for each group of tumors as measured by RPPA is shown on the y-axis (arbitrary units). The RPPA results for each group were compared by unpaired t tests, P values are shown below the bar graph. Thr308 Ser473 and P-AKT , each measured by RPPA, are shown in C and D, respectively. Melanomas with Similar comparisons of PTEN IHC results to levels of AKT Clonal PTEN expression (by IHC) were not analyzed because of an insufficient number of samples (n ¼ 2) from which to draw meaningful conclusions.

described (23). Tumor samples were assessed as part of a tissue microarray (TMA), which included three cores for each tumor. Complete absence of PTEN was defined as

C melanoma patients with BRAFV600 mutations.

Loss of function of PTEN is a frequent event in melanoma, particularly in tumors with BRAF(V600) mutations. The prevalence, pathologic features, and c...
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