A High-Throughput Panel for Identifying Clinically Relevant Mutation Proﬁles in Melanoma

Published OnlineFirst March 1, 2012; DOI: 10.1158/1535-7163.MCT-11-0676

Molecular Cancer Preclinical Development Therapeutics

Ken Dutton-Regester1,2, Darryl Irwin3, Priscilla Hunt3, Lauren G. Aoude1, Varsha Tembe6,7, Gulietta M. Pupo6,7, Cathy Lanagan4, Candace D. Carter7, Linda O'Connor4, Michael O'Rourke5, Richard A. Scolyer7,8,9, Graham J. Mann6,7, Christopher W. Schmidt4, Adrian Herington2, and Nicholas K. Hayward1

Abstract Success with molecular-based targeted drugs in the treatment of cancer has ignited extensive research efforts within the field of personalized therapeutics. However, successful application of such therapies is dependent on the presence or absence of mutations within the patient’s tumor that can confer clinical efficacy or drug resistance. Building on these findings, we developed a high-throughput mutation panel for the identification of frequently occurring and clinically relevant mutations in melanoma. An extensive literature search and interrogation of the Catalogue of Somatic Mutations in Cancer database identified more than 1,000 melanoma mutations. Applying a filtering strategy to focus on mutations amenable to the development of targeted drugs, we initially screened 120 known mutations in 271 samples using the Sequenom MassARRAY system. A total of 252 mutations were detected in 17 genes, the highest frequency occurred in BRAF (n ¼ 154, 57%), NRAS (n ¼ 55, 20%), CDK4 (n ¼ 8, 3%), PTK2B (n ¼ 7, 2.5%), and ERBB4 (n ¼ 5, 2%). Based on this initial discovery screen, a total of 46 assays interrogating 39 mutations in 20 genes were designed to develop a melanoma-specific panel. These assays were distributed in multiplexes over 8 wells using strict assay design parameters optimized for sensitive mutation detection. The final melanoma-specific mutation panel is a cost effective, sensitive, high- throughput approach for identifying mutations of clinical relevance to molecular-based therapeutics for the treatment of melanoma. When used in a clinical research setting, the panel may rapidly and accurately identify potentially effective treatment strategies using novel or existing molecularly targeted drugs. Mol Cancer Ther; 11(4); 888–97. 2012 AACR.

Introduction beyond 5 years (3). Recently, novel effective molecular- Melanoma is a highly aggressive malignancy and targeted therapies for the treatment of metastatic mela- accounts for the majority of all skin cancer-related deaths noma have emerged. (1). The high mortality rate for advanced stage metastatic One class of molecular-based drugs of current interest melanoma is largely due to the ineffectiveness of currently for treating metastatic melanoma is RAF inhibitors. RAF approved systemic treatment strategies (2). For example, mutations represent highly desirable targets because use of traditional chemotherapeutic approaches has approximately 60% of melanoma patients have consti- tutively active mitogen-activated protein kinase (MAPK) shown poor response rates; less than 10% of patients BRAF demonstrate a clinical response to standard treatment signaling due to mutations in (4). Although initial with dacarbazine and less than 5% patients survive investigations using the RAF inhibitor sorafenib showed no significant increase in survival compared with che- motherapy (5), the recent development of more selective Authors' Affiliations: 1Queensland Institute of Medical Research, Onco- BRAF inhibitors has reignited research into this class of genomics Laboratory; 2Faculty of Science and Technology, Queensland drug (6). Recently, a phase 3 randomized clinical trial 3 fi 4 University of Technology; Sequenom Inc. Asia Paci c; Queensland of PLX4032 (vemurafenib) showed improved rates of Institute of Medical Research, Cancer Immunotherapy Laboratory; 5Morris Tower, Brisbane, Queensland; 6University of Sydney at Westmead Millen- overall and progression-free survival in patients with nium Institute, Westmead; 7Melanoma Institute Australia (formerly the BRAF V600E mutations when compared with dacarba- Sydney Melanoma Unit), North Sydney; 8Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown; and 9Discipline of zine (7). Pathology, The University of Sydney, New South Wales, Australia Interestingly, the use of PLX4032 in tumors not harbor- Note: Supplementary material for this article is available at Molecular ing the V600E mutation has shown adverse biological end Cancer Therapeutics Online (http://mct.aacrjournals.org/). points including increased cell division and proliferation in vitro in vivo Corresponding Author: Ken Dutton-Regester, 300 Herston Road, Her- both and (8–10). Furthermore, mutations ston, 4006 Queensland, Australia. Phone: 6-17-3362 0323; Fax: 6-17-3845 occurring in MAPK kinase (MEK), a downstream effector 3508; E-mail: [email protected] of BRAF in the MAPK signaling pathway, have been doi: 10.1158/1535-7163.MCT-11-0676 shown to confer resistance to PLX4032 treatment (11, 2012 American Association for Cancer Research. 12). In addition, durable responses have yet to be achieved

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with PLX4032, suggesting that a number of mechanisms with 10% heat inactivated fetal calf serum (55Cfor can lead to acquired drug resistance (11–15). Thus, screen- 30 minutes), 100 U/mL penicillin, and 100 mg/mL ing for multiple mutations will be necessary for the devel- streptomycin at 37 Cin5%CO2.GenomicDNAwas opment of effective treatment strategies. extracted by QIAGEN QIAamp Blood Maxi Kits accord- Targeted approaches treating melanoma patients har- ing to the manufacturer’s instructions (QIAGEN Pty boring aberrations other than BRAF mutations are also Ltd). Tumor DNA was extracted from 20 to 30 mg of being investigated. One approach was highlighted in a fresh-frozen tumor using QIAamp DNA Mini Kits phase II trial of imatinib mesylate in which a clinical (QIAGEN) with on-column RNAse digestion. Briefly, response was observed in a melanoma patient whose tissue was pulverized in liquid nitrogen then incubated tumor harbored a KIT mutation (16). A number of case with ATL buffer (QIAGEN) and proteinase K for 96 reports have recently highlighted the efficacy of imatinib hours at 56C. treatment in KIT-mutated melanoma patients, and more recently, significant clinical responses in a subset of Mutation detection patients in a phase II clinical trial (17, 18). Mutation detection was carried out with the Seque- Due to the recent successes of a number of targeted nom MassARRAY system following standard protocols drugs in the treatment of metastatic melanoma, we sought (Sequenom). In brief, 20 ng of genomic DNA were used to develop a melanoma-specific mutation panel to facil- in a PCR reaction and cleaned postamplification with itate rapid identification of somatic mutation events rel- shrimp alkaline phosphatase. A single base pair exten- evant to targeted treatments. An extensive literature sion reaction using iPLEX Pro chemistry was carried search was conducted, including interrogation of the out, resin treated to remove contaminants and spotted Catalogue of Somatic Mutations in Cancer (COSMIC) onto SpectroCHIP II arrays. Mutant and wild-type database, to identify oncogenic mutations in melanoma. alleles were then discriminated via mass spectrometry A confirmation panel of 120 highly ranked mutations was using the Sequenom MassARRAY Analyser 4 platform. screened for prevalence in 271 melanomas before a final Mutations were detected by a minimum 10% threshold melanoma-specific mutation panel of 39 amino acid of the mutant allele peak and were all manually alterations was derived. Mutations selected for the final reviewed. The panel of stage III melanoma cell lines panel were "clinically relevant" in regards to having either was also analyzed using the Sequenom OncoCarta v1.0 documented evidence or potential clinical significance for panel, which consists of 238 mutations covering 17 targeted therapeutics. oncogenes (24).

Materials and Methods Assay design parameters Cell line and tumor samples As the confirmation panel’s purpose was to identify The first cohort of samples consisted of 61 cutaneous or frequently occurring mutation events from a large list of nodal melanoma metastases previously described (19, 20) mutations, standard iplex parameters (Assay designer as well as a panel of 43 stage III (American Joint Com- 4.0, Sequenom) were used with a maximum multiplex- mittee on Cancer) melanoma early passage cell lines ing level of 24 assays per well for cost-effective, high- established at the Queensland Institute of Medical throughput screening. The final melanoma-specific Research (unpublished data). In addition to this, 16 mutation panel, in contrast, used strict assay design matched tumor samples were included to assess the parameters optimized for sensitive mutant allele peak similarity of mutation profiles with the associated cell detection. A maximum multiplexing level of 12 assays line. Eleven previously described uveal melanoma cell per well with increased penalty scores for hairpin, self- lines (provided by Dr. Jerry Nierderkorn, UT Southwest- dimerization, heterodimerization, and false priming of ern Medical Centre, Dallas, Texas), were included to the extension primer were applied. In addition, if pos- identify mutations in this subtype of melanoma (21). A sible, mutant allele peaks were designed as the first second cohort comprised 98 fresh-frozen stage III and IV detected allele peak of an assay to reduce potential noise metastases, from the Melanoma Institute Australia (22), from salt adducts. Further details of the confirmation and 30 melanoma cell lines (of which 9 represented panel are provided in Supplementary Table S1. The final additional clones) established as previously described melanoma-specific mutation panel (MelaCARTA v1) is (23). Cell lines with corresponding matched tumors were available from Sequenom Inc. profiled to confirm authenticity using an AmpFISTR Profiler Plus PCR amplification kit (Applied Biosystems) Statistical analysis and analyzed on a 3100 Genetic Analyzer (Applied To assess the accuracy and sensitivity of the final mel- Biosystems). anoma-specific mutation panel compared with the Onco- Carta panel, the mutant allele frequencies obtained with Cell culture and DNA extraction each panel were compared. A 2-tailed paired Student t test The panel of 43 stage III melanoma cell lines was was used to determine whether any differences were cultured in filter-sterilized RPMI1640 supplemented statistically significant.

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Results ority mutations include those that occur frequently in Mutation detection with the OncoCarta mutation the population, occur frequently at single amino acid panel v1.0 positions, are relevant to targeted drugs currently To identify frequently occurring mutations to be includ- available or in clinical development, or are responsible ed in the melanoma-specific mutation panel, 43 stage III for tumorigenesis through the disruption of important melanoma cell lines were screened with the Sequenom biological pathways. For this reason, mutations that OncoCarta panel. Mutations were detected in 80% of cell had a reported mutation frequency of more than 1%, lines (34 of 43). BRAF and NRAS mutations were mutually occurred in genes with kinase domains, introduced exclusive, with mutations occurring in approximately charge changes in the amino acid sequence and that 60% (25 of 43) and 20% (9 of 43) of cell lines, respectively. did not occur in genes mutated in a typical fashion Four BRAF-mutated samples had an additional mutation associated with inactivation of tumor suppressor genes in CDK4, PIK3CA,orMET. Table 1 lists mutations and were preferentially selected. This strategy reduced the allelic ratios in the stage III melanomas. Ten mutations in list from 314 mutations to 110 highly ranked mutations 5 genes from the OncoCarta panel were included in the in 42 genes. Figure 1 shows a summary of the mutation final melanoma-specific mutation panel, thus eliminat- filtering process. The confirmation panel was used to ing excessive and potentially unnecessary genotyping not determine the significance of these mutations in a large relevant for melanoma. number of melanoma cell lines and tumors. Mutations identified in the confirmation panel Mutations identified from the COSMIC database and The confirmation panel detected 247 mutations in 271 detailed literature search samples (Table 2). The highest frequency of mutation The COSMIC database is a comprehensive resource of was in BRAF (n ¼ 154 or 57%), NRAS (n ¼ 55 or 20%), somatic mutations occurring in cancer, curated from data CDK4 (n ¼ 8 or 3%), PTK2B (n ¼ 7 or 2.5%), and ERBB4 in scientific literature, and large-scale tumor genomic (n ¼ 5 or 2%). BRAF mutations were mutually exclusive resequencing efforts (25). Using this resource, a compre- to NRAS mutations and the majority occurred at codon hensive list of mutations occurring in metastatic melano- 600, mostly resulting in a valine to glutamate substitu- ma was compiled along with associated population mutation, that is, V600E (137 of 271 or 50.5%). The most tion frequency data (Supplementary Table S2 lists muta- frequently occurring NRAS mutation (52 of 271, 19%) tions and references). was at amino acid 61 (2 Q61H, 21 Q61K, 9 Q61L, Atthetimeofdatabaseaccession,themajorityof and 20 Q61R). mutations in this list derived from a resequencing study Twenty-seven samples had more than 1 mutation; the of 518 kinases in 210 diverse cancers including 6 mel- majority were a BRAF mutation in association with anoth- anomas (26). Most mutations in these samples were er mutation (23 of 27, 85.1%). Samples with ERBB4 muta- found in MZ7-mel and CP66-mel (476 and 258 mutations did not segregate with BRAF (2 of 5) or NRAS tions, respectively, out of 900 mutations), which were mutations (1 of 5). Interestingly, of the 3 samples with deemed to have a "mutator phenotype" and were thus mutations in MEK, one (D28) also had a BRAF V600K excluded from our analysis as most of the mutations are mutation. likely "passenger" events. After removing duplicate and Eleven melanoma cell lines of uveal origin were synonymous changes, 87 mutations from the remaining screened. Consistent with previous reports (27, 28), 4 samples were included for consideration in the con- mutations in GNAQ (3 of 11, 27%) and GNA11 (2 of firmation stage. An additional 76 documented muta- 11, 18%) were mutually exclusive. With one exception, tions in COSMIC, not related to these 4 samples, were TB1, a cutaneous melanoma metastasis, mutations of also considered. GNAQ and GNA11 were identified only in tumors of For comprehensive coverage of mutations in melano- uveal origin. Three uveal melanomas (27%) had muta- ma, an extensive literature search was carried out with tions in BRAF, including cell line OCM1, which has PubMed to identify mutations not included in COSMIC. been documented (29, 30). No mutations were detected ERBB4, MITF, GNAQ, GNA11, MEK Mutations in , and in approximately 18% of all samples (48 of 271). A MMP the family were identified. The combination of complete list of results is provided in Supplementary approaches identified 314 mutations that were considered Table S3. for inclusion in the confirmation phase (Supplementary All mutations identified in the confirmation panel were Table S2). included in the final version of the melanoma-specific mutation panel (Table 3). Forty-six assays interrogating 39 Filtering strategy to reduce mutations screened in the mutations in 20 genes were designed into 8 wells with confirmation panel strict assay design parameters optimized for sensitive To reduce the number of mutations genotyped in the mutation allele detection. The final version of the mela- confirmation phase, mutations were ranked according noma-specific mutation panel was retested on all samples to criteria based on technology platform capabilities, and all mutations identified in the confirmation panel clinical significance, and translational value. High-pri- were reidentified.

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Table 1. Comparison between mutations identiﬁed with the OncoCarta and melanoma-speciﬁc mutation panels

OncoCarta Melanoma speciﬁc

Sample Gene Mutation WT% Mut% WT% Mut%

C001 NRAS Q61K 0.467 0.533 0.316 0.684 C002 NRAS Q61K 0.259 0.741 0.000 1.000 C004 BRAF V600E 0.528 0.472 0.540 0.460 C006 NRAS Q61L 0 1 0.000 1.000 C011 BRAF V600E 0.444 0.556 0.436 0.564 C012 BRAF V600E 0.739 0.261 0.726 0.274 C013 NRAS Q61L 0 1 0.000 1.000 C017 BRAF V600E 0.449 0.551 0.459 0.541 C021 — — ———— C022 — — ———— C025 — — ———— C027 NRAS Q61K 0.349 0.651 0.156 0.844 C028 BRAF V600E 0.686 0.314 0.692 0.308 C037 — — ———— C038 BRAF V600E 0.673 0.327 0.666 0.334 C042 BRAF V600E 0.541 0.459 0.546 0.454 C044 BRAF V600E 0.671 0.329 0.664 0.336 C045 BRAF V600E 0.553 0.447 0.532 0.468 C052 — — ———— C054 NRAS Q61K 0.593 0.407 0.463 0.537 C057 BRAF V600E 0.727 0.273 0.735 0.265 C057 CDK4 R24C 0 1 0.000 1.000 C058 BRAF L597S 0.317 0.683 0.292 0.708 C060 BRAF V600E 0.741 0.259 0.694 0.306 C062 BRAF V600E 0.552 0.448 0.565 0.435 C065 BRAF V600E 0.114 0.886 0.200 0.800 C067 — — ———— C071 BRAF V600E 0.688 0.312 0.691 0.309 C071 CDK4 R24C 0.466 0.534 0.466 0.534 C074 BRAF V600E 0.571 0.429 0.525 0.475 C077 — — ———— C078 BRAF V600E 0.842 0.158 0.844 0.156 C081 BRAF V600K 0.513 0.487 0.605 0.395 C083 NRAS Q61L 0.419 0.581 0.383 0.617 C084 — — ———— C086 — — ———— C088 BRAF V600K 0.273 0.727 0.332 0.668 C089 BRAF V600E 0.539 0.461 0.483 0.517 C089 MET T1010I 0.467 0.533 0.508 0.492 C091 BRAF V600E 0.592 0.408 0.585 0.415 C094 BRAF V600E 0.574 0.426 0.537 0.463 C096 NRAS Q61R 0.444 0.556 0.440 0.560 C097 BRAF V600E 0.63 0.37 0.541 0.459 C100 BRAF G469R 0.702 0.298 0.681 0.319 C100 PIK3CA R88Q 0.447 0.553 0.460 0.540 C106 NRAS Q61L 0.329 0.671 0.360 0.640 C108 BRAF V600K 0.219 0.781 0.293 0.707

NOTE. Dashes identify samples with no mutations. Abbreviations: WT%, percentage wild type allele; Mut%, percentage mutant allele.

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Compiled list of mutations occuring in melanoma from 3 different sources

1. Mutations from literature reports 2. COSMIC Database Approximate ERBB4 Prickett et al. Nat Genet, 2009 Sample Mutations GNA11 Van Raamsdonk et al. NEJM, 2010 number of LB2515-MEL GNAQ Onken et al. Invest Opth Vis Sci, 2008 16 COLO-829 mutations MEK Murugan et al. Cell Cycle, 2009 32 MZ7-MEL MEK Emery et al. PNAS, 2009 57 CP66-MEL MITF Cronin et al. Pigm Cell Mel Res, 2009 61 AKT3 Davies et al. Br J Cancer, 2008 MMP Palavalli et al. Nat Genet, 2009 MMP Bleeker et al. Hum Mutat, 2009 3. Mutations identified using Sequenom 1,000 CXCR4 Ierano et al. Cell Cycle, 2009 OncoCarta Panel EPHA3 Balakrishnan et al. Cancer Res, 2007 BRAF, NRAS, KIT, CDK4, PIK3CA, MET FAS Shin et al. Am J Pathol, 1999

Silent mutations removed. Duplicate mutation data removed and merged. 300

Mutations selected for final screening according to them: -occuring in 1% of all samples tested -occurring in genes encoding kinases -causing a charge change in the substituted amino acid 150 -not occuring in known tumor suppressor genes

Any mutation not identified in the samples screened were removed. Mutations with known clinical and functional significance included. 39

Figure 1. Filtering process for selecting mutations of the melanoma-specific panel. From a list of approximately 1,000 mutations, the panel was narrowed down to 39 mutations to allow for cost-effective, high-throughput screening. Mutations that occurred at high frequency and with clinical significance were prioritized for inclusion in the final melanoma-specific mutation panel.

Replication and concordance between panels/mutant Table 2. Summary of mutations detected with to normal allele peak ratios the melanoma-specific mutation panel To assess the accuracy of the final melanoma-specific mutation panel compared with the OncoCarta panel, GENE # Samples Percentage (%) themutantallelefrequencies obtained with each panel were compared. There was complete concordance BRAF 154 56.82 between the mutations identified, and overall, the NRAS 55 20.29 mutant allele ratios did not significantly differ (P ¼ CDK4 8 2.95 0.118, Student paired t test;Table1).Awiderangeof PTK2B 7 2.58 mutant allele peaks was detected in the complete ERBB4 5 1.84 cohort of samples, ranging from 5% to 100%. Tumor MET 4 1.48 samples generally had lower mutant allele peaks than MEK 3 1.10 cell lines (Fig. 2). This effect is likely due to the GNAQ 3 1.10 presence of contaminating stromal cells in the tumor AKT3 2 0.74 specimens. GNA11 2 0.74 Several cell lines in the series tested had either CTNNB1 2 0.74 additional clones or matched tumor samples that were KRAS 2 0.74 also screened with the melanoma-specific mutation KIT 1 0.37 panel. Cell lines with duplicate clones had 100% con- NEK10 1 0.37 cordance in mutations identified (n ¼ 21). All 16 cell PDGFRA 1 0.37 lines had comparable mutation profiles when com- PIK3CA 1 0.37 pared with their matched tumor samples, although EPHA10 1 0.37 2 cell lines differed in mutant to normal allele fre- Wild type 48 17.71 quency ratios from their parent tumors (Supplemen- tary Table S4).

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Table 3. Mutations included in final version of cacy observed in recent trials using RAF and tyrosine kinase inhibitors (7, 17, 18). The success of this strategy the melanoma-specific mutation panel (39 in relies heavily on the stratification of patients based on the total) spectrum of mutations harbored within their tumor. For effective use in a clinical research setting, the identifica- Mutation Mutation tion of mutations that confer drug sensitivity and absence Gene (AA) Gene (AA) of those that confer drug resistance in an accurate, high- AKT3 E17K KIT K642E throughput and timely manner is highly desirable. Here, BRAF G466 KIT L576P we describe the development of a melanoma-specific BRAF G469 KIT V559A mutation panel that can rapidly and accurately identify BRAF K601E KIT W557R clinically relevant mutations in metastatic melanoma. BRAF L397 KRAS G12 A very high concordance between the melanoma-spe- BRAF V600 KRAS Q61 cific panel and OncoCarta panel was observed when CDK4 R24C MEK P124L comparing mutation and allele frequencies, indicating CDK4 R24H MEK Q56P the remultiplexing process required to generate the mel- CTNNB1 S45 MET R1170Q anoma-specific panel did not affect its reproducibility. CXCR4 V160I MET T992I High concordance was also observed between matched EPHA10 E124K NEK10 E379K tumor and cell line samples; only 2 of 16 samples had EPHB6 G404S NRAS G12 slight differences in mutation profiles, possibly indicating EPHB6 R679Q NRAS G13 clonal selection during cell line generation. Alternatively, ERBB4 E452K NRAS Q61 the disparity may result from stromal contamination in ERBB4 R393W PDGFRA E996K the tumor samples leading to an observed difference in GNA11 Q209 PIK3CA R88Q mutant normal allele ratios. One benefit of using the GNA11 R183C PTK2B R429C Sequenom MassARRAY system for mutation analysis is GNAQ Q209 PTK2B R918Q the quantification of mutant to wild-type allele ratios, a GNAQ R183Q ROR2 A793S key feature for estimating mutant subpopulations of cells. KIT D820Y The platform allowed detection of samples with mutant allele frequencies at 10%. However, one mutation was NOTE: Asterisks indicate multiple amino acid changes reliably detected as low as 5% (MM595-Tumor) with the detected. inclusion of a number of duplicates. A skewing toward lower mutant allele peaks was observed in excised human tumors when compared with cell lines, consistent with Discussion stromal contamination. Sensitive mutation detection is an Recent success in molecular-based targeted therapies important feature in a clinical setting as a high degree of has provided new avenues for the treatment of a wide admixture of cell populations within a tumor has the variety of cancers based on the mutational profiles of the potential to disguise mutation events critical to choice of tumor. This approach is exemplified by the clinical effi- therapy. In an attempt to reduce cost while also maintaining high-throughput analysis, a confirmation panel of highly

40 ranked mutations was initially screened in 271 melanoma Cell line samples to identify frequently occurring mutations. As 35 Tumor the majority of mutations selected for the conﬁrmation 30 panel have only been identiﬁed in single tumors, screened

25 in small numbers of samples, and not replicated in independent cohorts of samples, it is possible a number of 20 these events may be patient-speciﬁc mutations or passen- 15 ger mutations not responsible for melanoma develop-

10 ment. This is shown by a high proportion of mutations identified in a large-scale sequencing screen of the protein 5 tyrosine kinase gene family (31) that were not identified 0 here in 271 melanoma samples during the confirmation Overall of percentgae samples tested None 0.2 0.4 0.6 0.8 1 stage (n ¼ 50 or 85%). Mutant allele frequency within an individual sample The final melanoma-specific panel consisted of mutations identified within the confirmation stage or that have direct clinical significance to current or novel molecular- Figure 2. Mutant allele frequencies detected between cell line and tumor samples. The mutant allele frequency of samples is grouped on the x-axis based targeted therapeutics. The clinical utility of these in 20% increments, whereas the y-axis shows the overall percentage of mutations is highlighted in Fig. 3. Not surprisingly, a high samples. rate of BRAF V600E mutations was observed in patients

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clinical trial of GSK2118436 (34), screening for additional

KIT MET ERBB4 clinically relevant mutations within the tumor through

Imatinib Lapatinib mutation panels may be important for overall response to PDGFRA BRAF RAS selected therapies. The sample with both V600K Imatinib? and MEK P124L is an interesting example in which screening only for the BRAF mutation would not have BRAF PIK3CA CDKN2A identified the ideal treatment strategy: a BRAF inhibitor PLX4032 BEZ235 Sorafenib would likely be ineffective due to the co-occurence of a GNAQ MEK mutation. GNA11 PTEN CDK4 Recently, mutations in GNAQ and GNA11 at amino AZD6244? MEK UCN-01? acid residues R183 and Q209 have been identified in up AZD6244 AKT to 83% of uveal melanomas (27, 28). Mutations in GNAQ RB1 GNA11 ERK and occurred here only in cell lines of uveal origin except for a GNA11 R183C mutation identified in a cuta-

Affected proliferation, cell division, apoptotic, invasive, neous, superficial spreading melanoma with a nodular and metastatic pathways component (TB1). As far as we are aware, this is the first documented GNA11 mutation in a nonuveal melanoma. From a clinical aspect, initial reports indicate that tumors Figure 3. Commonly affected genes and pathways in melanoma. A with mutations in GNAQ and GNA11 are likely to be number of mutations in these genes are included in the melanoma- responsible for the activation of the MAPK pathway and fi speci c mutation panel and have therapeutic targets that have been or may potentially be susceptible to treatment with MEK are currently under clinical investigation (denoted under gene name). Oval GNAQ GNA11 boxes represent oncogenes, whereas rectangular boxes represent tumor inhibitors (27). However, and mutant suppressor genes. uveal cells showed mild sensitivity to MEK inhibitors and complete resistance to BRAF inhibitor treatment in vitro (35). for whom BRAF inhibitor treatment would represent an An emerging treatment regimen in KIT-mutated mel- appropriate treatment strategy. Although not all mechan- anomas is through use of tyrosine kinase inhibitors such isms of RAF inhibitor resistance can be detected using the as Imatinib (17). TB349 was the only sample harboring a melanoma-specific mutation panel, such as upregulation KIT mutation (W557R) in our series. The low rate of KIT of COT or receptor tyrosine kinases (13), a number of mutations observed in this data set is possibly due to interesting mutations that could potentially confer resis- insufficient coverage provided by the melanoma-specific tance to inhibitors of the MAPK pathway were identified, mutation panel, compared with the wide variety of muta- as detailed below. tions reported to activate this gene (36). However, the An early report investigating mechanisms of resistance most frequently documented mutations identified in KIT to inhibitors of the MAPK pathway identified a P124L in melanoma were included in the final panel. Acral and MEK mutation from a resistant metastatic tumor in a mucosal melanomas are the subtypes most likely to har- patient treated with a MEK inhibitor, AZD6244, which bor mutations in KIT (10%–15% of cases); however, the was not present before treatment (11). Ex vivo functional frequency of KIT mutations in melanomas occurring in analysis revealed the mutation was responsible for chronically sun-exposed anatomic sites is probably much acquired resistance of AZD6244 treatment and likely lower than reported in earlier studies (37). This interpre- explained drug resistance in this patient. Interestingly, tation is supported by our results because while our the P124L mutation showed cross-resistance to PLX4720 cohort contained only 2 acral and 1 mucosal melanomas, in vitro, indicating MEK mutations may be clinically one third of the melanomas in the MIA subset arose from relevant to RAF inhibitor treatment. Screening for the primary melanomas with high sun damage scores or from MEK P124L mutation with the melanoma-specific muta- chronically sun-exposed body sites. tion panel revealed mutations in 3 samples, one of which Platelet-derived growth factor receptor A (PDGFRA) had an additional BRAF V600K mutation (D28). This mutations occur in a mutually exclusive pattern to suggests that MEK P124L mutations occur in a small KIT in 5% to 10% of gastrointestinal stromal tumors proportion of patients with metastatic melanoma and (GIST) and is a therapeutic target for imatinib (38). The possibly represents an alternative mechanism to the acti- most frequent mutation of PDGFRA in GIST occurs at vation of the MAPK pathway apart from commonly D842V and shows both in vitro and in vivo resistance observed BRAF and NRAS mutations. to imatinib; however, it is important to note that alter- Although some clinical trials of recent BRAF inhibitors native mutations in PDGFRA are imatinib respons- have enrolled only melanoma patients with BRAF V600E ive and represent a viable treatment strategy (38–40). mutations, there is evidence of positive drug response for Although early reports failed to identify PDGFRA muta- V600K mutated tumors in vitro and in vivo (7, 32, 33). If tions in melanoma (41, 42), recent studies indicate V600K-mutated tumors are susceptible to modern BRAF mutations in PDGFRA do occur but represent rare inhibitors, as is suggested by results of an early phase events (31, 43, 44). The melanoma-specific mutation

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panel revealed a PDGFRA E996K mutation in MM648 the spectrum of mutations occurring within these patients that had been previously detected in another sample was not investigated (49). It is interesting to speculate and may represent a mutation hotspot (43). Although a about the possibility of CDK4 inhibitor drugs such as rare event in melanoma, the identification of PDGFRA UCN-01 being most effective in tumors harboring CDK4 mutations warrants further investigation because of its mutations, although this has yet to be investigated exper- likely prediction of imatinib sensitivity in this subset of imentally. Lastly, the presence of CDK4 mutations in patients. BRAF V600E–mutated cell lines did not confer resistance Screening of the protein tyrosine kinase gene family to BRAF inhibitors in vitro; however, one report investi- in cutaneous metastatic melanoma revealed that novel gating a combination approach of CDK4/MEK inhibitors mutations of ERBB4 occurred in 19% of patients and led in BRAF V600E/p16 negative cell lines showed a signi- to increased kinase activity in vitro (42). Although a ficant increase in apoptosis compared with a respective majority of these mutations were tested in the confir- monotherapeutic strategy (50). Further investigation into mation panel (12 of 20 mutations), R393W and E452K the clinical significance of CDK4 mutations and pharma- were the only mutations in ERBB4 identified in the cologic treatment strategies in metastatic melanoma series of samples. This suggests that a majority of ERBB4 patients is warranted. mutations may be rare or patient-specific mutations, or In conclusion, we describe here the development and alternatively occur throughout the entire gene instead of validation of a melanoma-specific mutation panel that can in localized regions. However, the identification of rapidly and accurately determine the molecular profiles of E452K in 3 samples in this study suggests that E452 is tumors in a cost-effective and high-throughput manner. a likely mutation hotspot in ERRB4, occurring in a small Using a mass spectrometry approach on the Sequenom proportion of melanomas. This has clinical significance MassARRAY system is not only advantageous in terms of as in vitro analysis revealed ERRB4 E452K-mutated cell minimizing price and maximizing speed but also pro- lines had 10- to 250-fold enhanced sensitivity to lapa- vides a highly flexible format that will facilitate the addi- tinib, a tyrosine kinase inhibitor of the HER2 growth tion of novel mutations influencing therapeutic patient receptor pathway (42). Although the significance of response as they are identified. This will be of particular ERBB4 in the development and progression of melano- importance with the application of next generation ma has yet to be confirmed in vivo; ERBB4 mutations sequencing in identifying mutations associated with the represent rational therapeutic targets for ERBB receptor development of melanoma or drug-resistant mutations inhibitors in a subset of patients. acquired during treatment. The melanoma-specific muta- Cyclin-dependent kinase 4 (CDK4)isanimportant tion panel also allows for sensitive allele detection impor- kinase responsible for cell-cycle regulation and is a tant for the analysis of tumor samples in which contam- high penetrance melanoma predisposition gene mutat- inating stromal cells may affect the mutant wild-type ed in a small number of families worldwide (45). In allele ratio. Finally, the panel can quickly identify muta- addition, a small number of CDK4 somatic mutations tion profiles in patient samples and may be useful in a have been identified in melanoma tumors and cell clinical setting to determine effective treatment strategies lines; the majority of which result in a cysteine or using molecular-based targeted drugs. histidine substitution of an arginine residue at position 24 (46). Mutations occurring at this position abrogate Disclosure of Potential Conflicts of Interest the binding of inhibitor p16 and lead to constitutive D. Irwin is employed by Sequenom Inc. kinase activity of CDK4 and cell-cycle progression (47). The melanoma-specific mutation panel revealed 8 Authors' Contributions CDK4 Conception and design: K. Dutton-Regester, D. Irwin, G.M. Pupo, N.K. samples with either R24C or R24H mutations in Hayward and was the most commonly mutated gene after BRAF Development of methodology: K. Dutton-Regester, D. Irwin, C.W. and NRAS. Interestingly, all of these samples also Schmidt BRAF Acquisition of data: K. Dutton-Regester, D. Irwin, P. Hunt, L. Aoude, V. harbored V600E mutations. Tembe, G.M. Pupo, C. Lanagan, C.D. Carter, L. O’Connor, M. O’Rourke, R. Although the CDK4 mutations identified here have not A. Scolyer, G.J. Mann, N.K. Hayward Analysis and interpretation of data: K. Dutton-Regester, D. Irwin, G.M. been determined as somatic events in the samples tested, Pupo, R.A. Scolyer, G.J. Mann, N.K. Hayward the recurrence of these mutations suggests an important Writing, review, and/or revision of the manuscript: D. Irwin, L. Aoude, V. role in melanoma progression and possible clinical sig- Tembe, G.M. Pupo, R.A. Scolyer, G.J. Mann, C.W. Schmidt, A. Herington, N.K. Hayward nificance in a small number of patients. This subset of Administrative, technical, or material support: D. Irwin, P. Hunt, G.M. tumors may be susceptible to a CDK4 inhibitor strategy of Pupo, C. Lanagan, A. Herington which a number of drugs are currently under develop- Study supervision: D. Irwin, G.J. Mann, A. Herington, N.K. Hayward ment, or in clinical trials. Evidence of this approach was Acknowledgments observed in a phase I clinical trial of a CDK modulator The authors thank Drs. Mark Smithers and Andrew Barbour from the UCN-01, which resulted in a partial response in a patient Princess Alexandra Hospital, and Prof. John Thompson, Director MIA, on with metastatic melanoma (48). Although a larger phase II behalf of all surgeons of the Institute, as well as the entire MIA Biospecimen Bank team for collection of specimens and tumors. The authors also thank trial of UCN-01 in a cohort of 17 patients with metastatic Drs. Kavitha Gowrishankar and Helen Rizos for the derived cell line melanoma did not reveal significant disease progression; subclones.

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Dutton-Regester et al.

Grant Support The costs of publication of this article were defrayed in part by the pay- ment of page charges. This article must therefore be hereby marked adver- This work was supported by grants from the National Health and tisement Medical Research Council (NHMRC), Cancer Council Queensland, Can- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cer Institute NSW, and the Australian Centre for Vaccine Development. Establishment of QIMR cell lines was supported by a NHMRC grant to the Received August 30, 2011; revised January 23, 2012; accepted February Australasian Biospecimen Network (Oncology). 12, 2012; published OnlineFirst March 1, 2012.

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www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 897

A High-Throughput Panel for Identifying Clinically Relevant Mutation Profiles in Melanoma

Ken Dutton-Regester, Darryl Irwin, Priscilla Hunt, et al.

Mol Cancer Ther 2012;11:888-897. Published OnlineFirst March 1, 2012.

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