Revisiting Determinants of Prognosis in Cutaneous Melanoma

Review Article Revisiting Determinants of Prognosis in Cutaneous Melanoma Sarah A. Weiss MD 1,2 ; Douglas Hanniford PhD 2,3 ; Eva Hernando PhD 2,3 ; and Iman Osman MD 1,2,4 The American Joint Committee on Cancer staging system for cutaneous melanoma is based on primary tumor thickness and the presence of ulceration, mitoses, lymph node spread, and distant metastases as determinants of prognosis. Although this cutaneous melanoma staging system has evolved over time to more accurately reflect patient prognosis, improvements are still needed, because current understanding of the particular factors (genetic mutation, expression alteration, host response, etc) that are critical for predicting patient outcomes is incomplete. Given the clinical and biologic heterogeneity of primary melanomas, new prognostic tools are needed to more precisely identify patients who are most likely to develop advanced disease. Such tools would affect clinical surveillance strategies and aid in patient selection for adjuvant therapy. The authors reviewed the literature on prognostic molecular and immunologic markers in primary cutaneous melanoma, their associations with clinicopathologic and survival outcomes, and their potential for incorporation into current staging models. Overall, the studies considered in this review did not define prognostic markers that could be readily incorporated into the current staging system. Therefore, efforts should be continued in these and other directions to maximize the likelihood of identifying clinically useful prognostic biomarkers for cutaneous melanoma. Cancer 2015;121:4108-23. VC 2015 American Cancer Society. KEYWORDS: biomarkers, genetics, melanoma, messenger RNA, micrornas, prognostic, staging, tumor-infiltrating lymphocytes. INTRODUCTION Melanoma is increasing in incidence. In 2014, there were over 76,000 new melanoma cases and greater than 9000 melanoma-related deaths in the United States. 1 The current American Joint Committee on Cancer (AJCC) staging system for cutaneous melanoma is based on primary tumor thickness and the presence of ulceration, mitoses, lymph node spread, and distant metastases as determinants of prognosis. However, survival outcomes can vary widely even within the same stage. Given the clinical and biologic heterogeneity of primary melanoma, new prognostic tools are needed to more precisely identify which patients are most likely to develop advanced disease. Such tools would affect clinical surveillance strategies and aid in patient selection for adjuvant therapy. Here, we discuss the evolution of primary melanoma prognostic determinants, their limitations, and the current literature on melanoma prognostic markers from a molecular and immunologic perspective. The Evolution of Cutaneous Melanoma Staging Over Time In the mid-20th century, melanoma staging and, thus, prognosis were driven by thickness and level of invasion. In 1969, Clark et al 2 described 5 levels of primary melanoma tumor invasion that inversely correlated with overall survival (OS) in a study of 208 primary melanomas. 2 Breslow 3 also observed that tumor thickness combined with Clark level produced the most accurate prognoses in a cohort of 98 patients who had primary melanomas examined for Clark level, tumor thickness, and cross-sectional area (thickness times greatest tumor dimension). No melanoma recurrences or metastases from primary tumors <0.76 mm thick in Clark level II and III tumors occurred within 5 years of follow-up; thus, 0.76 mm was recommended as a cutoff point to stratify patients for lymph node dissection. 3 Corresponding authors: Iman Osman, MD, New York University Langone Medical Center, 522 First Avenue, Smilow 405, New York, NY 10016; Fax: (212) 263-9090; iman.osman@nyumc.org; Eva Hernando-Monge, PhD, New York University Langone Medical Center, 522 First Avenue, Smilow 305, New York, NY 10016; Fax: (212) 263-8211; eva.hernando-monge@nyumc.org 1 Department of Medical Oncology, New York University School of Medicine, New York, New York; 2 Interdisciplinary Melanoma Cooperative Group, New York University School of Medicine, New York, New York; 3 Department of Pathology, New York University School of Medicine, New York, New York; 4 Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, New York. Additional supporting information may be found in the online version of this article. DOI: 10.1002/cncr.29634, Received: May 19, 2015; Revised: July 9, 2015; Accepted: July 13, 2015, Published online August 26, 2015 in Wiley Online Library (wileyonlinelibrary.com) 4108 Cancer December 1, 2015

Determinants of Prognosis in Melanoma/Weiss et al Since those seminal studies, melanoma staging has continued to evolve as patient cohorts have expanded and additional prognostic features have been described. Today, the AJCC oversees melanoma staging guidelines, which incorporate tumor thickness and other histopathologic parameters into the TNM (tumor, lymph node, metastasis) criteria used for most solid tumors. In 2002, the AJCC made significant changes to the melanoma staging system, including increasing thickness thresholds for tumor classification from 0.75, 1.5, and 4 mm to 1, 2, and 4 mm and including ulceration status as a second determinant of tumor classification based on a multivariate analysis of 13,581 patients with primary melanoma. 4,5 In addition, in a subset of 4750 patients without clinically detected lymph node metastases, the Clark level was predictive of outcome in those who had thin melanomas (<1 mm), whereas ulceration was the most prognostic factor in those who had thicker melanomas (1 mm). 5 Other AJCC staging updates at that time included shifting thick T4 melanomas from stage IIIA to stage IIC, the inclusion of satellite metastases in lymph node staging, and exclusion of the size of lymph node metastases, which was no longer considered prognostic. 5-7 In 2010, the mitotic rate (1/mm 2 ) replaced the Clark level for characterizing T1 melanomas when a multivariate analysis of 4861 T1 primary melanomas demonstrated that thickness, mitotic rate, and ulceration significantly predicted survival outcomes, whereas Clark level did not. 7 The inclusion of mitoses in melanoma staging was considered practice-changing. Because of a 10% risk of sentinel lymph node (SLN) positivity for a primary tumor thickness from 0.76 to 1.0 mm with mitoses, surgeons may now be prompted to offer SLN biopsy to patients who have thinner melanomas if mitoses are present. 8 In addition, a lymph node was defined as positive if any isolated tumor cells were detected histologically or by immunohistochemistry (IHC), whereas at least 0.2 mm of tumor was previously required for positivity. This also changed prognostic discussions that clinicians have with these patients, who are now being upstaged to at least stage IIIA melanoma for even a single tumor cell noted in the lymph node, conferring a worse prognosis. 7,8 Limitations of the AJCC Staging System Although the AJCC cutaneous melanoma staging system has evolved over time to more accurately reflect patient prognosis, improvements are still needed. TNM staging does not always reliably predict patient outcomes by stage. For example, stage I melanomas 1 mm comprise the majority (approximately 80%) of melanoma diagnoses reported to the National Cancer Institute s Surveillance, Epidemiology, and End Results Cancer Registry and carry an excellent prognosis, with 5-year OS rates from 92% to 97%. 9,10 However, 15% of these patients die from melanoma. 10 Furthermore, patients who have stage IIC melanomas have a worse 5-year OS (53%) compared with those who have stage IIIA melanomas (78%); and patients who have stage III melanomas have a highly variable 5-year OS rate, which ranges from 78%, 59%, and 50% for stage IIIA, IIIB, and IIIC melanomas, respectively. 9,11 It has been hypothesized that adding measurements of host antitumor immune response and/or molecular features to staging may improve primary melanoma prognostication. New tumor-based, host-based, and/or blood-based biomarkers to address the inconsistencies of an ever-evolving, imperfect staging system are critically needed. For this report, we reviewed the literature on prognostic molecular and immunologic markers in primary cutaneous melanoma, their associations with clinicopathologic and survival outcomes, and their potential for incorporation into current staging models. TISSUE-BASED MOLECULAR GENETIC MARKERS Identifying tissue-based prognostic molecular markers in melanoma has been challenging. One major limitation is that most primary melanomas are preserved as formalinfixed, paraffin-embedded (FFPE) samples rather than fresh-frozen tissues because of the small size of the specimens. High-quality genetic material is less commonly extractable from FFPE samples, precluding their use with early generation sequencing methods. 12 The relative stability of messenger RNA (mrna) is also inconsistent in FFPE samples. 13 However, improved extraction methods; newer technologies; and novel, stable RNA molecules, such as microrna (mirna), have allowed exome-wide or genome-wide mutational analysis or RNA expression profiling from small amounts of genetic material isolated from FFPE samples. 12 Such technical and biologic advances have yielded new insights into melanoma biology and new molecules for prognostic assessment. Genes that were initially identified as genetically altered drivers of melanoma tumorigenesis (v-raf murine sarcoma viral oncogene homolog B [BRAF], neuroblastoma RAS viral oncogene homolog [NRAS], phosphatase and tensin homolog [PTEN], etc) were captured in candidate-based analyses focused on known proliferation and survival pathways. Next-generation sequencing (NGS) has afforded an unprecedented depth of knowledge of the mutational landscape of entire melanoma Cancer December 1, 2015 4109

Review Article genomes, which has confirmed and expanded on these findings. 14-17 However, challenges remain in identifying the genetic drivers of melanomagenesis and progression. A comprehensive analysis of whole-exome sequencing data from 21 tumor types (The Cancer Genome Atlas and the Broad Institute) calculated that sequencing information for >5000 samples is required to identify all enriched, mutated genes present in >2% of melanomas at 90% power, 18 highlighting the particular challenge of interpreting sequencing data for melanoma. Moreover, among the genes nominated from melanoma NGS studies, few reports have associated individual mutations with clinical outcomes or have studied their use as prognostic biomarkers in cutaneous melanoma. Nonetheless, there has been a movement to genetically define melanomas; and, in this report, we review the associations between patient prognosis and commonly mutated genes in melanoma. BRAF Activating BRAF mutations are present in 50% to 60% of melanomas, the majority of which are codon 600 valineto-glutamic acid (V600E) (approximately 90%) or valineto-lysine (V600K) (approximately 10%) mutations. 19 BRAF mutations have been described with high frequency in benign nevi, although they occur less frequently in radial growth phase than in vertical growth phase melanomas, suggesting that oncogenic BRAF mutations are associated with melanoma progression more strongly than with melanomagenesis. 20 There are conflicting data on BRAF mutation status as an independent prognostic marker in melanoma. A meta-analysis has indicated that patients who have BRAF-mutant tumors have a 1.7-fold increased risk of death compared with those who have BRAF wild-type (WT) tumors. 22 In contrast, many small studies of primary melanomas cite no independent association between BRAF mutation status and survival (Table 1), 22,23,25,28 although Ellerhorst et al did observe that BRAF-mutant primary melanomas may have more adverse pathologic characteristics (increased thickness and ulceration) compared with WT tumors and tend to present clinically with higher stage disease. 23 NRAS NRAS mutations, which occur in about 15% of cutaneous melanomas, most commonly at codon 61 (glutamine to lysine [Q61K] or to arginine [Q61R]), also lead to constitutive activation of the mitogen-activated protein kinase (MAPK) pathway. 22,23 Similar to BRAF mutations, the prognostic implications of NRAS mutations vary in the literature (Table 1). Devitt et al prospectively studied 249 primary melanomas and observed that patients with NRAS-mutant melanoma (codon 61) had shorter melanoma-specific survival (MSS) (hazard ratio [HR], 2.51; P 5.05) and trended toward worse recurrence-free survival (RFS) in multivariate analysis (HR, 2.08; P 5.09), but NRAS mutation status had no impact on OS (HR, 1.47; P 5.37). 24 NRAS-mutated primary melanomas tended to be thicker, had more mitoses, and more frequently were of the nodular subtype compared with BRAF-mutant or NRAS/BRAF-WT tumors. 24 Multiple other studies of primary melanomas have indicated no association of NRAS mutation status with RFS, MSS, or OS. 23,25 Most recently, investigators from the Genes, Environment, and Melanoma study reported on associations of BRAF and NRAS status with clinicopathologic variables and MSS in 912 patients with primary melanoma. The 7.6-year median follow-up was completed before 2011, which limits the possible confounders of targeted or immunotherapy administration in patients who progressed. Although there were no differences in 5-year OS between patients who had BRAF-mutant, NRAS-mutant, or WT tumors, there was a 3-fold increase in melanomarelated death in patients who had BRAF-mutant and NRAS-mutant tumors, specifically in the high-risk category (T2b or above), compared with those who had WT tumors. 27 V-kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene The v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (KIT) is a type III receptor tyrosine kinase for stem cell factor and functions as an oncogene involved in melanocytic differentiation, migration, proliferation, and survival. 29,30 KIT mutations or gene amplifications occur in 10% to 25% of melanomas arising from areas of acral, mucosal, or sun-damaged skin; they are rare (range, 0%- 2%) in cutaneous melanomas without chronic sundamage; and they can rarely coexist with BRAF mutations. 29,31,32 There is limited information on the prognostic significance of KIT in melanoma, although melanoma subtypes associated with KIT mutation have a worse overall prognosis compared with more common cutaneous melanomas. 33 PTEN The tumor suppressor PTEN functions as a phosphatase that, when inactivated, results in constitutive activation of 4110 Cancer December 1, 2015

Determinants of Prognosis in Melanoma/Weiss et al TABLE 1. Prognostic Implications of Genetic Mutations in Melanoma Study Population Clinicopathologic Associations Survival Associations Reference BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutation Primary melanomas (n 5 89) Truncal location, superficial spreading No association with development of Maldonado 2003 22 histologic subtype, younger age metastases or MSS Primary melanomas (n 5 223) Higher rate of ulceration No association with survival Ellerhorst 2011 23 outcomes Primary melanomas (n 5 249) Worse RFS in multivariate analysis Devitt 2011 24 adjusting for age, sex, tumor site, tumor stage, mitosis, and ulceration, but not in a second multivariate analysis, which also controlled for thickness Meta-analysis of 4 melanoma studies A 1.7-fold increased risk of death Safaee Ardekani 2012 21 Primary nodular melanomas (n 5 51) Higher rate of ulceration in primary No association with RFS or MSS Akslen 2005 25 and paired metastases (n 5 18) tumor Melanoma lymph node metastases Worse survival outcome Mann 2013 26 and their associated primaries (n 5 79) Primary melanomas (n 5 912) Younger age, superficial spreading histologic subtype, present mitoses A 3-fold-increased risk of melanoma-related death compared with WT for high-risk patients after adjustment for age, sex, study center, anatomic site, and TIL grade Thomas 2015 27 NRAS (neuroblastoma RAS viral oncogene homolog) mutation Primary melanomas (n 5 223) Thicker, upper extremity location No association with OS Ellerhorst 2010 23 Primary melanomas (n 5 249) Thicker, higher mitotic rates; trend Association with shorter MSS Devitt 2011 24 toward higher proliferation as measured by Ki-67 Primary melanomas (n 5 51) and No association with RFS or MSS Akslen 2005 25 paired metastases (n 5 18) Melanoma lymph node metastases and Worse survival outcome Mann 2013 26 their associated primaries (n 5 79) Primary melanomas (n 5 912) Lower TIL grade, presence of mitoses, anatomic site other than scalp/neck A 3-fold-increased risk of melanoma-related death compared with WT for high-risk patients after adjustment for age, sex, study center, anatomic site, and TIL grade Thomas 2015 27 Abbreviations: MSS, melanoma-specific survival; OS, overall survival; RFS, recurrence-free survival; TIL, tumor-infiltrating lymphocytes; WT, wild type. phosphoinositide 3-kinase/protein kinase B signaling and increased cell survival. 34 Up to 10% of cutaneous melanomas harbor loss-of-function alterations in PTEN, including mutations, deletions, and promoter methylation. 35 In a study of 127 primary melanomas, decreased PTEN protein abundance was associated with primary tumor ulceration, but not with survival outcomes. 34 In contrast, a recent study reported that patients who had tumors with PTEN methylation had worse OS compared with those who had PTEN-unmethylated tumors. 36 PTEN is inactivated by multiple mechanisms, and future outcome association studies ideally should incorporate all mechanisms that contribute to and define PTEN-inactivated tumors. Newly Identified Genetic Mutations NGS studies have expanded the known mutational landscape of melanoma and have nominated novel mutations as drivers of melanoma tumorigenesis. A literature review reveals that only a few studies to date have examined prognostic associations of these newly identified genetic mutations. A mix of recurrent and nonrecurrent mutations in protein phosphatase 6 (PPP6C) recently was identified in 9% to 12% of melanomas. 15,17 Consistent with previous reports, our group recently reported PPP6C mutations in 25 of 233 (10.7%) and 8 of 77 (10.4%) primary and metastatic melanoma samples, respectively. 37 In that report, we documented that, among patients with AJCC stage I through III melanoma, those who had stop mutations trended toward worse RFS, whereas those who had nonstop mutations trended toward improved RFS compared with patients who had WT PPP6C tumors. Although they are identified in only 5% to 9% of melanomas, mutations in Ras-related C3 botulinum toxin substrate 1 (RAC1) are predominantly codon 29 prolineto-serine (P29S) missense mutations that yield aberrant Cancer December 1, 2015 4111

Review Article RAC activity, which drives melanoma proliferation and tumorigenesis. 15,17 A recent study indicated that only 27 of 814 (3.3%) primary cutaneous melanomas harbored RAC1 P29S mutations, 38 suggesting that these mutations occur as later events in the natural history of melanoma and may be key drivers of metastatic spread. Consistent with this notion, RAC1 mutations were associated with lymph node spread at diagnosis in addition to other negative prognostic indicators. Unfortunately, that study did not report associations of RAC1 P29S mutation status with distant metastasis or patient outcomes, precluding a definitive assessment. Recurrent mutations in regulatory regions of several genes that aberrantly activate expression of these genes have also recently been described. 39-41 Of these, recurrent mutations in the promoter of telomerase reverse transcriptase (TERT) that create novel ETS family transcription factor binding sites have been documented in up to 70% of melanoma patient samples. TERT promoter mutations result in increased telomerase expression, thus favoring chromosomal stability through maintenance of telomere length, which may improve the fitness of cancer cells harboring such mutations. Two recent studies indicated that TERT promoter mutations (43% and 22% of patients) in primary melanomas were associated with key prognostic parameters (tumor thickness, ulceration, and mitotic rates) and poor disease-free survival (DFS) and OS. 42,43 Collectively, these findings support the potential relevance of newly identified genetic mutations as clinically useful prognostic factors, and further studies to analyze associations of genetic mutations with patient outcomes are warranted. Increased collaboration among research centers to stimulate sample and data pooling and to standardize statistical methods and analyses will aid in a more definitive assessment of genetic mutations as prognostic markers. EXPRESSION OF PROTEIN-CODING GENES Expression level-based analyses of mrnas and proteins provide immensely alluring molecular markers, because these potential markers measure in the tens of thousands. In theory, proteins have a distinct advantage of being the functional end products of protein-coding genes. Moreover, their stability, modifications, and localization multiply their potential as prognostic markers. However, tools to measure proteins genome-wide are still developing; and, to date, the protein-based (IHC) biomarker studies reported in melanoma have largely taken candidate approaches. Several comprehensive reviews and/or metaanalyses have examined protein-based biomarker studies in melanoma 44-47 ; thus, we refer readers to those excellent articles instead of addressing them in depth here. Briefly, we do note 3 important multimarker prognostic signature studies that have been developed with the use of tissue microarray-based IHC. 44,48 Gould Rothberg et al built a 5-protein signature (high nonnuclear/nuclear activating transcription factor 2 [ATF2], high nuclear cyclin-dependent kinase inhibitor 1 [p21], high total b-catenin, low nonnuclear/nuclear cyclin-dependent kinase inhibitor 2A [p16], and low total fibronectin; from 20 candidates evaluated) that was indicative of a favorable prognosis (OS) using immunofluorescence-based, automated quantitative analysis histochemistry in 192 AJCC stage II FFPE specimens. 44 Those authors validated their scoring method in a set of 226 additional patients with primary melanoma. In contrast, Meyer et al developed a 7-marker signature (B-cell lymphoma 2 [Bcl-2] associated X protein [Bax], Bcl-2 related apoptosis regulator [Bcl-X], PTEN, cyclooxygenase 2 [COX-2], b-catenin loss, methylthioadenosine phosphorylase [MTAP] loss, and the presence of cluster of differentiation 20 [B-lymphocyte antigen; CD20]-positive B lymphocytes, from 70 candidates evaluated) that was indicative of a poor prognosis (RFS and OS) in training (n 5 362) and validation (n 5 225) patient cohort sets. 48 Both studies identified b-catenin as a protective prognostic indicator. Of the remaining 6 markers reported by Meyer et al, none were evaluated by Gould Rothberg et al; and, of the remaining 4 reported by Gould Rothberg et al, p21 and p16 were evaluated by Meyer et al, although that study did not examine the prognostic impact of nuclear staining only (p21) or nonnuclear/nuclear ratios (p16). In addition, in a cohort of 395 primary melanomas, Kashani-Sabet et al confirmed a protein-based prognostic signature that contained nuclear receptor coactivator 3 (NCOA3), secreted phosphoprotein 1 (SPP1) (osteopontin), and regulator of G-protein signaling 1 (RGS1), which they previously identified as prognostic, in a complementary DNA microarray-based study. 49 A multimarker index score of expression was established as an independent predictor of disease-specific survival (DSS) and SLN status and was validated in an independent patient cohort. It is noteworthy that, across multiple studies, it has been demonstrated that osteopontin is a prognostic factor, which we further detail below. In contrast to protein, mrna-based biomarkers can be robustly and quantifiably measured genome-wide using several methodologies and should be easily translatable into a highly quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) based clinical assay. However, such 4112 Cancer December 1, 2015

Determinants of Prognosis in Melanoma/Weiss et al TABLE 2. Prognostic Molecular Markers and Signatures Selected Genes Study Population and Method Clinicopathologic or Survival Associations Reference MCM3, MCM4, MCM6, KPNA2 KRT9, DCD, PIP, SCGB1D2, SCGB2A2, COL6A6, KBTBD10, ECRG2, HES6 Fresh-frozen primary melanomas (n 5 83); microarray IHC Fresh primary melanomas (n 5 41); microarray, RT-qPCR CD24, EVL Nonmetastasizing (stage I/II; n 5 116) and metastasized (stage III/IV; n 5 72) primary melanomas; SAGE BAP1, MGP, SPP1, CXCL14, CLCA2, S100A8, S100A9, BTG1, SAP130, ARG1, KRT6B, KRT14, GJA1, EIF1B, RBM23, LTA4H, CRABP2, DSC1, ROBO1, AQP3, SPRR1B, CST6, TRYP1, ID2, TACSD2, PPL Primary cutaneous melanomas (n 5 268) with semirandom stratification into 2 cohorts; RT-qPCR Genes (n 5 254) differentially expressed based on <4-y or > 4-y DMFS (n 5 58); IHC validation for 8 of 23 genes analyzed, 4 of which were associated with OS in a validation cohort (n 5 176) Genes (n 5 92) differentially expressed in primary melanomas between short-term (OS, < 4y;n520) and long-term (OS, > 5 y; n 5 21) survivors RT-qPCR analysis of 92 previously identified genes from Brunner 2008 55 ; 11 genes associated with OS in univariate Cox analyses were further measured in 53 additional primary tumors; 9 genes retained a significant association with OS Top 50 differentially expressed genes used for class prediction in 28 independent primary tumors; CD24 and EVL, alone or combined with Breslow thickness; provided the strongest predictor of outcome 20 Genes selected from mining of public data sets for genes consistently altered in primary vs metastatic melanoma; 5 additional genes prognostic in uveal melanoma (BAP1; 3 0 and 5 0 ends) and 1 gene originally selected as 1 of 4 reference genes comprise RT-qPCR based, 28-gene prognostic signature; radial basis machine modeling stratified patients into low-risk or high-risk groups, which clearly separated in Kaplan-Meier DFS curves; multivariate Cox analyses with key prognostic parameters indicated that this signature may be independently prognostic Same as above Subset of above Follow-up study examined 28-gene signature in 217 patients with primary melanoma who had undergone SLNB; in this patient cohort, the expression signature performed as well or better as an independent predictor of DFS, DMFS, and OS HOXA1 targets genes NA HOXA1 proinvasion transcriptional signature identified; the signature classified patient samples from Winnepennninckx 2006 55 CD2 Stage II (n 5 47) and III (n 5 48) FFPE primary melanomas; Nanostring mirna mir-382, mir-516b FFPE primary melanomas (n 5 92, n 5 119); mirna microarray 134 mirnas FFPE primary melanomas (n 5 82, n 5 97); mirna microarray A 53-gene panel of immunoregulatory genes in patients with stage II and III melanoma predicted nonprogression, RFS, and DSS Reported mirnas associated with tumor thickness and recurrence status, but focused on functional contributions to metastatic spread; 2 metastasissuppressor mirnas (mir-382 and mir-516b) were not clearly associated with survival parameters in multivariate analyses (data not reported) Reported mirna associated with nodular vs superficial spreading melanoma subtypes in primary melanoma tissues; prognostic implications within each subtype were not reported Winnepennninckx 2006 56 Brunner 2008 57 Brunner 2013 58 Gschaider 2012 59 Gerami 2015 60 Gerami 2015 61 Wardwell-Ozgo 2014 62 Sivendran 2014 63 Hanniford 2015 64,65 Poliseno 2012 66 Cancer December 1, 2015 4113

Review Article TABLE 2. Continued Selected Genes Study Population and Method Clinicopathologic or Survival Associations Reference mir-200 family, mir-203 Fresh-frozen primary melanomas (n 5 23); RT-qPCR mir-30d FFPE primary melanomas (n 5 92); mirna microarray mir-1908, mir-199a-3p, mir-199a-5p Primary melanomas (n 5 71); RT-qPCR mir-15b Primary melanomas (n 5 128); RT-qPCR Eleven mirnas (of 159 measured) associated with tumor thickness; in validation patient set, study observed progressive loss of mir-200a, mir-200c, and mir-203 expression from nevi, to primary, to metastasis and at the invasive front of primary tumors Prometastatic mir-30d expression associated with stage, thickness, histologic subtype, and RFS and OS in primary melanoma samples mir-1908, mir-199a-3p, and mir-199a-5p were up-regulated in a melanoma metastasis in vivo selection model; study observed univariate associations with metastasis-free survival in patient samples Expression of proproliferative mir-15b associated with poor RFS and OS in multivariate analyses; mir-34a and mir-210 did not associate with survival parameters van Kempen 2012 67 Gaziel-Sovran 2011 68 Pencheva 2012 69 Satzger 2010 70 Abbreviations: AQP3, aquaporin 3 (Gill blood group); ARG1, arginase 1; BAP1, breast cancer 1 (BRCA1)-associated protein-1 (ubiquitin carboxy-terminal hydrolase); BTG1, B-cell translocation gene, antiproliferative); CD2, cluster of differentiation 2 (cell adhesion molecule); CD24, cluster of differentiation 24 (heat stable antigen CD24); CLCA2, chloride channel accessory 2; COL6A6, collagen, type VI, a6; CRABP2, cellular retinoic acid binding protein 2; CST6, cystatin E/M; CXCL14, chemokine (C-X-C motif) ligand 14; DCD, dermcidin; DFS, disease-free survival; DMFS, distant metastasis-free survival; DSC1, desmocollin 1; DSS, disease-specific survival; ECRG2, esophageal cancer-related gene 2; EIF1B, eukaryotic translation initiation factor 1B; EVL, Enah/Vasp-like; FFPE, formalin-fixed, paraffin-embedded; GJA1, gap junction protein a1, 43 kda; HES6, hes family basic helix-loop-helix transcription factor 6; HOXA1, homeobox A1; ID2, inhibitor of DNA binding 2, dominant negative helix-loop-helix protein; IHC, immunohistochemistry; KBTBD10, kelch repeat and BTB domain containing protein 10; KPNA2, karyopherin a2 (RAG cohort 1, importin a1); KRT14, keratin 14, type I; KRT6B, keratin 6B, type II; KRT9, keratin 9, type I; LTA4H, leukotriene A4 hydrolase; MCM3, minichromosome maintenance complex component 3; MCM4, minichromosome maintenance complex component 4; MCM6, minichromosome maintenance complex component 6; MGP, matrix Gla protein; mir, microrna; mrna, messenger RNA; NA, not available; OS, overall survival; PIP, prolactin-induced protein; PPL, periplakin; RBM23, RNA binding motif protein 23; RFS, recurrence-free survival; ROBO1, roundabout guidance receptor 1; RT-qPCR, quantitative reverse transcriptase-polymerase chain reaction; S100A8, S100 calcium-binding protein A8; S100A9, calcium-binding protein A9; SAGE, serial analysis of gene expression; SAP130, Sin-3A-associated protein, 130 kda; SCGB2A2, seroglobin, family 2A, member 2; SCGB1D2, secretoglobin, family 1D, member 2; SLNB, sentinel lymph node biopsy; SPP1, secreted phosphoprotein 1; SPRR1B, small proline-rich protein 1B; TACSD2, tumor-associated calcium transducer 2; TRYP1, tryparedoxin peroxidase. studies are limited by poor-quality mrna obtained in archival FFPE tissues. 13,50 Moreover, contamination from infiltrating cells and the surrounding microenvironment likely result in significant expression bias and complicate cross-study comparisons. There are numerous reports documenting mrna expression profiling of melanoma cell lines and metastatic melanoma tissues or comparisons across melanoma progression (nevi, primary, metastasis). 51-56 However, only a few studies have examined gene expression profiling or have developed mrna-based prognostic signatures in primary melanoma tissues from patients who had different outcomes (Table 2). Winnepenninckx et al reported mrna expression profiles for 83 fresh-frozen, primary cutaneous melanomas in which they defined a set of 254 genes that had differential expression based on 4-year distant metastasis-free survival (DMFS), and they identified high expression of minichromosome maintenance (MCM) genes as key predictors of reduced DMFS. 56 A follow-up study reanalyzed those data in the context of biologic pathways and revealed that high expression of DNA repair genes was associated with poor DMFS. 71 It is noteworthy that another recent study, in which a proinvasive homeobox A1 (HOXA1) transcriptional signature was defined, indicated that patients from the study by Winnepenninckx et al who had a high HOXA1 signature had poor DMFS, adding an intriguing underlying transcriptional mechanism to the original expression findings, although the authors did not note whether DNA repair pathway genes were highly represented in their signature. 62 A second primary cutaneous melanoma expression-profiling study identified 92 genes associated with OS 57 ; and a subsequent follow-up study, which further assessed those 92 genes, reported that a 9-gene signature (6 protective and 3 unfavorable genes) was associated with OS in a 91-patient cohort. 58 A recent report used serial analysis of gene expression to identify differentially expressed transcripts between 116 primary tumors that had not metastasized 4114 Cancer December 1, 2015

Determinants of Prognosis in Melanoma/Weiss et al (stage I/II) and 72 primary tumors that had already spread at the time of initial diagnosis (stage III/IV). 59 Subsequent multivariate class-prediction analyses of the top 50 differentially expressed genes identified CD24 (heat-stable antigen CD24) and Enah/Vasp-like (EVL) as strong predictors of metastatic risk in an independent set of 28 primary melanoma samples. Conway et al 72 performed complementary DNA-mediated annealing, selection, extension, and ligation expression profiling of 502 cancer genes on >350 FFPE cutaneous primary melanomas from 2 patient cohorts and observed that high osteopontin (SPP1) expression was associated with reduced RFS, which confirmed previous findings. 51,73,74 Osteopontin has been implicated in cell motility, proliferation, and activation of the nuclear factor-jb pathway, which may add mechanistic explanations for its prognostic capacity. 73 A subsequent reanalysis of those data by the same group associated the increased expression of DNA repair genes with poor RFS, 75 consistent with the described reanalysis of the data reported by Winnepenninckx et al. 71 Collectively, those studies reported from tens to hundreds of genes associated with metastatic risk and/or survival outcomes in primary cutaneous melanoma. No reported genes overlapped in more than 2 studies. Of the 50 genes reported by Gschaider et al, 59 7 genes overlapped with those reported by Winnepenninckx et al, 56 although it is noteworthy that the cellular source of those RNAs is likely to be keratinocytes, endothelial cells, or infiltrating immune cells, which would support the microenvironment and/or the host response as key prognostic markers for patient outcomes in primary cutaneous melanoma (Supporting Table 1; see online supporting information). Five genes in a combined list from Conway et al and Jewell et al overlapped with those reported by Winnepenninckx et al, and osteopontin overlapped between the studies by Conway/Jewell et al and Gschaider et al. None of the 92 genes that originally were associated with OS by Brunner et al 57 and further assessed in a follow-up study 58 overlapped with genes reported in the other studies, although we note that none of the other studies defined genes that were directly associated with OS. Two recent studies by Gerami and colleagues selected a set of 28 gene targets (melanoma metastasisassociated genes identified from data mining, previously defined prognostic uveal melanoma genes, and breast cancer 1-associated protein-1 [ubiquitin carboxy-terminal hydrolase] [BAP1]) for analysis as a prognostic molecular signature in melanoma. 60,61 In their first study, the gene set was measured in 268 primary melanoma patient samples, which were mostly randomly stratified to a training cohort (n 5 164) and a validation cohort (n 5 104). 61 In the second study, the analysis of gene set measurements was restricted to patients who had undergone an SLN biopsy (SLNB) procedure. Prognostic modeling was performed using radial basis machine modeling to separate patients into 2 risk classes, which were plotted on Kaplan- Meier survival curves and compared with AJCC stage, Breslow depth, ulceration, mitotic rate, and age or SLN status in univariate and multivariate analyses. Both studies indicated that the defined signature was an independent predictor of metastatic risk. Those studies are the basis of a clinical prognostic test (DecisionDx) offered by Castle Biosciences (Friendswood, Tex), which is discussed below. EXPRESSION OF MIRNA A newer class of RNA mirna has been of particular interest to cancer researchers over the last decade. Although fewer in number than mrnas and not translated to protein, mirnas are ideal potential biomarkers because they are quantifiable genome-wide and are highly stable in FFPE tissues, 76,77 although they are equally affected by sample purity as are mrna. mirna expression profiling has typically been performed using microarrays (genome-wide, but inconsistently quantitative) or RT-qPCR (fewer mirnas, but highly quantitative), although newer technologies, including RNA-sequencing and Nanostring, allow highly quantitative genome-wide analyses. Early studies of global mirna expression in primary melanoma did not focus on prognosis but, rather, on the identification of mirnas that play functional roles in melanoma tumorigenesis. 78-82 Our group reported 18 mirnas that were associated with post-recurrence survival, 83 and a subset of 6 of those mirnas (mir-150, mir-342-3p, mir-455-3p, mir-145, mir-155, and mir- 497) was defined as a post-recurrence survival signature. It is worth noting that mir-342-3p is located in an intron of its host gene EVL, which was recently associated with metastatic risk in primary melanoma. 59 Another recent study replicated the prognostic importance of mir-150 and mir-142 (from the 18 mirnas that were associated with postrecurrence survival) 83 in stage III lymph node metastases and patient sera. 84 We recently reported the identification of metastasissuppressive mirnas that we originally identified as differentially expressed in array-based global mirna profiling of primary cutaneous melanoma tissues (2 cohorts of 92 and 119 patient samples). 64 We observed that these mirnas were associated with thickness, recurrence status, and/or Cancer December 1, 2015 4115

Review Article RFS. From those data, we defined a 4-miRNA signature (mir-150, mir-15b, mir-16-5p, andmir-374b-3p) that, in combination with AJCC stage, was predictive of developing brain metastasis. 65 Our group also reported a series of 134 mirnas that were differentially expressed in nodular melanoma (n 5 56) versus superficial spreading melanoma (n 5 26) histologic subtypes, controlling for stage 66 ; however, the possibility that selected mirna signatures have independent prognostic importance for these subtypes has not yet been explored. van Kempen et al performed expression profiling of 159 mirnas using RT-qPCR in 23 fresh-frozen melanoma samples. 67 In their patient cohort, 11 mirnas were associated with tumor thickness. In a validation patient set, expression analysis of those 11 mirnas and further in situ hybridization revealed loss of mir-200 family (mir-200a and mir-2900c)and mir-203 expression during melanoma progression and at the invasive front in primary tumors. Of the mirnas that were consistently correlated with tumor thickness in our patient cohorts (n 5 27 mirnas), 10 were measured in the study by van Kempen et al; 4 of those 10 (mir-200b, mir-200c, mir-203, and mir-205) were correlated with thickness; and each maintained the same direction as in our data (Supporting Table 2; see online supporting information). Of the 11 mirnas reported by van Kempen and colleagues, there was evidence of a correlation with thickness for 9 in at least 1 of our data sets, and the directions of all correlations were maintained between studies. Several other studies identified mirnas that play functional roles in the proliferative or invasive/metastatic capacity of melanoma cells based on analyses of mirna expression and associations with patient outcomes. Our group previously reported that high expression in primary melanoma tissues of mir-30d, a prometastatic mirna that regulates glycosylation and immune suppression in melanoma, is associated with advanced stage, increased thickness, nodular histologic subtype, and poor RFS and OS. 68 Another recent study used in vivo selection in mice to generate highly metastatic melanoma cell line variants. 69 The mirnas mir-1908, mir-199a-3p, and mir-199a-5p, which are highly up-regulated in these metastatic variants and are critical drivers of melanoma progression, were inversely associated with metastasis-free survival in a cohort of 71 primary melanoma samples, although multivariate analyses were not performed, and patient characteristics were not reported. Several reports have defined loss of mir-211 expression as a key feature of melanoma cells, and its overexpression is suppressive of melanoma cell proliferation and invasion in vitro. 80,82,85 Surprisingly, however, no studies have yet reported any association of mir-211 expression in primary tumors with patient outcomes. In this regard, the results from 1 of our recently published data sets, which included a set of congenital nevi, are supportive of decreased abundance of mir-211 in primary melanoma compared with nevi. 64 However, mir-211 expression in those tumors was not clearly associated with prognostic parameters or patient outcomes in the data set (data not shown). Finally, Satzger et al reported that high expression of the proproliferative mirna mir-15b in a set of 128 primary melanoma tissues was significantly associated with poor outcomes (RFS and OS) in multivariate analyses. 70 Overall, these studies have demonstrated the potential utility of mirna expression-based prognostic assays; however, there is limited overlap of the key mirnas reported as prognostic in these studies, and sample sizes remain modest. Based on tissue and serum studies (described below), mir-150 and mir-15b are associated the most consistently with prognosis in patients with primary and metastatic melanoma. IMMUNE MARKERS OF THE HOST Tumor-Infiltrating Lymphocytes Immunologic markers are being studied as prognostic tools that may enhance outcome predictions and could be incorporated into traditional TNM melanoma staging. For example, the Immunoscore has been designed to prognosticate early stage colon cancer patients based on CD3-positive, CD8-positive, and CD45-positive lymphocyte densities within tumors. High Immunoscores in colon cancer are associated with improved RFS and OS. 86 Tumor-infiltrating lymphocytes (TILs) have been investigated across multiple malignancies for decades as a surrogate for antitumor immune response; however, current cancer staging systems do not incorporate TIL status. There is debate concerning whether TIL status is clearly associated with melanoma prognosis (Table 3). Clark et al reviewed 98 primary melanomas for 8 pathologic variables, including tumor-infiltrating lymphocytes (TILs), which were assigned 1 of 3 grades as measured in the vertical growth phase: brisk (lymphocytes infiltrate the entire vertical growth phase or tumor base), nonbrisk (lymphocytes are focally present in the vertical growth phase), or absent (lymphocytes may be present in fibrotic areas or near vessels but do not infiltrate the vertical growth phase). TIL grade was significantly correlated with 8-year OS. 87 Since then, data in the literature both 4116 Cancer December 1, 2015

Determinants of Prognosis in Melanoma/Weiss et al support 90,94-96 and refute 88,91-93 the prognostic relevance of absent, nonbrisk, and brisk TILs in primary melanomas. Azimi and colleagues scored 1865 primary melanomas using a different TIL grading system with a score that ranged from 0 to 3 and demonstrated that TIL grade was inversely associated with SLN status. TIL grade 0 was associated with a 27.8% rate of SLN positivity compared with a rate of only 5.6% for TIL grade 3. 94 Higher TIL grade also was significantly associated with longer RFS and MSS (survival rate, 100% vs 75% for TIL grades 3 vs 0, respectively; median follow-up, 43 months). Multivariate analysis demonstrated that the strongest predictors of RFS were thickness, ulceration, and TIL grade. 94 Similarly, in 887 patients who underwent SLN mapping, Taylor et al demonstrated that the absence of TILs was predictive of SLN positivity in both univariate and multivariate analysis; however, in contrast to the results of Azimi et al, there was no difference in DFS or OS. 91 There are several reasons for the discrepancies in TIL grade among studies. Studies differ by sample size, patient population, characteristics of the primary tumors, and even method of grading TILs. For example, Clemente et al examined 285 primary melanomas, of which 82% were thicker than 2 mm, and observed an association of TIL grade with survival. 89 In contrast, Barnhill et al studied 548 primary melanomas with a higher proportion of thinner tumors (43% measured < 0.76 mm in thickness) in which there was no significant difference in OS based on TIL grade. 88,89 It is believed that TILs are more frequent in thin melanomas 91 ; and; in a study of 293 highrisk primary melanomas (thickness > 4 mm), TIL grade did not correlate significantly with survival outcomes, although there was a trend toward improved RFS survival for the presence of TILs. 97 Furthermore, studies vary in the TIL groups they compare for their survival analyses. Some only compare absent versus present TIL grade 88,91 without separating out the brisk and nonbrisk groups, whereas others group nonbrisk and absent TIL grades 90 or do not address absent TILs at all, 93 which makes crossstudy comparisons challenging. The identification of balance between immunostimulatory and immunosuppressive T-cell populations within the lymphocytic infiltrates is an overlooked consideration. For example, in a study of CD3, CD8, CD20, and forkhead box P3 (Foxp3) IHC expression on lymph nodes that were positive for melanoma, the ratios of peritumoral to intratumoral TILs for both CD3 and CD8 expression were lower in patients who developed recurrent melanoma. 96 In metastatic melanoma, Erdag et al characterized immunotypes A, B, and C, which roughly compare with absent, nonbrisk, and brisk TIL grades, and demonstrated differences in the median OS rate (15, 23, and 130 months, respectively) as well as differences in the cellular composition of the immune infiltrate present in each immunotype. 99 Higher densities of CD8-positive T cells and CD45- positive leukocytes, T cells, and B cells were associated with improved survival. There was no correlation of Foxp3, a regulatory T-cell marker, with survival. The proportion of B cells was highest in immunotype C, and the proportion of macrophages was lowest in immunotype C. 99 Immunoregulatory Gene Expression Levels A newer area of investigation beyond pathologic TIL classification is aimed at identifying differentially expressed genes with immunoregulatory functions, with the hope of characterizing prognostic immune biomarkers. This is now possible with the advent of efficient, high-throughput technology, such as Nanostring, that can quantify gene expression levels for up to 800 genes of interest from small amounts of RNA isolated from FFPE tissue. Recently, a 53-gene immune panel proposed from an initial set of 446 genes was tested for prognostic value using Nanostring technology in RNA isolated in melanomas from 47 patients with stage II and III disease. 63 The gene signature was identified as predictive of nonprogression, RFS, and DSS. The candidate genes are important for T-cell and natural killer cell function and leukocyte migration, and they are involved in immune surveillance pathways. For example, C-C chemokine receptor type 5 (CCR5), CD8, CD3, and IKAROS family zinc finger 1 (IZKF1), were more highly expressed in patients who did not progress. Specifically, the most differentially expressed gene was CD2, a costimulatory molecule expressed on T cells and natural killer cells. Increased CD2-positive cells are correlated with melanoma nonprogression, as confirmed by IHC. 63 The Cancer Genome Atlas recently reported a distinct immunologically characterized subgroup of cutaneous melanomas that predict improved survival outcomes. Regional melanoma lymph nodes were examined for a lymphocyte score (LScore), which was calculated based on the density and location of TILs, with higher LScores correlating with prolonged survival and stratification into the immune subclass. In addition, elevated expression of the LCK (proto-oncogene, Src family tyrosine kinase) and SYK (spleen tyrosine kinase) nonreceptor tyrosine kinases involved in lymphocyte signaling were identified in the immune subgroup, and a correlation of increased LCK gene expression and improved survival was observed. 100 Although the differing study designs and patient populations challenge our understanding of the prognostic Cancer December 1, 2015 4117

Review Article TABLE 3. Tumor-Infiltrating Lymphocyte Grade in Primary Melanomas and Association With Survival Outcomes No. of Patients Survival Associations Comments Reference 264 Predicts 8-y OS (TILs: brisk, 88%; Clark 1989 87 nonbrisk, 75%; absent, 59%) 548 No survival differences Compared absent vs present TILs only Barnhill 1996 88 285 Predicts 5-y OS (TILs: brisk, 77%; Clemente 1996 89 nonbrisk, 53%; absent, 37%) 259 Predicts 5-y OS (TILs: brisk, 100%; Groups with nonbrisk and absent TILs Tuthill 2002 90 nonbrisk and absent, 71%) were included in the analysis 887 No survival differences Groups with nonbrisk and brisk TILs Taylor 2007 91 were included in the analysis 1251 No survival differences Mandala 2009 92 515 Predicts 5-y OS (TILs: brisk, 95%; Did not address absent TILs Burton 2011 93 nonbrisk, 84%) 1865 Predicts 5-y OS Used their own TIL grading system Azimi 2012 94 assigning a score from 0 to 3 2845 Predicts 5-y OS (TILs: brisk, 97%; nonbrisk, 94%; absent, 93%) Thomas 2013 95 Abbreviations: OS, overall survival; TILs, tumor-infiltrating lymphocytes. value of TILs, the role of the host antitumor immune response in melanoma cannot be ignored. Future studies characterizing immune phenotypes and perhaps an Immunoscore or quantitative molecular markers of host responses are needed to better assess the correlation of TILs with clinical outcomes. Gene expression analyses of immunoregulatory genes like that by Sivendran et al may identify key immunologic markers that can be linked with current known AJCC prognostic factors or molecular markers for more accurate outcome predictions. SERUM MARKERS Although the serum marker lactate dehydrogenase is incorporated into the AJCC staging system for the substaging of metastatic (stage IV) melanoma, no serumbased biomarkers have been identified that would meet criteria for incorporation into the staging system for earlier stage melanoma (predictive of relapse) or to provide better prognostic or therapeutic guidelines in advanced melanoma. Melanoma-inhibitory activity protein (MIA) is produced and secreted by melanoma cells. In a cohort of 84 patients with stage I and II cutaneous melanoma who were followed prospectively, high versus low serum MIA concentrations were associated with increased recurrence (66% vs 5%) and reduced DFS at 4 years (30.3% vs 94.5%). 101 Serum levels of S100B, a protein involved in microtubule assembly and inhibition of the tumor suppressor p53, increase progressively in each stage of melanoma and associate negatively with survival. 102,103 C-reactive protein (CRP) is an acute-phase reactant that is elevated in numerous clinical scenarios, including states of infection, inflammation, and malignancy. The measurement of elevated serum CRP levels may be superior to serum lactate dehydrogenase measurements in identifying patients who progress to metastatic melanoma. 104 In multivariate analyses, serum CRP levels measured at the time of primary melanoma resection and then sequentially thereafter revealed an association between elevated CRP levels and worse OS, MSS, and DFS in patients with stage I and II melanoma, suggesting that serum CRP may be a valuable prognostic biomarker in melanoma. 105 Two studies by our group examined the utility of serum-based mirna abundance for prediction and surveillance of recurrence in patients with melanoma. 106,107 Friedman et al defined a 5-miRNA signature from sera isolated in blood obtained at diagnosis from 80 patients with primary melanoma that classified patients into high-risk and low-risk groups, which separated well in Kaplan-Meier RFS curves. In a follow-up study, we also developed a refined, 4-miRNA, serabased signature (mir-150, mir-15b, mir-30d, and mir-425) adjusted for AJCC stage to define low-risk and high-risk patients, which stratified patients by RFS and OS into 2 cohorts (n 5 201 and n 5 82, respectively). 106 Finally, there is interest in measuring circulating tumor DNA (ctdna) in patients with advanced melanoma as an early indicator of response to treatment. 108,109 Although ctdna measurements are not currently used in clinical practice, prospective studies will address whether ctdna can be used as a blood-based biomarker to detect melanoma recurrence. 4118 Cancer December 1, 2015