RAS Mutations Are Associated With the Development of Cutaneous Squamous Cell Tumors in Patients Treated With RAF Inhibitors

Abstract

Purpose

RAF inhibitors are effective against melanomas with BRAF V600E mutations but may induce keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cSCCs). The potential of these agents to promote secondary malignancies is concerning. We analyzed cSCC and KA lesions for genetic mutations in an attempt to identify an underlying mechanism for their formation.

Methods

Four international centers contributed 237 KA or cSCC tumor samples from patients receiving an RAF inhibitor (either vemurafenib or sorafenib; n = 19) or immunosuppression therapy (n = 53) or tumors that developed spontaneously (n = 165). Each sample was profiled for 396 known somatic mutations across 33 cancer-related genes by using a mass spectrometric–based genotyping platform.

Results

Mutations were detected in 16% of tumors (38 of 237), with five tumors harboring two mutations. Mutations in TP53, CDKN2A, HRAS, KRAS, and PIK3CA were previously described in squamous cell tumors. Mutations in MYC, FGFR3, and VHL were identified for the first time. A higher frequency of activating RAS mutations was found in tumors from patients treated with an RAF inhibitor versus populations treated with a non–RAF inhibitor (21.1% v 3.2%; P < .01), although overall mutation rates between treatment groups were similar (RAF inhibitor, 21.1%; immunosuppression, 18.9%; and spontaneous, 17.6%; P = not significant). Tumor histology (KA v cSCC), tumor site (head and neck v other), patient age (≤ 70 v > 70 years), and sex had no significant impact on mutation rate or type.

Conclusion

Squamous cell tumors from patients treated with an RAF inhibitor have a distinct mutational profile that supports a mechanism of therapy-induced tumorigenesis in RAS-primed cells. Conceivably, cotargeting of MEK together with RAF may reduce or prevent formation of these tumors.

INTRODUCTION

At least 50% of all melanomas carry an activating mutation in the BRAF oncogene.¹ In the advanced setting, the treatment of these melanomas with the selective RAF inhibitors vemurafenib (formerly PLX4032 or RG7204) and GSK2118436 has yielded response rates of 50% to 80%^2–4 and an improvement in overall survival when compared with conventional chemotherapy.⁵ Similar to patients treated with other small-molecule kinase inhibitors, patients treated with a selective RAF inhibitor frequently experience skin toxicities.⁶ However, a striking distinction of these agents has been the development of skin tumors in the form of keratoacanthomas (KAs) or cutaneous squamous cell carcinomas (cSCCs) in up to approximately 25% of patients.^2,4,5 These lesions most frequently develop within 8 to 12 weeks of beginning therapy. Similar treatment-related skin neoplasms have been described with the structurally unrelated multikinase inhibitor sorafenib.^7,8 Sorafenib has been reported to have pan-RAF inhibitory properties,⁹ although the overall cellular potency of this compound against RAF proteins is much less pronounced when compared with selective inhibitors.¹⁰ Perhaps not surprisingly, sorafenib-induced skin tumors occur much less frequently and are more delayed in onset.^7,8 Together, these observations suggest that RAF inhibition may play a direct role in the development of skin tumors.

The concept that a targeted therapy that blocks an oncogenic pathway in one cell type may promote tumorigenesis in another is both novel and potentially concerning. Given that RAF inhibitors will likely gain widespread use in melanoma and perhaps other cancers, deciphering the molecular basis of inhibitor-induced cutaneous neoplasms is essential. One potential mechanism is suggested by recent preclinical experiments demonstrating that while RAF inhibitors inhibit mitogen-activated protein kinase (MAPK) signaling in BRAF-mutant cancer cells, they may also cause a paradoxical increase in MAPK signaling in the context of mutated or activated RAS. Toward this end, RAS mutations have previously been identified in actinic keratoses^11–13—premalignant skin lesions with the potential to transform into cSCCs.¹⁴ We therefore hypothesized that RAS activation in certain cutaneous cell subpopulations might interact with RAF inhibitor therapy to promote cell proliferation, ultimately resulting in KAs and cSCCs.

To test this hypothesis, we used a mass spectrometric genotyping platform (OncoMap) to generate mutational profiles for KA and cSCC lesions that developed in patients treated with an RAF inhibitor. As a comparator, we evaluated similar tumors that developed spontaneously or in the setting of immunosuppressive therapy.

METHODS

Tumor Specimens

Archival formalin-fixed paraffin-embedded (FFPE) cSCC and KA tumor specimens were collected from four international centers: the University of Essen, Essen, Germany; the Peter MacCallum Cancer Centre, East Melbourne, Australia; the University Hospital Zurich, Zurich, Switzerland; and the Gustave Roussy Institute, Villejuif, France. These samples were enriched for tumors that developed in patients undergoing treatment with an RAF inhibitor (vemurafenib or sorafenib) or immunosuppressive therapy for solid organ or bone marrow transplantations. Relevant clinical data were obtained from patients' medical records, and all samples were de-identified before analysis. The study was conducted with the approval of local institutional review boards.

DNA Preparation

Each tumor specimen was independently reviewed by two dermatopathologists to confirm the diagnosis. Tumor-rich areas (> 70% purity) were dissected and scraped from consecutive unstained FFPE slides. Genomic DNA was extracted by using the Qiagen DNeasy extraction kit (Qiagen, Valencia, CA) per the manufacturer's instructions. DNA quality was assessed by quantification with Picogreen and polymerase chain reaction amplification of fragments 100 to 200 base pairs (bp) long.

Mass Spectrometric Genotyping

High-throughput mutation profiling was performed on each sample by using the OncoMap platform. As previously described,¹⁵ this approach interrogated 396 mutations across 33 known oncogenes and tumor suppressor genes. Genomic DNA obtained from tumor samples was initially screened by using iPLEX (Sequenom, San Diego, CA) genotyping, and candidate mutations were validated by using homogeneous mass extension chemistry on unamplified DNA. The cancer gene mutations interrogated by OncoMap were selected on the basis of a combination of historically documented mutation frequencies and their potential as therapeutic targets.^15,16 Primers and probes were designed by using Sequenom MassARRAY Assay Design 3.0 software (Sequenom), as described previously.^15,16

Statistical Methods

For categorical comparisons, we performed either Fisher's exact test or the χ² test by using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). All tests were two-sided, and a threshold of P < .05 was used to define statistical significance.

RESULTS

Characteristics of Clinical Tumor Samples

A total of 237 FFPE clinical tumor specimens were evaluated for this study, consisting of 191 cSCCs and 46 KAs (Table 1). One hundred sixty-five samples were classified as “spontaneous” (ie, they originated in patients who did not receive either significant immunosuppressive therapy or small-molecule RAF inhibitor therapy); included were samples from four patients receiving cytotoxic chemotherapy, two patients taking thalidomide, one patient receiving interferon, and one patient who was HIV-positive. Patients undergoing significant immunosuppressive therapy contributed 53 samples (47 samples from solid organ transplantations, three from bone marrow transplantations, and one sample each from patients with severe psoriasis, aplastic anemia, and unknown), and 19 samples were derived from patients receiving small-molecule RAF inhibitors. Seven patients enrolled in phase I and II studies of vemurafenib contributed 10 lesions (four lesions from one patient), with lesions being excised between 48 and 107 days (mean, 70 days) after commencing vemurafenib. Nine samples were from patients receiving sorafenib for 3 to 9 months. Not unexpectedly, surrogate markers of higher ultraviolet radiation exposure (increased age, head and neck site) were statistically favored in the spontaneous versus nonspontaneous cohorts (P < .01), likely reflecting the lack of an additional precipitant when compared with the treatment groups.

Table 1.

Clinicopathologic Characteristics of Squamous Cell Tumor Samples

Characteristic	RAF-Inhibitor Treated(n = 19)		Immunosuppressed(n = 53)		Spontaneous(n = 165)		Total(N = 237)
	No.	%	No.	%	No.	%	No.	%
Sex
Male	10	52.6	41	77.4	113	68.5	164	69.2
Female	9	47.4	12	22.6	52	31.5	73	30.8
Age, years
Range	44-89		42-83		26-104		26-104
Mean	64.6		64.2		76.9		73.1
SEM	2.6		1.3		0.9		0.8
Site
Head and neck	5	26.3	24	45.3	102	61.8	131	55.3
Trunk	2	10.5	3	5.7	5	3.0	10	4.2
Upper limb	7	36.8	13	24.5	24	14.5	44	18.6
Lower limb	5	26.3	10	18.9	25	15.2	40	16.9
Genital	—		—		5	3.0	5	2.1
Unknown	—		3	5.7	4	2.4	7	3.0
Histology
cSCC	9	47.4	48	90.6	134	81.2	191	80.6
KA	10	52.6	5	9.4	31	18.8	46	19.4

Abbreviations: cSCC, cutaneous squamous cell carcinoma; KA, keratoacanthoma; SEM, standard error of the mean.

Mutations in cSCC and KA Detected by Mass Spectrometric Genotyping

The OncoMap platform identified mutations in 38 of the 237 samples tested. Five samples exhibited co-occurring mutations; thus, a total of 43 mutations were detected (Fig 1; Table 2). The overall frequency of mutations was not significantly different between samples in the RAF inhibitor therapy, immunosuppression therapy, or spontaneous groups (21.1%, 18.9%, and 17.6%, respectively) nor did the frequency vary with patient age, sex, or tumor site. Furthermore, there was a similar rate and range of mutations between the cSCCs and KAs.

Fig 1.

Mutually exclusive and co-occurring mutations in human squamous cell tumors. Each column describes an individual sample with a detected mutation. Affected genes are listed in rows, with single mutations indicated by black bars and co-occurring mutations indicated by red bars. Histology (cutaneous squamous cell carcinoma [cSCC] and keratoacanthoma [KA]) and treatment cohort (RAF-inhibitor, gold; immunosuppressed, gray; spontaneous, blue) are detailed on the bottom two rows.

Table 2.

Characteristics of Samples With Detected Mutations

Patient	Sex	Age (years)	Histology	Site	Mutation Type	Patient Cohort	Treatment
1	Male	89	cSCC	Arm	HRAS Q61L	RAF kinase inhibitor	Vemurafanib
2	Female	54	KA	Back	HRAS Q61L	RAF kinase inhibitor	Vemurafanib
3	Male	71	KA	Chest	HRAS G12D; BRAF V600E	RAF kinase inhibitor	Vemurafanib
4	Male	53	KA	Thigh	HRAS Q61L	RAF kinase inhibitor	Sorafenib
5	Male	59	cSCC	Ear	CDKN2A P114L; TP53 R248W	Immunosuppression	Methotrexate, cyclosporin, efalizumab, adalimumab
6	Male	82	KA	Lower arm	CDKN2A R80*	Immunosuppression	Cyclosporin
7	Male	67	cSCC	Lower leg	TP53 R273H	Immunosuppression	Cyclosporin
8	Male	68	cSCC	Face	TP53 R248Q	Immunosuppression	Drugs not specified
9	Male	74	cSCC	Leg	TP53 R248W	Immunosuppression	Cyclosporin
10	Female	61	KA	N/A	TP53 R248Q	Immunosuppression	Tacrolimus, mycophenylate mofitil, azathioprine
11	Male	60	cSCC	Lower arm	HRAS Q61K	Immunosuppression	Cyclosporin
12	Male	76	cSCC	Hand	KRAS G12D	Immunosuppression	Cyclosporin
13	Female	42	cSCC	Chest	PIK3CA E545K	Immunosuppression	Rapamycin, sirolimus
14	Male	80	cSCC	Hand	CDKN2A H83Y; TP53 R248Q	Spontaneous	No treatment
15	Male	69	cSCC	Head	CDKN2A R58*; TP53 R248W	Spontaneous	No treatment
16	Male	60	cSCC	Leg	CDKN2A R80*; PIK3CA E545K	Spontaneous	Methotrexate, cisplatin, fluorouracil, bleomycin
17	Male	81	cSCC	Face	CDKN2A P114L	Spontaneous	No treatment
18	Male	79	cSCC	Face	CDKN2A P114L	Spontaneous	No treatment
19	Male	72	cSCC	Scalp	CDKN2A R58*	Spontaneous	No treatment
20	Male	104	cSCC	Head	CDKN2A R58*	Spontaneous	No treatment
21	Male	71	cSCC	Head	CDKN2A H83Y	Spontaneous	No treatment
22	Male	77	cSCC	Ear	CDKN2A P114L	Spontaneous	No treatment
23	Male	65	cSCC	Hand	CDKN2A E88*	Spontaneous	No treatment
24	Male	81	cSCC	Lower arm	TP53 R248W	Spontaneous	No treatment
25	Male	83	cSCC	Ear	TP53 R248W	Spontaneous	No treatment
26	Female	88	KA	Cheek	TP53 R248W; FGFR3 G370C	Spontaneous	No treatment
27	Female	77	KA	Cheek	TP53 R273C	Spontaneous	No treatment
28	Male	81	cSCC	Cheek	HRAS Q61L	Spontaneous	No treatment
29	Male	75	cSCC	Ear	HRAS Q61L	Spontaneous	No treatment
30	Female	77	cSCC	Genital	HRAS Q61L	Spontaneous	No treatment
31	Male	75	cSCC	Auricle	HRAS G12V	Spontaneous	No treatment
32	Female	26	KA	Lip	HRAS Q61L	Spontaneous	No treatment
33	Male	77	cSCC	Arm	PIK3CA E542K	Spontaneous	No treatment
34	Female	81	cSCC	Forehead	PIK3CA E545K	Spontaneous	No treatment
35	Female	62	KA	Lower Leg	PIK3CA H1047R	Spontaneous	No treatment
36	Female	78	cSCC	Upper arm	MYC A59V	Spontaneous	No treatment
37	Male	57	cSCC	Face	FGFR3 G370C	Spontaneous	No treatment
38	Male	52	cSCC	Lower leg	VHL P81S	Spontaneous	No treatment
39	Male	61	cSCC	Arm	BRAF V600E	RAF kinase inhibitor	Vemurafanib

Abbreviations: cSCC, cutaneous squamous cell carcinoma; KA, keratoacanthoma; NA, not available.

^*Denotes stop codon.

Mutations were detected across eight different cancer-related genes. In keeping with previous studies,¹⁷) the most frequently involved genes were TP53 (n = 11), CDKN2A (n = 12), and the RAS isoforms HRAS (n = 10) and KRAS (n = 1). Not previously identified in cSCCs and KAs were mutations in PIK3CA (n = 5), FGFR3 (n = 2), MYC (n = 1), and VHL (n = 1).

RAS Mutations Occur More Frequently in Tumors From Patients Treated With RAF Inhibitors

Tumors from the cohort of patients treated with an RAF inhibitor were enriched for HRAS mutations despite similar rates of total mutations between groups. Known activating mutations in HRAS (Q61L, G12D) were identified in 30% (95% CI, 10% to 61%) of samples from patients treated with vemurafenib and 11% (95% CI, < 0.01% to 46%) of samples from patients treated with sorafenib. Combined, HRAS mutations were found in four of the 19 samples from patients treated with an RAF inhibitor compared with six of 218 HRAS mutations (21.1% v 2.8%; P < .01) and a single KRAS mutant (all RAS mutations, 21.1% v 3.12%; P < .01) in samples treated with a non-RAF inhibitor. No NRAS mutations were identified in this study. Furthermore, in the cohort of patients treated with an RAF inhibitor, no activating mutations were identified in 11 receptor tyrosine kinases that are commonly mutated in human cancers and that function upstream of RAS (CSF1R, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FLT3, KIT, MET, PDGFRA, and RET).

Surprisingly, we also identified BRAF V600E mutations in two samples from patients treated with vemurafenib for BRAF V600E-mutant metastatic melanoma (samples 3 and 39). Further immunohistochemical studies for AE1/AE3 (squamous cell carcinoma), S100, and melan-A (melanoma) identified clearly separate populations of malignant squamous cells and melanocytes in close proximity, with one sample showing evidence of metastatic melanoma cells within the lymphovascular space (Appendix Fig A1, online only). The BRAF mutations in these samples were therefore attributable to melanocytic contamination, and these mutations were not included in our statistical analysis.

DISCUSSION

Recent preclinical studies have identified the potential for selective RAF inhibitors to augment MAPK pathway activation in the context of activated RAS. In these systems, signaling occurred preferentially through C-RAF, with RAF inhibitors thought to induce a conformational change in C-RAF heterodimers or B-RAF homodimers that resulted in pathway hyperactivity.^18–21 In our study, we found that squamous cell tumors arising in patients treated with an RAF inhibitor are enriched for RAS mutations when compared with those from untreated patients. This finding suggests that the formation of these tumors is not due to a direct mutagenic event of RAF inhibitor therapy but is caused, at least in part, by a pro-proliferative interaction between RAF inhibitors and latent RAS mutant keratinocytes.

Unlike in other common skin cancers such as melanoma or basal cell carcinoma, a predominant mutational driver has yet to be identified in cSCC. H-, K-, and NRAS mutations occur infrequently in cSCC from the general^11,13,22,23 and immunosuppressed²⁴ populations and are reported at similarly low frequencies (1% to 12%) in premalignant actinic keratoses.^11–13,25 The presence of RAS mutations in these premalignant lesions (common in sun-damaged skin) suggests that ultraviolet radiation can induce RAS mutations in keratinocytes but that these mutations are not sufficient to induce malignant transformation. When RAS-activated keratinocytes are exposed to RAF inhibition, additional checks and balances may be exceeded, resulting in abnormal cell growth and, ultimately, progression to either KA or cSCC. This model would fit the clinical observations that the KA and cSCC lesions most commonly develop in the first 8 to 12 weeks after initiating RAF inhibitor therapy and are more frequent in patients with increased sun exposure, supporting the existing presence of a predisposing lesion. In some cases, these tumors have spontaneously regressed on treatment discontinuation. The mechanisms for this regression are not clear but could involve altered signaling, activation of a senescence-like program, or induction of an immune response to the KA and cSCC lesions. Regarding the latter, some studies have noted the presence of cytotoxic T lymphocytes, Langerhans cells, and CD30⁺ cells within KA lesions.^26–29

So far, the cutaneous squamous cell tumors that have developed in patients receiving vemurafenib and GSK2118436 have all been well differentiated and have not metastasized or recurred after complete excision.^4,30 Sorafenib is a multikinase inhibitor whose effects against RAF isoforms are considerably less pronounced in vivo than those of vemurafenib and GSK2118436, which may explain the lower incidence of skin tumors seen with this drug.^7,9 Notably, sorafenib is ineffective against BRAF-mutant melanoma; this reduced in vivo potency may relate in part to its higher affinity for the inactive conformation of this kinase than the active form acquired with a BRAF V600E mutation.³¹ In the context of patients with advanced melanoma and its associated poor prognosis, development of KA or cSCC as a treatment-related adverse effect has been considered acceptable to both patients and clinicians. However, the confirmation of a clinically significant interaction between selective RAF inhibitors and RAS-activated cells raises several additional considerations.

The most concerning of these is the potential for secondary malignancies other than those of the skin. RAS mutations are estimated to occur in 30% of all human cancers, with a significant further proportion having either activating mutations or overexpression of upstream receptor tyrosine kinases (such as HER2, c-KIT, and EGFR³²). Furthermore, apart from occurring in actinic keratoses, RAS mutations also occur as early genetic events in a range of premalignant lesions. For instance, KRAS mutations represent early genetic events in colon carcinogenesis: they are present in up to 50% of colonic adenomas.^33,34 They are also found at increasing frequency with progressively higher grades of pancreatic intraepithelial neoplasia, correlating with increasing risk of progressing to carcinoma³⁵ and are more commonly detected in the bronchial washings of smokers compared with those of nonsmokers.³⁶ In principle, exposure of these cell subpopulations to RAF inhibitors could promote clonal expansion and propel them toward permanent malignant transformation.

Why have no such extracutaneous tumors been detected thus far? One possible reason might be that the median treatment duration of vemurafenib and GSK2118436 (6 to 9 months) has not been long enough for these lesions to manifest. Alternatively, some such events may have been misinterpreted as melanoma progression. It is also possible that RAS-mutant cell populations in other organs may not undergo the sustained proliferation typical of fully malignant neoplasms. Concerns of possible tumorigenic complications may become heightened by the inevitable transition of these agents into the adjuvant setting, in which treatment duration could last 1 to 2 years, and the clinical impact of any secondary malignancies might be increased. Thus, for patients participating in the initial adjuvant studies of these agents, careful surveillance will be essential. Aggressive histologic characterization (which may include tumor mutational profiling) may be needed for new lesions that arise during adjuvant treatment using selective RAF inhibitors.

Our findings also illustrate how proposed strategies to overcome resistance and potential strategies to prevent secondary tumor development may converge going forward. To date, several mechanisms of secondary RAF inhibitor resistance have been postulated. These include acquired mutations in NRAS and overexpression of PDGFR-β and IGFR, both of which can operate upstream of RAS.^37,38 These models may suggest a switch to C-RAF–driven MAPK signaling³⁹ that is operant in some cases. Conceivably, such resistance mechanisms might be circumvented through the development of more potent RAF inhibitors that abrogate the mechanism of RAF activation observed with existing compounds, or alternatively by blocking signaling downstream of both B-RAF and C-RAF by targeting MEK or ERK. Clinical studies of concomitant RAF and MEK inhibitors have commenced in an attempt to prolong the effectiveness of (or overcome resistance to) RAF blockade in BRAF V600E-mutant melanomas. Preliminary results suggest that this combination may reduce the incidence of RAF inhibitor–induced KA and cSCC lesions.⁴⁰ We speculate that such combinations may also suppress proliferation of RAS-activated nonmelanoma cell populations elsewhere in the body.

More generally, our study provides one of the largest mutational studies of cSCCs and KAs reported to date. Consistent with the previous literature, the most frequently mutated genes were TP53 and CDKN2A.^41,42 However, we detected substantially lower rates of mutations in these genes (4.6% and 5.1%, respectively) compared with those previously reported (44.4% and 24.5%, respectively), most likely reflecting the known limitation of genotyping-based platforms in detecting mutations in tumor suppressor genes. Inactivating mutations are more diverse and therefore harder to cover with multiplexed, mutation-specific assays when compared with the limited number of functional activating mutations in oncogenes. For instance, our assays covered only 11% of TP53 and 24% of CDKN2A mutations previously identified in cSCC, whereas 100% of previously described H-, K-, and N-RAS mutations were assessed. Of note, PTCH1 was reportedly mutated in more than 5% of cSCC samples; however, this finding was limited to only one study,⁴³ in which all three nonsilent mutations occurred in patients with a history of multiple basal cell carcinomas (PTCH1 was not assessed in our study). We identified novel mutations in four genes: PIK3CA, FGFR3, MYC, and VHL, but these occurred in no more than 2% of samples. No difference was identified in the rate or types of mutations between cSCCs and KAs. The histologic and biologic distinction between these entities remains an area of controversy for dermatopathologists.^44,45

Finally, although we have identified mutations in RAS in roughly 20% of squamous cell tumors that developed during therapy with an RAF inhibitor, tumorigenic mechanisms operant in RAS-negative KA and cSCC lesions remain unclear. It is possible that the frequency of RAS mutations in treatment-induced KAs and cSCCs will rise as larger patient cohorts treated at maximal RAF inhibitor doses are analyzed. Additional mechanisms may involve activation of upstream effectors (eg, receptor tyrosine kinases) by gene amplification or overexpression, which were not examined here. The application of more comprehensive mutation profiling technologies such as targeted, massively parallel sequencing may shed additional light on the full spectrum of genomic alterations that drive the biology of these squamous cell tumors. Several other mechanisms have been proposed by which tumors may develop in the presence of sorafenib, with most being related to its multikinase activity.^46,47 How such mechanisms might relate to those linked to selective RAF inhibitors remains unclear.

In summary, exposure to selective RAF inhibitors may lead to pro-proliferative effects on RAS-primed cells. This has already manifested clinically in the form of squamous cell tumors, but the potential may also exist to promote growth of other extracutaneous neoplasms (and to promote resistance in melanoma) by the same mechanism. Cotargeting of MEK (or, in the future, ERK) together with RAF may block this effect. Thus, compound MAP kinase pathway inhibition may simultaneously enhance antitumor efficacy and restrict pro-neoplastic adverse effects of single-agent RAF inhibition.

Appendix

Fig A1.

Tumor samples with BRAF V600E mutations demonstrating colocalized cutaneous squamous cell tumors and melanoma. (A) Sample 3: hematoxylin and eosin (H+E) stain demonstrating classical keratoacanthoma histology, with an interspersed S100-positive melanocytic population. (B) Low-power and (C) high-power images of H+E stains from sample 39. AE1/AE3-positive cells (squamous cell carcinoma) are demonstrated immediately adjacent to S100-positive and melan-A–positive cells (melanoma).

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: None Consultant or Advisory Role: Caroline Robert, GlaxoSmithKline (C), Roche (C); Grant A. McArthur, GlaxoSmithKline (U), Roche (U); Levi A. Garraway, Foundation Medicine (C), Novartis (C), Daiichi Sankyo (C) Stock Ownership: Levi A. Garraway, Foundation Medicine Honoraria: Caroline Robert, GlaxoSmithKline, Roche; Levi A. Garraway, Boehringer Ingelheim, Constellation Pharmaceuticals, Merck, Novartis Research Funding: Levi A. Garraway, Novartis Expert Testimony: None Other Remuneration: None

AUTHOR CONTRIBUTIONS

Conception and design: Patrick A. Oberholzer, Damien Kee, Caroline Robert, Günther F.L. Hofbauer, Grant A. McArthur, Dirk Schadendorf, Levi A. Garraway

Financial support: Levi A. Garraway

Provision of study materials or patients: Damien Kee, Caroline Robert, Günther F.L. Hofbauer, Grant A. McArthur, Dirk Schadendorf

Collection and assembly of data: Patrick A. Oberholzer, Damien Kee, Piotr Dziunycz, Antje Sucker, Nyam Kamsukom, Robert Jones, Christine Roden, Clinton J. Chalk, Kristin Ardlie, Emanuele Palescandolo, Adriano Piris, Laura E. MacConaill, Caroline Robert, Günther F.L. Hofbauer, Grant A. McArthur

Data analysis and interpretation: Patrick A. Oberholzer, Damien Kee, Grant A. McArthur

Manuscript writing: All authors

Final approval of manuscript: All authors