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Correspondence to: Leslie Castelo-Soccio, MD, PhD, Children’s Hospital of Philadelphia, 3550 Market St, Second Floor, Philadelphia, PA 19104 E-mail: [email protected] REFERENCES 1. P€ uttgen KB. Diagnosis and management of infantile hemangiomas. Pediatr Clin North Am. 2014;61:383-402. 2. McMahon P, Oza V, Frieden IJ. Topical timolol for infantile hemangiomas: putting a note of caution in ‘‘cautiously optimistic.’’. Pediatr Dermatol. 2012;29:127-130. 3. Berk D, Lehman P, Franz TJ, et al. On topical timolol gel-forming solution for infantile hemangiomas. Pediatr Dermatol. 2013;30: 160-161. 4. Beal BT, Chu MB, Siegfried EC. Ulcerated infantile hemangioma: novel treatment with topical brimonidine-timolol. Pediatr Dermatol. 2014;31:754-756. http://dx.doi.org/10.1016/j.jaad.2015.10.021

Lack of TERT promoter mutations in melanomas with extensive regression To the Editor: The recently discovered TERT promoter mutations have been shown to play an important role in melanoma pathogenesis.1 These mutations imply an increased gene expression, with the restoration of telomerase activity and the stabilization of telomeres length.2 Interestingly, some authors have described absence of telomerase activity and shortening of telomeres in regressing swine melanoma cell lines.3 Therefore, although most studies have focused on immunologic response to explain spontaneous regression, telomere dysfunction has been proposed as an additional mechanism.4 We sought to determine whether melanomas with and without regression harbor different prevalences of TERT promoter mutations. A case-control study was designed in a series of 110 primary melanoma samples diagnosed between January 2012 and December 2014. Ethical approval from the Institutional Review Board and informed consent from all patients were obtained. Clinical characteristics and histologic parameters were collected. Regression was classified as absent (47%; 52/110), partial with less than 50% of the specimen involved (41%; 45/110), or extensive with more than 50% involvement (12%; 13/110). Genomic DNA was extracted from formalin-fixed and paraffin-embedded sections using the GeneRead DNA FFPE Kit (Qiagen), according to manufacturer’s protocol. Mutational status of TERT promoter region was determined by polymerase chain reaction and Sanger sequencing as has been previously described.5 The relationship between mutational status and clinicopathologic features was evaluated

Fig 1. Association of TERT promoter mutation status and clinicopathologic features. TERT promoter mutations are associated with sex, location, histological subtype, Breslow thickness, ulceration, regression, and mitosis. P values were derived from 2-test and considered statistically significant if \.05.

Fig 2. Distribution of TERT promoter mutations according to regression. TERT promoter mutations were significantly associated with melanomas without regression. None of the melanomas with extensive regression harbored recurrent mutations.

using 2 and logistic regression analysis. P values \.05 were considered statistically significant. TERT promoter mutations were detected in 30% (33/110) of melanoma samples. The somatic changes 146C[T and 124C[T were detected in 45% (15/33) and 27% (9/33) of the samples, respectively. Mutations

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Table I. Association of TERT promoter mutations with clinicopathologic characteristics TERT promoter status Variables

Age \40 40-65 [65 Sex Male Female Sunburns in melanoma localization No Yes Solar lentigines No Yes Anatomic site Head/neck Upper extremities Trunk Lower extremities Acral Histologic subtype Lentigo maligna melanoma Superficial spreading Nodular Acral Other Breslow thickness \1 mm 1-2 mm 2-4 mm [4 mm Ulceration No Yes Regression No \50% [50% Mitoses/mm2 \1 [1 Tumor stage I-II III-IV

Wildtype N (%)

Mutated N (%)

Total N

5 (83.3) 39 (76.5) 33 (62.3)

1 (16.7) 12 (23.5) 20 (37.7)

6 51 53

46 (80.7) 31 (58.5)

11 (19.3) 22 (41.5)

49 (70) 28 (70)

OR

95% CI

P value

1.89

0.90-3.95

.219

57 53

2.97

1.26-6.98

.011

21 (30) 12 (30)

70 40

1.00

0.43-2.33

35 (79.5) 42 (63.6)

9 (20.5) 24 (36.4)

44 66

2.22

0.91-5.40

.740

9 6 40 14 8

(64.3) (35.3) (83) (63.6) (88.9)

5 11 8 8 1

(35.7) (64.7) (17) (36.4) (11.1)

14 48 48 22 9

1.11

0.79-1.55

.007

6 58 4 8 1

(66.7) (73.4) (33.3) (88.9) (100)

3 21 8 1 0

(33.3) (26.6) (66.7) (11.1) (0)

9 79 12 9 1

0.98

0.57-1.70

.038

52 11 10 4

(81.3) (61.1) (55.6) (40)

12 7 8 6

(18.8) (38.9) (44.4) (60)

64 18 18 10

1.87

1.25-2.79

.014

64 (74.4) 13 (54.2)

22 (25.6) 11 (45.8)

86 24

2.46

0.96-6.29

.049

32 (61.5) 32 (71.1) 13 (100)

20 (38.5) 13 (28.9) 0 (0)

52 45 13

0.42

0.21-0.84

.025

20 (87) 57 (65.5)

3 (13) 30 (34.5)

23 87

3.50

0.96-12.76

.046

65 (69.9) 12 (70.6)

28 (31.1) 5 (29.4)

93 17

0.46

0.20-1.06

.954

were more common in women (P ¼ .011; OR ¼ 2.97 [1.26-6.98]) and were also associated with increased Breslow thickness (P ¼ .014; OR ¼ 1.87 [1.25-2.79]), non-acral skin (P ¼.007; OR ¼ 1.11 [0.79-1.55]), nodular subtype (P ¼ .038; OR ¼ 0.98 [0.57-1.70]), ulceration (P ¼ .049; OR ¼ 2.46 [0.96-6.29]), and increased mitotic rate (P ¼.046; OR ¼ 3.50 [0.96-12.76]) (Fig 1). Regarding regression, mutations were significantly associated with melanomas without regression (P ¼ .025;

1.00

OR ¼ 0.42 [0.21-0.84]). Interestingly, none of the melanomas with extensive regression harbored recurrent mutations (Fig 2). Results of the univariate analysis are list in Table I. In the multivariate analysis female gender (P ¼ .003; OR ¼ 5.20 [1.75-15.52]), increased thickness (P ¼.003; OR ¼ 2.31 [1.33-4.03]), and nodular subtype (P ¼ .025; OR ¼ 0.43 [0.20-0.90]) were independently associated with TERT promoter mutations.

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Several studies have demonstrated that TERT promoter mutations are associated with increased Breslow thickness, ulceration, nodular subtype, BRAF concomitant mutations, and poorer survival.5 Our findings provide further evidence that telomerase may be involved in the phenomenon of regression, as melanomas with regression are less frequently associated with TERT mutations than melanomas without regression. In fact, a role for telomere dysfunction in melanoma regression was already pointed out by Bastian, who proposed that cancer cells undergo a process termed ‘‘crisis.’’4 In this stage, there is an extensive genomic remodeling and cells stabilize their telomeres through activation of telomerase, so they are able to escape crisis and acquire an immortal phenotype. Conversely, in melanomas with regression there is less telomerase activity and telomere attrition becomes critical, resulting in genomic instability and leading to permanent cell cycle arrest and cell death. Improving our knowledge on telomerase biology and spontaneous regression of melanoma may have potential prognostic and therapeutic implications for the future. Blanca de Unamuno Bustos, MD,a Rosa Murria Estal, o, BSc,c Vicente Oliver MSc,c Gema Pe rez Sim d Martınez, MD, PhD, Margarita Llavador Ros, MD,b Sarai Palanca Suela, PhD,c* and Rafael Botella Estrada, MD, PhDa* Department of Dermatology,a Department of Pathology,b and Molecular Biology Laboratory, Service of Clinical Analysis,c University Hospital La Fe, and Department of Dermatology, University General Hospital of Valencia,d Spain *Senior investigators. Funding sources: This study was supported in part by the Health Research Institute La Fe for having granted the project IISLaFe 2014/0370 and the Carlos III Health Institute for having granted the project PIE13/00046. Conflicts of interest: None declared. Correspondence to: Sarai Palanca Suela, PhD, Molecular Biology Laboratory, Service of Clinical Analysis, University Hospital La Fe, Avda de Fernando Abril Martorell, n.106, 46026 Valencia (Spain) E-mail: [email protected] REFERENCES 1. Huang FW, Hodis E, Xu MJ, et al. Highly recurrent TERT promoter mutations in human melanoma. Science. 2013; 339(6122):957-959.

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2. Gomez DE, Armando RG, Farina HG, et al. Telomere structure and telomerase in health and disease (review). Int J Oncol. 2012;41:1561-1569. 3. Pathak S, Multani AS, McConkey DJ, et al. Spontaneous regression of cutaneous melanoma in Sinclair swine is associated with defective telomerase activity and extensive telomere erosion. Int J Oncol. 2000;17:1219-1224. 4. Bastian BC. Hypothesis: a role for telomere crisis in spontaneous regression of melanoma. Arch Dermatol. 2003; 139:667-668. 5. Heidenreich B, Nagore E, Rachakonda PS, et al. Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma. Nat Commun. 2014;5:3401. http://dx.doi.org/10.1016/j.jaad.2015.10.003

High specificity and sensitivity of NRAS Q61R immunohistochemistry (IHC) in melanomas To the Editor: BRAF and NRAS are the most frequently mutated genes in melanoma (40%-50% and 20% of cases, respectively).1 Determining BRAF and NRAS tumor status is mandatory for patient treatment with targeted therapies and is commonly achieved by DNA sequencing. Immunohistochemistry (IHC) has recently appeared as a reliable surrogate technique for molecular BRAF V600E mutation screening.2 Ilie et al3 in this journal, and others,4 reported that IHC had 100% specificity and sensitivity for detecting the most frequent NRAS mutation, Q61R. We aimed to confirm these results on a different IHC platform (Bond-III, Leica Biosystems, Nussloch, Germany) in a selected series of 70 stage-IV melanomas previously screened by targeted next-generation sequencing for presence of NRAS and BRAF mutations.2 After antigen retrieval (1008C for 60 minutes 1 12 minutes’ cooling with ER2 solution), slides were incubated with the anti-NRAS Q61R antibody, clone SP174 (1:100 dilution, SpringBio Science, Pleasanton, CA) for 20 minutes at 378C and revealed using the Bond Polymer Refine Red detection kit (Leica Biosystems). Of the 22 of 70 cases with NRAS Q61R mutation, all but 1 displayed a moderate to strong cytoplasmic staining in more than 80% of tumor cells (Fig 1). The 1 remaining case showed heterogenous staining, with strongly stained areas adjacent to faintly stained areas. Tumor cellularity was estimated at ;80% for this case, whereas NRAS Q61R mutation allelic ratio was estimated at ;25% using next-generation sequencing. Faintly stained areas may therefore represent a tumor subclone without NRAS Q61R mutation. The remaining 48 cases (10 NRAS Q61L, 9 NRAS Q61K, 2 NRAS Q61H, 13 BRAF V600E, 1 BRAF V600K, and 13 cases with no NRAS or BRAF mutation) showed no staining. We confirmed 100%