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Distinct Patterns of Acral Melanoma Based on Site and Relative Sun Exposure

Open ArchivePublished:September 01, 2017DOI:https://doi.org/10.1016/j.jid.2017.08.022
      Acral melanoma is distinct from melanoma of other cutaneous sites, yet there is considerable variation within this category. To better define this variation, we assessed melanomas occurring on dorsal (n = 21), volar (n = 9), and subungual/interdigital (n = 13) acral skin as well as acral nevi (n = 24) for clinical, histologic, and molecular features. Melanomas on dorsal acral surfaces demonstrated clear differences compared with volar and subungual/interdigital melanomas. The latter two groups exhibited significantly less frequent BRAF mutations (P = 0.01), were significantly less likely to have the superficial spreading histologic subtype (P = 0.01), occurred in older patients (P = 0.05), and had more frequent involvement in non-Caucasians (P = 0.01). These differences can be explained by differing levels of UV exposure. Subungual/interdigital melanomas had the most diverse group of oncogenic mutations including PIK3CA (2/13), STK11 (2/13), EGFR (1/13), FGFR3 (1/13), and PTPN11 (1/13). In addition, subungual/interdigital melanomas had a significantly higher frequency of copy number aberrations (67%) than other subgroups (P = 0.02), particularly in CDK4 and cyclin D1, and were less likely to have BRAF mutations or a superficial spreading histologic subtype (P = 0.05) compared with volar acral melanomas. Although based on a limited sample size, differences between volar and subungual/interdigital melanomas in our study may be the result of differing levels of UV exposure.

      Abbreviation:

      SSM (superficial spreading melanoma)

      Introduction

      Melanomas from acral sites are often considered a distinct subgroup with unique clinical, morphologic, and genetic characteristics. However, there can be considerable variation. Some authors exclusively refer to acral melanoma as cases that occur on the glabrous skin of the palms and soles or on subungual sites. Others include melanomas from the dorsal surfaces of the hands and feet. Acral melanoma generally occurs at a later age (60 years or older), has equal incidence in darker- and lighter-skinned individuals, and has a characteristic WHO histologic pattern known as the acral lentiginous subtype. Patients often have a poorer prognosis compared with melanomas from other sites (
      • Carrera C.
      • Gual A.
      • Diaz A.
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      • Nogues S.
      • Vilalta A.
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      Prognostic role of the histological subtype of melanoma on the hands and feet in Caucasians.
      ,

      Teramoto Y, Keim U, Gesierich A, Schuler G, Fiedler E, Tuting T, et al. Acral lentiginous melanoma—a skin cancer with unfavourable prognostic features. A study of the German Central Malignant Melanoma Registry (CMMR) in 2050 patients. Brit J Dermatol, in press.

      ). Compared with sun-exposed skin, BRAF mutations are less frequent.
      In this study, we assessed the clinical, histologic, and molecular features of 43 melanomas from acral skin. Our findings show different genetic and epidemiologic patterns in acral melanomas from the dorsal surface of the hand and foot compared with melanomas from subungual/interdigital spaces or melanomas from volar surfaces. Melanomas arising in the most UV-protected sites (subungual/interdigital) are the most genomically diverse, with a variety of non-BRAF-related mechanisms to activate cellular proliferation.

      Results

      Melanoma from dorsal acral sites

      Twenty-one melanomas were identified on dorsal acral skin, with 16 from the foot and 5 from the hand. The mean age was 55 years and included 19 women and 2 men. All patients with available demographic information identified as Caucasian (n = 19). The stages of disease included 9 melanomas in situ (43%), 7 T1a/b (33%), 3 T2a/b (14%), 1 T3a/b (5%), and 1 T4a/b (5%). WHO histologic subtypes included 16 superficial spreading (SSM) (76%), 1 lentigo maligna (5%), 2 acral lentiginous (10%), and 2 nodular (10%) melanomas. A majority of cases were BRAF mutated (67%, 14/21). Other oncogenic mutations identified included NRAS (4/21), KIT (2/21), FGFR3 (1/21), and IDH1 (1/21). One case had a mutation in TP53 (1/21). Five of 19 cases demonstrated chromosomal copy number alterations in the targeted loci (5/19, 26%) (Table 1). Copy number gains included TERT, CDK4, AUKRA, CCND1, PAK1, and GAB2. Copy number deletions included CDKN2A, PTEN, and NF1.
      Table 1Variants identified on next generation sequencing and FISH
      Oncogenes are shown in green and tumor suppressor genes are shown in red. FISH results are shown on the far right, with gains in blue and losses in yellow. Melanomas with insufficient tissue for FISH are shaded dark gray.
      Abbreviations: A, age, D, dorsal, FISH, fluorescence in situ hybridization; G, gender, I, interdigital; MIS, melanoma in situ; R, race; SU, subungual; T, subtype; V, volar.
      1This patient had a germline mutation in MLH1 and had several other mutations in genes not found in other melanomas including MET A1261T, SMO W535R, FGFR2 G305R, HNF1A R272C, CDH1 T73A, ERBB2 D880N, and SMAD4 R100G.

      Melanoma from volar acral sites

      Nine melanomas were included from volar acral surfaces. The mean age was 59 years and included seven women and two men. Ethnicity data were available for eight patients, five of whom were identified as Caucasian (63%), two as Hispanic (25%), and one was identified as African American (13%). The melanoma stages included two melanomas in situ (22%), four T1a/b (44%), and three T2a/b (33%). WHO histologic subtypes included three SSM (33%) and six acral lentiginous subtype (66%). Mutations in oncogenes included NRAS (3/9), BRAF (2/9), EGFR (1/9), and KIT (1/9). Tumor suppressor mutations included TP53 (1/9) and ATM (1/9). Copy number aberrations were found in one of nine cases (11%), which was a KIT-mutated T1a melanoma exhibiting gains in TERT (Table 1).

      Melanoma from subungual or interdigital acral sites

      Thirteen acral melanomas were found to involve the nail unit or interdigital web spaces. The mean age was 67 years with eight women and five men. Ethnicity data were available for 12 patients. Seven were Caucasian (54%), two were Hispanic (15%), two were African American (15%), and one was Asian (8%). The melanoma stages included three melanoma in situ (23%), one T1a/b (8%), three T2a/b (23%), three T3a/b (23%), and three T4a/b (23%). All cases were of the acral lentiginous (n = 11, 85%) or nodular (n = 2, 15%) subtype. Mutations in oncogenes included PIK3CA (2/13), NRAS (2/13), EGFR (1/13), BRAF (1/13), IDH2 (1/13), PTPN11 (1/13), FGFR3 (1/13), and ALK (1/13). Mutations in tumor suppressor genes included TP53 (2/13), STK11 (2/13), and APC (1/13). Eight of 12 melanomas tested exhibited copy number alterations (67%). Seven of these eight cases displayed gains in CCND1 or CDK4 (Table 1).

      Acral nevi

      Twenty-four acral nevi were assessed from 16 females and 8 males with a median age of 39 years (range 25–70 years). Nine were found in sun-exposed locations (38%), whereas seven were on sun-shielded surfaces (29%) and eight were on the digits (33%). Cases included 12 routine compound or intradermal nevi, 7 dysplastic nevi, 4 MANIAC nevi, and 1 compound nevus with a congenital pattern. Regardless of location or diagnosis, a majority of acral nevi were BRAF mutated (71%, n = 17). Three cases demonstrated mutations in NRAS, and no mutations were identified in four cases.

      Group comparison

      We compared age, race, histologic subtype (SSM vs. non-SSM), frequency of BRAF mutations, and frequency of chromosomal copy number aberrations between melanomas from dorsal acral surfaces and those from other acral sites (volar or subungual/interdigital). Patients with dorsal acral melanomas more frequently had a younger mean age (P = 0.05), were Caucasian (P = 0.01), had SSM histology (P < 0.01), and had BRAF mutations (P = 0.01). We compared the same variables between the volar acral melanomas and the subungual/interdigital melanomas. Although most volar acral melanomas were of the acral lentiginous melanoma histologic subtype, there was a statistically higher likelihood of the SSM (P = 0.05) subtype to occur among the volar cases compared with the subungual/interdigital cases. Volar acral melanomas were less likely than subungual/interdigital melanomas to have chromosomal copy number aberrations (P = 0.02).

      Discussion

      In this study, we showed that melanomas from dorsal acral sites are distinct in terms of epidemiology, morphology, and genetic drivers from melanomas from other acral sites. Melanomas found on the dorsum of the hand or foot had a younger average age (P = 0.05), were exclusive to Caucasians (P = 0.01), and were far more frequent among female patients (19/21, 90%), likely related to more UV exposure with open-toed footwear. BRAF mutations were far more frequent in this group of acral melanomas (P = 0.01) as was the SSM histologic subtype (P < 0.01) (Figure 1a–c ). A precursor nevus was found in 5 of 21 cases (24%) in a relatively similar frequency to melanomas from the intermittently sun-damaged skin of the trunk, which has been estimated at 30% (
      • Duman N.
      • Erkin G.
      • Gokoz O.
      • Karahan S.
      • Kayikcioglu A.U.
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      Nevus-associated versus de novo melanoma: do they have different characteristics and prognoses?.
      ).
      Figure 1
      Figure 1Histologic and FISH findings in a superficial spreading MIS, subungual acral lentiginous MIS, and an ALM. Case 83: superficial spreading MIS (b) on the left dorsal foot of a 53-year-old female patient with a BRAF V600E mutation (a) and copy number gains in TERT on FISH (c). Case 29: subungual acral lentiginous MIS (d, e) on the left great toe of an 82-year-old female patient with no mutations identified on next generation sequencing but significant copy number gains in CCND1on FISH (f). Case 60: acral lentiginous melanoma (h, i) found on the finger of a 68-year-old female with a PIK3CA H1047L mutation (g). Notice the acral lentiginous growth pattern with confluent, single atypical melanocytes in the basal layer of the epidermis without significant pagetosis. Scale bars = 0.2 mm in all micrographs. ALM, acral lentiginous melanoma; FISH, fluorescence in situ hybridization; MIS, melanoma in situ.
      This profile suggests that melanomas from the dorsal hand and foot are most similar to melanomas from other sites of intermittently sun-damaged skin and likely have a similar UV-driven pathway of mutagenesis. The predominance of BRAF mutations in acral nevi from all sites but only in acral melanomas from the dorsum of the foot or hand further supports the concept that BRAF-mutated melanocytic neoplasms are highly dependent on UV-mediated mutagenesis for acquisition of additional mutations and progression to melanoma (
      • Shain A.H.
      • Garrido M.
      • Botton T.
      • Talevich E.
      • Yeh I.
      • Sanborn J.Z.
      • et al.
      Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.
      ).
      When compared with melanoma from the dorsal hand or foot, melanomas involving volar or subungual/interdigital sites exhibited demographic features more consistent with those typically associated with acral melanoma including a high percentage of non-Caucasian patients (P = 0.01), older age (P = 0.05), acral lentiginous growth pattern, a low frequency of BRAF mutations (P = 0.01), and infrequent precursor nevi (2/22). Although volar acral and subungual/interdigital melanomas had many similar features, the two groups presented some notable differences. For example, the SSM histologic subtype that is typical of melanomas in intermittently sun-exposed skin was seen in 3 of 9 volar cases compared with 0 of 13 subungual/interdigital cases. In addition, chromosomal copy number aberrations previously described in melanomas from acral sites were seen in only a small percentage of our volar acral (11%) melanomas. Conversely, 67% of melanomas from subungual or interdigital sites had chromosomal copy number aberrations (P = 0.02), which in seven of eight cases included CCND1 or CDK4 (Figure 1d–f). A high frequency of chromosomal copy number changes in CCND1 or CDK4 has also been reported in other melanomas from highly sun-protected sites such as sinonasal melanomas (
      • Chraybi M.
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      • Andre J.
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      Oncogene abnormalities in a series of primary melanomas of the sinonasal tract: NRAS mutations and cyclin D1 amplification are more frequent than KIT or BRAF mutations.
      ).
      Although our sample size limits the ability to draw definitive conclusions, one possible explanation for this difference is that although volar skin is relatively sun protected, it is still possible to have episodes of intense intermittent sun exposure to volar acral surfaces, for example, with indoor or outdoor tanning. Conversely, there are studies that have shown that the nail plate can filter nearly 100% of UVB and 98% of UVA (
      • Stern D.K.
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      • Quijije J.
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      UV-A and UV-B penetration of normal human cadaveric fingernail plate.
      ). Others have similarly observed varied frequencies of UV-mutational patterns in volar melanomas (
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      • Fenwick K.
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      • MacKay A.
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      Genomic characterisation of acral melanoma cell lines.
      ,
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      The mutational burden of acral melanoma revealed by whole-genome sequencing and comparative analysis.
      ). Hence, although the majority of volar and subungual/interdigital melanomas are of the acral lentiginous melanoma subtype, there is a clinical and scientific basis to suspect a higher likelihood of UV-induced SSM-type melanomas occurring on volar acral surfaces compared with subungual/interdigital sites.
      Another factor that may be different in comparing volar acral melanomas and subungual/interdigital melanomas is the role of trauma. Some investigators have reported preferential involvement of volar melanomas to weight-bearing areas of the foot (
      • Costello C.M.
      • Pittelkow M.R.
      • Mangold A.R.
      Acral melanoma and mechanical stress on the plantar surface of the foot.
      ). In our study, six of seven volar melanoma cases with sufficiently detailed site descriptions to determine the exact volar location occurred in weight-bearing areas of the foot such as the ball or the heel.
      A majority of the volar acral and subungual/interdigital melanoma cases lacked mutations in BRAF and demonstrated more diversity in oncogenes involved compared with dorsal acral melanomas, which is further consistent with the supposition that volar acral and especially subungual/interdigital melanomas are most reliant on non-BRAF and non-UV-mediated pathways to malignancy. Two subungual/interdigital melanomas demonstrated mutations in PIK3CA, a known oncogenic driver that leads to constitutive activation of the phosphatidylinositol-3-kinase /acutely transforming retrovirus AKT8 in rodent T-cell lymphoma pathway in a wide variety of malignancies. Activating mutations in PIK3CA have been identified in a small proportion of melanoma cases from the trunk and some desmoplastic melanomas (
      • Janku F.
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      Mutations of PIK3CA are rare in cutaneous melanoma.
      ,
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      • Sanborn J.Z.
      • et al.
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      ,
      • Silva J.M.
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      ). The PIK3CA gene encodes for the catalytic subunit of the phosphatidylinositol 3-kinase protein, which acts via the acutely transforming retrovirus AKT8 in rodent T-cell lymphoma to activate a variety of antiapoptotic factors and simultaneously promote G1-S cell cycle progression through activation of the mechanistic target of rapamycin pathway (Figure 2) (
      • Halilovic E.
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      ). Mutations in the mechanistic target of rapamycin have been shown to be significantly more common in acral melanoma than in other subtypes (
      • Kong Y.
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      ). Both mutations in PIK3CA map to the critical C-terminal alpha-helix of the catalytic/kinase domain (p110 subunit) (Figure 3). The H1047L variant previously identified in melanoma and, to our knowledge, the previously unreported M1040V mutation lie in the same critical protein domain, further suggesting that these variants are true oncogenic drivers. These mutations may be of considerable therapeutic significance, as there are FDA-approved targeted inhibitors of PIK3CA.
      Figure 2
      Figure 2Melanoma signaling pathway depicting degrees of UV-driven mutagenesis and mutational diversity among acral sites. Schematic showing the RAS/RAF/MEK pathway as well as the PI3K/AKT pathway. Genes or proteins are shown with their relative mutation distributions among dorsal, volar, and subungual melanomas with dorsal in red, volar in blue, and subungual in green. The sun symbol indicates UV predominant pathways. Activation of the PI3K/AKT pathway, primarily through silencing of PTEN, is a frequent finding in BRAF-mutated melanomas. Mutations in the catalytic subunit of PI3K (PIK3CA) were found with high frequency in our sun-shielded melanomas. This mutation is relatively uncommon in sun-exposed melanoma, and may represent an alternative means of PI3K/AKT pathway activation in sun-shielded tumors. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; ERK, extracellular signal–regulated kinase; MEK, mitogen activated protein kinase; mTOR, mechanistic target of rapamycin; PI3K, phosphatidylinositol-3-kinase.
      Figure 3
      Figure 3Crystal structures showing identified mutations in their respective domains. Structures are labeled and color coded by their major domains and mutations are represented by red spheres. (a) The crystal structure of PIK3CA (Protein Data Bank Code 4ZOP) is shown with the M1040V and H1047L mutations localizing to the same C-terminal alpha-helix within the catalytic domain of PIK3CA (gray). (b) The crystal structure of the EGFR tyrosine kinase domain (Protein Data Bank Code 2J6M) is shown with the T790M and L704F mutations. (c) The crystal structure of SHP2 (Protein Data Bank Code 2SHP), encoded by PTPN11 gene, is shown with a Q510L mutation. (d) The crystal structure of STK11 (Protein Data Bank Code 2WTK) is shown with E57K and P281R mutations. Both mutations are in the serine/threonine-protein kinase domain.
      Subungual/interdigital cases were the only melanomas found to have mutations in STK11 (2/13), a gene that acts primarily as a tumor suppressor and is involved in several cell processes including cell polarity, energy metabolism, checkpoint DNA repair, and protein translation (
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      ). It acts via the AMP-activated protein kinase pathway to decrease the activity of the mechanistic target of rapamycin (Figure 2). STK11 mutations have been identified in pancreatic, cervical, and non-small-cell lung cancer. Mutations in STK11 are relatively uncommon in melanoma, occurring in only 10% of tumors (
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      ). Missense variants at codons 55 and 56 and other substitutions at codon 281 of STK11 have been shown to be pathogenic (
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      ). The E57K and P281R variants identified in this study are both found in the catalytic domain of the kinase (Figure 3) and to our knowledge previously unreported.
      One T3b subungual melanoma exhibited a Q510L mutation in PTPN11. PTPN11 mutations have been identified in desmoplastic melanoma, and substitutions at Q510 have been implicated in several malignancies, including neuroblastoma and acute myeloid leukemia (
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      A T4b interdigital melanoma was found to have a N646D mutation in FGFR3, a gene shown to be mutated in up to 70% of urothelial carcinomas (
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      ). A similar FGFR3 mutation was identified in a dorsal BRAF-mutated melanoma. Both mutations are located in a region of the kinase domain of the protein (Figure 3) where high frequencies of missense mutations have been identified in other malignancies (
      • Weinstein J.N.
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      ). FGFR3 activation decreases apoptosis and leads to activation of the mitogen-activated protein kinase pathway (
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      Activating mutations in PIK3CA and FGFR3 have been found with high frequency in solar lentigos, whereas germline mutations in PTPN11 and STK11 can lead to LEOPARD and Peutz-Jegher syndromes, two conditions characterized by abnormal lentigines and mucocutaneous pigmentation (
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      ). Interestingly, both early acral lentiginous melanomas and solar lentigines show increased single melanocytes predominantly along the basal layer of the epidermis (Figure 1g–i). Hence, it is plausible that in some cases this shared early morphology is the result of a common early oncogenic driver mutation in PIK3CA, FGFR3, PTPN11, or loss of STK11. Conversely, melanomas with BRAF mutations, which predominate in nevi and melanomas from intermittently sun-exposed skin, have more frequent nesting at earlier stages and more frequent pagetosis (
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      ). In our experience assessing many histopathologic samples of melanonychia striata, a histopathologic diagnosis of lentigo or atypical basal melanocytic hyperplasia resembling a solar lentigo is a considerably more frequent finding than subungual nevus and hence may be a precursor to subungual melanomas (
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      Mutations in the oncogene EGFR were seen in one volar and one subungual/interdigital melanoma. Recently,
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      ). Although uncommon in sun-exposed melanoma, activating EGFR mutations are prevalent in non-small-cell lung cancer. The T790M EGFR mutation, found in a volar melanoma, leads to acquired resistance to tyrosine kinase inhibitors by enhancing receptor adenosine triphosphate affinity in lung cancer but has also been shown to cause tumorigenesis as a de novo mutation (
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      ). The L704F is of uncertain significance. As there are known inhibitors to EGFR, activating mutations may be of therapeutic significance.
      Three germline variants of uncertain significance were also identified. Patients with germline variants in ATM (P604S) and SMO (R400H) were diagnosed with melanoma at ages significantly younger than the average of all included patients (35 and 42). Heterozygous missense mutations in ATM, including the variant identified in our study, have been postulated to increase the risk for breast cancer and aggressive Hodgkin’s lymphoma (
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      • Feilotter H.E.
      • Mulligan L.M.
      • Nagai M.A.
      ATM gene mutations in sporadic breast cancer patients from Brazil.
      ,
      • Offit K.
      • Gilad S.
      • Paglin S.
      • Kolachana P.
      • Roisman L.C.
      • Nafa K.
      • et al.
      Rare variants of ATM and risk for Hodgkin's disease and radiation-associated breast cancers.
      ). An 80-year-old patient was found to have a germline mutation in MLH1 (S406N), a gene implicated in Lynch syndrome. This mutation is of uncertain significance based on previous studies (
      • Johnston J.J.
      • Rubinstein W.S.
      • Facio F.M.
      • Ng D.
      • Singh L.N.
      • Teer J.K.
      • et al.
      Secondary variants in individuals undergoing exome sequencing: screening of 572 individuals identifies high-penetrance mutations in cancer-susceptibility genes.
      ), yet this patient’s tumor tissue exhibited a significantly higher mutational burden than any other included cases, indicating that the variant likely affected the protein’s DNA repair function and may have played a role in tumorigenesis.
      In summary, within the category of acral melanoma, there is considerable variation dependent on the degree of sun exposure. Dorsal surface acral melanomas exhibit characteristics of sun-exposed melanomas of other sites. Melanomas from subungual/interdigital web spaces and volar acral melanomas were characterized by older age of onset, infrequent WHO classification of SSM, infrequent BRAF mutations, and occurrence in non-Caucasian and Caucasian populations. Furthermore, our data suggest that subungual/interdigital melanomas may differ slightly from volar acral melanomas, constituting a class of even greater UV independence because of UV filtration through the nail plate. These tumors had the greatest oncogenic diversity and the lowest frequency of BRAF mutations (1/13). We identified a variety of pathogenic oncogenes and tumor suppressor genes and a high frequency of copy number gains in CCND1 or CDK4 (67%) when compared with the other subgroups. Many of the genes involved such as PIK3CA, PTPN11, STK11, and FGFR3 have also been associated with lentigines. Interestingly, early forms of subungual melanoma often have morphologic overlap with lentigines and do not show early nesting and other features associated with BRAF-mutated melanomas. Importantly, some mutations found in sun-protected melanomas such as the EGFR T790M and the PIK3CA mutations have known targeted inhibitors, and thus potential therapeutic significance. Hence, when assessing for potential therapeutic targets, it is especially important in cases of sun-protected acral melanomas to use a broad next generation sequencing panel, compared with sun-exposed melanomas where BRAF mutations predominate.

      Materials and Methods

      After Northwestern Institutional Review Board approval, we searched our dermatopathology database for melanomas and nevi from acral sites. We identified 90 patients with melanoma from acral sites and excluded 47 patients who did not have adequate tumor and control tissue for analysis. Each case was evaluated for clinical, histologic, and genetic features.

      Statistical analysis

      Student’s t-test and Fisher’s exact tests (SPSS Statistics 23, IBM, Armonk, NY) were used to compare dorsal acral and nondorsal acral melanomas for age, ethnicity (Caucasian vs. non-Caucasian), histologic subtype (SSM vs. non-SSM), frequency of BRAF mutations, and frequency of chromosomal copy number aberrations. The same variables were compared between subungual/interdigital and volar acral melanomas. P-values < 0.05 attained statistical significance.

      Fluorescence in situ hybridization

      Fluorescence in situ hybridization was performed using probes targeting loci commonly aberrant in acral melanoma including TERT (5p15.33), AURKA (20q13.2), CCND1 (11q13.3), CDK4 (12q14.1), CDKN2A (9p21.3), PAK1 (11q14.1), PTEN (10q23.31), NF1 (17q11.2), and GAB2 (11q14.1). Probes were prepared via the manufacturer’s protocol (Empire Genomics, Buffalo, NY). Slides were examined using the Olympus BX41 microscope and CellSens Standard for nuclei detection and image processing. Fluorescence in situ hybridization enumeration was performed as previously described, and cases were considered positive if greater than 30% of enumerated cells showed evidence of copy number gains of a given locus for oncogenes or greater than 30% had deletions for tumor suppressor genes (
      • Gerami P.
      • Jewell S.S.
      • Morrison L.E.
      • Blondin B.
      • Schulz J.
      • Ruffalo T.
      • et al.
      Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma.
      ,
      • Gerami P.
      • Li G.
      • Pouryazdanparast P.
      • Blondin B.
      • Beilfuss B.
      • Slenk C.
      • et al.
      A highly specific and discriminatory FISH assay for distinguishing between benign and malignant melanocytic neoplasms.
      ).

      Next generation sequencing

      We evaluated 50 genes using the Ion Torrent PGM and the Ion Torrent AmpliSeq Cancer Hotspot Panel v2 (Life Technologies, Grand Island, NY). Sequencing methodology was as previously described for genital melanoma and nevi cases (
      • Yelamos O.
      • Merkel E.A.
      • Sholl L.M.
      • Zhang B.
      • Amin S.M.
      • Lee C.Y.
      • et al.
      Nonoverlapping clinical and mutational patterns in melanomas from the female genital tract and atypical genital nevi.
      ).

      Variant calling

      Data generated by the PGM were analyzed using the Ion Torrent Variant Caller plug-in (Life Technologies). Annotated variants were reviewed using the Ion Reporter version 5.2 online software (Life Technologies) to evaluate variant effect. Intronic mutations and/or synonymous mutations were excluded. Nonsynonymous exonic variants were included if they occurred with a frequency of >1%, were not seen in normal controls, and had at least five reads at the mutant allele. Nonsynonymous mutations were visualized using the Integrative Genomic Viewer (
      • Robinson J.T.
      • Thorvaldsdottir H.
      • Winckler W.
      • Guttman M.
      • Lander E.S.
      • Getz G.
      • et al.
      Integrative genomics viewer.
      ) to exclude strand bias, homopolymers, and mispriming. A two-tailed Fisher’s exact test was used to compare the read distributions between tumor and normal samples with a threshold of P < 0.05 for statistical significance (
      • Dees N.D.
      • Zhang Q.
      • Kandoth C.
      • Wendl M.C.
      • Schierding W.
      • Koboldt D.C.
      • et al.
      MuSiC: identifying mutational significance in cancer genomes.
      ). Statistical analysis was performed using SAS 9.4 (SAS, Cary, NC). The pathogenicity of all mutations identified as somatic variants was analyzed using the Sorting Intolerant From Tolerant (SIFT) score (
      • Kumar P.
      • Henikoff S.
      • Ng P.C.
      Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.
      ) as well as the Clinvar (
      • Landrum M.J.
      • Lee J.M.
      • Benson M.
      • Brown G.
      • Chao C.
      • Chitipiralla S.
      • et al.
      ClinVar: public archive of interpretations of clinically relevant variants.
      ) and COSMIC databases. Mutations found in both tumors and normal controls were considered germline variants. Germline variants were evaluated using the ExAC database (
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • Samocha K.E.
      • Banks E.
      • Fennell T.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      ) and those found in greater than 1% of the population were considered polymorphisms and excluded.

      CastPCR on EGFR T790M

      Next generation sequencing revealed EGFR T790M mutations with a very low allele frequency in eight cases. Only one of eight samples passed our variant calling criteria above. Considering the potential therapeutic significance, castPCR was performed for validation. Twenty nanograms of DNA templates from tumor or normal samples was mixed with 1x Taqman genotyping master mix (Life Technologies), nuclease-free water, and 1x Taqman EGFR T790M mutant assay or 1x Taqman EGFR reference assay in a 20-μl reaction. Three different wild-type DNA and three technical replicates were used for ΔCt cutoff determination. All samples were loaded onto a 96-well plate and underwent real-time PCR on a QuantStudio 7 Flex Real-Time PCR System (Life Technologies). The PCR reaction started with an incubation step at 95 °C for 10 minutes, followed by 5 cycles of 92 °C for 15 seconds and 58 °C for 1 minute and then 40 cycles of 92 °C for 15 seconds and 60 °C for 1 minute.
      Amplification plots were generated by the QuantStudio Real-time PCR System v1.3 using 0.2 as Ct (threshold cycle). ΔCt value from wild-type DNA was normalized by the Mutation Detector Software v2.0 to determine the ΔCt cutoff for other tested samples. The mutation was detected when the normalized ΔCt was lower than the ΔCt cutoff. In our experiment, samples with Ct lower than 37 and ΔCt cutoff lower than 9.61 were designated EGFR T790M positive. CastPCR resulted in one EGFR T790M-positive sample, which was consistent with our next generation sequencing result (Table 2, Figure 4).
      Table 2Consistency of results from NGS and CastPCR for EGFR T790M detection
      Sample typeEGFR T790M allele frequency from NGSCastPCR ΔCt cutoffCastPCR normalized ΔCt
      Tumor1.90%9.619.35
      Negative controln/a9.6113.16
      Abbreviation: NGS, next generation sequencing.
      Figure 4
      Figure 4Amplification plot showing signals for tumor sample versus negative control. Cast-PCR for validating the EGFR T790M mutation in an acral melanoma patient. Of note, the tumor sample showed a stronger amplification signal than the negative control.

      ORCID

      Conflict of Interest

      PG has served as a consultant for Myriad Genomics, DermTech Int., and Castle Biosciences and has received honoraria for this. All other authors state no relevant conflicts of interest. This work is original and has not been previously published.

      Acknowledgments

      This work was partially supported by the IDP Foundation and the Melanoma Research Foundation (SP0043559).

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      Linked Article

      • Clinical, Epidemiological, and Molecular Heterogeneity in Acral Melanoma
        Journal of Investigative DermatologyVol. 138Issue 2
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          Acral melanoma comprises a poorly characterized and distinct type of melanoma, in terms of differing roles of UVR, molecular substrate, distribution among all ethnicities, and poor prognosis. Haugh et al. explore clinical, histological, and molecular aspects of acral melanomas and provide insights into the complexity of these tumors.
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