AMP case report: NGS in the diagnosis of RAS opathies in histologically uninformative skin biopsy samples

The definitive treatment of nevus sebaceus is full-thickness dermal and epidermal excision; however, controversy exists on whether prophylactic excision is warranted.^12,13 In addition to surgery, laser and photodynamic therapy have also been explored for treatment of nevus sebaceus, with varying degrees of success.¹³

Although secondary transformation of affected tissue is typically seen post puberty, it is not uncommon for malignancy to arise within nevus sebaceus during childhood.⁷ Secondary neoplasms occur in about 24 percent of sebaceous nevi and are primarily benign, most commonly trichoblastomas and syringocystadenoma papilliferum. However, malignant neoplasms such as basal cell carcinoma and squamous cell carcinoma have also been reported,¹³ with basal cell carcinoma being the most common malignant neoplasm arising in sebaceous nevi.¹⁴ These observations support ongoing, lifelong clinical assessment of lesions.

Molecular studies have provided opportunities to better understand the underlying genetic causes of nevus sebaceus and Schimmelpenning syndrome. Studies using Sanger and whole exome sequencing have revealed postzygotic activating mutations are frequent, with HRAS c.37G>C being the most common, followed by KRAS c.35G>A.^1-3

While most cases of sebaceous nevi can be diagnosed on histologic examination, occasional cases, such as presented here, demonstrate that molecular techniques may be helpful for confirmation of diagnosis and may spare patients additional procedures. Possible scenarios for molecular testing include when microscopic assessment is inconclusive in the setting of reasonable clinical suspicion, or when skin biopsy cannot be performed in the setting of suspected Schimmelpenning syndrome. Possible substrates for molecular testing can include biopsy material, peripheral blood, or even lesional tissue obtained from scratched-off scurf.⁵ In such clinical scenarios, appropriate genetic testing can help prevent multiple biopsies or invasive procedures.

Methods. DNA sequencing was performed using the UW-OncoPlex assay on a HiSeq 2500 sequencing system (Illumina, San Diego) with 2 × 101 bp paired-end reads as previously described.⁶ Sequencing libraries were prepared from DNA obtained from FFPE samples. Libraries were hybridized to a custom design of complementary RNA (cRNA) biotinylated oligonucleotides targeting approximately 1.5 megabases of DNA from 262 genes involved in neoplasia. Sequence alignment and variant calling were performed against the reference human genome (University of California, Santa Cruz, hg19); alignment processing was performed using Burrows-Wheeler Aligner; and nucleotide variants were detected using Genome Analysis Toolkit (Broad Institute, Cambridge, Mass.), VarScan 2, and custom software. The UW-OncoPlex assay is capable of detecting all classes of mutations, including single-nucleotide variants, indels (1–100+ bp), and structural rearrangements, with a sensitivity of 10 percent minor allele frequency.

1. Groesser L, Herschberger E, Ruetten A, et al. Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome. Nat Genet. 2012;44(7):783–787.
2. Levinsohn JL, Tian LC, Boyden LM, et al. Whole-exome sequencing reveals somatic mutations in HRAS and KRAS, which cause nevus sebaceus. J Invest Dermatol. 2013;133(3):827–830.
3. Sun BK, Saggini A, Sarin KY, et al. Mosaic activating RAS mutations in nevus sebaceus and nevus sebaceus syndrome. J Invest Dermatol. 2013;133(3):824–827.
4. Hucthagowder V, Cottrell CE, Schaffer A. Targeted next-generation sequencing identifies HRAS mutation in Schimmelpenning-Feuerstein-Mims syndrome. Poster presented at: Annual Clinical Genetics Meeting; March 8–12, 2016; Tampa, Fla.
5. Wang H, Qian Y, Wu B, Zhang P, Zhou W. KRAS G12D mosaic mutation in a Chinese linear nevus sebaceous syndrome infant. BMC Med Genet. 2015;16:101.
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7. Goldstein GD, Whitaker DC, Argenyi ZB, Bardach J. Basal cell carcinoma arising in a sebaceous nevus during childhood. J Am Acad Dermatol. 1988;18(2 Pt 2):429–430.
8. Aslam A, Salam A, Griffiths CE, McGrath JA. Naevus sebaceus: a mosaic RASopathy. Clin Exp Dermatol. 2014;39(1):1–6.
9. Jackson R. The lines of Blaschko: a review and reconsideration: observations of the cause of certain unusual linear conditions of the skin. Br J Dermatol. 1976;95(4):349–360.
10. Happle R. Mosaicism in human skin: understanding the patterns and mechanisms. Arch Dermatol. 1993;129(11):1460–1470.
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13. Moody MN, Landau JM, Goldberg LH. Nevus sebaceous revisited. Pediatr Dermatol. 2012;29(1):15–23.
    14. Fergin PE, Chu AC, MacDonald DM. Basal cell carcinoma complicating naevus sebaceus. Clin Exp Dermatol. 1981;6(1):111–115.

Test yourself

Here are three questions taken from the case report. Answers are online now at www.amp.org/casereports and will be published next month in CAP TODAY.

1. Which of the following is the most commonly mutated gene in Schimmelpenning syndrome?

a) KRAS

b) HRAS

c) NRAS

d) PTCH

2. Which of the following is the most common malignant neoplasm arising within nevus sebaceus?

a) Squamous cell carcinoma

b) Trichoblastoma

c) Basal cell carcinoma

d) Keratoacanthoma

3. Which of the following sequencing methods have been used to identify the underlying nevus sebaceus mutation?

a) Sanger sequencing

b) Whole exome sequencing

c) Targeted next-generation sequencing

d) All of the above

Dr. Khoobyari is a resident in the departments of Pathology and Laboratory Medicine, and Dr. Konnick is an assistant professor in the Department of Laboratory Medicine and associate director of the genetics and solid tumor laboratory, all at the University of Washington, Seattle.