Spitzoid Melanoma with ALK Activation: A Novel Mechanism

1. Spitzoid melanoma with histopathologic features of ALK gene rearrangement exhibiting ALK copy number gain: A novel mechanism of ALK activation in spitzoidneoplasia M. Farah, P. Nagarajan, J.L. Curry, Z. Tang, K. Tae-Beom, P.P. Aung, C.A. Torres-Cabala, AK. Eterovic,J.A. Wargo, V.G. Prieto, and M.T. Tetzlaff The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA British Journal of Dermatology. DOI: 10.111/bjd.16881

2. Lead researcher M.T. Tetzlaff

3. Introduction What’s already known? • Spitzoid neoplasms frequently pose diagnostic difficulty in distinguishing the relatively more common indolent lesions from the rare metastasizing spitzoid melanoma. • Recent studies have elaborated a molecular-genetic framework by which to categorize these tumours, and it is now known that certain discrete alterations correlate with specific clinical and/or pathologic features. • Spitzoid lesions with ALK gene rearrangement exhibit a distinctive plexiform pattern of growth with intersecting fascicles of amelanotic spindled melanocytes.

4. Case Presentation • An 87-year-old gentleman presented with a solitary 3-cm exophytic erythematous nodule on his mid-upper back of 2 years duration. • Punch biopsy revealed intradermal proliferation of spindled cells arranged in intersecting fascicles with occasional clefts and numerous mitotic figures (5/mm2). • On IHC, the tumour cells were diffusely and strongly positive for S100, SOX10 and H3K27Me3, focally positive for MITF, and negative for anti-melanocytic cocktail (anti-HMB45, anti-MART1, and anti-tyrosinase), anti-cytokeratin cocktail, SMA and desmin.

5. Case Presentation Morphologic and immunophenotypic findings of the skin biopsy. • H&E; 20x • H&E; 200x • H&E; 400x; arrow: mitotic figure • S100 IHC; 400x • SOX10 IHC; 400x • H3K27me3 IHC; 400x

6. Methods • ALK IHC study was performed on 5 µm-FFPE tissue sections using D5F3 (cell signaling) Ab/1:100. • FISH was performed on 5 µm-FFPE sections using a break apart ALK kit (Abbott-Vysis) and examined using Olympus Fluorescent Photomicroscope.

7. Methods • Fifty nuclei were assessed using 100x objective. • Nuclei harboring split-signals ≥ 2 signal diameters apart were scored as positive for ALK rearrangement if > 50% of 50 cells were positive. • ALK copy number gain was defined as ≥3 ALK signals detected in > 20% of 50 cells.

8. Methods • DNA from tumour and normal tissue was extracted using the Qiagen FFPE extraction kit. • Somatic mutations were identified using a sequencing platform consisting of 323 cancer-related genes. • Functional consequences of somatic variants were assessed by comparison to dbSNP, COSMIC, and TCGA databases and annotated them using VEP, Annovar, and CanDrA.

9. Results • ALK IHC showed diffuse weak to moderate cytoplasmic ALK positivity. ALK IHC; 400x

10. Results • FISH demonstrated that 36% of the tumour cells exhibited >3 intact ALK signals, confirming ALK copy number gain. • No ALK rearrangement was detected. FISH; arrows: cells with additional ALK copies

11. Results • NGS analysis identified numerous somatic mutations in the tumour cells affecting genes previously described as altered in spitzoid melanomas, including NF1, CDKN2A, PTEN, and LRP1B. • Mutations affecting BRAF, NRAS, KIT, GNAQ or GNA11 were not identified.

12. Discussion • Around 50% of spitzoid neoplasms harbour mutually exclusive gene fusions activating receptor tyrosine kinases: ROS1 (in 17% of tumours), NTRK1 (16%), ALK (10%), BRAF (5%), and RET (3%). • These rearrangements result in high expression of constitutively active kinases that stimulate multiple oncogenic signalling pathways. • Chromosomal rearrangements resulting in ALK fusions were detected in up to 15% of spitzoid neoplasms, including 3% of spitzoid melanomas.

13. Discussion • ALK-translocation-associated Spitz tumours exhibit distinctive plexiform growth pattern on histology, nearly identical to the morphology observed in the reported case. • ALK rearrangements position the ALK kinase domain under the control of a strong and constitutively activated promoter resulting in strong ALK expression by IHC. • ALK copy number gains occur in a minority of tumour cells and does not alter transcriptional control of the ALK promoter resulting in a weaker ALK expression, as seen in the reported case.

14. Discussion • Another mechanism of ALK activation in melanoma independent of structural genomic aberrations is an alternative transcription initiation (ATI) site which produces a novel ALK isoform, ALKATI. • Histologically, ALKATI expressing melanomas lack the distinctive morphology of ALK-rearranged tumours. • The co-occurrence of ALKATI with alterations of the ALK gene locus is uncommon.

15. ConclusionsWhat does this study add? • This is the first reported case of a plexiformspitzoid neoplasm exhibiting ALK copy number gain instead of ALK rearrangement. • ALK copy number gain represents a novel mechanism of ALK activation among spitzoid tumours and is associated with the same characteristic histopathologic growth pattern seen among ALK-rearranged spitzoid neoplasms.

16. The Team P.P. Aung Z. Tang P. Nagarajan M. Farah J.L. Curry C.A. Torres-Cabala A.K. Eterovic J.A. Wargo V.G. Prieto M.T. Tetzlaff K. Tae-Beom (not pictured)

17. Call for correspondence • Why not join the debate on this article through our correspondence section? • Rapid responses should not exceed 350 words, four references and one figure • Further details can be found here