Genomic aberrations in spitzoid melanocytic tumours and their implications for diagnosis, prognosis and therapy

Abstract

Histopathological evaluation of melanocytic tumours usually allows reliable distinction of benign melanocytic naevi from melanoma. More difficult is the histopathological classification of Spitz tumours, a heterogeneous group of tumours composed of large epithelioid or spindle-shaped melanocytes. Spitz tumours are biologically distinct from conventional melanocytic naevi and melanoma, as exemplified by their distinct patterns of genetic aberrations. Whereas common acquired naevi and melanoma often harbour BRAF mutations, NRAS mutations, or inactivation of NF1, Spitz tumours show HRAS mutations, inactivation of BAP1 (often combined with BRAF mutations), or genomic rearrangements involving the kinases ALK, ROS1, NTRK1, BRAF, RET, and MET. In Spitz naevi, which lack significant histological atypia, all of these mitogenic driver aberrations trigger rapid cell proliferation, but after an initial growth phase, various tumour suppressive mechanisms stably block further growth. In some tumours, additional genomic aberrations may abrogate various tumour suppressive mechanisms, such as cell-cycle arrest, telomere shortening, or DNA damage response. The melanocytes then start to grow in a less organised fashion and may spread to regional lymph nodes, and are termed atypical Spitz tumours. Upon acquisition of even more aberrations, which often activate additional oncogenic pathways or alter cell differentiation, the neoplastic cells become entirely malignant and may colonise and take over distant organs (spitzoid melanoma). The sequential acquisition of genomic aberrations suggests that Spitz tumours represent a continuous biological spectrum, rather than a dichotomy of benign versus malignant, and that tumours with ambiguous histological features (atypical Spitz tumours) might be best classified as low-grade melanocytic tumours. The number of genetic aberrations usually correlates with the degree of histological atypia and explains why existing ancillary genetic techniques, such as array comparative genomic hybridisation (CGH) or fluorescence in situ hybridisation (FISH), are usually capable of accurately classifying histologically benign and malignant Spitz tumours, but are not very helpful in the diagnosis of ambiguous melanocytic lesions. Nevertheless, we expect that progress in our understanding of tumour progression will refine the classification of spitzoid melanocytic tumours in the near future. By integrating clinical, pathological, and genetic criteria, distinct tumour subsets will be defined within the heterogeneous group of Spitz tumours, which will eventually lead to improvements in diagnosis, prognosis and therapy.

Keywords: BAP1; BRAF; Biomarkers; RAS; Spitz tumours; classification; diagnosis; genetics; genomics; melanocytic tumours; melanoma; molecular biology; pathology; precision oncology; spitzoid neoplasms; targeted therapy.

Fig. 1

Frequent genomic aberrations in cutaneous melanocytic tumours. (A) Common acquired naevi show BRAF hotspot mutations in the vast majority of cases. Congenital naevi frequently harbour NRAS hotspot mutations. (B) Blue naevi and related melanocytic neoplasms share activating mutations of GNAQ and GNA11. While GNAQ mutations dominate in cutaneous proliferations, GNA11 and GNAQ mutations account each for ~40% of uveal melanomas. (C) Cutaneous melanomas show BRAF hotspot mutations in ~50%, NRAS mutations in ~25% and rarely KRAS and HRAS mutations, and NF1 loss in ~10% of cases. The ‘triple wild-type melanoma’ subtype is a heterogeneous subgroup of melanoma with infrequent driver mutations such as KIT, CTNNB1, or genomic rearrangements. (D) Compared to cutaneous melanoma and to common and blue naevi, Spitz tumours show different chromosomal aberrations: translocations involving the kinases ALK, ROS1, NTRK1, BRAF, RET, MET are observed in up to 50% of cases, but are rarely observed in other subtypes of melanocytic tumours. HRAS mutations are often associated with a desmoplastic histological phenotype (desmoplastic Spitz naevus). Aberrations of BAP1 occur frequently with activating BRAF mutations and are associated with an epithelioid morphology.

Fig. 2

Clinical and histological appearance of melanocytic tumours with BAP1 loss. (A) A 65-year-old man with a BAP1 germline mutation. Numerous flat and homogeneously brown-pigmented melanocytic skin lesions dominate the clinical picture. (B,C) Melanocytic tumours with BAP1 loss usually present as inconspicuous, small, skin-coloured to slightly pink plaques or dome-shaped papules. (D) Wedge-shaped, symmetrical tumour with melanocytes in nests and sheets. (E) The neoplastic cells present with well-defined cytoplasmic borders, moderate amounts of amphophilic cytoplasm, and slightly enlarged, round to oval nuclei with condensed chromatin without atypia. (F) BAP1 immunohistochemistry is negative in the naevoid melanocytes, but positive in nuclei of epidermal keratinocytes and scattered lymphocytes. (G) Shave biopsy of a predominantly dermal melanocytic tumour. (H) Large epithelioid and atypical melanocytes (same magnification as E; note the difference in size of the neoplastic cells). The neoplastic cells show well-defined cytoplasmic borders and abundant amphophilic cytoplasm. The large nuclei show vesicular chromatin with prominent nucleoli, and vary in size and shape. (I) The nuclei of the large, epithelioid melanocytes are negative for BAP1 immunohistochemistry, whereas the admixed lymphocytes are positive. These two cases illustrate the two extremes in the biological spectrum of melanocytic neoplasms with BAP1 inactivation, ranging from clearly benign lesions with only very slightly atypical naevoid cells (D–F) to borderline malignant tumours with significant atypia (G–I).

Fig. 3

Desmoplastic Spitz tumours frequently show HRAS mutations and gains of the short arm of chromosome 11. (A) Quite symmetrical, intradermal tumour with low cellularity from the retro-auricular region of a 43-year-old man. (B) Desmoplasia with single cells and clusters of spindle-shaped and epithelioid melanocytes between collagen bundles. (C) Large, epithelioid melanocytes between collagen bundles with vesicular chromatin and prominent nucleoli. (D) The array CGH profile indicates a gain of the short arm of chromosome 11, which harbours the HRAS gene. (E) The electropherogram shows a mutation affecting codon 61 of the HRAS gene (HRAS^Q61R).

Fig. 4

Plexiform Spitz tumours frequently show ALK translocations. (A) Relatively symmetrical, exophytic, predominantly intradermal melanocytic tumour with focal epidermal hyperplasia from the buttock of a 14-year-old boy. (B) Plexiform growth pattern and intersecting fascicles of fusiform melanocytes. (C) Proliferation of cytologically fairly bland spindle and epithelioid melanocytes. Note mitosis in the centre. (D,E) The neoplastic melanocytes are positive for ALK immunohistochemistry, with staining of the cytoplasm. (F) Illustration of the TPM3-ALK kinase fusion. The ALK gene is located on chromosome 2p23; the TPM3 gene on chromosome 1q21. Because of the genomic rearrangement, the TPM3 exons 1–8 are fused with the ALK exons 20–29 containing the transforming tyrosine kinase domain. The in-frame fusion junction of the chimaeric transcript is confirmed by Sanger sequencing.

Fig. 5

Spitz tumours with a ROS1 kinase fusion. (A) Exophytic, compound melanocytic tumour with irregular epidermal hyperplasia and permeative lymphocytic infiltrate from the lower left arm of a 19-year-old woman. (B) Epidermal and dermal nests of (C) epithelioid melanocytes with minor atypia. (D) The neoplastic melanocytes are positive for ROS1 immunohistochemistry. (E) Illustration of the PPFIBP1–ROS1 kinase fusion. The ROS1 gene is located on chromosome 6q22, and the PPFIBP1 gene on chromosome 12p11. Owing to genomic rearrangements, PWWP2A exons 1–8 are fused to ROS1 exons 35–43 containing the tyrosine kinase domain. Sanger sequencing over the junction confirms the chimaeric transcript.

Fig. 6

Spitz tumours with a NTRK1 kinase fusion. (A) Oval-shaped, compound melanocytic tumour with moderate epidermal hyperplasia from the upper arm of a 9-year-old girl. (B) Large epithelioid melanocytes with vesicular nuclei and prominent nucleoli, and moderate nuclear pleomorphism. (C,D) The neoplastic melanocytes are positive for NTRK1 immunohistochemistry and show cytoplasmic staining. (E) The LMNA–NTRK1 kinase fusion is caused by a 743 kb intrachromosomal deletion on chromosome 1q, joining the first two exons of LMNA with exon 11–17 of NTRK1. Sanger sequencing confirms the in-frame junction of the fusion transcript.

Fig. 7

Pigmented spindle cell naevus with a BRAF fusion. (A) Symmetrical and well-circumscribed compound proliferation with slight epidermal hyperplasia from the lower right leg of a 17-year-old woman. (B) Elongated melanocytic nests with numerous melanophages in the papillary dermis. (C) Spindled and epithelioid pigmented melanocytes. (D) Interphase FISH with break-apart probes flanking the BRAF locus confirms the BRAF rearrangement (individual green and red signals). The paired red/green signals indicate the wild-type BRAF alleles.

Fig. 8

Molecular pathways of proliferation, survival and senescence. Extracellular growth factors trigger the dimerisation of receptor tyrosine kinases, leading to autophosphorylation and activation of intracellular signalling cascades such as the MAPK/ERK or the PI3K/AKT/mTOR pathway. Among a variety of other functions, these signalling pathways increase cell proliferation and survival. Many of the components, especially of the MAPK/ERK pathway, show genetic aberrations in spitzoid melanocytic tumours, which lead to a constitutive pathway activation. However, the activation of oncogenic pathways epigenetically de-represses the CDKN2A locus, encoding the proteins p16 and p14. Oncogene activation and cell proliferation may also lead to telomere shortening and increased concentrations of reactive oxygen species (ROS), which causes a DNA-damage response and activates p53 and p21. The signalling cascades of the p16 and p53 pathway converge on the tumour suppressor retinoblastoma (Rb) and on cell cycle inhibitors. After initial cell proliferation, these signalling pathways cause a durable proliferative arrest, termed senescence.

Fig. 9

Tumour progression model of Spitz tumours. Most tumours, including melanocytic neoplasms, develop through sequential acquisition of genomic aberrations. This tumour progression model suggests a continuous biological tumour spectrum rather than a clear dividing line between benign and malignant. The acquisition of genomic aberrations usually correlates with increased histological atypia and, consequently, tumours with conflicting histological criteria (atypical Spitz tumours) show more genomic aberrations than benign (Spitz naevi), but fewer than malignant (spitzoid melanoma) tumours. The order of genetic aberrations described here is likely to be a common sequence, because cell proliferation (induced by mitogenic genetic aberrations) is associated with a high probability to acquire additional genetic aberrations. However, the genetic changes may also occur in a different order and the sequence displayed here denotes that spitzoid melanomas have additional mutations, but not that all melanomas arise from naevi. Spitzoid melanoma arising without an obvious antecedent naevus may suggest that aberrations in the fail-safe mechanisms develop before the mitogenic driver. Spitz naevi usually only have a strong proliferation signal such as activating HRAS mutations or kinase fusions. These mitogenic genetic aberrations initiate tumour formation, but after initial cell proliferation, multiple fail-safe mechanisms stably block further growth (Spitz naevus). Atypical Spitz tumours abrogate some of these fail-safe mechanisms by gaining additional genomic aberrations so that the cells may continue to grow or to survive in distant organs, such as the lymph nodes. For example, aberrations of CDKN2A, CDK4, or CCND1 undermine the cell-cycle arrest, and TERT promoter mutations may prevent telomere shortening, and consequently senescence. The acquisition of these aberrations is reflected by an increase of cytological and histological atypia. Spitzoid melanomas acquire even more genetic and epigenetic aberrations, which may activate additional oncogenic pathways, affect the chromatin landscape, or reduce cell differentiation, so that the neoplastic cells may colonise and replace the infiltrated organs.

Fig. 10

Array comparative genomic hybridisation (aCGH) as an ancillary diagnostic tool in evaluating Spitz tumours. The aCGH profile of atypical Spitz tumours usually shows more genomic aberrations than of Spitz naevi, but not as many as spitzoid melanoma. (A) The aCGH profile of benign Spitz naevi shows usually a flat line indicating no chromosomal gains or losses (here the profile of the X- and Y-chromosome indicates that the tumour is from a female patient: 2 X-chromosomes, no Y-chromosome). (B) Atypical Spitz tumours at the benign end of the biological spectrum show mild histological atypia, and usually no more than gains or losses of one or two chromosomes or chromosome arms (here loss of the entire chromosome 3). (C) Atypical Spitz tumours at the malignant end of the biological spectrum show a considerable amount of histological atypia, including anisonucleosis and pleomorphism. In aCGH, gains and losses of several chromosome parts can be observed (here loss of the entire chromosome 6, a gain on the long arm of chromosome 7, a small loss and a larger gain on chromosome 10). (D) Spitzoid melanoma shows severe histological atypia, pleomorphism, and numerous, atypical mitoses; aCGH shows numerous chromosomal aberrations (here loss of parts of chromosome 1, 3, 6, 15, and loss of the entire chromosome 9 and 22).

Fig. 11

Fluorescence in situ hybridisation (FISH) as ancillary diagnostic tool for ambiguous melanocytic tumours. Melanoma FISH assays are often based on 4 probes, which are labelled with 4 different fluorescent dyes (red, green, orange, aqua) and bind to 4 specific chromosomal regions. (A) In normal cells without chromosomal gains and losses 2 signals of each probe are detected. (B) In cells with chromosomal aberrations, which affect the FISH probe-binding regions, losses or gains of the FISH signals can be observed (here 4 aqua, 3 green, and 1 red and orange signal). (C) FISH of a melanoma showing several cell nuclei with a varying number of FISH probes indicating chromosomal aberrations and chromosomal instability. (D) Binding sites of the original 4 FISH probes described by Gerami et al.: RREB1 (6p25), MYB (6q23), CCND1 (11q13) genes and of centromere 6. This aCGH profile of an AST shows loss of chromosome arm 9p, which would not be detected by FISH. (E) Modified and commercially used assay with FISH probes for RREB1 (6p25), MYC (8q24), CDKN2A (9p21), centromere 9 (CEP 9) and CCND1 (11q13). The corresponding aCGH profile detects several chromosomal aberrations involving chromosome 1, 3, 7, 9, 13, 18, 19, all of which would be missed by FISH. These examples illustrate that melanoma FISH has major blind spots. On the other hand, the detection of a chromosomal aberration by FISH does not prove that the tumour is malignant (see Fig. 9). Consequently, FISH is not very helpful in the diagnosis of ASTs.

Fig. 12

Integrated molecular diagnostics with targeted next-generation sequencing for ambiguous melanocytic tumours. The tumour metastasised to the regional lymph nodes. (A) Predominantly intradermal melanocytic tumour with large nests and prominent desmoplasia. (B) Large, epithelioid melanocytes with vesicular chromatin and prominent nucleoli between collagen bundles; mitosis. (C) A targeted next-generation sequencing assay, termed MSK-IMPACT, reveals a loss of chromosome 1p, and partial gains on chromosome 1q and 11p, which is characteristic for desmoplastic Spitz tumours. The log2 ratio was calculated across all targeted regions by comparing the coverage in tumour versus matched normal DNA. (D) Mutational profiling of 341 genes revealed 5 somatically acquired mutations, including an activating HRAS^Q61K mutation, which is in line with the 11p gain, and a non-frameshift insertion of 6 nucleotides in exon 1 of ARID1B. The 3 missense mutations exchange a single amino acid in the genes NSD1, FLT1, and CHEK2. These mutations are of uncertain functional significance and need further evaluation.