The Morpho-Molecular Landscape of Spitz Neoplasms

Spitz neoplasms are a heterogeneous group of melanocytic proliferations with a great variability in the histological characteristics and in the biological behavior. Thanks to recent discoveries, the morpho-molecular landscape of Spitz lineage is becoming clearer, with the identification of subtypes with recurrent features thus providing the basis for a more solid and precise tumor classification. Indeed, specific mutually exclusive driver molecular events, namely HRAS or MAP2K1 mutations, copy number gains of 11p, and fusions involving ALK, ROS, NTRK1, NTRK2, NTRK3, MET, RET, MAP3K8, and BRAF genes, correlate with distinctive histological features. The accumulation of further molecular aberrations, instead, promotes the increasing malignant transformation of Spitz neoplasms. Thus, the detection of a driver genetic alteration can be achieved using the appropriate diagnostic tests chosen according to the histological characteristics of the lesion. This allows the recognition of subtypes with aggressive behavior requiring further molecular investigations. This review provides an update on the morpho-molecular correlations in Spitz neoplasms.


Introduction
In 1948, the American pathologist Sophie Spitz published a landmark case series of melanocytic proliferations called "juvenile melanomas" or "melanomas of childhood" characterized by a combination of distinctive architectural and cytological features in association with peculiar epidermal changes ( Figure 1) [1,2]. Such lesions are symmetric, often showing a dome-shaped, wedge-shaped or plaque-like silhouette, with sharp lateral borders and maturation towards the deep part [1,2]. The pattern of growth is predominantly nested. Nests may display varying cellularity, size, and shape are arranged in parallel to the rete ridges [1,2]. The overlying epidermis is hyperplastic and may be separated by the junctional nests and single melanocytes by clefts [1,2]. Pagetoid spread of the melanocytes into the epidermis, when present, usually occurs in bundles or nests of cells [1,2]. A variable number of small to large dull eosinophilic globules composed of amorphous filaments (so-called Kamino bodies) may be scattered throughout [1,2]. There is a predominance of enlarged epithelioid and/or spindle melanocytes with abundant eosinophilic or amphophilic cytoplasm with "ground-glass" appearance, round to oval to spindle nuclei with finely dispersed chromatin and distinct nucleoli [1,2]. Nuclear pseudoinclusions and multinucleated melanocytes may be encountered. Most lesions are amelanotic or paucimelanotic [1,2]. The mitotic activity is usually low. The inflammatory cell infiltrate is generally perivascular and dispersed throughout. These melanocytic proliferations typically occurred on the extremities of children and young adults and, despite the tendency to loco-regional nodal involvement, were hallmarked by an indolent clinical course, thus justifying their distinction from the adult melanoma in which the prognosis was dismal [1,3]. With time, it became evident that using these histological criteria it was possible to distinguish a group of benign melanocytic lesions -then named Spitz nevus (SN)-from melanoma and that these may be present also in adults [4]. However, cases occur that are more difficult to differentiate from melanoma because showing SN characteristics along with worrisome histological features, such as increased size, asymmetry, epidermal ulceration, lack of maturation, solid growth, diffuse pagetoid spread, hypodermic extension, marked cytological atypia, increased mitotic activity, deep and atypical mitoses. In 1959 Albert Bernard Ackerman recognized that melanocytic proliferations with Spitz histology encompassed a broad morphological spectrum of neoplasms ranging from completely benign, namely SN, to their fully malignant counterpart, namely malignant Spitz tumor (MST), passing through an intermediate category, namely atypical Spitz tumor (AST; Table 1) [5][6][7]. It must be highlighted that many MST diagnoses are achieved in the context of known synchronous metastasis or are initially defined as AST and classified as fully malignant after the detection of distant metastasis during the clinical follow-up. Indeed, it is well known that distinguishing AST from MST histologically is very difficult and at times impossible, even with the aid of common ancillary analyses. Spitz neoplasms may present at any age and at any site but most frequently affect the lower extremities and the face of patients under the age of 30 [8]. They are quite infre-quent, accounting for about 1% of all resected melanocytic lesions and with an estimated annual incidence of little more than 1 case per 100,000 [8,9]. AST do not exceed the 6-8% of the number of SN and MST are very rare [10]. In the footsteps of Ackermann, the term "Spitzoid" was coined referred to melanocytic neoplasms sharing (at least some of) the distinctive SN features. This has led to confusion in the classification of melanocytic lesions since the term Spitzoid has been applied even to lesions with only the epidermal modifications typical of SN or to those with a small subpopulation of enlarged epithelioid and/or spindle melanocytes.
Until a few years ago, the genetic determinants of Spitz neoplasms development were largely unknown [11][12][13]. Thanks to the recent discoveries on the molecular landscape of the melanocytic Spitz lineage, the taxonomy of these tumors and the understanding of their biological behavior is becoming clearer [14]. Overall, both nevi and melanomas share the activation of some growth-promoting signaling pathways (considered the driver molecular events), mainly PI3K-AKT and RAF-MEK1/2-ERK1/2 [15,16]. In benign nevi these are almost the only molecular aberrations present [15,16]. In malignant melanomas, instead, there is also a variable number of additional molecular alterations (considered the promoting molecular events) able to block tumor-suppression mechanisms and to trigger further oncogenic signals, such as 9p21 deletion, 6p25 copy number gain, TP53 mutations, and TERT-promoter mutation [15,[17][18][19] Thus, the driver molecular events seem to determine the specific histotype of each melanocytic neoplasm, while the type and amount of promoting aberrations seem to define their morphological and clinical aggressiveness [15,16]. According to this interpretation, the neoplasms regarded as melanocytic tumors of uncertain malignant potential result from the combination of the same driver molecular alterations of nevi and melanomas with a limited number of additional promoting genetic events determining a malignant potential lower than that of full-blown melanoma [15,16]. This seems to be true also in the Spitz setting. Indeed, the drivers of most common nevi and malignant melanomas are BRAF and NRAS activating mutations, but these alterations are virtually absent in Spitz neoplasms [20][21][22][23][24]. Instead, these harbor oncogenic HRAS or MAP2K1 mutations or kinase gene fusions involving ALK, BRAF, MET, NTRK1, NTRK2, NTRK3, RET, ROS1, and MAP3K8 in a mutually exclusive pattern [5,[11][12][13][25][26][27][28][29][30][31][32][33][34][35][36][37]. The integration of such molecular data in the histology-based tumor classification of melanocytic neoplasms provides a way to identify the true Spitz neoplasms in the heterogeneous group of Spitzoid lesions [14]. Moreover, the existence of consistent genotype-phenotype relationships among the different subtypes of Spitz neoplasms (Table 2) can be used for the selection of the appropriate ancillary analyses to support a histological diagnosis of AST or MST and to better assess their malignant risk.
This review resumes the state-of-art in the knowledge of genotype-phenotype correlations in the field of Spitz neoplasms, especially focusing on subtypes harboring HRAS or MAP2K1 mutations, copy number gains of 11p, or fusions involving ALK, ROS, NTRK1, NTRK2, NTRK3, MET, RET, MAP3K8, and BRAF genes.

Spitz Neoplasms with HRAS Mutations or 11p Copy Number Gains
The RAS proto-oncogene family comprises three members, namely KRAS, NRAS, and HRAS, respectively located on the short arm (p) of chromosome 12, 1, and 11, and encoding the proteins KRAS4A, KRAS4B, NRAS, and HRAS [38,39]. These proteins are implicated in the signal transduction from the cell surface to the nucleus through the PI3K-AKT and RAF-MEK1/2-ERK1/2 pathways stimulating growth, differentiation, proliferation, and survival of the cell [40,41]. Missense single nucleotide point mutations usually occur in hotspot regions of the RAS active site leading to the production of aberrant proteins able to trigger downstream signaling without the need of extracellular cues [39]. Interestingly, the oncogenic role of RAS seems to be histotype-specific since different tumors are related to mutation of a precise RAS isoform [40]. This is particularly evident in melanocytic lesions where common nevi and malignant melanomas harbor almost exclusively NRAS mutations while Spitz neoplasms HRAS mutations [12,22,24]. Compared to the other RAS isoforms, HRAS appears to have a higher affinity for the PI3K-AKT pathway that is believed to be responsible for conferring the enlarged epithelioid or spindle phenotype to the melanocytes in Spitz neoplasms [42][43][44]. Copy number gains of the 11p region encompassing HRAS leads to overexpression of the protein product with similar results. Activating HRAS mutations and copy number gains of 11p occur in an exclusive or concurrent way in about 20% of Spitz neoplasms [22,[45][46][47][48]. Most HRAS mutations commonly involve the codons 59-61 in exon 3 (mainly Q61R and Q61L) and rarely affect the codons 12 and 13 in exon 2 (mainly G13R) [5,13,22,[48][49][50][51][52][53][54][55][56]. The detection of HRAS mutation requires sequencing analysis (classic or NGS), while FISH or CGH analysis is needed for the identification of 11p copy number alterations. The monoclonal antibody SP174 recognizes with high sensitivity the RAS Q61R mutant protein; unfortunately, however, it cross-reacts with both KRAS, NRAS, and HRAS isoforms resulting useless from a diagnostic point of view [57,58] Antibodies against the wild type HRAS protein may be of aid in highlighting Spitz neoplasms with HRAS overexpression due to 11p copy number gains.
Spitz neoplasms with HRAS aberrations tend to be predominantly intradermal symmetric lesions with infiltrative base characterized by epithelioid and spindle cells with abundant eosinophilic or amphophilic cytoplasm and slightly to moderately pleomorphic vesicular nuclei, intermingled with thick collagen bundles (desmoplasia) (Figure 2) [12,13,48]. Marked cytological atypia can be present in about 40% of HRAS-mutated cases [33]. Usually, mitoses are rare and not atypical. However, it must be highlighted that, on one hand, not all the Spitz neoplasms with these genomic alterations are desmoplastic and, on the other hand, desmoplasia has been reported also in Spitz neoplasms with ROS1, ALK, and BRAF gene fusions [13,59]. Most Spitz neoplasms with activating HRAS mutations and/or copy number gains of 11p are readily recognizable as SN, but cases occur with AST features; the prognosis is favorable [22,46,60,61].
Clinically, ALK-fused Spitz neoplasms tend to be large and solitary papules or nodules arising on the extremities of young patients [3,11,70]. Histologically, most of these lesions share a distinctive pattern substantiated by a compound wedge shape silhouette with a bulbous and/or infiltrative base ( Figure 3) [11,71,76]. The presence of non-pigmented, large, spindle melanocytes with pericellular clefts, amphophilic cytoplasm, vesicular nuclei, and prominent nucleoli, growing in plexiform intersecting fascicles, appears as a hallmark of ALK fusions [11,33,71,76]. Nevertheless, it is important to note that, although the described pattern is extremely sensitive in the prediction of ALK fusions, it is not equally specific. Other molecular subtypes of Spitz neoplasms may show overlapping features, such as NTRK1-fused cases [76]. Another pattern reported to be quite characteristic for ALKfused Spitz neoplasm is the angiomatoid one [33]. The epidermis is often hyperplastic and pagetoid spread is usually absent [11]. Nuclear pleomorphism is usually mild and rarely moderate [11,33,71,76]. Worrisome features, such as ulceration, deep mitoses, and perineural invasions have been described [11,[68][69][70]77]. Of note, combined Spitz neoplasms are more commonly ALK-fused [33].

Spitz Neoplasms with ROS1 Fusions
ROS1 proto-oncogene is located on the long arm (q) of chromosome 6 and encodes a tyrosine kinase receptor implicated in the PI3K-AKT, RAF-MEK1/2-ERK1/2, and JAK3-STAT3 pathways [78]. According to the analysis of large series of Spitz neoplasms, ROS1 fusions were found in 7-17% of cases [13,31]. Among these lesions, several fusion partners were reported, with PWWP2A (37% of cases) and TPM3 (31% of cases) being the most common in the series by Gerami et al. [59]. Other less common fusion partners of ROS1 among Spitz neoplasms are: PPFIBP1, CLIP1, ERC1, FIP1L1, HLA-A, MYH9, ZCCHC8, CAPRIN1, KIAA1598, MYH9, and MYO5A [5,13,59,73,79]. Immunohistochemistry with monoclonal antibody D4D6 against ROS1 is a fast, low-cost, and well-performing screening test for the identification of ROS1-fused Spitz neoplasms. Indeed, it showed 100% sensitivity and specificity when compared with FISH [80]. Although different ROS1 immunohistochemical patterns have been observed, such as diffuse or sparse granular cytoplasmic staining, dot-like staining, and nuclear staining, no specific correlations with the cellular localization of the various ROS1 fusions have been found [80]. In addition to FISH, NGS can be used to identify ROS1 fusions.
Clinically, ROS1-fused Spitz neoplasms are pink to red papules distributed throughout the body, mainly occurring in young adults of both sexes [59]. Histologically, there are not distinctive features specifically associated with ROS1 fusions. Indeed, many characteristics are shared with other molecular subtypes of Spitz neoplasms, particularly with those NTRK1-fused [59]. Nevertheless, a certain histological signature is enriched since most of the reported ROS1-fused lesions show a compound plaque-like or nodular silhouette with prominent expansile junctional nesting, with possible adnexal involvement and transepidermal elimination, composed of pure spindle melanocytes or of mixed spindle and epithelioid melanocytes, with mild to moderate nuclear pleomorphism, evidence of maturation, lack of pigmentation, associated with numerous Kamino bodies [13,33,59,79]. Recently, plexiform and angiomatoid patterns have been reported as more common in ROS1-fused Spitz neoplasms than in other molecular subtypes [33]. Cell sizes range from intermediate to large [13,59]. Mitoses may be not uncommon [79]. As above mentioned, cases occur in which ROS1 fusions have been found in desmoplastic SN [59].
Of great interest, in all published series, the presence of ROS1 fusions was associated with favorable outcome (no recurrence, no distant metastasis, and negative sentinel lymph node biopsy), although having been identified in cases diagnosed as MST [13]. No adverse events were observed in the available follow ups; thus, it is entirely reasonable to admit that ROS1-fused Spitz neoplasms usually have an indolent course [59].

Spitz Neoplasms with NTRK Fusions
The NTRK proto-oncogene family comprises three members, namely NTRK1, NTRK2, and NTRK3, respectively located on chromosomes 1q, 9q, and 15q and encoding the cell surface receptor tyrosine kinase proteins TRKA, TRKB, and TRKC (collectively referred as TRK proteins) involved in the PI3K-AKT, RAF-MEK1/2-ERK1/2, and PLCγ1 pathways [81][82][83]. These receptors are normally expressed in the nervous system and can be activated by the binding with several ligands, such as NGF, BDNF, and NT-3/4 [81]. TRK activation determines the autophosphorylation of the intracellular tyrosine residues and consequently the transmission of the signal through different pathways regulating the transcription of genes involved in neuronal survival and differentiation [81]. Fusions involving the NTRK gene family results in the production of TRK chimeric proteins with oncogenic properties since they couple constitutive expression with ligand-independent activation provided by the kinase domain preservation [83].
Current guidelines for NTRK fusion detection in solid tumors state that a two-step testing approach must be followed [89]. Firstly, cases must be immunohistochemically screened using the monoclonal antibody EPR17341 that reacts against a C-terminal epitope conserved in wild-type and in all the chimeric TRK proteins (hence the definition of pan-TRK immunohistochemistry) [89][90][91]. Secondly, all the immunohistochemically positive cases must be analyzed using RNA-based NGS to prove the NTRK fusion [89]. This algorithm has been tested on a large AST series confirming the reliability of pan-TRK immunohistochemistry as screening test [88]. However, the application of RNA-based NGS to verify the presence of the gene fusion showed some weakness and it has been suggested to also perform FISH in all pan-TRK positive cases not confirmed by NGS analysis [88]. The staining pattern of pan-TRK can also provide information about the probable underlying NTRK fusion. Indeed, strong and diffuse nuclear immunostaining is quite specific for ETV6-NTRK3 fusion, while linear immunostaining in dendritic processes of the melanocytes directs towards MYO5A-NTRK3 fusion [29].

Spitz Neoplasms with NTRK2 Fusions
The only reported NTRK2-fused SN belonged to histological variant of pigmented spindle cell nevus (aka nevus of Reed). The lesion is junctional, with hyperplastic epidermis, large nests with peripheral clefts composed of pigmented spindle melanocytes with abundant eosinophilic cytoplasm and elongated or oval nuclei [30]. Neither nuclear pleomorphism nor mitoses are observed [30]. Kamino bodies are widely distributed [30].

Spitz Neoplasms with NTRK3 Fusions
NTRK3-fused Spitz neoplasms are mostly compound or dermal, with epidermal hyperplasia and dome-shaped silhouette [29]. Depending on the underlying fusions, the lesions show different morphology. Indeed, cases with ETV6-NTRK3 fusions are predominantly composed of epithelioid melanocytes with distinct cell borders, abundant eosinophilic cytoplasm, and pleomorphic nuclei, arranged in large coalescing and lobulated nests [29,33]. The constituent melanocytes of MYO5A-NTRK3-fused lesions, instead, are homogeneously spindled and organized in a fascicular to plexiform growth pattern [29]. Palisading resembling Verocay bodies and rosettes-like structures are occasionally seen [29]. Lesions with MYH9-NTRK3 fusion are characterized by moderately large epithelioid melanocytes syncytially arranged with central desmoplastic stroma and peripheral collagen trapping [29].
The prognosis of Spitz neoplasms harboring NTRK1 or NTRK3 fusions is invariably favorable. Although rare cases of neoplastic cellular deposits in regional lymph nodes occur, no distant metastases and adverse outcomes have been reported so far [68,86].
The few RET-fused Spitz neoplasms so far described are mainly compound symmetric lesions with epidermal hyperplasia, a plaque-like silhouette, large expansile nests of dyscohesive, intermediate-sized, and monotonous predominantly epithelioid melanocytes characterized by mild to moderate nuclear atypia [13,93].
Despite RET fusions have been found in SN, AST, and MST, the prognosis is favorable since the available follow up were uneventful [13,31,73,85,93].

Spitz Neoplasms with MET Fusions
MET proto-oncogene resides on chromosome 7q and encodes a tyrosine kinase receptor involved in the PI3K-AKT, RAF-MEK1/2-ERK1/2, PLCγ1, and β-catenin pathways [26,67]. Even though only a handful of MET-fused Spitz neoplasms have been reported up to date, the list of identified partner genes is quite long: TRIM4, ZKSCAN1, LRRFIP1, PPFIBP1, EPS15, and DCTN1 [26,31,87]. In this setting, NGS and FISH are the available options for the recognition of the MET fusions.
The morphologic features of Spitz neoplasms harboring MET fusions are not specific. Indeed, most of the reported cases are compound or intradermal, symmetric, and domeshaped lesions with epidermal hyperplasia, large nests of intermediate to large epithelioid or spindle melanocytes with pericellular clefting [26].
MET fusions have been found in SN, AST, and MST, but all cases with available follow up behaved indolently [26,31,87].

Spitz Neoplasms with MAP2K1 Mutations
MAP2K1 proto-oncogene is located on chromosome 15q and encodes MEK1, the serinethreonine and tyrosine kinase directly downstream of RAF, which in turn phosphorylates ERK in the RAF-MEK1/2-ERK1/2 pathway [34]. The molecular background of MAP2K1 mutated lesions typically consists of in-frame deletions, that lead to an impaired MEK activation through RAF-dependent, RAF-regulated (conferring resistance to RAF inhibitors), or RAF-independent (insensitive to allosteric MEK inhibitors) possible mechanisms [37,94]. As for Spitz neoplasms, most MAP2K1 mutations involve exons 2 and 3, especially as in-frame deletions (p.E102_I103del. and p.I103_K104del), removing an autoinhibitory domain of the protein thus leading to a constitutive activated state unresponsive to feedback inhibition by RAS and RAF (class II in-frame deletions) [36]. However, these alterations seem to be extremely rare in this context. To date, only few cases of MAP2K1-mutated Spitz  [33][34][35][36][37]. Sanger or NGS analysis is needed for the detection of the mutations.
Clinically, Spitz neoplasms harboring MAP2K1 mutations occur as small, flat or slightly elevated, pigmented lesions on the lower extremities of young patients, with a substantial female preponderance (M: F ratio 1: 2) [34][35][36][37]. Despite the attempts made to find relevant genotype-phenotype correlations within the framework of these lesions, this purpose has been greatly frustrated due to the very small sample size of the series, with the consequent wide variability of the observations [35,36]. Nevertheless, some morphologic features recur among the histological descriptions. Spitz neoplasms with MAP2K1 mutations seem to be hallmarked by a tendency toward a compound or intradermal wedge shape silhouette, with plexiform architecture and with convergence around the adnexa and the neurovascular bundles of nests composed of large epithelioid cells with vesicular nuclei and moderate to severe nuclear pleomorphism [34][35][36]. Other histological findings comprise heavy pigmentation, stromal accumulation of melanin and melanophages, lack of epidermal hyperplasia, and poor maturation [36,37]. Of interest, Donati and co-workers also described the association between the cytological features of a Spitz neoplasm and the architecture of a dysplastic nevus, matching the description of the so-called SPARK nevus [37]. It has been suggested that this morphological heterogeneity of Spitz neoplasms harboring MAP2K1 mutations resides in the effects of the secondary genetic hit on the phenotype [36]. Indeed, most cases harbor also other passenger mutations in well-known oncogenes and tumor-suppressor-genes, such as BRAF, IDH1, BAP1, and NF1 [36].
MAP2K1 mutations can be found in both benign and malignant Spitz lesions but are more common in AST and MST [34,36,37]. An overt malignant phenotype is mainly observed in cases with concurrent molecular aberrations involving HRAS, CDKN2A, ARID1A, or NOTCH2, or with copy number gains of 6p [35]. Nevertheless, no recurrences or adverse outcomes have been noted during the follow up of the patients [34][35][36][37].
From a molecular point of view, AST and MST with MAP3K8 fusions regularly harbor additional genetic aberrations, mainly 9p21 deletion [5,[31][32][33][34]. This can be easily showed by focal or diffuse homogeneous lack of immunostaining of the melanocytes for p16 [5,31,33,34]. Of note, Kervarrec et al. found that AST and MST with severe cellular atypia and p16 loss have a very high probability to be MAP3K8-fused [33].
Clinically, BRAF-fused Spitz neoplasms usually occur as pink papules mainly on the lower extremities followed by the upper extremities [104]. The age range is wide, but most patients are young, even if slightly older than those with other subtypes of Spitz neoplasms [104]. There is a clear female predominance (M:F ratio 1:2) [104]. Histologically, these lesions are mostly compound or dermal, with epidermal hyperplasia, plaque-like, wedge shaped, or nodular silhouette, and are composed of intermediate to large epithelioid melanocytes with amphophilic cytoplasm, vesicular nuclei, and prominent nucleoli [5,11,33,68,79,84,104,106]. Nuclear pleomorphism is frequently marked [5,11,33,68,79,84,104,106]. Spitz neoplasms harboring BRAF fusions may show a typical pattern characterized by a superficial hyper-cellular dermal component with sheet-like architecture along with a deep hypo-cellular dermal component with prominent desmoplasia [11,13,33,104,106]. Moreover, nevoid feature has been observed more frequently in BRAF-fused Spitz neoplasms than in the other subtypes, excluded the NTRK1fused one [33].

Conclusions
The morpho-molecular landscape of Spitz neoplasms is becoming clearer, with the identification of specific subtypes with recurrent characteristics thus providing the basis for a more solid and precise tumor classification. Histological features may already guide the choice of the immunohistochemical and/or molecular investigations to be performed in AST and MST to identify the underlying driver genetic alterations. This allows to confirm the Spitz nature of the lesions and to highlight the need of further molecular analyses in cases with either MAP2K1 mutations or MAP3K8 and BRAF fusions since these subtypes is commonly associated with aggressive behavior. Future studies should provide long follow up data to substantiate this approach.
Author Contributions: Conceptualization, R.C.; writing-original draft preparation, C.A.D.P. and R.C.; writing-review and editing, R.C.; supervision, R.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.