Introduction

The family of Spitz neoplasms is defined in the most recent edition of the World Health Organization Classification of Skin Tumors (4th edition) as a melanocytic neoplasm with a characteristic Spitz fusion or a mutation in HRAS with Spitzoid morphologic features. Recent studies have attempted to correlate specific clinical and morphologic findings in the various fusion subgroups such as ALK, NTRK1, NTRK3, MAPK, BRAF, and ROS1 [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Genomic fusions involving the ROS1 oncogene are seen in 7–17% of Spitz neoplasms [17, 18]. However, thus far only one study of six cases has described the morphologic features of ROS1 Spitz neoplasms [13].

In this study, we report the clinical, histologic, and molecular findings in 17 ROS1 fusion Spitz neoplasms in order to better characterize this subset of Spitz neoplasms. We compared a number of morphologic features in this set of ROS1 fusions to a control set of 99 non-ROS1 Spitz melanocytic neoplasms which have also been assessed by next generation sequencing (NGS). We describe characteristic morphologic features and report those morphologic features statistically more frequent in ROS1 Spitz compared to other subtypes of Spitz neoplasms. We also report for the first time the occurrence of ROS1 fusions in two cases of desmoplastic Spitz nevi (SN).

Materials and methods

Case selection and genomic sequencing

Study approval and waiver of consent for use of archived tissue were obtained through the Northwestern Institutional Review Board. The dermatopathology database at Northwestern was searched for SN, atypical Spitz tumor (AST), and Spitz melanomas (SM) in which a ROS1 fusion was identified by NGS. We identified eight cases matching the above criteria. The paired normal tissue were identified for Case #1, #2, #5, and #6. Additionally, nine cases were contributed from the personal consultation files of KJ Busam at Memorial Sloan Kettering Cancer Center in New York. We also identified 99 cases consisting of 20 SN, 53 ST, and 26 SM. Each diagnosis was made at the time of clinical presentation based on morphology with incorporation of FISH or array CGH in select cases. The control group included 59 fusions consisting of the following genes: ALK (n = 14), MAP3K8 (n = 12), BRAF (n = 6), NTRK1 (n = 10), NTRK3 (n = 6), RET (n = 4), MET (n = 1), RASGRF (n = 1), RAF1 (n = 1), MAP3K3 (n = 1), FGFR (n = 1), ERBB4 (n = 1), and PRKDC (n = 1). Additionally, there were five MAP3K8 truncations. Lastly there were mutations in 19 cases in the following genes: BRAF (n = 8), NRAS (n = 4), HRAS (n = 5), GNAQ (n = 1), and ROS1 (n = 1). In 16 cases no known fusions or mutations were identified.

“Spitzoid” morphology was identified according to the World Health Organization Classification of Skin Tumors (4th edition) and other relevant literature [19,20,21,22]. NGS with a 1171 cancer related gene panel for DNA and a whole transcriptome sequencing on each case was performed with using the Tempus xO platform and variant-calling [23, 24]. The 1711-gene assay is validated and designed to target therapeutically actionable genes.

Tumor classification and clinicopathologic features

In total there were 17 cases with ROS1 fusions. The clinical features including age, sex, and site of the tumors were summarized from the medical record. Morphologic features were assessed by two board certified dermatopathologist experienced in the assessment of melanocytic tumors. The following morphologic features were evaluated: silhouette (plaque, wedge, or nodular), cytology (epithelioid, spindled, or both), nuclear atypia (mild, moderate, or severe), pigmentation (absent, focal, or extensive), host inflammatory reaction (absent, non brisk, or brisk), cell size (small, intermediate, large), mitotic figures per mm2, and for the absence or presence of Kamino body, maturation, ulceration, epidermal hyperplasia, plexiform growth, epithelioid sheets, pagetosis, nesting in the adnexa, and desmoplasia.

Mild nuclear atypia was defined as a slightly larger nucleus than conventional nevomelanocytes. Moderate atypia was defined as a nuclear size similar to the size of keratinocytes with a hyperchromatic nuclear membrane, visible nucleolus, and variable chromatin quality. Severe nuclear atypia was defined as a nuclear size larger than keratinocytes with a hyperchromatic nuclear membrane, prominent and/or multiple nucleoli, and coarse chromatin. For host inflammatory reaction, a brisk response was defined as a diffuse infiltration of lymphocytes across the entire base of the tumor; a non-brisk response was defined as a focal infiltration of lymphocytes that does not cover the entire base [25]. For cell size, the size of melanocytes was compared to the basal keratinocytes [26]. Cells about the size of basal keratinocytes were considered small, those moderately larger than basal keratinocytes were intermediate in size and cells nearly twice the size of basal keratinocytes were considered large. Clinical information including age, gender, and site of tumor was also included for analysis.

Statistical analysis

All statistical analyses were performed in R Studio v1.2.5001 to compare morphologic features across the groups Spitz neoplasms. Fisher’s exact test or Chi square test was used to compare associations in categorical variables. Student’s t test was used to compare mean values. A p value of <0.05 was considered statistically significant. All tests were two sided.

Results

Clinical findings in ROS1 fusion Spitz neoplasms

The final diagnosis from the time of clinical care in the set of 17 ROS1 Spitz neoplasms was Spitz nevus in ten cases and Spitz tumor in seven cases. In none of the cases was a diagnosis of Spitz melanoma favored. The patient ages ranged from 3 to 58 with a mean age of 19 years old. There were ten female and seven male patients. The body site of involvement was highly variable with four in the head/neck region, three on the upper extremities, three on the trunk, and seven on the lower extremities. Grossly, all cases were pink to red papules. In 14 cases the clinical impression was available. In seven cases the clinician suspected an atypical Spitz nevus and in one of these cases a dermoscopic description of radial streaming was provided. In two cases the clinical impression was dermatofibroma, in two cases it was benign nevus, in two cases it was pyogenic granuloma and in one case it was cyst.

Follow-up was available for 13 of 17 cases (Table 1). The average follow-up time was 23 months and ranged from 4 to 95 months. In 12 cases the lesions were re-excised with clear margins with no evidence of recurrence. One of these 12 cases also had a sentinel lymph node biopsy (SLNB) which was negative. In one case the original biopsy was incisional and no further re-excision was performed. There was persistent tumor at a follow exam 4 months later.

Table 1 Summary of clinical data in 16 cases of Spitz neoplasms with ROS1 fusions.
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Morphologic and immunohistochemical findings in ROS1 fusions Spitz neoplasms

The low power silhouette on the 16 ROS1 cases was mostly that of either a plaque like (n = 7) or nodular pattern (n = 7). Two cases had a wedge shaped silhouette and one was polypoid. In 12 cases the cytomorphology was a mixed pattern of epithelioid and spindle cells while in four cases there was a predominance of spindle cells. In all cases the atypia was mild or moderate with none of the cases having high grade nuclear atypia (P = 0.006) (Fig. 1). This was statistically significant with ROS1 cases being less likely to have high grade nuclear atypia than the group of non-ROS1 Spitz neoplasms. The cell sizes were also all small to intermediate with none of the cases having large cells and this was also statistically significant (P = 0.001). Maturation was present in all cases and this was also statistically significant (P = 0.044). There was also a tendency for lower mitotic rate 1.3/mm2 (P = 0.001) (Table 2). Kamino bodies were also more common in this type of Spitz (8/17) than non-ROS1 Spitz (P = 0.025).

Fig. 1: Hematoxylin and Eosin staining on Case 1.
figure 1

a At 40× one can appreciate the plaque-like silhouette of this ROS1 fusion Atypical Spitz Tumor. b At 100× the epidermal hyperplasia with a predominance of nests with spindle-shaped melanocytes can be seen in a back-to-back pattern crowding the epidermis. c At 200× one can appreciate the transepidermal elimination of small nests into the stratum corneum. d 400× demonstrates the Spitzoid cytomorphology with relatively low-grade nuclear atypia.

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Table 2 Comparison of clinical and morphologic findings in ROS1 and non-ROS1 fusion Spitz neoplasms.
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Thirteen of 17 cases had overlying epidermal hyperplasia. Fourteen of 17 cases were completely amelanotic. Lobulated nests were seen in two cases and nesting in the adnexa in five cases. Five cases had notable pagetosis in the epidermis. None of these features were statistically significant compared to non-ROS1 Spitz neoplasms. Nine of 17 cases had floating nests defined as nests situated above the basal layer and in three cases there was transepidermal elimination of nests (Figs. 1, 2). Myxoid changes were not identified in any of the cases. Two cases were characterized by prominent stromal desmoplasia, and were morphologically best characterized as a desmoplastic Spitz nevus (Fig. 3).

Fig. 2: Hematoxylin and Eosin staining on Case 2.
figure 2

a, b At 40× and 100×, respectively, a plaque-like Atypical Spitz Tumor with epidermal hyperplasia and back-to-back expansile nests. c At 200× one can appreciate some floating nests in the epidermis. d At 400× one can appreciate the relatively bland cytology of the Spitzoid melanocytes.

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Fig. 3: Hematoxylin and Eosin staining on Case 13.
figure 3

a Low power shows a symmetric paucicellular Spitzoid neoplasm in a desmoplastic stroma. b Higher magnification shows small nests and individual units of Spitzoid melanocytes entrapped in a sclerotic stroma consistent with a diagnosis of desmoplastic Spitz nevus.

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Immunohistochemical staining for ROS1 was performed in 16 cases. Fifteen of the 16 cases showed strong positive staining (Fig. 4). In one case only a blush staining was seen which was not convincingly positive.

Fig. 4: An example of a strong positive IHC staining for ROS1.
figure 4

a Low power showing plaque-like silhouette of a ROS1 Fusion Spitz nevus. b IHC staining for ROS1 shows strong and uniform staining throughout the nevus. c Higher magnification shows nests of epithelioid and spindle-shaped melanocytes with bland cytomorphology lacking significant atypia.

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Genomic findings in ROS1 fusion Spitz neoplasms

The fusion partner was identified in 16 of the 17 cases in the study. The most common genomic fusions among the 16 ROS1 cases were a PWWP2A–ROS1 fusion seen in six cases and a TPM3–ROS1 fusion also seen in five cases. Other recurrent fusion partners included a PPFIBP1–ROS1 fusion seen in two cases, and fusions partners involving MYH9–ROS1, CAPRINI1–ROS1, and MYO5A–ROS1 were each seen in one case (Table 3).

Table 3 Genomic fusions in ROS1 Spitz neoplasms.
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Three cases had copy number aberrations identified by NGS and SNP arrays. Two cases had copy number aberrations identified by NGS and one case had a copy number aberration identified by SNP array. Copy number loss of BCL11B, FGF3, CARD11, FBXO11, FLT4, GRIN2A, HGF, MGMT, MYCN, MYOD1, NPM1, NTRK3, PLAG1, PTPRT, RET, TERT, and TLX1 were identified in case 2. This case was negative for copy number alterations when tested by a SNP array platform. Copy number gains of HOXA9, JUN, and MDM2 were identified in case 6. Case 10 had an isolated loss at 6q.

Discussion

Among two studies sequencing a large number of Spitz neoplasms the frequency of ROS1 fusions varied from 7 to 17% [17, 18]. The vast majority of these cases were diagnosed as either Spitz nevus or Spitz tumor. In this study ten were diagnosed as Spitz nevus and seven as Spitz tumor. We did not identify any cases that met the criteria of a Spitz melanoma. In the study from Wiesner et al. where kinase fusions in Spitz neoplasms were first described [17], 3 of 24 ROS1 fusions were designated as Spitz melanoma, but no adverse clinical outcome was reported. This study from Wiesner et al. is the larger series on ROS1 fusions but does not discuss morphologic features. Thus far there is only one study involving 6 cases of ROS1 fusions which were all designated as Spitz tumors by Donati et al. which discusses morphologic features [13].

While there is limited clinical outcomes information available on Spitz tumors with ROS1 fusions, among the 13 cases with follow-up in this study and the six cases from Donati et al. there are no reported recurrences or metastases after complete excision of the primary tumors. One case in our series had a SLNB which was also negative. Thus, preliminary evidence suggests that most Spitz tumors with ROS1 fusions are likely indolent or at least in a much lower risk category compared to Spitz neoplasms with BRAF or MAP3K8 fusions which seem to constitute much of the more aggressive variants of Spitz neoplasms [9,10,11, 15, 27,28,29].

We did not identify morphologic features which could allow for a definitive diagnosis of a ROS1 fusion by microscopic review alone but there were some characteristic features. This included a tendency for plaque-like or nodular silhouette without a deeply infiltrative component with a combination of epithelioid and spindle cell cytomorphology. Statistically significant features included lack of high grade cytologic atypia in all cases, lack of larger cell type, presence of maturation, frequent Kamino bodies and lower mitotic rate. These findings are consistent with the fact that all cases were diagnosed as Spitz nevus or Spitz tumor and none were thought to be Spitz melanoma.

In our cases, 13/17 had epidermal hyperplasia and 5/17 had notable epidermal pagetosis. Two cases had lobulated nests and four had nesting in the adnexa. None of these features were statistically significant as they can be seen in a broad spectrum of Spitz subtypes. In particular many of these features can overlap with NTRK1 fusion Spitz neoplasms. Donati et al. reported transepidermal elimination of nests and myxoid changes as being present in all six cases. Another highly characteristic feature was floating nests seen in 9 of 17 cases with transepidermal elimination of nests in three cases. We did not identify significant mucinous changes though a colloidal iron was not performed. Although none of these features are totally specific, one might anticipate a ROS1 fusion in compound plaque-like Spitz neoplasm with prominent intraepidermal component, Kamino bodies, with small to intermediate sized cells with low grade cytology, pagetosis, and floating nests within the epidermis.

An interesting and novel observation is the detection of a ROS1 fusion in two desmoplastic SN. This illustrates the wide spectrum of microscopic features associated with ROS1 fusions, but it also documents that the desmoplastic phenotype among SN is not limited to HRAS aberrations. Gains of 11p (location of HRAS) and/or HRAS mutations have previously been thought to be typical of desmoplastic SN. While they likely represent the most common aberration associated with a desmoplastic Spitz nevus, we hereby document two cases with a ROS1 kinase fusion associated with a desmoplastic phenotype.

In the 17 cases in this series, six different fusion partners were identified. This included PWWP2A (n = 6), TPM3 (n = 5), PPFIBP1 (n = 2), MYO5A (n = 1), CAPRINI1 (n = 1), and MYH9 (n = 1). PWWP2A was also the most frequent fusion partner in the series from Donati et al. A figure showing the chimeric protein model and the breakpoint of the fusions can be found in Fig. 5. Previous in vivo studies show rising levels of phosphorylation produced by this fusion protein indicating that the ROS1 kinase is being constitutively activated [17].

Fig. 5: Diagrams of chimeric structure in the ROS1 fusion Spitz neoplasms.
figure 5

Functional domains are displayed. Breakpoints were indicated by the black arrows above the each schematic. Recurrent ROS1 fusion breakapart were identified in intron 34 and 35. Positive and negative strands of DNA sequence were marked with “+” and “–”, respectively. Transcribed DNA strand was highlighted in red. Arrows at the end of sequence indicate the direction of transcription.

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ROS1 fusions have been identified in 9% SM and 1.3% in melanomas from previous studies [17, 30]. There are no cases of ROS1 fusion melanoma in the TCGA database. ROS1 fusions are also seen in a subset of 1–2% non-small cell lung cancers. More recently ROS1 fusions were identified in 9 of 130 gliomas from an infant population [31]. Also, rare cases of ROS1 fusions in angiosarcoma, thyroid, and breast cancer have been reported [32,33,34]. Interestingly in melanocytic neoplasms with ROS1 fusions the tumors seem to have an indolent clinical behavior.

In conclusion, this study describes the largest series to date on ROS1 fusion Spitz neoplasms. They seem to represent a lower grade group of tumors with generally indolent behavior. We could not find specific morphologic aberrations that were predictive of the molecular aberration but identified a number of features that were enriched in the group of ROS1 fusion tumors. They included a plaque or nodular silhouette with a cellular intraepidermal component, frequent Kamino bodies, a slight predisposition toward spindle cytology, a lower grade of cytologic atypia, and floating nests/transepidermal elimination of nests. We also report for the first time the association of a desmoplastic phenotype with ROS1 fusions.