Update on cutaneous mesenchymal tumors in the 5th edition of WHO classification of skin tumors with an emphasis on new fusion-associated neoplasms

Published in 2023, the 5th edition of the WHO classification of skin tumors has undergone some significant changes in the mesenchymal tumors section. The main focus of this review will be the novel tumor entities included in this latest edition while the inclusion of other selected changes, such as alterations in terminology and novel (mainly molecular) findings added to established entities will be only briefly mentioned. All novel tumor entities, their salient clinicopathological and molecular features, and key differential diagnostic considerations are summarized in Table 1.

This group comprises three recently identified tumor types characterized by distinct genetic alterations: tumors with CRTC1::TRIM11, ACTIN::MITF, and MITF::CREM fusions [1‐8]. However, in the WHO classification, these tumors were placed in different chapters. ACTIN::MITF- and MITF::CREM-rearranged tumors are categorized as melanocytomas in the melanocytic tumors chapter while neoplasms with CRTC1::TRIM11 fusions are classified as malignant soft tissue tumors with uncertain differentiation. Notably, in contrast to the highly malignant nature of clear cell sarcoma, i.e., a tumor in many aspects similar to these three novel tumors, the latter exhibit a more favorable prognosis [1, 3‐6]. Despite shared pathogenesis, some of their clinicopathological features differ significantly. Nevertheless, there are also several recurrent features shared by all three tumor types.

All three tumors tend to involve the dermis, with variable extension into the subcutis, and typically lack an intraepidermal component. Additionally, they exhibit a similar immunophenotype characterized by frequent, albeit variable, expression of HMB-45 and Melan A (most cases show positivity with at least one of these markers). Notably, tumors with CRTC1::TRIM11 and MITF::CREM fusions consistently demonstrate diffuse expression of SOX10 and typically express S100 protein, while ACTIN::MITF tumors display variable S100 protein expression and are usually negative for SOX10 (although only two cases were tested) [1‐8].

The tumors share similar morphology and immunophenotype, leading to a common differential diagnosis, including cellular blue nevus, melanoma, clear cell sarcoma, and PEComa. CRTC1::TRIM11 and MITF::CREM tumors have identical differential diagnoses and thus will be discussed only in the section on CRTC1::TRIM11 tumors. ACTIN::MITF tumors also have a similar differential with the previously mentioned entities but due to a slightly different immunophenotype, it warrants a separate discussion.

While the specific molecular mechanisms vary to some extent and have been previously explained in detail [4, 5], they ultimately lead to the overexpression of MITF in both CRTC1::TRIM11 and ACTIN::MITF, as well as MITF::CREM-associated tumors. MITF, part of the MIT/TFE gene family alongside TFEB, TFE3, and TFEC genes, serves as a key regulatory protein in the development of melanocyte lineage. It not only regulates the expression of melanocyte proteins, such as HMB-45, but it also supports melanocyte survival and proliferation, implying its potential involvement in tumorigenesis [4, 9]. In melanocytes, its expression is triggered physiologically by a signaling pathway activated by melanocyte-stimulating hormone (MSH). Elevated MSH level increases cAMP, activating cAMP-dependent transcription factors from the CREB family, which comprises CREB1, ATF1, and CREM. These proteins directly recognize CREB in the MITF promoter, impacting MITF expression [1, 3, 4, 10, 11]. Interestingly, all three discussed fusions seem to activate this signaling pathway at various levels. ACTIN::MITF rearrangement results in MITF overexpression most likely by directly impacting this protein [4]. In contrast, similar to EWSR1::ATF1 fusion in clear cell sarcoma, both CRTC1::TRIM11 and MITF::CREM fusions likely lead to MITF overexpression through the induction of the CREB signaling cascade [1, 3, 5, 6]. However, it is unclear at this point whether melanocytic differentiation of these tumors (i.e., expression of S100, SOX10, HMB-45, Melan A, etc.) is attained via origin in a soft tissue melanocyte or via their distinct genetic alterations resulting in the activation of the MITF pathway or both [12]. Nevertheless, some of the experts in this field seem to favor the latter possibility which appears as a much more logical explanation to us as well [12].

The subgroup characterized by CRTC1::TRIM11 fusion is relatively well-documented, with over 40 reported cases so far [2, 5, 7, 8]. Typically found on the distal extremities and trunk, this tumor presents as a well-defined dermal nodule, occasionally extending into subcutis. Almost all cases lacked an intraepidermal component apart from one case which, most likely as a result of an upward growth of a primarily dermal tumor, exhibited junctional nests of tumor cells, similar to what has been described in rare cases of clear cell sarcoma [13‐15]. Individual cases have been observed in submucosal regions [5]. The tumor consists of short intersecting fascicles or nests of relatively uniform epithelioid to spindle cells with eosinophilic cytoplasm, moderate to severe atypia, prominent nucleoli, and limited mitotic activity (< 5 mitoses/10 HPF), without atypical forms (Fig. 1). Binucleated or multinucleated cells are occasionally observed, yet wreath-like multinucleated giant cells, which can be observed in clear cell sarcoma, are absent. Small areas of necrosis are infrequently detected. Immunohistochemically, the tumor consistently exhibits positivity for SOX10 and in approximately 80% of cases demonstrates expression of S100 protein, albeit often limited in extent. Furthermore, at least 60% of reported cases show positivity for HMB-45 and Melan A, with at least one of these markers being positive in 80% of cases [2, 5, 7].

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As previously mentioned, the main entities in the differential diagnosis include cellular blue nevus, melanoma, clear cell sarcoma, and PEComa. Cellular blue nevus typically presents with heavy pigmentation and often features a conventional blue nevus component at the periphery of the lesion. Mutations in GNAQ or GNA11 confirm the latter diagnosis in challenging cases. Cutaneous melanoma commonly exhibits an intraepidermal component, along with significantly higher pleomorphism and mitotic activity, including common atypical forms. The most frequently observed mutations in melanomas include BRAF, NRAS, NF1, etc. Moreover, genetic alterations such as a high mutational burden and a UV signature are another characteristic features of melanoma [16]. In both CRTC1::TRIM11 and MITF::CREM tumors, clear cell sarcoma poses the most challenging differential diagnosis due to their striking similarities. Clear cell sarcoma, typically localized around the ankle, may be distinguished by the presence of multiple wreath-like multinucleated giant cells. However, accurate differentiation often requires molecular methods. Detection of EWSR1 gene rearrangement and/or confirmation of an EWSR1::ATF1 or EWSR1::CREB1 gene fusion confirms the diagnosis of clear cell sarcoma. Distinguishing CRTC1::TRIM11 and MITF::CREM tumors from PEComa can be easily achieved by SOX10 staining, which is negative in the latter [1, 3, 5, 6].

The prognosis of these tumors is relatively favorable. However, out of 38 cases with follow-up information, 6 showed an adverse event either in the form of a local recurrence (n = 3/6), lymph node metastasis (5/6, unconfirmed histologically in one case), or pulmonary metastasis (3/6, unconfirmed histologically in one case) [5, 8, 17‐19].

Finally, a very recent study reported three cases with a novel MED15::ATF1 fusion. Briefly, all three patients were children (5–16 years old) and the tumors occurred on the arm, cheek, and scalp. All were centered to the dermis but one of the cases showed epidermotropism as well. Most of their morphological and immunohistochemical features were otherwise similar to CRTC1::TRIM11 tumors. Despite limited follow-up, one of the cases showed an early spread to regional lymph nodes [12].

This group of tumors represents a so far understudied subset, with only three documented cases reported in the literature, one of which was reported by our institution [1, 3, 6]. Among these cases, two were identified in newborns (one female and one of unknown gender), while the third occurred in a 37-year-old woman. Tumor locations varied, with two cases affecting the scalp and the remaining case arising on the dorsum of the hand. Given the limited available data, the prognosis of these tumors is not well established. From the three reported cases, one recurred locally 9 months after an incomplete excision and the patient had no evidence of disease 23 months later. The other two reported cases had no evidence of disease 9 and 39 months after complete excision.

Histology of all tumors exhibited infiltrative growth patterns, lacking well-defined margins, predominantly involving the dermis with partial subcutaneous extension (Fig. 2A). Although two cases closely abutted the epidermis, no intraepidermal component was noticed (Fig. 2A, B). All documented lesions consisted of highly cellular proliferation of epithelioid to spindle cells arranged in solid sheets or small solid nests or bundles which were particularly evident at the periphery. While clear cells predominated in one case, the remaining two cases consisted of cells with predominantly eosinophilic cytoplasm. Tumor cells demonstrated moderate nuclear atypia, prominent nucleoli, and increased mitotic activity (ranging from 4 to 11 mitoses/10 HPF), including atypical forms (Fig. 2C). Similar to CRTC1::TRIM11 tumors, diffuse strong expression of S100 protein (Fig. 2D), SOX10 (Fig. 2E), and MITF (Fig. 2F) was observed in all tested cases, with HMB-45 and Melan A positivity detected in two out of three cases. The differential diagnosis closely mirrors that of CRTC1::TRIM11 tumors; however, distinguishing between the two groups is very challenging [1, 3, 6]. Morphologically, the primary distinction often lies in the sharper demarcation and more fascicular arrangement of cells in CRTC1::TRIM11 tumors. Nonetheless, molecular methods may be indispensable for a reliable distinction.

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Limited information for this tumor subtype is available, derived from a single publication that included seven cases [4] and one additional very recent study primarily focusing on PEComas [20]. Nonetheless, the clinicopathological characteristics of these tumors appear distinct and were also observed in one additional case identified at our institution. Among the eight cases with clinical information known to us to date, all occurred in women of different ages (15–75 years, median 40 years), with seven affecting the extremities and one observed on the breast. Notably, no recurrence or metastasis were documented among the three cases with available clinical data, with follow-up durations ranging from 7 to 17 years [4].

This tumor typically manifests as a dermal lesion which, due to a highly infiltrative growth pattern, often extends into the subcutis (Fig. 3A). It consists of solid sheets of large epithelioid cells with clear cytoplasm which characteristically dissect the surrounding collagen bundles (so-called checkerboard-like pattern of growth [20]) (Fig. 3B). Cytomorphology is characterized by mild to moderate atypia, variably prominent nucleoli, and minimal mitotic activity (0–1 mitosis/10HPF) (Fig. 3C). Immunohistochemically, variable expression of S100 protein, tyrosinase (Fig. 3D), HMB-45 (Fig. 3E), or Melan A is commonly present, although some cases were also completely negative for all these markers. SOX10 was negative in one case published [4] as well as in our case (Fig. 3F). Regarding the genetic background, this tumor is associated with the alteration of MITF gene, which is fused either with the ACTB or ACTG1 gene [4]. Both of these genes belong to the ACTIN family and so the fusions observed are collectively referred to as ACTIN:MITF in the literature.

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Thanks to their characteristic morphology and minimal mitotic activity, this tumor can be reliably differentiated from blue nevus, clear cell sarcoma, and melanoma. However, distinguishing it from PEComa is more challenging and in some cases impossible without the use of molecular methods. Typical cases with clear cell morphology may be difficult to distinguish particularly from the TFE3-rearranged PEComa subtype, which is composed of similar looking clear cells and has a similar immunophenotype (TFE3 gene belongs to the same MIT/TFE signaling pathway as MITF). Importantly, two recently reported cases showed predominantly pink granular cytoplasm, making a distinction from conventional PEComas with TSC1/2 mutations (or more rarely other molecular alterations) impossible on morphological grounds [20]. Generally, the main distinguishing feature is the frequent positivity for S100 protein, observed in five out of eight cases, three of which showed diffuse positivity. In contrast, S100 protein expression is relatively rare (and focal if present) in PEComas. Nevertheless, the striking similarity between ACTIN::MITF-rearranged tumors and PEComas is intriguing and raises a question whether ACTIN::MITF tumors represent a standalone entity, a distinct subtype of PEComa or whether ACTIN::MITF fusions may be shared by two different tumor types [4, 20].

To date, 17 cases of this tumor have been documented in the English literature, affecting individuals across all age groups, with a slightly higher prevalence among women. The tumor shows a strong predilection for the acral location, particularly the lower limbs; one intraosseous case has been also described in the tibia [21]. It is a benign tumor which frequently recurs unless completely excised [22‐24].

Histologically, the tumor typically consists of two components: densely packed cellular bundles of monomorphic bland spindle cells and prominent hyalinized stroma (Fig. 4A–C). Mitotic activity is usually absent. Immunohistochemically, a strong diffuse nuclear expression of ERG is observed, with intensity similar to that of capillary endothelium (Fig. 4D). In contrast, markers such as keratins, CD34, CD31, and others are typically negative; SMA may be occasionally weakly positive. In all cases, EWSR1::SMAD3 fusion was present. EWSR1 break-apart FISH probe can be used to confirm the diagnosis in challenging cases [23, 24].

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In terms of differential diagnosis, it is crucial to distinguish this tumor from cellular spindle cell sarcomas such as monophasic synovial sarcoma, dermatofibrosarcoma protuberans, or low-grade fibromyxoid sarcoma. However, it is more commonly confused with benign spindle cell tumors such as myofibroma/myopericytoma, perineurioma, fibromatosis, and especially cellular fibrous histiocytoma. Almost all of these entities can be distinguished from EWSR1::SMAD3-rearranged fibroblastic tumor based on typical morphological and immunohistochemical features [24]. The only exception is cellular fibrous histiocytoma, which can closely resemble EWSR1::SMAD3-rearranged tumor morphologically and regularly show diffuse expression of ERG [25]. However, the intensity of ERG expression is typically weaker than that observed in the endothelium of surrounding vessels (or that observed in EWSR1::SMAD3-rearranged tumor) and comparison with these structures is diagnostically useful.

First characterized in 2014, this tumor typically affects adolescents or young adults and demonstrates a preference for the skin and subcutaneous tissue of the proximal limbs, particularly the thigh [26‐30].

It commonly presents as a well-circumscribed tumor, although small infiltrative foci may be observed (Fig. 5A). Morphologically, the tumor consists of sheets of epithelioid cells (Fig. 5B, C) or spindle cell bundles (Fig. 5D). Perhaps the most striking features are the abundant, markedly eosinophilic, granular-to-glassy cytoplasm and marked nuclear pleomorphism with frequent nuclear pseudoinclusions (Fig. 5C). Despite this, the mitotic activity is generally low, and atypical mitoses are usually absent, although exceptions exist [26‐30]. Rare cases may show collagen entrapment at the periphery, predominantly spindle cell morphology and somewhat less pronounced atypia, therefore mimicking atypical fibrous histiocytoma [31].

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By definition, the tumor shows diffuse strong expression of CD34 (Fig. 5E), along with frequent but usually focal expression of cytokeratins (Fig. 5F) which is seen in approximately 70–80% of cases [26, 27, 30]. Nuclear and predominantly diffuse expression of WT-1 is observed in about two-thirds of cases (clone 6F-H2 against the N-terminus of WT1) [26]. A valuable diagnostic marker for this tumor is the SynCam3/CADM3 antibody (Fig. 5G), which is a highly specific and sensitive marker for these tumors based on the available studies [26, 29, 30] as well as in our experience. Approximately half of these tumors harbor PRDM10 fusions with CITED2, MED12, ARHGAP32, or RAD30 genes being the fusion partners [26, 29, 30]. There are no significant differences in clinical characteristics between cases with and without fusion.

Recognition of this tumor is crucial due to its deceptively malignant appearance, often leading to confusion with undifferentiated pleomorphic sarcoma. Despite worrisome morphological features, this lesion has an indolent clinical behavior with low recurrence rate and very rare regional lymph node metastasis [26‐30]. Apart from undifferentiated pleomorphic sarcoma, the tumor is also frequently misdiagnosed as pleomorphic liposarcoma, dermatofibrosarcoma protuberans, or myxoinflammatory fibroblastic sarcoma. Correlating clinical features (superficial location, typically young patients), morphological appearance (characteristic cytoplasmic features, minimal mitotic activity), and immunophenotype is usually sufficient to differentiate these lesions.

Soft tissue tumors with kinase gene alterations traditionally comprised a heterogeneous group of mesenchymal neoplasms which, besides others, included dermatofibrosarcoma protuberans, inflammatory myofibroblastic tumor, and infantile fibrosarcoma (IFS) [32]. While the canonical gene rearrangement of IFS, the ETV6::NTRK3 fusion, was described more than two decades ago [33, 34], the recent widespread adoption of RNA sequencing (RNA-seq) has led to the description of several alternative gene rearrangements such as those of NTRK1/2/3, BRAF, RET, and others [32, 35‐38]. The same technical breakthrough has also led to the emergence of a novel and not yet fully defined group of mesenchymal tumors with kinase gene alterations that morphologically and molecularly partially overlap with IFS [39, 40]. This group is currently included in the WHO classification of soft tissue and bone tumors as the emerging entity “NTRK-rearranged spindle cell neoplasms” (NTRK-SCN) [41, 42]. These tumors may arise at any anatomical site and affect both children and adults (although the younger age group more commonly). Morphologically, they include spindle cell tumors that form a continuous morphological spectrum which based on cellularity, degree of atypia, and mitotic activity can be roughly divided into three subgroups [39]. The first subgroup consists of clinically benign spindle cell lesions exhibiting low cellularity, minimal atypia, and very few mitoses (further referred to as the low-grade (LG) group), while the second contains morphologically high-grade lesions (high-grade (HG) group) that can follow an aggressive clinical course. In addition, some cases are difficult to assign to one of these categories as they fall somewhere in between these two extremes (intermediate-grade (IG) group). The LG group may exhibit one of several often co-occurring morphological patterns such as lipofibromatosis-like neural tumor pattern [43], prominent stromal and perivascular hyalinization pattern (Fig. 6A) [44, 45], myopericytic/haemangiopericytic pattern [46], and others [41, 47]. Tumors in the IG group may retain similar architecture as tumors from the LG group or they may vaguely resemble LG malignant peripheral nerve sheath tumors [41, 44]. Tumors from the HG group morphologically overlap with IFS, adult fibrosarcoma, and/or HG MPNST (Fig. 6B) [32, 41, 44]. Although some tumors display an uncharacteristic immunoprofile, many cases across the LG, IG, and HG spectrum exhibit immunohistochemical co-expression of CD34 (Fig. 6C) and S100 protein (Fig. 6D) [39, 41]. Additionally, the expression of the CD30 antibody (Fig. 6E) appears to be diagnostically useful as well [48]. Although panTRK immunohistochemistry is relatively sensitive for mesenchymal tumors with NTRK1/2/3 fusions [49‐52], it demonstrates imperfect specificity in skin and soft tissue mesenchymal tumors, as it can be positive in other neoplasms such as those with neural or myogenic differentiation [51, 52]. The molecular background of NTRK-SCN is characterized by various gene fusions and less often other alterations in kinase genes such as NTRK1/2/3, BRAF, RAF1, RET, ALK, or MET [41, 44, 53‐61]. The list of these aberrations is broad and ever-expanding but in effect, they all lead to the constitutive activation of the MAP kinase signaling pathway [32].

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Overall, NTRK-SCN may recur and metastasize in 7–26% and 5–20% of cases, respectively [39, 44, 62]. Accurate diagnosis of NTRK-SCN is clinically significant as complete excision can prevent recurrences and thus potential progression of LG lesions to more aggressive HG variants. Additionally, the availability of targeted therapy for advanced or metastatic cases of NTRK-SCN also makes recognition of this entity crucial [39].

Due to the broad morphological spectrum of these lesions, the differential diagnosis is diverse [41]. Tumors on the LG spectrum must be distinguished from other LG spindle cell neoplasms, such as lipofibromatosis, dermatofibrosarcoma protuberans, solitary fibrous tumor, or neurogenic tumors. The most challenging differential in cases from the HG spectrum are tumors with fibrosarcoma-like morphology such from malignant peripheral nerve sheath tumors or fibrosarcoma arising in dermatofibrosarcoma protuberans. In most cases, molecular studies are necessary for a confirmation of the diagnosis of NTRK-SCN.

Alterations in terminology and the incorporation of novel findings into existing lesions, particularly regarding their molecular genetic background were the most significant other changes. However, only those which we believe will have the highest impact on routine clinical practice will be mentioned.