NTRK expression is common in xanthogranuloma and is associated with the solitary variant

Previously identified mutually‐exclusive driver genes in juvenile xanthogranuloma (JXG) and adult xanthogranuloma (AXG) include mutations in MAP kinase pathway genes such as MAP2K1, BRAF, ARAF, KRAS, NRAS, PIK3CD as well as fusions in BRAF and ALK, with a subset of cases with no identified driver yet. NTRK fusion has been identified in rare cases.


| INTRODUCTION
Juvenile xanthogranuloma (JXG) is a non-Langerhans cell histiocytosis that commonly presents in the skin, however, on occasion, it can occur in extracutaneous sites. 1 Similarly, these lesions can rarely present in adult patients and thus are termed adult xanthogranuloma (AXG). 2 JXG and AXG typically occur as solitary lesions, however, they can also present in a disseminated fashion. 1,2JXG has a classic clinical appearance, presenting as a yellowish papule, often involving the head and neck, torso, or extremities. 3Dermoscopy can be helpful as lesions classically demonstrate a "setting sun" pattern with additional findings of global symmetry, yellow globules, shiny white streaks, and irregular distribution of vascular structures. 4n histologic examination, xanthogranulomas demonstrate a relatively circumscribed proliferation of histiocytes within the dermis with associated scattered inflammatory cells.Mononuclear cells can demonstrate a spindled appearance, and Touton-type giant cells and foamy cells are often present. 5Lesional cells are usually positive for CD163, CD68, Factor XIIIa, and CD4 while negative for S100. 6,7Conversely, the differential diagnosis of Langerhans cell histiocytosis (LCH) classically demonstrates reactivity for CD1a, Langerin (CD207), and S100. 8e typical clinical course of xanthogranulomas is that of spontaneous resolution of a solitary lesion with rare instances of recurrence. 9In adults spontaneous resolution is uncommon.Extracutaneous and disseminated disease can rarely occur, most commonly affecting the eye, liver, spleen, and kidney.Factors associated with possible systemic involvement include a large number of cutaneous lesions or younger age. 7To our knowledge, the presence of predictive biomarkers (immunohistochemical, molecular, or serologic) for systemic involvement is not well described.
The molecular landscape of histiocytoses has been further expanded by combined whole-exome and transcriptome sequencing, identifying recurrent kinase fusions involving BRAF, ALK, NTRK1, and activating MAP2K1 and ARAF mutations in BRAF wild-type non-LCH. 14These efforts have revolutionized the treatment paradigm for these disorders as MEK and RAF inhibitors can target such kinase fusions.
6][17][18] In regards to histiocytoses, rare cases of NTRK association have been reported.LMNA-NTRK1 fusion has been identified in a single patient with ECD, 10,14 IRF2BP2-NTRK1 fusion in a patient with disseminated progressive nodular histiocytosis, 19 and LMNA-NTRK1 fusion in a patient with generalized eruptive histiocytosis. 20 regards to xanthogranulomas, a case of multiple AXG harboring an LMNA-NTRK1 fusion has been reported 21 and six further cases with NTRK1 fusion are presented in Durham et al. 22 However, NTRK and its role in JXG and AXG has yet to be further and fully characterized.

| MATERIALS AND METHODS
Cases of histopathologically confirmed JXG or AXG in the archives of the Dermatopathology Section at Indiana University Department of Pathology from 2013 to 2022 with an available tissue block were retrieved.In the eight cases lacking Touton cells, immunostains for both CD68 and CD163 were performed in five cases and immunostains for one of those markers in the other three cases to confirm histiocytic lineage.S100 and CD1a were performed in all eight cases lacking Touton cells and were negative (see Table 2).Fourteen cases of non-neoplastic histiocytic diseases were retrieved, including four cases of sarcoid, four cases of granuloma annulare, two ruptured cysts with histiocytic foreign body response, a granulomatous dermatitis associated with rheumatoid arthritis, a disrupted pilosebaceous unit, a lymphohistiocytic infiltrate, and a scar with histiocytic foreign body response.
Immunostaining for pan-NTRK was performed on the Dako Link 48 autostainer, using the pan-NTRK clone EPR17341 from Abcam, at 1:500 with high pH antigen retrieval and a red chromogen.The two index cases were stained for pan-NTRK using the same clone under identical conditions on a Dako Omnis immunostainer, using a brown chromogen.Other immunostaining was performed on the Dako Omnis platform as follows: CD163 from Leica, clone 10D6; S100 from Dako, polyclonal GA504; Langerin from Cell Marque, clone 12D6; CD1a from Dako clone IR069; CD68 from Dako, clone KP1.
Sequencing of xanthogranulomas was performed at Tempus Labs (Cases 1 and 2) using the XT assay.The positive control for pan-NTRK immunostaining was an NTRK1-LMNA kinase fusion soft tissue tumor that had been previously confirmed by DNA and RNA sequencing at Tempus Labs using the XT assay.Cases 49 and 50 were sequenced at Foundation Medicine using the Foundation One assay.Three additional cases of histiocytic neoplasia with non-NTRK driver mutations included in the study as negative controls were: a case of ECD with MCL1 amplification tested with the Tempus XT assay; a case of Erdheim-Chester with ALK fusion tested with the Foundation One assay and confirmed by ALK immunostaining, and a case of Langerhans cell sarcoma with p53 mutation tested with the Tempus XT assay.

| Evaluation of the pan-NTRK immunostain
We optimized an immunostain using an NTRK1-LMNA kinase fusion spindle cell neoplasm that had been identified by next-generation sequencing (NGS) (Figure 1A).We evaluated the immunostain in sections of non-neoplastic histiocytic diseases and demonstrated completely negative staining in all 14 cases (Figure 1B).We tested the immunostain in two cases of disseminated JXG with non-NTRK driver mutations and demonstrated negative results (Figure 1C,D).We also evaluated the immunostain in three other cases of histiocytic neoplasia with non-NTRK driver mutations and demonstrated negative results (not shown).

| Additional cases of xanthogranuloma
We tested an additional 48 cases with pan-NTRK immunostain.These cases had a mean of 46 months of follow-up (range 0-116 months).We identified 21 additional positives and 27 negatives (Table 1).We scored the level of reactivity on a scale of 3+ to 1+ (Figure 3A-D).Two cases that were negative by immunostain were also negative by NGS (Cases 49 and 50; Table 1).Consequently, in all four cases where both NGS and pan-NTRK immunostaining were done, the results were concordant.
Including the index cases, pan-NTRK immunostain was, therefore, positive in 23 of 50 cases (46%).In these cases, the level of  1).There were seven cases of multiple (multifocal or disseminated JXG), all seven of these were negative for pan-NTRK by immunostain (7 of 7, 100%) with confirmation by NGS in two.

| Histopathologic features and immunophenotype
We compared the histopathologic features of the NTRK-positive cases to the NTRK-negative cases and found them histopathologically indistinguishable (Figure 4A-D; Table 2).We also compared the immunophenotype and found no distinguishing features between the two groups.

| Pan-NTRK identifies components of the neoplastic process
Because the pan-NTRK immunostain identifies cells containing the fusion protein, which drives the neoplastic process and is negative in other cell types in our study, we reasoned that the immunostain may be capable of identifying other components of the neoplastic process.The stain confirmed that histiocytes, but also Touton-type giant cells (Figure 5A), spindle cells (Figure 5B), intraepidermal histiocytes (Figure 5C), and histiocytes trapped within central areas of fibrosis (Figure 5D) are likely all part of the neoplastic process.

| The pan-NTRK immunostain is useful as a screening tool in histiocytic neoplasia
The pan-NTRK immunostain was entirely negative in non-neoplastic histiocytic diseases (14 of 14 cases) and was also negative in histiocytic neoplasia lacking an NTRK fusion by NGS (5 of 5 cases).We encountered no cases in which NGS and the pan-NTRK immunostain gave conflicting results.Nevertheless, it would be desirable to confirm the presence of a fusion in the other cases in this cohort, and these studies are anticipated.
Pan-NTRK immunostain has previously proven to be a useful screening tool in various neoplasms with NTRK fusion.In 2017, Hechtman et al. found that pan-NTRK immunostaining is a time and tissue-efficient means of screening driver-negative malignancies, secretory carcinoma, and congenital fibrosarcoma. 23Other subsequent studies have shown similar results while also comparing various IHC clones.An analysis of pediatric mesenchymal tumors with NTRK fusion compared the screening capability of separate antibodies, pan-Trk (EPR17341) and TrkA (EP1058Y).Pan-Trk was found to be 97% sensitive and 98% specific, while TrkA was 100% sensitive and 63% specific, respectively.
Case 1 was identified by an NGS screening approach which demonstrated a TPM3-NTRK1 gene fusion.The presence of the protein was confirmed by pan-NTRK immunostain (Figure2A,B).Case 2 was identified by pan-NTRK immunostain, with the presence of a PRDX1-NTRK1 fusion confirmed by NGS (Figure2B,C).

4. 3 |F
The pan-NTRK immunostain identifies other cell types involved in the neoplastic process The pan-NTRK immunostain identifies neoplastic cells but is negative in non-neoplastic cells.The stain suggests that the Toutontype giant cells, as well as spindle cells, are likely part of the neoplastic process (Figure 5A,B).Adjacent to ulceration, I G U R E 4 NTRK-positive xanthogranulomas are histopathologically indistinguishable from others in the cohort.Index Case 2, an infiltrate of histiocytes, lymphocytes, and eosinophils in the dermis.(A) Hematoxylin and eosin (H&E), Â100.(B) H&E, Â200.(C) H&E, Â400.(D) Touton cells and foam cells are variably present, H&E Â400.T A B L E 2 Histologic and immunophenotypic features of patients with xanthogranuloma.

4 . 4 | 5
intraepidermal neoplastic cells were identified with the pan-NTRK immunostain in Cases 1 and 8 (Figure5C) but not in the 20 nonulcerated cases.The one exception was Case 5, in which intraepidermal pan-NTRK-positive cells were present.This lesion was not ulcerated histopathologically but was clinically described as a crusted papule, possibly representing incipient ulceration.A dense intraepidermal neoplastic infiltrate of histiocytes that lack desmosomes and do not contribute to the structural integrity of the epidermis may therefore be the mechanism of ulceration in the cases that ulcerate.Limitations of this studyThe presence of an NTRK1 fusion has been confirmed by NGS in only two cases, and the absence of a fusion has been confirmed by NGS in T A B L E 2 (Continued) Pan-NTRK immunostain positive: blue, pan-NTRK immunostain negative: gray.Abbreviations: neg, negative; pos, positive.Pan-NTRK immunostain identifies components of the neoplastic process.(A) Touton-type giant cells are positive and therefore likely neoplastic rather than reactive, pan-NTRK immunostain, Â400.(B) Spindled cells are positive and therefore likely neoplastic rather than reactive, pan-NTRK immunostain, Â400.(C) Neoplastic histiocytes are present within the epidermis adjacent to the ulcer, pan-NTRK immunostain, Â400.(D) Focally, neoplastic histiocytes are trapped within central fibrosis, pan-NTRK immunostain, Â400.only five cases.It remains possible that NTRK fusions other than NTRK1 such as NTRK3 are involved in some of our cases.It is also possible that some of our cases could have a positive pan-NTRK immunostain with a different underlying mechanism such as amplification of the gene or changes in methylation, although this appears to be very rare in NTRK-mediated neoplasia in other cell types.15 5 | CONCLUSIONNTRK expression is common in xanthogranuloma and appears to delineate a solitary, more indolent variant of the disease.