Utility of sequencing for ATP6AP1 and ATP6AP2 to distinguish between atypical granular cell tumor with junctional component and melanoma

Granular cell tumor (GCT) is a S100+ neoplasm with atypical and malignant variants. Similar to melanocytic neoplasms, the tumors make nests and can have junctional components raising a differential diagnosis of melanoma. Nevi and melanomas may also have granular cell cytoplasm. MelanA is useful in distinguishing melanocytic from granular cell lineage, but increasingly MelanA/SOX10 negative melanomas have been recognized by correlation with molecular methods.

Granular cell tumor (GCT) is a tumor of presumed schwannian histogenesis with atypical and malignant variants.The characteristic histopathologic finding is granular cytoplasm.Neoplasms in the granular cell lineage make nests, similar to melanocytic neoplasms.Immunostains for S100 and SOX10 are typically positive, in keeping with schwannian histogenesis.More specific melanocytic markers such as MelanA and HMB45 are typically negative.Criteria have been proposed for distinguishing benign, atypical, and malignant GCTs 1,2 ; however, these are difficult to apply consistently and there are case reports of GCTs with benign histopathologic features going on to metastasize.A multifocal benign variant also exists. 3In addition there are reports of atypical and malignant GCTs with junctional components that raise a differential diagnosis of melanoma. 4][7][8][9][10][11] Melanoma-specific immunostains such as MelanA and HMB45 are usually positive in melanoma and negative in GCT, 12 but also have limited sensitivity for melanoma in the range of 75%-92%, and as low as 17%-21% in spindle cell/desmoplastic melanoma. 13MelanA can rarely be positive in GCTs. 12Microphthalmia-associated transcription factor is positive in the majority of GCTs and cannot be used as a marker to discriminate between GCTs and melanocytic tumors. 12Other markers for GCT such as PGP9.5, inhibin, and calretinin are not specific and may be positive in both GCTs and melanoma. 125][16][17][18][19][20] Recently, recurrent loss of function mutations in the vacuolar ATPase complex genes ATP6AP1 and ATP6AP2, which regulate endosomal pH, have been identified in 72% of GCTs, including atypical and malignant GCT. 21These were frameshift mutations and premature stop codons.In addition to mutations in ATP6AP1 and ATP6AP2, a subsequent study found mutually exclusive mutations in six different genes encoding various components of the vacuolar ATPase complex. 22 practical terms, the potential morphologic overlap of GCT and melanoma is infrequently a diagnostic problem, as most GCTs feature banal, benign-appearing cytologic features.We have recently encountered several cases with sufficient morphologic overlap to seek additional molecular means of establishing a definitive diagnosis.This is important because an atypical GCT and a melanoma of similar thickness would be expected to have distinctly different prognosis, clinical work-up, and therapy.

| METHODS
All available cases of GCT, atypical or malignant GCT in the archives of the Dermatopathology Section, Department of Pathology, Indiana University from 2010 to 2022 were retrieved and reviewed.Immunohistochemical studies were performed on select cases with monoclonal antibodies against S100 (Dako), Sox-10 (Cell Marque), MelanA (Dako), PGP9.5 (Cell Marque) on a Dako Omnis stainer using formalin-fixed paraffin-embedded tissue sections cut at 4 μm sections.DNA sequencing of 648 genes and full-transcriptome RNA sequencing were performed on the two index cases at Tempus Labs, Chicago, IL, as previously described. 23This study was conducted under an institutional IRB and did not require ethical approval.

| Index case 1
A 57-year-old man with no relevant previous history presented with a solitary lesion on his back.Biopsy demonstrated a neoplasm with a junctional component, nesting, maturation in the deep dermis, granular cytoplasm, and mitotic figures at 1/10HPF (Figure 1A,B).Some spindling was also present.There was no necrosis.S100 (Figure 1C,D However, the assay also includes RNA whole transcriptome sequencing, and, on further interrogation of the RNA data, a frameshift mutation conferring loss of function was identified in ATP6AP1 (pN406fs), with a variant allele fraction of 40%.A diagnosis of atypical GCT was therefore established, with a recommendation for re-excision.

| Index case 2
A 32-year-old woman with no relevant previous history presented with a solitary lesion on her abdomen.Biopsy revealed a neoplasm with pseudo-epitheliomatous hyperplasia, nesting, and granular cytoplasm (Figure 2A,B).There was mild nuclear pleomorphism and mitotic figures at 1/10HPF were visualized.Immunostaining for S100 was positive (Figure 1C), and demonstrated nests of cells closely approximating the epidermis, but not convincing junctional nests as were seen in index case 1. Melan A (Figure 1D), as well as HMB45, and tyrosinase were negative.NGS using the Tempus XT platform was pursued; the DNA study did not identify melanoma-associated driver mutations or other pathogenic mutations.The RNA data were again further interrogated and demonstrated a premature stop codon in

| Additional cases of GCT with junctional component
Hematoxylin and eosin (H&E) stains and immunostains from all additional cases of cutaneous GCT diagnosed in the period 2010-2022 with available slides were reviewed.There were 19 GCTs, 3 atypical GCTs, and 1 malignant GCT, for a total of 23 cases.No additional true junctional components were identified; however, in one case, nests of granular cells were present closely approximating an epidermis showing pseudoepitheliomatous hyperplasia (Figure 3).Maturation of the dermal component was identified in no additional cases.

| DISCUSSION
Neoplasms with granular cell lineage may have an atypical and malignant variant.There have been reports of junctional nested components in GCTs. 4 This, along with the immunoprofile (S100, SOX10+) can raise a differential of melanocytic lineage.The issue can be important in the setting of atypical GCT where a differential diagnosis of melanoma with granular cell change is possible.We encountered two atypical GCTs with junctional nesting and with dermal maturation in one.We confirmed a diagnosis of granular cell lineage by means of RNA whole transcriptome sequencing, identifying a frameshift mutation in ATP6AP1 (pN406fs) in index case 1 and a premature stop codon in ATP6AP2 pY326* in index case 2. Other more limited molecular studies that would have potential utility in this differential would include BRAF, NRAS sequencing or BRAF V600E immunostain.If any of these studies were positive, it would imply a diagnosis of melanoma, while negative studies would not fully exclude melanoma.

| Specificity of ATP6AP1/2 frameshift and premature stop codon mutations
Loss of function mutations in ATP6AP1/2 have been identified in 72% of 82 tumors with granular cell lineage in the seminal paper of Pareja et al. 21Both of these genes are located on the X chromosome and in females the mutation is present on the active/non-methylated X chromosome, so that a single inactivating mutation would be sufficient to cause its complete loss of function. 21In all patients tested, the mutations were detectable by RNA sequencing.A search of the cancer genome atlas identified mutations in ATP6AP1 in only 0.27% of 6285 unrelated tumors, and in ATP6AP2 in only 0.25% of cases.Importantly the mutations identified in these cases were predominantly missense single nucleotide variants (SNVs) with only 0.04% frameshift or truncating mutations (ATP6AP1) and 0.02% for ATP6AP2. 21We also searched the Cosmic database for mutations in ATP6AP1/2 in cutaneous melanoma.Of 1784 skin samples tested for ATP6AP1, there were 24 melanomas with mutations identified in ATP6AP1, but these were all SNV or silent mutations, with no frameshift or premature stop codons identified.Of 1784 skin samples tested for ATP6AP2, there were 22 melanomas with SNV or silent mutations identified.A single melanoma had a premature stop codon in ATP6AP2, pS95*, but this lesion also had a melanoma-associated driver mutation in NRAS Q61K and two pathogenic mutations in ARID2.Frameshift and premature stop codons in ATP6AP1 and ATP6AP2 therefore appear to be highly specific for granular cell lineage, and capable of excluding melanoma, in the absence of known melanoma-associated driver mutations.
We identified a junctional component in GCT in our two index cases and 1 of 23 additional cases of GCT retrieved from our files.
Our literature review identified an additional report of junctional component in three GCTs including an atypical GCT. 4 We found that the differential diagnosis with melanoma is not typically an issue in the setting a bland, granular cell morphology.The differential diagnosis with melanoma becomes an issue in the context of atypical or malignant GCT.In one of our two index cases, we found that maturation was present, further mimicking a melanocytic neoplasm.This does not appear to have been commented on previously in the literature to our knowledge.Histopathologic criteria for atypical and malignant GCT have been proposed 1,2 but are hard to apply.In addition, there are reports of benign-appearing GCTs with metastasis.For this reason, there has been interest in using NGS to help make these distinctions, with mutations identified in TGFB and MAPK pathways in malignant GCT. 24We did not identify any recurrent DNA changes distinguishing atypical GCT from the benign variant in our cases.It might be feasible to distinguish these entities based on RNA expression but currently, there are insufficient data.
Pareja et al. reported results of immunofluorescence using antibodies to ATP6AP1 and ATP6AP2 and demonstrated loss of expression in GCTs with loss of function mutations in these genes. 21This raises the possibility that the same antibodies could be used to demonstrate granular cell lineage by immunoperoxidase on a clinical basis.
Whether this could have utility in the differential with melanoma would need additional study.

| CONCLUSION
We present two index cases of atypical GCT with a junctional component and maturation in one case, raising a differential diagnosis of melanoma with granular cell change.We reviewed a larger series of 23 GCTs from our archives and identified a junctional component in one additional case.We show the potential utility of sequencing for ATP6AP1/2 in excluding melanoma, and the specificity of loss of function mutations in these genes in the differential diagnosis with melanoma.

5 (
) and Sox10 immunostains were positive.S100 and PGP 9.Figure1E,F) both identified the junctional component.S100 and PGP 9.5 both demonstrate maturation, meaning smaller nests and single cells toward the deep margin of the lesion.MelanA, HMB45, and tyrosinase immunostains were negative.A differential diagnosis of melanoma with granular cell change versus atypical GCT was considered.Initial attempts to confirm or exclude the possibility of melanoma with granular cell change included sequencing BRAF and NRAS using the Therascreen platform (Qiagen).These studies were both negative and next generation sequencing (NGS) using the Tempus Xt platform was then employed.DNA sequencing demonstrated no known melanoma drivers.Drivers known to be associated with granular cell lineage in 70% of cases (ATP6AP1, ATP6AP2) are not sequenced in the DNA capture assay format of the Tempus assay.

F I G U R E 3
Additional granular cell tumor identified by review of case archives with nests of granular cells closely approximating an epidermis showing pseudoepitheliomatous hyperplasia.(A) Hematoxylin and eosin 200Â.(B) S100 immunostain, 200Â.