Mutations involving TGFB and MAPK may be associated with malignancy in granular cell tumors

Granular cell tumors (GrCTs) are mesenchymal neoplasms of presumed schwannian differentiation that may present as solitary or multifocal lesions with excision usually being curative. A minority of cases, however, show histological features associated with an increased risk for metastasis and are highly aggressive leading to death in about a third of cases. While benign and malignant cases have been shown to harbor mutations in the H + ATPase genes, there is only limited data examining molecular aberrations associated with malignancy. The departmental archives were searched for cases of atypical/malignant GrCTs. Clinical and histopathological features were noted. Whole‐exome sequencing was performed. Three cases of malignant GrCTs and one case of atypical GrCTs were included. All three malignant tumors metastasized to distant sites with a median disease‐free survival of 16 months and an overall follow‐up time of 35 months. Whole‐exome sequencing showed mutations involving TGFβ and MAPK pathways in all four tumors. Although the cohort size is small, our preliminary findings suggest that mutations involving the TGFβ and MAPK pathways may be associated with tumor progression or malignant transformation in GrCT pathogenesis.

pleomorphism. A tumor was considered malignant if it fulfilled three or more of these criteria. Meanwhile, a tumor showing one or two criteria was classified as atypical. Finally, cases without any of these features were classified as benign and these findings correlated highly with clinical outcome. Benign multicentric, and atypical GrCTs were devoid of metastasis and tumor-related deaths. However, 39% of patients with malignant GrCT died of disease, 29% were alive with disease, and only 32% were disease-free (median interval of 7 years).
Later, additional studies suggested that refinement of these criteria might be useful 4 and that specifically tumors within the soft tissues may behave slightly more aggressively than those limited to skin or viscera. 5

| Case selection and clinicopathologic evaluation
This study was approved by both institutional review boards and patients consented to a banking protocol that allowed for next-generation sequencing (WUSTL 201203042, Mount Sinai 01-0138-U). All four cases were identified during routine sign-out at both institutions. Follow-up information was calculated from the date of pathologic diagnosis to the last available follow-up. Clinical chart review was performed to assess for age, gender, tumor size, treatment method, clinical impression, disease-free, and overall follow-up and status at last follow-up. Slides were reviewed by pathologists subspecialized in bone and soft tissue pathology and assessed for histologic features following the Fanburg-Smith criteria. 3

| Whole-exome sequencing
For WES, exome was captured with an IDT exome reagent and libraries were constructed by Kapa hyper amplified libraries. 100Â coverage data was achieved. Raw fastq files were trimmed by using Trimmomatic v 0.39 6 and aligned against reference sequence hg38 via BWA-MEM to generate BAM files. 7 Duplicated reads and base quality score recalibration (BQSR) were marked and processed by using GATK 4.2. For the paired tumor/normal samples, somatic SNVs and small indels were detected using VarScan2, 8 Strelka2, 9 MuTect2, 10 and Pindel. 11 For the tumor-only sample, MuTect2 was used for somatic variant calling. Variant filtering and annotation were performed using Variant Effect Predictor (VEP). 12 Common variants found in the 1000 Genomes MAF and GnomAD MAF > 0.05% were filtered out. Waterfall somatic variant plots were created with maftools v2.10.0. 13 Polyphen and SIFT were used to classify mutations.

| Whole-genome sequencing
For whole-genome sequencing (WGS), libraries were constructed by Kapa hyper-amplified libraries. Sequence alignment, duplicated reads marking, and base quality score recalibration (BQSR) marking were performed with the same pipeline as in WES methods. HMMCOPY package 14 was utilized and copy number prediction with correction for GC and mappability bias for HTS data was used for the copy number calculation.

| Histologic features
The histologic features are summarized in Figure 1A cytoplasm with small nuclei, abundant granular cytoplasm, and low mitotic count (1/10 hpf); however, necrosis was present. All cases were positive for S100 protein and SOX10 by immunohistochemistry, while negative for HMB45 and Melan A.
F I G U R E 1 Histologic features of cases 1-4. Each row represents an individual case. In addition to typical features seen in granular cell tumors such as eosinophilic granular cytoplasm, case 1 showed spindling of tumor cells (A) and prominent nuclear pleomorphism and high nuclear to cytoplasmic ratio (B). Tumor cells in case 2 were epithelioid, round to oval cells (C) with prominent nucleoli, occasional pleomorphism, and high nuclear to cytoplasmic ratio (D). Case 3 showed a very cellular tumor (E) composed of epithelioid cells with granular cytoplasm exhibiting sheet-like growth and tumor necrosis but without obvious pleomorphism, spindling or prominent nucleoli (F). Case 4 showed sheets of cells (G) with nuclear pleomorphism, prominent nucleoli, and increased mitotic activity (H).
F I G U R E 2 Legend on next page.

| Molecular findings
Cases 1 and 2 showed loss-of-function mutations in ATP6AP1, while cases 3 and 4 did not. All four cases showed mutations in several cancer-related genes with the top 50 genes shown in Figure S1. Importantly, all four tumors showed mutations involving the TGFβ and/or MAPK pathway ( Figure 2B,C). In brief, GrCT1 showed a mutation involving the MAPK1 gene. GrCT2 showed mutations involving TGFBR2. GrCT3 showed a mutation in LTBP2, which encodes for a protein that is a member of the latent transforming growth factorbeta binding proteins. Finally, GrCT4 showed mutations in MAP3K15 and TGFBR1. Importantly, each individual mutation was considered pathogenic with moderate or high likelihood by Polyphen and/or SIFT and all mutations have been manually reviewed using the integrative genomic viewer (IGV). Detailed molecular data can be found in Tables S1-S6.
In addition to TGFβ and/or MAPK pathway variants, copy number alternations were also identified in this cohort of malignant GrCTs ( Figure S2). GrCT2 showed chr1q, chr17q, and chr20q gain as well as chr9p and chr16q loss. GrCT4 showed Chr5p, chr8p, chr10p and chr19q loss. Interestingly, chrX gain was identified in both GrCT2 and GrCT4. In contrast, the atypical tumor, GrCT3, had fewer copy number alterations and only showed partial Chr12q and chr21q gain. This data suggest that there is more chromosomal instability in malignant tumors compared to atypical tumors. GrCT1 tumor was not sent for WGS as there was not sufficient tissue.

| DISCUSSION
Malignant GrCTs are very uncommon and often clinically aggressive lesions. Importantly, this malignant distinction should be reserved for tumors that are histologically reminiscent of benign GrCT and which demonstrate at least three additional features associated with malignancy including necrosis, spindling, prominent nucleoli, increased mitotic activity (>2/10 hpf), high nuclear to cytoplasmic ratio, and pleomorphism. 3 Other models of classifications, including the observation of a clear transition from aconventional GrCT to a highly cellular malignant nerve sheath tumor as a requirement, have been proposed. 16 We present four cases of malignant and atypical GrCTs, based on malignant transformation both seem to support each other and promote growth and metastasis with TGFβ switching from tumor suppressor to a pro-metastatic factor, 19-21 as previously described in colorectal cancer and pancreatic cancers 22 and breast cancer. 23 Further, MAPK signaling has also been shown to play a role in the pathogenesis of sarcomas including osteosarcoma 24 and correlate with worse prognosis in undifferentiated pleomorphic sarcoma. 25 Meanwhile, high levels of TGFβ were associated with aggressive behavior in non-GIST sarcomas such as synovial sarcoma and MPNST. 24 Additionally, identifying TGFβ and/or MAPK mutations may prove to be useful for treatment purposes as well since receptor tyrosine kinase inhibitors are more and more accessible and could fill the gap of unmet needs for better, more specific drug treatment. In fact, a single case of metastatic malignant GrCT demonstrated a significant response to pazopanib, a VEGF inhibitor that is upstream of several key pathways, including the Raf-MAP kinase pathway. 26 Larger studies will be necessary to further validate these findings as well as clinical trials evaluating drugs targeting TGFβ and MAPK pathways to treat patients with malignant GrCT. While such trials may be difficult to perform due to the rarity of this entity, exploration of drugs that target these pathways may improve outcomes for patients with this aggressive and poorly understood cancer.

ACKNOWLEDGMENTS
The authors thank the Division of Oncology at Washington University School of Medicine for support for this research.

CONFLICT OF INTEREST STATEMENT
ACH: Consulting for Springworks Therapeutics and AstraZeneca/ Alexion; research support from Tango Therapeutics.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.