Next-Generation Sequencing of Sporadic Schwannomas Reveals Critical Importance of NF2 Alteration

Introduction: As opposed to recent advances in the genetics of schwannoma syndromes, the genetic basis of sporadic schwannoma formation has not been fully characterized. Consistent with studies on other tumor types, next-generation sequencing can potentially lead to insights in the somatic genetic events underlying sporadic schwannoma formation.

Methods: Sporadic schwannoma samples were identified and obtained from tumor banks at the Brigham and Women’s Hospital, the Massachusetts General Hospital, and the University of Sydney with appropriate institutional review. Case records were reviewed and patients with evidence of neurofibromatosis were excluded. Next-generation sequencing was performed at the Broad Institute (Cambridge, MA) and integrated analyses were performed.

Results: We performed whole-genome sequencing (average coverage 37X) of two vestibular schwannomas and whole-exome sequencing (average coverage 88X) of 41 additional schwannomas, including 32 vestibular, 1 trigeminal, and 7 peripheral tumors, for a total of 43 sporadic schwannomas characterized by next-generation sequencing. Two of the seven peripheral schwannomas were recurrent lesions and another two were segmental schwannomas from the same patient. Radiation therapy had been applied prior to initial resection for two of the cranial schwannomas.

We detected SCNAs through an integrated analysis of read depth and allelic fractions of somatic mutations genome-wide to determine local absolute allele-specific copy numbers genome-wide as well as overall ploidy and purity. The most common copy number alteration was loss of heterozygosity (LOH) of chromosome 22, detected in 26/43 samples. In 19 cases, this was hemizygous loss of chromosome 22. The other seven samples exhibited copy-neutral LOH (cnLOH).

The mutation pattern of sporadic schwannomas was most consistent with sporadic mutations from endogenous cellular processes, especially cytosine deamination. Schwannomas also harbored relatively few non-synonymous mutations (median of 16). Significance analysis demonstrated that only one gene, NF2, was significantly mutated in sporadic schwannoma; non-synonymous somatic NF2 mutations were observed in 23 samples with 29 individual events. These mutations contributed to biallelic inactivation of NF2 in 20 samples, nearly half the overall cohort, through combinations with chromosome 22 LOH (17 cases, including five cases of copy-neutral LOH) or the presence of compound NF2 mutations (three cases). Among the 23 cases without clear biallelic inactivation of NF2, 11 exhibited alterations of at least one allele (mutation in three cases; copy loss in six cases, and copy-neutral LOH in two cases). A total of 32/43 samples exhibited some somatic alteration of NF2.

To further examine the role of NF2 and chromosome 22 loss in sporadic schwannoma formation, we performed additional focused, deep sequencing on 71 sporadic schwannomas using a panel of genes known to be involved with schwannoma tumorigenesis. 65 of 71 samples (92%) demonstrated some alteration of NF2 (59 with at least one mutation and 6 with copy-loss).

Conclusion: Alterations in NF2 via mutation or loss of chromosome 22 represent an important majority of tumor-driving events in sporadic schwannoma. Additional research therefore must emphasize improved understanding and targeting of the NF2 tumor suppressor pathway.