Targeted Sequencing in Rosai-Dorfman Disease from Microdissected Specimens Reveals Higher Incidence of MAP2K1 Mutations

Rosai-Dorfman Disease (RDD) is a rare, non-Langerhans histiocytic neoplasm microscopically characterized by nodal sinus histiocytosis with emperipolesis. The clinical manifestations of RDD are heterogenous but commonly feature lymphadenopathy and violaceous cutaneous lesions; involvement of the central nervous and skeletal systems can also be observed.

While previously thought to be an inflammatory disorder, RDD was formally defined by the World Health Organization as a clonal neoplasm in 2016. The most common molecular alterations in RDD are mutations within the mitogen-activated protein kinase (MAPK) pathway, which has prompted a multitude of studies into its signaling and now widely-utilized targeted agents, including BRAF and MEK inhibitors, in histiocytic disorders. Prior research evaluating the presence of these mutations have not included stringent microdissection of tissue to enrich for disease involvement and separate non-neoplastic populations from true RDD. Accordingly, previous studies may have underestimated the incidence of these targetable mutations. In addition, low-level potential mutations in archival FFPE specimens that may reflect deamination artifacts are frequently reported in the available literature. Given that the sensitivity of molecular diagnostic testing can be highly influenced by tumor purity, we hypothesized that tissue microdissection may identify a higher incidence of mutations affecting the MAPK pathway than previously reported.

In this study, we obtained residual surgical specimens from patients diagnosed with Rosai-Dorfman Disease over a 20-year period at a single institution (n = 16). We microdissected areas enriched with histiocytes and minimal intervening inflammatory cells (requiring at least 10% histiocytes) with the aid of Diff Quik staining, the use of a dissection microscope and Pinpoint Slide DNA Isolation System (Zymo Research). Extracted DNA specimens were then sequenced using a targeted 50-gene panel (Oncomine Focus Solid Tumor Assay) with a limit of detection of 5% variant allele frequency (VAF, with greater than 750X average base depth). The VAF threshold of ≥ 5%, based on the minimum percentage of neoplastic cells, was utilized to distinguish true mutations from deamination artifact. Of the 16 submitted specimens, 14 yielded adequate sequencing data (all passing specimens > 1,200X sequencing depth). Four specimens (29%) demonstrated pathogenic MAP2K1 mutations (F53L, G128D, Y130C, E203K) and two (14%) demonstrated KRAS mutations (both A146P). All of these mutations were mutually exclusive with one another. Deamination artifacts were also detected in older specimens with allele frequencies < 5%. The incidence of MAP2K1 mutations was higher in the current study than in previous studies (Garces et al. 2017), though not statistically significant, at 29% (4/14) compared to 16% (8/49; p=0.15).

These results highlight the value of stringent tissue microdissection for identifying targetable molecular alterations in RDD and suggest that such approaches may help uncover additional neoplastic drivers and/or mutations implicated in therapeutic resistance.

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