Precision medicine for patients with salivary gland neoplasms: Determining the feasibility of implementing a next-generation sequencing-based RNA assay in a hospital laboratory

Abstract

Objective: Diagnosing neoplasms of the salivary gland is challenging, as morphologic features of these tumors are complex, and well-defined diagnostic categories have overlapping features. Many salivary gland neoplasms are associated with recurrent genetic alterations. The utilization of RNA-based targeted next-generation sequencing (NGS) panels for the detection of cancer-driving translocations and mutations is emerging in the clinical laboratory. Our objective was to conduct a proof-of-concept study to show that in-house molecular testing of salivary gland tumors can enhance patient care by supporting morphologic diagnoses, thereby improving therapeutic strategies such as surgical options and targeted therapies.

Material and methods: Residual formalin-fixed paraffin-embedded salivary gland neoplasm specimens from a cohort of 17 patients were analyzed with the Archer FusionPlex Pan Solid Tumor v2 panel by NGS on an Illumina NextSeq550 platform.

Results: We identified structural gene rearrangements and single nucleotide variants in our patient samples that have both diagnostic and treatment-related significance. These alterations included PLAG1, MAML, and MYB fusions and BRAF, CTNNB1, NRAS, and PIK3CA mutations.

Conclusion: Our RNA-based NGS assay successfully detected known gene translocations and mutations associated with salivary gland neoplasms. The genetic alterations detected in these tumors demonstrated potential diagnostic, prognostic, and therapeutic value. We suggest that incorporating in-house ancillary molecular testing could greatly enhance the accuracy of salivary gland fine needle aspiration cytology and small biopsies, thereby better guiding surgical decisions and the use of targeted therapies.

Keywords: RNA sequencing; fine-needle aspiration; next-generation sequencing; precision medicine; salivary gland.

Figure 1:

Adenoid cystic carcinoma with high-grade transformation with a detected MYB::NFIB fusion and PIK3CA p.H1047R mutation. (a) Hematoxylin and eosin stain, 20× objective. (b) Hematoxylin and eosin stain, 4× objective. (c) Hematoxylin and eosin stain demonstrating the dedifferentiation component invading soft tissue, 10× objective. (d) Schematic view of the detected MYB::NFIB fusion demonstrating anchored primer regions spanning exon 10 of NFIB and exon 14 of MYB. The “+” denotes the gene-specific anchored primer (first strand) and (-) the universal primer (second strand). The arrow represents the gene-specific primer.

Figure 2:

Myoepithelial carcinoma ex pleomorphic adenoma with a detected LIFR::PLAG1 fusion. (a) Hematoxylin and eosin stain, ×10 objective. (b) Hematoxylin and eosin stain, ×20 objective. (c) Schematic view of the detected LIFR::PLAG1 fusion demonstrating anchored primer regions spanning exon 2 of PLAG1 and exon 1 of LIFR. The “+” denotes the gene-specific anchored primer (first strand) and () the universal primer (second strand). The arrow represents the gene-specific primer.

Figure 3:

Mucoepidermoid carcinoma, well-differentiated ex-pleomorphic adenoma with a detected CTNNB1::PLAG1 fusion. (a) Hematoxylin and eosin stain, 20× objective. (b) Schematic view of the CTNNB1::PLAG1 fusion demonstrating anchored primer regions spanning exon 3 of PLAG1 and exon 1 of CTNNB1. The “+” denotes the gene-specific anchored primer (first strand) and (-) the universal primer (second strand). The arrow represents the gene-specific primer.

Figure 4:

Salivary duct carcinoma with a detected BRAF p.V600E mutation. (a) Hematoxylin and eosin stain, ×10 objective. (b) Hematoxylin and eosin stain, ×40 objective.

Figure 5:

Low-grade mucoepidermoid carcinoma with a detected CRTC1::MAML2 fusion. (a) Hematoxylin and eosin stain, ×4 objective. (b) Hematoxylin and eosin stain, ×10 objective. (c) Schematic view of the detected CRTC1::MAML2 fusion demonstrating anchored primer regions spanning exon 1 of CRTC1 and exon 2 of MAML1. The “+” denotes the gene-specific anchored primer (first strand) and (–) the universal primer (second strand). The arrows represent the gene-specific primer.

Figure 6:

Intermediate-grade mucoepidermoid carcinoma with a detected CRTC1::MAML2 fusion. (a) Hematoxylin and eosin stain, ×20 objective. (b) Schematic view of the detected CRTC1::MAML2 fusion demonstrating anchored primer regions spanning exon 1 of CRTC1 and exon 2 of MAML2. The “+” denotes the gene-specific anchored primer (first strand) and (–) the universal primer (second strand). The arrows represent the gene-specific primer.

Figure 7:

Adenoid cystic carcinoma ex pleomorphic adenoma with a detected HRAS p.Q61L mutation. (a) Focus of the adenoma component juxtaposed with benign salivary gland, hematoxylin and eosin stain, ×20 objective. (b) Focus of the adenoid cystic carcinoma component, hematoxylin and eosin stain, ×10 objective.

Figure 8:

Basal cell adenoma with a detected CTNNB1 p.I35T mutation, hematoxylin and eosin stain, ×10 objective.