FAST-FNA molecular diagnostic assay facilitates rapid diagnosis and ntrk biomarker testing of salivary gland tumors.

Abstract

e18089 Background: Primary epithelial salivary gland tumors (SGTs)are classified into 31 tumor types. Diagnosing SGTs through fine needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. In this study, we developed an innovative, multiplexed cycling technology which allows for the rapid cellular analyses of single cells obtained from FNA which can facilitate the rapid molecular analyses and diagnosis of SGTs. Methods: Antibodies against 29 cell surface markers associated with 7 SGT subtypes were validated and chemically modified via custom linker–bio-orthogonal quencher probes (FAST). Single cell homogenates and a FNA sample set representing 7 SGT subtypes were profiled by FAST cyclic staining, imaging, and automated computational analysis. A prediction model was generated utilizing a training set of 151,926 single cells obtained from primary SGTs (N = 26) and validated on a separate cohort of SGTs (N = 30). Companion biomarker testing, such as NTRK, was also assessed with the FAST technology. Results: The customized FAST-FNA SGT molecular diagnostic and biomarker panel was validated on human cell lines and specificity confirmed by both flow cytometry and clinical immunohistochemical staining on primary SGTs. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell suspension samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single tumor marker increased the diagnostic accuracy of the SGTs, 0.82 as compared to 0.65-0.74, respectively. The results of the assay were highly reproducible and independent of the cell number sampled. NTRK expression in SGTs was also assessed by the FAST assay within hours, highlighting the potential therapeutic application of this technology. Conclusions: High dimensional single-cell profiling and molecular diagnostic testing on FNA samples are feasible at relatively low cost and as a rapid test. Application of the novel multiplexed single cell technology improves the diagnostic accuracy of SGT biopsy and facilitates multiplex biomarker testing from FNA samples. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision making.