Analysis of circulating small extracellular vesicle-associated miRNA in head and neck squamous cell carcinoma (HNSCC): Biomarker discovery in pre-operative neoadjuvant nivolumab.

2577 Background: Standard treatment options for HNSCC include chemotherapy, radiation, surgery, and immunotherapy. Immune Checkpoint Inhibitors (ICI) are safe and improve survival in platinum-refractory and metastatic HNSCCs. Further studies are needed to efficiently stratify patients who may benefit from ICIs. MYC-V1 is a family of oncogenes, induced by MYC signaling, that is negatively correlated with response to ICI in HPV+ HNSCC. Non-invasive Biomarkers (NiBs) identifying patients with low MYC-V1 expression may aid in predicting response to ICIs. Small extracellular vesicles (sEVs) are membrane-bound mediators of intercell communication that are present in circulation, serving as a promising source of NiBs. The Let-7 family of tumor suppressors are negative regulators of the MYC-V1 gene family and exhibit low expression in HNSCC compared to healthy controls. Let-7 family members, such as Let-7d and Let-7c, may play a role in ICI responsiveness via PD-L1 and MYC mRNA degradation, respectively. Here, we analyze the miRNA profile of sEVs from patients enrolled in a randomized Phase I clinical trial undergoing Neoadjuvant Nivolumab therapy. (NCT03238365). Methods: Plasma was collected before and after treatment (N=24) with ICI. 12 patients were included and stratified based on HPV status (6 HPV-, 6 HPV+) and pathologic treatment response (pTR) (6 Responders [R], 6 Nonresponders [NR]). pTR >20% of tumor surface area as graded by two pathologists was R; 0% was NR. sEVs were isolated via immunoprecipitation targeting tetraspanins CD63/9/81. miRNA was isolated and NGS was performed. Analysis was performed using Python and Biomni. Log2FC (FC) compared expression levels between groups and a False Discovery Rate (FDR) <.05 was significant. Results: 189 miRNAs were detected in HNSCC sEVs. Analysis comparing HPV+ R vs NR revealed 48 miRNAs with significantly different levels between these pretreatment groups. 47 were more abundant in R; 1 was less abundant. miRNAs found at higher levels in HPV+ R include the Let-7 family. Let-7d was most abundant (FC: 2.26, FDR 0.04), with a family (Let-7a,b,d,f,g,i) FC range of 1.63-2.26 (FDR 0.03-0.05). Following treatment with ICI, HPV+ patients had increased Let-7c levels (FC 4.71, FDR 0.02) compared to pretreatment. Analysis of HPV– R vs NR showed 0 miRNA that met statistical significance. Conclusions: Here, we show that ICI-responsive HPV+ HNSCC is associated with high levels of circulating, sEV-contained Let-7 family members and that Let-7c is increased in response to ICI in those same patients. We propose that a MYC-V1-suppresive, PD-L1-destabilizing mechanism may be present in patients with higher than average Let-7 expression, conferring sensitivity to ICI, and that sEV-associated Let-7 levels may serve as NiBs to identify these patients as favorable ICI candidates prior to treatment.