Evaluation of Targeted and Immunotherapeutic Approaches Using an Organoid Model of Sinonasal Intestinal-Type Adenocarcinoma (ITAC)

Introduction: Sinonasal intestinal-type adenocarcinoma (ITAC) is rare tumors representing about 8 to 25% of sinonasal and skull base malignancies. Sinonasal ITAC is highly prevalent in European counties, strongly associated with exposure to wood dust, and typically arises in the ethmoid cavity. Sinonasal ITAC is locally aggressive and may invade nearby structures including the skull base, dura, orbit, optic nerve, and others. Standard treatment oftentimes includes surgical resection followed by adjuvant radiation and the 5-year overall survival rate is 45 to 73%. Cell lines, organoids, or xenograft models of ITAC are scarce, thus hindering functional, molecular, or mechanistic studies. We recently developed a sinonasal ITAC organoid model from a 50-year-old male patient with T1N0M0 high-grade sinonasal tumor. This organoid model has been passaged >40 times and closely resemble the histologic structure of the primary tumor and retains a similar pattern of copy number variations. We hypothesized that whole-exome sequencing and tumor immune microenvironment evaluation would reveal potential targetable and immunotherapeutic approaches that could be evaluated using a unique organoid model of sinonasal ITAC.

Methods: Targetable mutations in the primary tumor and organoid model were identified through whole-exome sequencing analysis. Organoid viability experiments were conducted by measuring ATP levels following exposure to cisplatin and small molecule inhibitors targeting WEE1 kinase (AZD1775) and the receptor tyrosine kinase EGFR/ErbB2 (AZD8931). IC50 values were calculated by generating killing curves with data normalized to vehicle and baseline controls. Multiplex immunofluorescence of the patient’s primary tumor tissue was performed to assess the presence of immune cell compartments. T cell killing assays were performed by culturing tumor infiltrating lymphocytes (TIL) from the primary tumor and combining TIL with ITAC organoids at various effectors to target ratios. Apoptotic cell death was measured by flow cytometry via staining for caspase3/Sytox.

Results: Whole-exome sequencing analysis identified potential targetable mutations in APC (frame shift), FGFR1, ERBB3 and JAK3 (all missense). Organoid viability experiments with targeted small molecular inhibitors AZD8931 (mean IC50 0.016 μmol/L, SEM 0.0049), AZD1775 (mean IC50 0.2349 μmol/L, SEM 0.1142) and chemotherapeutic agent cisplatin (mean IC50 3.692 μmol/L, SEM 0.7523) demonstrated that ITAC organoids are susceptible to targeting these pathways ([Fig. 1A]–[D]). Multiplex immunofluorescence of the patient’s primary tissue detected the presence of CD8+, CD4+ FOXP3 cells, and CD11b myeloid cells and PD-L1 staining was observed on both immune cells and tumor cells ([Fig. 2A, B]). In ITAC organoid and autologous TIL co-culture experiments TILs were able to kill the sinonasal ITAC organoid cells at increasing effector to target ratios ([Fig. 2C], p < 0.05).

Conclusion: Collectively these data identify potential targetable pathways in sinonasal ITAC and reveal that these tumors may also be susceptible to approaches that enhance TIL.