Background
ASCC is a rare malignancy commonly associated with human papillomavirus (HPV) infection for which there have been no significant advances in therapy in 5 decades. The small number of patients included in clinical trials coupled with lack of preclinical models have made it challenging to study novel therapeutics to improve survival outcomes. There are currently no human ASCC organoids in the literature. Our objective was to generate the first ASCC patient-derived tumour organoid (PTDO) biobank.
Methods
Viable ASCC tumour biopsies were verified, digested, embedded in extracellular matrix, and cultured in complete medium. Characterisation, validation were performed by histology, immunohistochemical, whole exome sequencing (WES) and transcriptomic profiling.
Results
We established the first 7 ASCC organoids from patients with primary, local persistent, recurrent, re-recurrent and metastatic disease. Five PDTOs were generated from the anal canal, 1 derived from a vaginal vault local re-recurrence, and 1 originated from a liver metastasis. HPV genotyping and p16 immunostaining identified the majority of samples to be HPV driven cancers (6/7). WES of matched organoid and tumour identified oncogenic alterations in genes PIK3CA, KMT2D, EP300 and TP53 with 97% concordance (range 0.94-1.00) rate. Differentially expressed genes in p16 positive PDTOs were significantly enriched for those involved in the Gene Ontology Biological Process (GO: BP) of negative regulation of viral genome replication and inflammatory response. In contrast, differentially expressed genes in the p16 negative PDTOs were for GO:BP DNA replication dependent nucleosome organisation and chromatin silencing at RDNA pathways with multiple shared genes involving histones. PDTOs subjected to conventional chemotherapy agents and irradiation displayed responses that correlated with clinical outcomes. Molecular directed therapies with alpelisib (PI3-kinase inhibitor) or abemaciclib (CDK4/6 inhibitor) demonstrated efficacy against PDTOs with PIK3CA mutation or p16 loss, respectively. Preliminary co-culture studies with HER2 CART demonstrated cytotoxicity, which was enhanced by anti-PD-1 blockade.
Conclusion
This proof of concept study demonstrates that generation of PDTOs is feasible in primary, relapsed and metastatic ASCC. The organoids faithfully recapitulate the parent tumours genomic, transcriptomic and molecular characteristics. This patient informed PDTO translation platform is an invaluable resource to interrogate vulnerabilities in ASCC, particularly in relapse settings.