Integrated Molecular and Functional Characterization of Cervical Small-Cell Neuroendocrine Carcinoma Using a 3D Organoid Model

Cervical small-cell neuroendocrine carcinoma (SCNEC) is a rare cervical cancer with high metastatic potential and is frequently associated with high-risk human papillomavirus (HPV) infection. Because of its low incidence, SCNEC remains understudied and treatment options are limited, posing major therapeutic challenges. This study aimed to characterize SCNEC at the molecular and functional levels to support more informed therapeutic strategies. Organoids and spheroids were generated from a cervical SCNEC biopsy, and a matched organoid-derived xenograft was established in immunodeficient mice. Model fidelity was evaluated by histopathology and immunohistochemistry. HPV status was assessed by p16 immunostaining and HPV18 PCR, and viral-host integration sites were inferred using whole-exome sequencing (WES) junction reads. WES was also used to compare shared somatic variants and copy-number alterations across the patient tumor, organoid, and xenograft. Drug responses were assessed in organoids and spheroids following exposure to a panel of chemotherapeutic agents and a targeted inhibitor. Organoids exhibited robust growth, morphologic maturation, and efficient recovery after cryopreservation. The organoids and matched xenografts faithfully recapitulated SCNEC, with preserved neuroendocrine differentiation (CD56, synaptophysin, and NSE positivity), a high Ki-67 proliferative index (>80%), and strong p16 expression. HPV18 status was conserved across the primary tumor, organoids, and xenografts, with an integration site at chr8 (8q24.21) associated with increased MYC expression. WES revealed strong cross-model concordance, including 26 shared somatic variants with a canonical <i>PIK3CA</i> hotspot mutation (p.E542K) and conserved oncogenic copy-number gains of <i>PIK3CA</i>, <i>TERT</i>, and <i>MYC</i>, as well as copy number loss of <i>TP53</i>. Functional assays showed dose-dependent loss of viability following exposure to conventional cytotoxic agents or an mTOR pathway inhibitor. This study presents the first integrated molecular and functional analyses of patient tumors and matched organoid and xenograft models in cervical SCNEC. These models offer robust resources for mechanistic studies and may enable precision therapeutic strategies for this rare malignancy.