Application of organoid culture from HPV18-positive small cell carcinoma of the uterine cervix for precision medicine

Abstract

Background: Small cell carcinoma of the uterine cervix (SCCC) is a rare and highly malignant human papillomavirus (HPV)-associated cancer in which human genes related to the integration site can serve as a target for precision medicine. The aim of our study was to establish a workflow for precision medicine of HPV-associated cancer using patient-derived organoid.

Methods: Organoid was established from the biopsy of a patient diagnosed with HPV18-positive SCCC. Therapeutic targets were identified by whole exome sequencing (WES) and RNA-seq analysis. Drug sensitivity testing was performed using organoids and organoid-derived mouse xenograft model.

Results: WES revealed that both the original tumor and organoid had 19 somatic variants in common, including the KRAS p.G12D pathogenic variant. Meanwhile, RNA-seq revealed that HPV18 was integrated into chromosome 8 at 8q24.21 with increased expression of the proto-oncogene MYC. Drug sensitivity testing revealed that a KRAS pathway inhibitor exerted strong anti-cancer effects on the SCCC organoid compared to a MYC inhibitor, which were also confirmed in the xenograft model.

Conclusion: In this study, we confirmed two strategies for identifying therapeutic targets of HPV-derived SCCC, WES for identifying pathogenic variants and RNA sequencing for identifying HPV integration sites. Organoid culture is an effective tool for unveiling the oncogenic process of rare tumors and can be a breakthrough for the development of precision medicine for patients with HPV-positive SCCC.

Keywords: cervical cancer; human papillomavirus; integration site; organoid; precision medicine.

FIGURE 1

Established organoid maintains the histological architecture and neuroendocrine marker positivity of the original tumor. (A) Macroscopic view of a resected tumor (arrowheads) at the uterine cervix. (B) Simplified schematic representation of the modified Matrigel bilayer organoid culture protocol. (C) Established organoid of small cell carcinoma of the uterine cervix. (D–F) Histological examination of the original tumor (D) and organoid [(E): Passage 2, (F): Passage 4] by hematoxylin and eosin staining, immunohistochemical staining, and Alcian blue staining (×400).

FIGURE 2

Organoid‐replicated genomic landscape of the primary tumor. (A) Somatic mutation profiles by whole exome sequencing in the original tumor and organoid. (B) Comparison of VAF of genetic alterations detected in the original tumor and organoid. (C) Copy number variation profiles of the original tumor and organoid. The panel displays the total copy number log ratio with chromosomes alternating in blue and gray. The median log ratio of each segment is shown in red. The purple line indicates the log ratio of the diploid state. The bottom bar shows the associated cellular fraction (cf). Dark blue indicates high cf. Beige indicates a normal segment. Cf‐em: the cellular fraction of the segment.

FIGURE 3

Validation of the integration site. (A) The integration site was estimated by the ViFi tool, and the viral‐human fusion sequence was validated by PCR using designed primers. The PCR products were analyzed by 2% agarose gel electrophoresis, cloned, and then sequenced. (B) Validation of the orientation of viral‐human fusion by strand‐specific PCR. cDNA synthesis was performed using strand‐specific primers (Primer 1: primer of human side, Primer 2: primer of HPV18 side) and amplified by PCR. (C) Identified structure of the integration site. (D) Comparison of MYC expression between the SCCC organoid and four cervical cancer cell lines, i.e., C33a (HPV‐negative), HeLa (HPV18‐positive), CaSki (HPV16‐positive), SiHa (HPV16‐positive), evaluated by RT‐qPCR analysis using the delta–delta Ct method. (E) Comparison of MYC expression between the original SCCC tissue and 13 HPV‐associated cervical cancer some cases of which HPV integration sites were identified. The table on the right shows the integration site for each case estimated by the ViFi tool. AC, adenocarcinoma; ASC, adenosquamous carcinoma; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; ND, not determined; SCC, squamous cell carcinoma; SCCC, small cell carcinoma of the uterine cervix; ViFi, Viral Integration and Fusion Identification

FIGURE 4

Drug sensitivity testing of the SCCC organoid to MEK inhibitor and MYC inhibitor. (A) Dose–response curves of the SCCC organoid treated with trametinib and everolimus (left graph), cisplatin, and etoposide (right side). Mean ± SD of the technical triplicates is shown for each drug. (B) SCCC organoids were treated with trametinib at 10 nM for 72 h (control = 0.4% dimethyl sulfoxide), followed by staining with propidium iodide for cell cycle distribution, and then analyzed using flow cytometry. Three independent experiments were performed. (C) Dose–response curves of the SCCC organoid and cervical cancer cell lines treated with MYCi975. Mean ± SD of the technical triplicates is shown for each drug. SCCC, small cell carcinoma of the uterine cervix; SD, standard deviation

FIGURE 5

Drug sensitivity testing of the small cell carcinoma of the uterine cervix organoid to MEK inhibitor. (A) Established organoid‐derived mouse xenograft mouse model. (B) Histological examination of the organoid‐derived mouse xenograft. (C) The median tumor volume and weight at the beginning of the treatment and days after implantation in each group were not significantly different. (D) Kaplan–Meier curve between a control group and a trametinib‐treated group.

FIGURE 6

Workflow of our research.