Circulating tumor cell-based molecular classification to reveal heterogenous subclones in gestational trophoblastic neoplasia.

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Background: Gestational trophoblastic neoplasia (GTN) is caused by cancerous transformation of placental trophoblastic cells, which can undergo hematogenous spread via circulating tumor cells (CTCs). Due to limited accessibility to tumor tissues, the molecular classification and metastatic evolutionary dynamics of GTN progression remains poorly defined. Methods: We performed molecular subtyping and evolutionary trajectory analysis using single-cell omic profiling of CTCs and patient-matched tumor cells from GTN blood and tissue biopsies respectively. Experimental in vitro and in vivo assays were employed to test the function of disease-driving genes involved in GTN progression. Blood and tissue-based clinical analyses from independent patient samples were carried out to demonstrate the molecular heterogeneity of GTN subclones within individual patients. Results: Three distinct GTN molecular subtypes were identified and validated in clinical samples, including cilia-like subtype marked by FOXJ1 positivity, tuft cell-like subtype driven by POU2F3, and a senescent subset with elevated BHLHE41 expression. Experimental analyses using GTN cell line models confirmed a central role of POU2F3 in driving epithelial–mesenchymal transition (EMT), migration, invasion, and tumorigenesis in vivo . Furthermore, BHLHE41 overexpression is sufficient to drive p53/p21 activation and senescence induction, characterized by β-Galactosidase formation and elevated expression of the senescence-associated secretory phenotype (SASP) signatures. In a prospective cohort of patient blood samples, CTCs overexpressing POU2F3 or BHLHE41 were significantly elevated in the GTN group compared to the benign hydatidiform moles (HM) cases, implicating their translational potential as single-cell liquid biopsy for accurate molecular subtyping and prediction of GTN progression in the absence of tissue biopsies. Conclusions: This study uncovers key molecular subtypes and functional states of GTN using CTC-based single-cell omic analysis, offering a noninvasive approach for disease classification and real-time monitoring in the absence of tumor tissue biopsies.