Frozen tumor sections collected at T1 and T2, were homogenized using TissueLyser LT (Qiagen, Benelux, B.V.) in Trizol Reagent. The PureLink RNA Mini Kit (Life technologies) was adopted to extract and purify total RNA. The concentration and purity of RNA were determined at 260/280 nm using Nano Drop One Spectrophotometer (Thermo Fisher). Bulk long-read RNA sequencing was performed using the Oxford Nanopore Technologies (ONT; Oxford, UK) PCR-cDNA Sequencing Kit (SQK-PCB114-24). Sequencing libraries were prepared according to the manufacturer's protocol. Briefly, 700 ng of full-length RNA per sample was used as input for complementary DNA (cDNA) synthesis and strand switching with kit-supplied oligonucleotides. Double-stranded cDNA (dscDNA) was generated by PCR amplification (10 μL cDNA/sample) using primers containing 5′ tags, which enable ligase-free attachment of rapid sequencing adapters. The resulting libraries were loaded onto R10.4.1 flow cells (FLO-PRO114M) and sequenced on P2 Solo PromethION devices using MinKNOW acquisition software (v24.11.10).