Direct specimen collection during routine operation improves nucleic acid quality in genetic analysis samples for head and neck tumors: a retrospective study

Abstract Background The success of sequencing analysis and genetic treatment is influenced by sample quality. Preserving nucleic acid integrity in head and neck squamous cell carcinoma (HNSCC) samples is challenging owing to poor formalin fixation caused by lengthy surgical procedures and demineralization. New sampling and fixation methods are required to prevent the loss of important variants. We aimed to improve nucleic acid preservation in HNSCC specimens using a new collection method. Methods A total of 128 samples from 44 patients with HNSCC were included: 32 genetic analysis samples (GAS), which were collected from the tumor surface in the operation room and immediately placed in a 25 mL bottle with 10% neutral buffered formalin solution; 43 primary tumor components (Primary); 14 decalcified tumor samples (DC); 32 metastatic tumors in lymph nodes (LN); and seven parakeratinized components (PKC) from HNSCC. The quality of nucleic acids in the GAS, Primary, DC, LN, and PKC samples was compared using the DNA integrity number (DIN), RNA integrity number (RIN), the percentage of RNA fragments with > 200 nucleotides (DV200), and methyl green-pyronin (MGP) staining. The next-generation sequencing (NGS) metrics of the Primary, LN, and PKC from three HNSCC samples were also assessed. Results The DIN was significantly higher in the GAS than in the Primary, LN, and DC groups ( p < 0.05). The RIN decreased in the order LN, GAS, Primary, and DC. DV200 was significantly higher in the GAS than in the primary and DC groups ( p < 0.05). On MGP staining, the preserved DNA and RNA were visualized in the GAS, Primary tumors from 2022, and LN, but not in DC. The PKC samples contained DNA, but RNA was not detected. NGS, using DNA extracted from the PKC samples, reliably detected mutations. No significant differences were detected in most NGS metrics among the Primary, LN, and PKC samples. Conclusions GAS should be collected during routine hospital activities, especially for head and neck tumors. Moreover, when the volume of viable materials is limited, PKC should be considered for genetic analysis. Finally, MGP staining is useful for pre-pathological analysis.