In this retrospective immunohistological pilot study, ameloblastoma recurrence was observed in 23 (24.0%) out of 96 patients during the follow-up period (median [Q1, Q3]: 617.5 [314.0, 1106.0] days). The independent prognostic factors associated with ameloblastoma recurrence were recurrent tumors (vs. primary tumor) and MICA/B expression.

Ameloblastoma, benign odontogenic epithelial tumor mainly originating from undifferentiated enamel tissue, ranks among the most common benign tumors affecting the mandible and maxilla, accounting for approximately 1% of all oral area tumors [3, 19]. Ameloblastoma presents clinical challenges owing to its dual nature. Classified as “benign” tumor with a slow growth pattern, it exhibits “malignant tumor-like” behavior, including local invasiveness, frequent recurrence rate, and potential for metastasis [3].

The overall recurrence rate for ameloblastoma was 22–31% [1, 4, 20]. Our study’s overall recurrence rate of 24% is consistent with this range. The factors known to affect recurrence rate are the type of surgery, tumor recurrence (primary vs. recurrent), tumor size, clinicopathological type, and tumor location [2,3,4,5]. Surgical factors are considered to strongly influence ameloblastoma recurrence rates [2,3,4,5]. A recent systematic review reported that the recurrence rates for ameloblastoma following conservative surgery ranged from 33 to 93%, whereas those following radical surgery ranged from 7 to 22% [4]. Despite the advantages of radical surgery in terms of recurrence control, radical surgery necessitates a margin of over 1 cm [3, 4]. This led to a recent preference for conservative surgery, which preserves anatomical form and provides cosmetic and functional benefits [1, 21]. A recent meta-analysis failed to demonstrate the superiority of radical surgery over conservative surgery for ameloblastoma recurrence prevention [3, 21]. Moreover, the changing preferences of recent patients, who prioritize their quality of life, have added to the complexity of the decision-making process for surgeons. In the present study, a higher preference for conservative surgery (96.9%) over radical surgery (3.1%) was observed, which is presumed to be a reflection of patient choices. Due to the low incidence of radical surgery in this study, the impact of this factor was not assessed, which represents a limitation of our study.

Recurrent tumors, as demonstrated in our final model, are an independent risk factor for ameloblastoma recurrence, suggesting that primary tumors are associated with a lower risk of ameloblastoma recurrence. Hresko et al. conducted a retrospective analysis on patients with ameloblastoma who had a clinical follow-up period exceeding 3 years [22]. The study found that of the 69 patients with primary ameloblastoma, 24 experienced recurrences. Notably, these 24 patients further experienced a cumulative total of 35 recurrence episodes, underscoring an increased recurrence risk in patients that had previously experienced recurrence.

Tumor immunotherapy has emerged as a cornerstone in the oncological arsenal, complementing traditional modalities like surgery, chemotherapy, and radiation [23]. NK cells play a pivotal role in tumor immunosurveillance due to their unique ability to recognize and eliminate transformed cells without the need for prior sensitization [23, 24]. Within this realm, the NKG2D ligand-mediated pathway is gaining prominence as a potential target for immunotherapy, attributed to its selective expression of “stress-induced ligands” on tumor cells and the potent activation of NK cells by NKG2D [23]. To distinguish transformed tumor cells from healthy normal ones, NK cells recognize specific ligands on the surface of target tumor cells [25]. Cells that have sustained damage or undergone tumorigenesis express NKG2D ligands on their surface, making them more susceptible to detection and eradication by NK cells [9, 25]. Although human NKG2D ligands such as MICA/B and ULBP1-6 have been recognized, understanding of the roles of ULBP4-6 is limited [25,26,27]. In this study, NKG2D ligands, including MICA/B (36.5%), ULBP1 (86.5%), ULBP2 (100%), and ULBP3 (99.0%), were found to be frequently expressed in ameloblastoma cases. Furthermore, the expression of MICA/B was found to be lower in the recurrence group (8.7%) than in the recurrence-free group (45.2%, p = 0.003), whereas there was no significant difference in the expression of ULBPs (ULBP1, p = 0.504; ULBP2, not applicable; ULBP3, p = 0.240). The intensity of NKG2D ligand expression differed in MICA/B between the recurrent and recurrence-free groups, whereas no significant difference was observed in ULBPs. Our final model also demonstrated that positive MICA/B expression is an independent prognostic factor for ameloblastoma recurrence, suggesting that the expression of MICA/B is associated with a reduced risk of ameloblastoma recurrence. The association between NKG2D ligands and disease prognosis has mainly been investigated in malignant tumors, demonstrating that positive NKG2D ligand expression is associated with a favorable outcome, as observed in our study [10,11,12]. In an experiment with tissue samples from patients with hepatocellular carcinoma, Kamimura et al. showed that while the expression of ULBP1 did not influence overall survival, loss of ULBP1 expression was associated with reduced RFS (95% CI of adjusted HR: 1.537–16.261, p = 0.008) [10]. In a large-scale study involving 574 breast cancer tissue samples, high expression of NKG2D ligands was associated with advantageous clinicopathological parameters, and the presence of MICA/B (HR [95% CI]: 0.60 [0.448, 0.810], p = 0.001) and ULBP2 expression (HR [95% CI]: 0.63 [0.454, 0.869], p = 0.005) was indicative of extended recurrence-free periods in breast cancer [11]. In a study involving 462 patients with colorectal cancer, reduced MICA/B expression was associated with higher tumor grade (p = 0.037) and MICA/B expression emerged as an independent predictor associated with improved overall survival (p = 0.012) [12].

Although NKG2D ligands are typically cell surface proteins, our study observed several cases with nuclear or cytoplasmic reactivity (Fig. 1). Previous research suggests that the subcellular localization of NKG2D ligands is regulated to prevent autoimmunity (e.g., cell membrane versus cytoplasm) [28]. Another study also found that viral infection can induce the translocation of NKG2D ligands from the cell surface to other areas, such as the endoplasmic reticulum (ER) or cis-Golgi, as an immune evasion mechanism [29]. These findings indicate that NKG2D ligands can also be present in the cytoplasm as well as on the cell surface [28, 29]. We considered that if the protein is present in the rough endoplasmic reticulum (RER), it could appear as nuclear staining under the light microscope. The intracellular localization of NKG2D ligands is not yet fully understood. Consequently, the simultaneous staining patterns (cell membrane, cytoplasm, and nuclear) observed in several of our cases highlight the need for further research in this area to better understand these observations and their implications.

Obesity has been associated with unfavorable outcomes in some cancers, although the exact mechanism is unclear [30]. Specifically, higher recurrence rates have been observed in patients with breast, colorectal, prostate, and gastroesophageal cancers [30]. In this study, we analyzed the association between the recurrence of ameloblastoma and body mass index; however, we did not find any significant correlation between them.

Recent meta-analyses suggest a potential association between anesthesia and cancer recurrence and prognosis [31, 32]. Furthermore, in our previous preclinical in vitro studies, we observed that sevoflurane, a widely used inhalation anesthetic, could influence NKG2D-mediated immunosurveillance in non-small-cell lung cancer and breast cancer cell lines [25, 33]. However, in this study involving ameloblastoma patients, no significant relationship was found between the type of anesthetic agent and the recurrence rate of ameloblastoma. This may be due to the relatively short anesthetic duration of approximately one hour in the included cases or because the impact of the anesthetic agent on ameloblastoma recurrence is limited. Further evaluation is necessary to accurately assess this relationship.

The present study has several limitations. First, given the low incidence of ameloblastoma, at 0.92 per million person-years [34], this investigation was structured as a retrospective pilot study rather than full-scale prospective research. Recognizing the limitations of pilot studies, including risks of bias and over-interpretation of results, we have highlighted the need for more comprehensive and advanced full-scale research in this area. Second, due to the low preference for radical surgery in the study, surgical factors impact on ameloblastoma recurrence was not assessed. Third, the recruitment period for the study spanned over 7 years due to the low prevalence rate of ameloblastoma. This extended period may introduce bias due to the evolving surgical and anesthetic skills during this time. Fourth, as NKG2D ligand-mediated cancer immunotherapies are still in the pre-clinical stage [23], in vitro diagnostic (IVD) products for measuring NKG2D ligand expression have been validated as research use only (RUO) products, not as general purpose reagent (GPR) products, in line with USA regulatory standards [35]. Caution must be exercised in interpreting the findings of this study. Fifth, our study focused on examining the relationship between NKG2D ligand expression and recurrence-free survival, without exploring the detailed molecular mechanisms. In this study, an analysis of mRNA expression was not performed. It is important to note that the level of NKG2D ligand expression is not solely determined by its mRNA-mediated transcription and translation, but is also influenced by numerous other factors. Notably, to escape NKG2D-mediated immune surveillance, tumor cells release extracellular matrix (ECM) degrading enzymes such as matrix metalloproteinases (MMPs) or a disintegrin and metalloproteinases (ADAMs) [23]. These enzymes facilitate the shedding and removal of NKG2D ligands from the surface of tumor cells [23]. Additional research is essential to comprehensively elucidate the intricate mechanism, which includes the transcription and translation processes of NKG2D ligands and their subsequent shedding facilitated by ECM degrading enzymes. Sixth, we did not use image analysis devices for the quantification of immunohistochemistry. This decision was based on the fact that, compared to nuclear staining, image analyzers tend to be less accurate in detecting cytoplasmic staining than experienced pathologists. To address this, two oral pathologists independently interpreted the immunohistochemistry readings. Seventh, this study did not separately consider histopathological type. This is due to the current consensus that there is no correlation between histopathological type and tumor behavior or prognosis [36]. Lastly, because our study followed a retrospective design, it cannot establish definitive cause-and-effect relationships between prognostic factors and RFS in ameloblastoma.

In conclusion, our study showed that recurrent cases and loss of MICA/B expression are independent risk factors for early ameloblastoma recurrence following surgical resection. Our results suggest that diminished MICA/B expression may impair NKG2D-mediated immunosurveillance, potentially contributing to the early recurrence of ameloblastoma. Further in-depth and extensive research is required to understand the molecular mechanisms behind NKG2D ligand expression in ameloblastoma and to understand the cause-and-effect relationships between NKG2D ligand expression and ameloblastoma recurrence.