Surgery remains the cornerstone of treatment for oral malignancies, with the primary objective being complete removal of the tumor along with a sufficient margin of healthy tissue to reduce the risk of local and regional recurrence1. Surgical margins are critical in determining prognosis; margins containing tumor cells within 1 mm of the edge are considered positive, margins less than 5 mm are defined as close, and margins with at least 5 mm of healthy tissue are considered negative.2 Despite careful surgical planning, studies show that a substantial proportion of resection margins are inadequate, with rates as high as 85% for close or positive margins and up to 40% for positive margins. The deep, submucosal margin is most frequently involved. Inadequate margins are clinically significant as they strongly correlate with higher rates of local recurrence and negatively affect disease-specific survival3. Patients with positive margins often require additional interventions, including repeat surgery or adjuvant therapies such as radiotherapy or chemoradiotherapy. These additional treatments are associated with increased morbidity, extended recovery times, higher resource utilization, and elevated healthcare costs, highlighting the importance of precise margin assessment during the initial procedure.4Currently, the most commonly used intraoperative method to assess surgical margins is frozen section analysis. While this technique has been in use for decades, it has several limitations. Only a fraction of the resection specimen is analyzed, which introduces sampling error and results in a sensitivity of approximately 50%. Consequently, a notable percentage of cases initially deemed margin-negative may be reported as positive on final histopathology.5 Even when positive margins are identified intraoperatively, the success of revising the margins to achieve true negative status is limited, with studies showing successful intraoperative reresection in only about 58% of cases.6,7 Furthermore, achieving negative margins through intraoperative revision does not necessarily confer the same prognostic benefit as achieving negative margins during the initial resection. These limitations underscore the need for more reliable methods of real-time surgical guidance to improve patient outcomes.Fluorescence molecular imaging (FMI) has emerged as a promising approach to address these challenges. This technique involves the use of tumor-specific fluorescent tracers to enhance visualization of malignant tissue, allowing surgeons to identify tumor boundaries that are not apparent under conventional white-light imaging. A common target for these tracers is the epidermal growth factor receptor (EGFR),8 which is frequently overexpressed in oral malignancies. Early-phase clinical studies have demonstrated that EGFR-targeted fluorescent tracers are safe and can provide real-time intraoperative visualization of tumor tissue, including areas at the deep surgical margin that are prone to incomplete resection. Pre-dosing with unlabeled antibody prior to administration of the tracer has been shown to improve imaging contrast, likely by reducing rapid plasma clearance and minimizing non-specific binding to normal tissues.9 Current evidence suggests that this approach could potentially overcome the limitations of frozen section analysis by providing a more comprehensive and rapid assessment of the resection site, enabling immediate correction of inadequate margins. By allowing surgeons to achieve negative margins during the primary procedure, FMI has the potential to reduce the need for secondary surgeries and adjuvant treatments, thereby minimizing patient morbidity and optimizing resource utilization.10,11 Overall, the development and implementation of FMI could significantly enhance surgical precision and improve long-term oncological outcomes in patients undergoing resection for oral malignancies.This technology represents a shift toward more personalized and precise surgical management, providing real-time feedback to the operating surgeon and potentially transforming the standard of care for margin assessment in oral cancer surgery.12,13 Its integration into routine clinical practice could ultimately lead to lower recurrence rates, improved survival, and reduced treatment-related complications.

AIM

To assess the feasibility and accuracy of EGFR-targeted fluorescence molecular imaging for real-time intraoperative margin evaluation in oral cancer surgery.

This prospective observational study included 105 patients diagnosed with primary oral cancer and scheduled for curative surgical resection and carried out in the department ofbiochemistry, Govt. Medical college, Kota. The total duration of the study was 1 yr from may 2023 to April 2024. Patients aged 18 years and above with histopathologically confirmed oral cancer, ECOG performance status 0–2, and who provided written informed consent were included. Exclusion criteria included known allergy to the EGFR-targeting tracer, pregnancy or lactation, and severe comorbidities precluding surgery. Preoperative evaluation included a detailed history and physical examination, imaging studies (CT, MRI, or PET-CT) to assess tumor size and nodal involvement, routine laboratory investigations, and confirmatory tumor biopsy.

Table 1:Age-wise Distribution of Patients

In this study, the majority of patients were in the 51–60 years age group, accounting for 38% of the study population. Patients aged >60 years and 41–50 years contributed equally with 24% each, while only 14% were below 40 years.

Table 2:Sex-wise Distribution of Patients

In this study, males constituted the majority with 62% of the cases, while females accounted for 38%. This shows a male predominance in the patient population.

Table 3:Site-wise Distribution of Tumors

The most common tumor site observed was the tongue, accounting for 33% of cases, followed by the buccal mucosa/cheek at 24%. Mandibular gingiva (17%), floor of mouth (15%), and maxillary gingiva (11%) were less frequently involved. This distribution highlights the tongue as the predominant site for tumors in the study population.

Table 4:Tumor Stage and Lymph Node Status of Patients

Most patients in this study presented with T2 tumors (43%), followed by T3 (29%), T1 (19%), and T4 (9%). Regarding lymph node status, the majority were N0 (52%), while N1 and N2 nodes were involved in 33% and 15% of patients, respectively.

Table 5:Tumor Site-wise positive Margin Analysis and FMI Detection

The study assessed tumor margins in 105 patients across various sites, with a total of 540 margins evaluated. Fluorescence molecular imaging (FMI) detected all 68 positive margins, achieving 100% detection across tongue, buccal mucosa, mandibular and maxillary gingiva, and floor of mouth tumors.

Table 6:Tumor Site-wise Negative Margin Analysis and FMI Detection

Out of 540 tumor margins evaluated in 105 patients, 169 were negative, with fluorescence molecular imaging (FMI) detecting 113 of them, achieving an overall detection rate of 66.8%. Detection varied across sites, with the tongue and buccal mucosa showing the highest number of negative margins identified by FMI.

Table 7:Tumor Site-wise Close Margin Analysis and FMI Accuracy

Out of 540 tumor margins evaluated in 105 patients, 303 were classified as close margins, with fluorescence molecular imaging correctly identifying 221 of them, achieving an overall accuracy of 73%. Detection varied across tumor sites, with the tongue and buccal mucosa showing the highest number of correctly identified close margins

The age-wise distribution of patients in this study shows that the maximum proportion belonged to the 51–60 years group, making up 38% of the total. Patients aged above 60 years and those between 41–50 years were equal in proportion, each contributing 24%. Younger patients, below 40 years of age, formed the smallest group with only 14%. This indicates that middle-aged and elderly individuals were more frequently affected. The findings suggest a higher disease burden in the 5th and 6th decades of life.

The sex-wise distribution of patients in this study revealed a male predominance. Out of the total cases, 62% were males, while females constituted 38%. This indicates that the condition was more commonly observed in men compared to women. The difference highlights possible gender-related risk factors or exposure differences. Such findings are important for understanding demographic variations in disease occurrence.

The site-wise distribution of tumors in this study showed that the tongue was the most commonly affected site, accounting for 33% of cases. The buccal mucosa or cheek followed with 24% of patients. Tumors of the mandibular gingiva and floor of mouth were seen in 17% and 15% of cases, respectively. The maxillary gingiva was the least involved site, contributing 11% of cases. Overall, the findings indicate that the tongue is the predominant site for tumor occurrence in this patient population.

The tumor stage and lymph node status of patients in this study showed that most tumors were diagnosed at the T2 stage, accounting for 43% of cases, followed by T3 tumors at 29%. Early-stage T1 tumors were seen in 19% of patients, while advanced T4 tumors were the least common at 9%. Analysis of lymph node involvement revealed that the majority of patients (52%) had no regional lymph node metastasis (N0). N1 involvement was observed in 33% of cases, and N2 in 15%, indicating progressive nodal spread in a smaller proportion. These findings suggest that while many patients present with moderately advanced tumors, over half still have no lymph node involvement. Understanding tumor stage and nodal status is crucial for treatment planning and prognostication.Similarly,In a study by de Wit, J.G., Vonk, J. et al¹⁴ the median age of patients was 68 years (range 29–90) and 33 were female (51%). Primary tumors were located in the tongue (n = 30), mandibular gingiva (n = 19), maxillary gingiva (n = 6), floor of mouth (n = 5), cheek (n = 4), buccal fold (n = 1), and glossotonsillar sulcus (n = 1). Final histopathology showed 14 tumor-positive margins, 37 close margins, and 71 tumor-negative margins

The margin analysis across different tumor sites in this study involved a total of 540 margins from 105 patients. The tongue was the most common site with 175 margins evaluated, of which 22 were positive, all detected by fluorescence molecular imaging (FMI). Buccal mucosa/cheek tumors had 125 margins with 16 positive, and mandibular gingiva tumors had 99 margins with 13 positive, all accurately detected by FMI. Maxillary gingiva tumors had 66 margins with 8 positive, while floor of mouth tumors had 9 positive margins out of 77 evaluated. Overall, FMI achieved 100% detection of all 68 positive margins across all tumor sites. These findings highlight the effectiveness of FMI in intraoperative identification of positive tumor margins, ensuring precise surgical excision.

The analysis of negative tumor margins in this study included 540 margins from 105 patients. Of these, 169 margins were negative, and fluorescence molecular imaging (FMI) detected 113, corresponding to an overall detection rate of 66.8%. The tongue had 55 negative margins, of which 37 were identified by FMI, while the buccal mucosa/cheek had 39 negative margins with 26 detected. Mandibular gingiva and maxillary gingiva showed 31 and 21 negative margins, with 21 and 14 detected by FMI, respectively. The floor of mouth had only 2 negative margins, with 3 detected, indicating some variation in detection efficiency. Overall, FMI demonstrated good, though not complete, sensitivity in identifying negative margins across all tumor sites.

The study analyzed 303 close tumor margins among a total of 540 margins from 105 patients. Fluorescence molecular imaging (FMI) correctly identified 221 of these close margins, achieving an overall accuracy of 73%. The tongue had the highest number of close margins at 98, with 72 correctly detected by FMI. Buccal mucosa/cheek tumors had 70 close margins, of which 51 were accurately identified. Mandibular and maxillary gingiva showed 55 and 37 close margins, with 40 and 27 correctly detected, respectively. The floor of mouth had 43 close margins, with 31 identified by FMI, demonstrating its overall effectiveness in detecting close margins across tumorsites.In a study by de Wit, J.G., Vonk, J. et al¹⁴ assessing 66 surgical specimens, 113 surgical planes were evaluated within minutes after excision, similar to our assessment of 105 patients and 540 margins. Fluorescent molecular imaging identified all 14 tumor-positive margins, achieving 100% sensitivity, while 16 of 37 close margins and 10 of 73 negative margins had detectable fluorescence. An SBR ≥2 was established as the optimal cut-off for tumor-positive margin detection, yielding a specificity of 85.9% and a negative predictive value of 100%. Detection of close margins showed lower sensitivity at 43.2% but maintained high specificity (85.9%). These findings are comparable to our study, where FMI achieved 100% detection of positive margins and 66.8% detection of negative margins, with 73% accuracy for close margins. Overall, both studies highlight the effectiveness of fluorescence imaging in intraoperative margin assessment, particularly for tumor-positive margins.

The study demonstrates that oral cancer predominantly affects middle-aged and elderly patients, with a male predominance and the tongue being the most common tumor site. Most tumors were moderately advanced (T2–T3), with over half of the patients showing no lymph node involvement. Fluorescence molecular imaging (FMI) proved highly effective in intraoperative assessment, achieving 100% detection of tumor-positive margins, 66.8% detection of negative margins, and 73% accuracy for close margins. These results indicate that FMI can significantly enhance the precision of surgical excision, reduce the risk of residual disease, and aid in better treatment planning. Overall, the study confirms the clinical utility of FMI in improving surgical outcomes in oral cancer.

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