Clinical Experience with Autofluorescence Guided Oral Squamous Cell Carcinoma Surgery

1. Introduction

Oral squamous cell carcinoma is a serious and relatively frequent disease of the oral cavity and, unfortunately, also belongs to the most common malignancies in the orofacial region [1]. The estimated age-standardized rate incidence in Europe is 16.9, in the European Union 17.0, followed by the mortality of 7.1 and 6.7 respectively per 100 000 for the year 2020 [2]. The situation in the Czech Republic is comparable to the EU average (Chart 1). The surgical treatment, especially in the early stage of the disease, has the best curative results [3]. The radicality of the procedure is crucial for the prognosis and the treatment success [4,5,6,7,8,9,10,11]. It is essential to determine the area of a tissue affected by tumor cells from the healthy one during the surgery to ensure the radicality.

New effective examination and imaging techniques are being developed that allow the surgeon to better visualize the boundaries of the primary tumor before and during the surgery more precisely [12,13,14,15,16,17,18,19,20,21,22,23,24]. With the help of these techniques, it is possible, among other things, to have a positive impact on margin surveillance. An ideal investigative technique for this purpose should meet certain characteristics. High sensitivity and specificity while maintaining complete non-invasiveness should be the essence. Furthermore, it is important that this examination is feasible intraoperatively, achieves stable results, is reproducible, quick and simple, as well as economically sustainable and applicable in a wide range of practice. Optical methods fulfill most of these properties, but so far, they themselves are burdened with a number of shortcomings [25]. We assumed that the use of the selected optical examination method, despite its shortcomings, would bring improvement in the treatment results of our patients suffering from OSCC in our study. For further investigation, the direct autofluorescence method was chosen.

Direct autofluorescence is a technique used for screening or better determination of potentially malignant changes of oral mucosa. In 2006, thanks to extensive research efforts, the VELscope system was registered in Canada and also certified by the FDA in the United States. Similar to some other systems working on the principle of natural autofluorescence (Identafi 3000, Sapphire Plus Lesion Detection), the VELScope device uses a non-invasive method of examination using a handpiece emitting bright blue light (400 - 460 nm), enabling direct visualization of the oral mucosa in real time. This illumination leads to the excitation of endogenous mucosal and submucosal fluorophores [26], which emit a green light, that could be registered through the semipermeable handpiece filter. The visible loss of physiologic fluorescence signifies dysplastic changes of the epithelium, but could be seen also in hyperemia, traumatization, hyperkeratosis and other benign changes, that lower the specificity [27,28,29,30]. We evaluate the hypothesis, that the use of direct autofluorescence (VELscope system tumor mucosal surrounding examination) is enhancing the ability to reach the pathologically free margin (pFM) in our study.

In order to ensure global comparability and to clearly declare what evaluation criteria were used, it is necessary to properly define the quality of the resection margin. Despite the prevailing belief that leaving part of the tumor cells in the patient's body is the most common reason for OSCC treatment failure, there is still no clear definition of an adequate resection margin [31,32]. Resection margins are mostly classified as either positive (pPM), that means tumor "cut- through", close (pCM), or negative (pFM), with different definitions of the healthy tissue rim range [33]. The distance between the tumor border and surgical margin to gain the pFM vary in some studies [34] [11]. According to International Collaboration on Cancer Reporting (ICCR) the definition of a resection margin >5 mm is clear and 1-5 mm is close and <1 mm is positive [11]. Similar to this definition is a statement of the National Comprehensive Cancer Network (NCCN), where pFM is 5 mm or more from the invasive tumor front, pCM is defined as a distance from the invasive tumor front to the resected margin that is less than 5 mm and a pPM means a carcinoma in situ or an invasive carcinoma at the margin of resection [35]. Some role in this uncertainty plays probably shrinkage of the histological specimen. There are some studies to address margin shrinkage in patients with head and neck cancer, which was on the order of 20 % to 25 % [36,37]. In accordance with established international practice, the ICCR model was used to assess the condition of surgical margins.

2. Materials and Methods

This pilot retrospective randomized study was conducted at the Department of Stomatology, University Hospital Pilsen, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic in cooperation with the Sikl´s Department of Pathology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic. For better objectivity the whole study design, data processing and the results emerged from the study were discussed with the European front specialists from the University Clinic of Oral and Maxillofacial Surgery, Medical University of Innsbruck, Innsbruck, Austria. We collected two groups (a study group and a random control group) comprising a total number of 122 patients cured in the period of the years 2016 – 2022 at our department. The inclusion criteria of the study group were as follows: age over 18 years, histologically verified oral squamous cell carcinoma with no sign of inflammation or traumatization of the surrounding mucosa and no previous surgery (except for a small primary biopsy to confirm the diagnosis), radiotherapy or chemotherapy for head and neck cancer, signed informed consent, indication for primary surgical treatment and tumor localization at oral anatomical sites that can be directly visualized using both white light and fluorescence visualization device (this includes ICD-10 site codes: C02.0-C06.9)[38] and VELScope examination before surgery. Other parameters taken into account were tumor site, sex, age, TNM classification and grade according to the Union for International Cancer Control (UICC). The inclusion criteria of the control group were the same except for the VELscope examination. Simple randomization was used for the distribution of the patients into mentioned groups. All of the patients underwent standard preoperative examination including staging based on clinical examination and imaging (ultrasonography, computed tomography, magnetic resonance or hybrid positron emission tomography). The patients signed a detailed informed consent form, with the privacy policy agreement. The design of this study was approved by the Committee of Ethics in Research of Department of Stomatology, University Hospital Pilsen, Faculty of Medicine in Pilsen, Charles University under the code 333/2020. This study was conducted according to the Declaration of Helsinki.

Preoperative evaluation of tumor margins with the help of VELscope device (model No. V1, LED Dental, Inc., 201-15047 Marina Driva White Rock, BC V4B IC5, Canada) was then provided by an experienced surgeon trained and calibrated with the VELscope system in each patient of the study group. It is recommended to provide this examination in a dark room to avoid other illumination interference and to gain the best contrast of examined field. During this procedure, a field of loss of autofluorescence was marked by permanent (tattoo) or transient (gentian violet - directly before surgery) staining, or marked by monopolar electrocoagulation device directly at the beginning of the surgery in general anesthesia (marking modality was chosen according to the patient compliance and surgeons’ preference in each case), and the discrepancy between the mark and day-light visible tumor boundary was measured (Figure 1).

In an attempt to shorten the surgical time and thus also the time of general anesthesia for the patient, we developed a technique for permanent tattoo marking, which can be done even a few days before the operation. An insulin syringe was filled with a small amount of the conventional tattoo dye and a little superficial mucosal scratch by the syringe needle was directly stained by the chosen dye. For instant specimen orientation and better cooperation with the pathologist different colors for each specimen site were used (Figure 2). The procedure was provided under topical anesthesia (lidocaine spray 10 g / 100 ml) for higher comfort for the patient. The interference between the marking and the histological margin examination was avoided by the rim of tissue excised behind the marks, as descripted subsequently. None of the patients had a peri- or post procedural painful perception, we noticed no health complications or negative tumor site affection by this procedure.

Cold steel or a gentle high-frequency electro surgery excision at least 3 mm behind the marks in the study group was performed (in the control group, the excision was provided according to the international conventional recommendation at least 10 mm behind the naked-eye visible tumor boundary) and the specimen was sent to the dedicated pathologist for histological examination at the Sikl´s Department of Pathology. After hematoxylin-eosin staining and immunohistochemical examination, typing, grading, perineural and intravascular invasion and the tumor to margin distance were evaluated. The histological outcome, VELscope findings and other data were processed by a professional statistician. The main emphasis was placed on evaluating the increase in the extent of visibility of tumor changes when comparing white light versus VELscope and, above all, on the differences in the quality of the resection margin of both groups from a histological point of view. As mentioned before, the ICCR model was used for evaluating the status of surgical margins.

3. Results

3.1. Groups characteristics

Gender distribution was 40 males and 21 females in the study group and 36 males and 25 females in the control group suffering from OSCC (Table 1).

The mean age in the study group was 65.3 years with a range from 37 to 90 years of age and 64.3 years with a range from 34 to 88 years in the control group respectively (Chart 2).

The most affected site of the oral cavity was the tongue in both groups (Table 2).

The stage and grade status of all tumors were evaluated and the situation in both groups is presented in appropriate tables (Table 3, 4).

Statistical analysis did not reveal any significant difference between the two groups in terms of the features described above.

3.2. Treatment outcomes comparison

• The loss of physiologic fluorescence resulted in a resection enlargement of 4.68 mm in average (1 – 12 mm) compared to the polychromatic light and the tactile tumor borders assessment.
• The histological examination revealed no case of pPM (n = 0; 0 %), six cases of pCM (n = 6; 9.84 %) and fifty-five specimens were pFM (n = 55; 90.16 %) in the mucosal margins in the study group. The situation in the control group was as follows: pPM (n = 7; 11.48 %), pCM (n = 14; 22.95 %), pFM (n = 40; 65.57 %) (Chart 3). Although the autofluorescence technique has no influence on the deep surgical margins, we present the deep margin situation for comprehensiveness – in the study group we encountered pPM in eight cases (n = 8; 13.11 %), pCM in ten cases (n = 10; 16.39 %) and pFM in forty-three cases (n = 43; 70.49 %), in the control group situation is as follows: pPM (9; 14.75 %), pCM (6; 9.84 %), pFM (46; 75.41 %) (Chart 4).

3.3. Observed effect of autofluorescence assistance

• As expected, our study proved a statistically significant difference between the two groups in the mucosal layer only (see table 5). There was no significant difference in deep margins. Despite of the larger resection in the study group, there was no significant increase in postoperative morbidity with regard to either swallowing or speech. No patient experienced major complications in the postoperative period and no patient was discharged with a feeding tube or tracheostomy. The overall treatment outcome in terms of surgical tumor eradication was significantly better in the study group compared to the control group.

Table 5. Comparison of mucosal margin status in both groups.

Margin status	Frequency – amount / percentil		Total
	Study group	Control group
MpFM	55 / 90.16	40 / 65.57	54
MpCM	6 / 9.84	14 / 22.95	56
MpPM	0 / 0.00	7 / 11.48	12
Total	61	61	122

Table 5. Comparison of mucosal margin status in both groups.

Margin status	Frequency – amount / percentil		Total
	Study group	Control group
MpFM	55 / 90.16	40 / 65.57	54
MpCM	6 / 9.84	14 / 22.95	56
MpPM	0 / 0.00	7 / 11.48	12
Total	61	61	122

4. Discussion

A multimodal treatment with an accent on radical surgical tumor excision has the best curative results [39,40]. Early detection, prompt staging and individual setting of the required treatment plan can lower the risk of curative failure, recurrence and enormous impairment of the quality of life of the patient [4]. New examination techniques, as well as the modern surgical approaches, came into the clinical practice thanks to the scientific progress [41]. One of the modern techniques described in our study – the mucosal autofluorescence – has the potential to help clinicians to early detect a mucosal malignity in the oral cavity and in addition to determine the tumor boundaries more precisely [42,43,44,45]. The surgeon’s main goal in the surgical treatment of OSCC is to achieve the R0 resection, which means ideally the pFM [40]. The presence of residual tumor cells is convicted to be the most important prognostic factor. The high number of studies comparing local recurrence rate with margin status in OSCC found a strong correlation, although the absolute number of local recurrences and the criteria used to define positive margins vary significantly among the particular studies [46,47,48]. Some studies surprisingly failed to demonstrate any correlation between the recurrence and margin status [49]. It is still not clear, how wide the distance between surgical margin and tumor should be. It is crucial to obtain adequate surgical margins of the tumor, however, the surgeon must find a balance between the radicality of the operation and the effort to preserve the function of the orofacial system and thus the quality of life and limit the cosmetic disability [3,50,51,52,53,54,55,56]. Many scientific publications deal with the problem of positive resection margins, unfortunately there are currently no globally valid guidelines. The setting of a minimum safety margin is discussed in a number of studies and the range varies between 10 – 2 mm, the median recommended clinical distance for the resection of OSCC is 5 mm, although a clear basis for this distance is currently lacking. Mainly because of the specimen shrinkage and “invisible” carcinoma cells spread, the excised rim of macroscopically unchanged mucosa should be more than the mentioned pFM distance. It is recommended to keep at least 10 mm distance from the tumor to avoid impairing the histological margin status during the surgery [57]. The intraoperative evaluation of resection margins can be made by several methods. The use of frozen sections (FS) used to be the gold standard, it was accepted worldwide but, as underlined by Yahalom et al. [58], the procedure is not standardized. The issue of discussion is also the site where the FS should be taken if it is the resection bed or the specimen margin [59]. The other problem is the risk of incorrect identifying of the area to be re-excised in the event of positive margins [60] and time consumption during surgery. For this reason, the endeavor of finding a more favorable method for intraoperative determination of adequate resection margins is very topical and research is very dynamic in this regard. There are some more methods, that have been suggested for better achieving pFM in mucosal layer OSCC surgical treatment or oral mucosa malignity detection and we present a list of some of them for comparison below:

Contact endoscopy - allows an in vivo microscopic examination of upper aerodigestive tract mucosa with a rigid endoscope. It is a noninvasive technique and provides information on a microscopic diagnosis and lesion margins. A sensitivity of 80 %, a specificity of 100 % and an accuracy of 93 % for contact endoscopy in the diagnosis of malignancy is reported [61,62,63].

Narrow band imaging is a video endoscopic system for the examination of mucous membranes. Thanks to narrow band filters, only two specific bands of visible light, which are typical for the absorption peak of hemoglobin, are allowed to pass through. The observed wavelengths increase the visibility of microvascular abnormalities that could be related to preneoplastic and neoplastic mucosal changes [64,65,66,67,68].

Staining with Lugol’s iodine solution or toluidine blue is one of the methods, that can reduce the number of positive margins by pointing up the tumor margins [69,70,71,72,73]. Vital staining utilizes the enhanced affinity of some dyes to certain cell structures present in dysplastic or malignant cells, making them more apparent. The widest use has the toluidine blue in the oral cavity mucosa. This basic metachromatic stain has an affinity toward DNA and RNA and demonstrates an invasive malignancy, carcinoma in situ (CIS) and dysplasia by staining abnormal tissues blue [73,74].

Using touch imprint cytology, the properties of the cells located on the surface of the resection margin can be evaluated intraoperatively. It is a fast, simple, cheap and relatively accurate examination technique [75].

Optical coherence tomography is another technique for distinguishing between positive and negative surgical margins [76,77]

Some publications mention a molecular definition of surgical margins, from protein markers to DNA-based techniques, that can evaluate the margins at the subcellular level and can explain, for example, the local recurrence in optical microscope pFM [78,79,80]. Various monoclonal antibodies and other ligands are already being clinically used or researched in this area, which have the ability to mark the structures of tumor cells in a certain way and make them visible intraoperatively, for example by means of fluorescence [12,13,17,18].

The last-mentioned technique is a tissue reflectance. The ViziLite device, which uses reflectance is adapted for use in the oral cavity. The principle of this method is, that the abnormal cells reflect the light (high nucleus - cytoplasm ratio, keratinization excess, hyperparakeratinization), while normal cells absorb the light and are depicted in a bluish color [30].

We verified the potential of autofluorescence to detect the dysplastic and malignant mucosal changes in our study. The limitations of this technique and the methodology of our study is that the detection capability stays only on the superficial mucosal layer. In our study, we identified some possible sources of bias that need to be taken into account. A frequent weakness of clinical studies is that the compared groups are not identical, but statistically very similar, which is also the case in our study. Another problem is the burden of the natural autofluorescence method with a relatively low specificity despite a very high sensitivity. We partially eliminated this weakness by the fact that the histological nature of the disease was known and further by using the set inclusion parameters. According to published studies, the other presented optical examination techniques also achieve promising results, but there is a lack of scientific works relevantly comparing individual methods with each other. Further research is needed in this area. The biggest limitation of autofluorescence in the way of use we are investigating is the limitation to the mucosal layer. In this regard, it would be appropriate to combine the method with another similarly non-burdensome and simple examination technique that detects deep tumor margins. Such a technique could be, for example, the adjuvant perioperative use of ultrasonography, which also achieves very good results [19,20,21]. In this sphere, we consider further research to be extremely interesting, as the potential of both methods could be exponentially enhanced. On the other hand, we developed a technique of preoperative marking of the pathological mucosal changes by a permanent tattoo, which can help the surgeon to set the resection line more precisely. We gain a better specimen orientation, which leads to surgical treatment success enhancement, more precise orientation for better pathologist communication and better identification of risk field in case of reexcision need by this technique.

The potential of this technique for better detection of mucosal malignancy is well known and have been multiple published. The role of autofluorescence in setting the margins into the R0 region or even better in achieving the pFM is being worldwide researched. The results of some studies are promising, but it is hard to compare the results because of its nonhomogenity. Some presented works are limited to the early stage of OSCC only, or do not depict the resection margin changes clearly [44,45,81,82,83]. The further investigation in this field is needed to assess the potential of this technique and to create a recommendation for the standardized use of autofluorescence in OSCC surgery in the sense of a guideline.

5. Conclusions

Our findings support the hypothesis of improving the surgery outcomes in the study group, we have discovered the statistically significant difference between the study and the control group. We proved the efficacy of mucosal autofluorescence in setting the sufficient mucosal surgical margins in our study. If combined with the permanent tattoo marking of the pathological changes visible under VELscope as described above, this technique can enhance the surgeon´s ability to successfully treat the patient with OSCC even more.