The primary role of OSCC biopsy is diagnostic but recently, there is increasing advocacy for a prognostic role for it [3, 4, 16, 17]. Only optimal (at least > 5 mm deep) biopsies can serve this role. Biopsies close to 10 mm in depth have been suggested to accurately predict the true DOI in at least 80% of OSCC patients, although surgeons consistently take biopsies < 5 mm in depth irrespective of their experience, tumor accessibility and size . Despite good accessibility, lack of obstructive structures (e.g. bone) and minimal risk of damage to important structures, the mean depth of OTSCC biopsies was still < 5 mm . Many biopsies in the present study were sufficiently wide but relatively shallow, especially those from KSA. DOI is now a clearly defined parameter as part of the pathologic T staging of OSCC by AJCC/UICC. Clinicians have even advocated estimating DOI in OSCC using bimanual palpation for clinical staging . Stromal features like TSR and TILs are better assessed in deeper biopsies where the invasive front including cancer cells growth pattern may be more clearly identified. A shallow biopsy may only be useful for the estimation of these features in superficially invasive tumors (Fig. 3).
In this study, only 62% of biopsies had determinable invasive front. There was a significant contrast between the Finnish and Saudi-Arabian biopsies in relation to detecting the invasive front with far more of invasive front observed in the Finnish samples. The value of imaging methods in evaluating the DOI has been reported, although it is possible to overestimate it by as much as 3 mm when compared with histopathological assessment [19, 20]. A combination of clinical estimation and preoperative imaging evaluation of DOI, supplemented by further biopsy evaluation will greatly increase the accuracy of the tumor staging. Notably however, in some cases, it will still not be possible to measure DOI from biopsies due to problems like tissue fragmentation, distorted plane of sectioning, and samples with only epithelial components and no connective tissue interface. Even in resection samples, technical difficulties that may hamper the precise measuring of DOI have been highlighted in some studies [21, 22]. Dhanda and colleagues  have suggested that standardization of all oral cancer biopsies using the punch technique will more likely improve the stromal to epithelial ratio in addition to being a more proficient technique than the scalpel biopsy. All the biopsies assessed here were taken by scalpel blades, mostly in a wedge form with the surface forming the base of a triangle or the larger end of a trapezium. Using this method often leaves a very small area for interface analysis if the biopsy is taken from the central parts of the tumor. To reduce this problem, the general advice is to take the biopsy at the periphery of the lesion, especially in verrucopapillary lesions, alongside an adjacent “normal” tissue. However, taking more than one specimen may seem preferable, including the most severe (avoiding necrosis), internal regions with the deepest cancer growth [23, 24]. Some of these recommendations are illustrated in Fig. 4.
The possibility of assessment of stromal histological prognostic features of OTSCC (e.g. TSR, TILs) offers an intriguing prospect in its preoperative staging and management planning . The prognostic potential of these features in OTSCC has been well-documented [10, 11]. In this study, 43% of cases included in the evaluation of stromal features were found not to be suitable for such an assessment. A lot more cases would have been found suitable if a biopsy technique that could reliably sample both epithelial and stromal tissue is instituted . Many clinicians still rely on scalpel biopsies, and it may be sufficient that the only procedural modification needed is to make deeper incisions .
An important finding in this study is that not only is it possible that digital analysis can be used for assessing the stromal features in biopsies but that its agreement with light microscopic assessment is quite significant. In the biopsies, percentage agreement for the stromal features was between 75% and 87%. All the digital measurements for this study were made by an image analyst with no prior pathology training. A pathologist was only on hand to guide on where measurements should be made. It could be argued that if a pathologist proficient in the use of digital measurement actually did the measurement, the agreement rate could be much higher. Many histopathology laboratories now have access to digital pathology which could be ultimately integrated into histopathology workflow and not only limited to research, teaching and external quality assurance practice . Ultimately using Qupath for stromal assessment could improve the preoperative tumor staging and risk assessment stratification to ensure that the most appropriate treatment is rendered, and obviate the need for several unplanned interventions thereafter. Broadly, while further refining of digital measurement is obviously needed, the conclusion is that it can be effectively used.
Regarding the comparison between biopsies and resections, only TSR showed significant agreement when the two were compared. The explanation is that TSR measurement is based on finding a single spot on the tumor slide for measurements to be taken. Conversely, TILs measurement depends on scanning the whole of the slide’s tumor invasive front to arrive at the aggregated score. Pathologists are generally familiar with variations in lymphocytic host response in different parts of a resection. TILs in biopsies may therefore not be truly representative of the real picture. It provides only a snapshot of the TILs population in the area of the tumor from which the biopsy was taken. Similarly, due to lack of uniformity all around the tumor, discordant evaluation of the WHO histological grade in biopsies and resections has been previously reported . In OSCC resections, a novel TILs digital evaluation system using WSI regional classification using a deep convolutional neural network and binary classifier of tumor-lymphocyte co-localization to estimate TILs abundance was found to be a strong prognostic indicator of disease-free survival . In general, if TILs score is high in a biopsy, it is an indication that it will be high in the resection while low value in a biopsy does not always imply low value in the resection. TSR appears not to be affected by the issues noted with TILs and therefore could possibly be added to the previously reported tumor histological factors of prognostic importance that can be evaluated in biopsies (Table 4).
In conclusion, this study showed that stromal prognostic features can be evaluated in optimal and representative OTSCC biopsies, using both light microscopy and digital measurements. There is relatively good agreement using both methods despite the small sample size available for this pilot study, and also when compared with corresponding resection specimen. Digital evaluation needs further refining and familiarity to oral and maxillofacial pathologists for easy and relatively accurate evaluation of stromal features. Finally, we suggest that clinicians should take representative deep biopsies (≥ 5 mm), and clinical pathologists should evaluate BD and TSR scores from those biopsies.