Image-guided surgery in oral cancer: toward improved margin control.

PURPOSE OF REVIEW: The aim of this review is to discuss recent studies on the assessment of tumor extension and resection margins by different intraoperative techniques allowing for image-guided surgery of oral cancer. RECENT FINDINGS: There are different in-vivo and ex-vivo intraoperative techniques to improve margin control of which intraoperative ultrasound and targeted fluorescence-guided resections have high potential clinical value and are closest to clinical implementation. SUMMARY: In oral cancer surgery, resection margins, particularly deep margins, are often inadequate. Intraoperative frozen section does not improve resection margin control sufficiently. Specimen-driven intraoperative assessment for gross analysis of suspected margins reduces the amount of positive resection margins substantially but leaves still room for improvement. Mucosal staining methods, optical coherence tomography and narrow band imaging can only be used for superficial (mucosal) resection margin control. Spectroscopy is under investigation, but clinical data are scarce. Intraoperative ex-vivo imaging of the resection specimen by magnetic resonance and PET/computed tomography may be used to assess resection margins but needs more research. Intraoperative in-vivo ad ex-vivo ultrasound and targeted fluorescence imaging have high potential clinical value to guide oral cancer resections and are closest to clinical implementation for improved margin control.

MET ectodomain shedding is associated with poor disease-free survival of patients diagnosed with oral squamous cell carcinoma.

Ectodomain shedding unleashes the aggressive nature of the MET oncogene product. Using specific C- and N-terminal MET antibodies (D1C2 and A2H2-3), MET protein status (i.e., no MET, decoy MET, transmembranous C-terminal MET with or without the ectodomain) was investigated in oral squamous cell carcinoma. For the cancers showing transmembranous C-terminal MET, the impact of ectodomain shedding on prognosis was investigated. To examine ectodomain shedding, reduced lysates of oral squamous cell carcinoma cell lines were immunoblotted using D1C2 and an ELISA was performed on culture media using A2H2-3. In addition, reduced lysates of fresh frozen tissues of 30 oral squamous cell carcinoma were immunoblotted using D1C2 and immunohistochemistry was performed on corresponding formalin-fixed paraffin-embedded tissues using both antibodies on parallel sections. To examine MET protein status, differences between membranous D1C2 and A2H2-3 immunoreactivities were scored using parallel tissue microarray sections representing 156 oral squamous cell carcinoma. The prognostic value of ectodomain shedding was examined using Cox regression analysis for disease-free survival and overall survival. Ectodomain shedding was observed in all cell lines, 43% (n = 13) of fresh frozen and 50% (n = 15) of formalin-fixed paraffin-embedded cancers (27% overlap, n = 8). The tissue microarray showed no MET in 23% (n = 36), decoy MET in 9% (n = 14), and transmembranous C-terminal MET in 68% (n = 106) of examined cancers. Within the latter group, ectodomain shedding occurs in 36% (n = 38) of the cases and is independently associated with poor disease-free survival (HR = 2.41; 95% CI, 1.35-4.30 and P = 0.003)-though not overall survival (HR = 1.64; 95% CI, 0.92-2.94 and P = 0.095)-after correcting for factors known to influence survival. In conclusion, MET ectodomain shedding occurs in transmembranous C-terminal MET positive oral squamous cell carcinoma and is independently associated with disease-free survival. These findings might aid in designing companion diagnostics for targeted therapies directed against MET.

Tumor classification with MALDI-MSI data of tissue microarrays: A case study.

With mass spectrometry imaging (MSI) on tissue microarrays (TMAs) a large number of biomolecules can be studied for many patients at the same time, making it an attractive tool for biomarker discovery. Here we investigate whether lymph node metastasis can be predicted from MALDI-MSI data. Measurements are performed on TMAs and then filtered based on spectral intensity and the percentage of tumor cells, after which the resulting data for 122 patients is further preprocessed. We assume differences between patients with and without metastasis are expressed in a limited number of features. Two univariate feature selection methods are applied to reduce the dimensionality of the MALDI-MSI data. The selected features are then used in combination with three classifiers. The best classification scores are obtained with a decision tree classifier, which classifies about 72% of patients correctly. Almost all the predictive power comes from a single peak (m/z 718.4). The sensitivity of our classification approach, which can be generically used to search for biomarkers, is investigated using artificially modified data.

Liquid Biopsy: A Future Tool for Posttreatment Surveillance in Head and Neck Cancer?

The prognosis of head and neck squamous cell carcinoma (HNSCC) is largely based on disease stage. Despite improvements in treatment, recurrence rates are still considered high. Currently, disease progression or regression after curative treatment is monitored by clinical evaluation combined with flexible endoscopy and/or imaging. However, specificity of imaging is low due to the posttreatment effects. Detection of circulating tumor DNA (ctDNA) from blood samples of HNSCC patients is a minimally invasive technique that could lead to an earlier detection of recurrence. In addition, digital droplet PCR (ddPCR) could be used to sensitively detect these mutational targets. Future study on ctDNA using ddPCR in blood samples of HNSCC patients is recommended during the follow-up stage to detect recurrences in a timely manner.

Oropharyngeal squamous cell carcinomas differentially express granzyme inhibitors.

OBJECTIVES: Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCCs) have an improved prognosis compared to HPV-negative OPSCCs. Several theories have been proposed to explain this relatively good prognosis. One hypothesis is a difference in immune response. In this study, we compared tumor-infiltrating CD3+, CD4+, CD8+ T-cells, and granzyme inhibitors (SERPINB1, SERPINB4, and SERPINB9) between HPV-positive and HPV-negative tumors and the relation with survival. METHODS: Protein expression of tumor-infiltrating lymphocytes (TILs) (CD3, CD4, and CD8) and granzyme inhibitors was analyzed in 262 OPSCCs by immunohistochemistry (IHC). Most patients (67%) received primary radiotherapy with or without chemotherapy. Cox regression analysis was carried out to compare overall survival (OS) of patients with low and high TIL infiltration and expression of granzyme inhibitors. RESULTS: HPV-positive OPSCCs were significantly more heavily infiltrated by TILs (p < 0.001) compared to HPV-negative OPSCCs. A high level of CD3+ TILs was correlated with a favorable outcome in the total cohort and in HPV-positive OPSCCs, while it reached no significance in HPV-negative OPSCCs. There was expression of all three granzyme inhibitors in OPSCCs. No differences in expression were found between HPV-positive and HPV-negative OPSCCs. Within the group of HPV-positive tumors, a high expression of SERPINB1 was associated with a significantly worse overall survival. CONCLUSION: HPV-positive OPSCCs with a low count of CD3+ TILs or high expression of SERPINB1 have a worse OS, comparable with HPV-negative OPSCCs. This suggests that the immune system plays an important role in the carcinogenesis of the virally induced oropharynx tumors.

Beneath the surface: A systematic review on intraoperative imaging techniques for deep margin assessment in oral squamous cell carcinoma.

Resection margins of oral squamous cell carcinoma (SCC) are often inadequate. A systematic review on clinical intraoperative whole-specimen imaging techniques to obtain adequate deep resection margins in oral SCC is lacking. Such a review may render better alternatives for the current insufficient intraoperative techniques: palpation and frozen section analyses (FSA). This review resulted in ten publications investigating ultrasound (US), four investigating fluorescence, and three investigating MRI. Both US and fluorescence were able to image the tumor intraorally and perform ex-vivo imaging of the resection specimen. Fluorescence was also able to image residual tumor tissue in the wound bed. MRI could only be used on the ex-vivo specimen. The 95 % confidence intervals for sensitivity and specificity were large, due to the small sample sizes for all three techniques. The sensitivity and specificity of US for identifying < 5 mm margins ranged from 0 % to 100 % and 60 % to 100 %, respectively. For fluorescence, this ranged from 0 % to 100 % and 76 % to 100 %, respectively. For MRI, this ranged from 7 % to 100 % and 81 % to 100 %, respectively. US, MRI and fluorescence are the currently available imaging techniques that can potentially be used intraoperatively and which can image the entire tumor-free margin, although they have insufficient sensitivity for identifying < 5 mm margins. Further research on larger cohorts is needed to improve the sensitivity by determining cut-off points on imaging for inadequate margins. This improves the number of adequate resections of oral SCC's and pave the way for routine clinical implementation of these techniques.

Intraoperative Techniques That Define the Mucosal Margins of Oral Cancer In-Vivo: A Systematic Review.

BACKGROUND: This systematic review investigates techniques for determining adequate mucosal margins during the resection of oral squamous cell carcinoma (SCC). The primary treatment involves surgical removal with ≥5 mm margins, highlighting the importance of accurate differentiation between SCC and dysplasia during surgery. METHODS: A comprehensive Embase and PubMed literature search was performed. Studies underwent quality assessment using QUADAS-2. RESULTS: After the full-text screening and exclusion of studies exhibiting high bias, eight studies were included, focusing on three margin visualization techniques: autofluorescence, iodine staining, and narrow-band imaging (NBI). Negative predictive value (NPV) was calculable across the studies, though reference standards varied. Results indicated NPVs for autofluorescence, iodine, and NBI ranging from 61% to 100%, 92% to 99%, and 86% to 100%, respectively. Autofluorescence did not significantly enhance margins compared to white light-guided surgery, while iodine staining demonstrated improvement for mild or moderate dysplasia. NBI lacked comparison with a white light-guided surgery cohort. CONCLUSIONS: We recommend studying and comparing the diagnostic accuracy of iodine staining and NBI in larger cohorts of patients with oral SCC, focusing on discriminating between SCC and (severe) dysplasia. Furthermore, we advise reporting the diagnostic accuracy alongside the treatment effects to improve the assessment of these techniques.

Feasibility of an MR-based digital specimen for tongue cancer resection specimens: a novel approach for margin evaluation.

Objective: This study explores the feasibility of ex-vivo high-field magnetic resonance (MR) imaging to create digital a three-dimensional (3D) representations of tongue cancer specimens, referred to as the "MR-based digital specimen" (MR-DS). The aim was to create a method to assist surgeons in identifying and localizing inadequate resection margins during surgery, a critical factor in achieving locoregional control. Methods: Fresh resection specimens of nine tongue cancer patients were imaged in a 7 Tesla small-bore MR, using a high-resolution multislice and 3D T2-weighted Turbo Spin Echo. Two independent radiologists (R1 and R2) outlined the tumor and mucosa on the MR-images whereafter the outlines were configured to an MR-DS. A color map was projected on the MR-DS, mapping the inadequate margins according to R1 and R2. We compared the hematoxylin-eosin-based digital specimen (HE-DS), which is a histopathological 3D representation derived from HE stained sections, with its corresponding MR-images. In line with conventional histopathological assessment, all digital specimens were divided into five anatomical regions (anterior, posterior, craniomedial, caudolateral and deep central). Over- and underestimation 95th-percentile Hausdorff-distances were calculated between the radiologist- and histopathologist-determined tumor outlines. The MR-DS' diagnostic accuracy for inadequate margin detection (i.e. sensitivity and specificity) was determined in two ways: with conventional histopathology and HE-DS as reference. Results: Using conventional histopathology as a reference, R1 achieved 77% sensitivity and 50% specificity, while R2 achieved 65% sensitivity and 57% specificity. When referencing to the HE-DS, R1 achieved 94% sensitivity and 61% specificity, while R2 achieved 88% sensitivity and 71% specificity. Range of over- and underestimation 95HD was 0.9 mm - 11.8 mm and 0.0 mm - 5.3 mm, respectively. Conclusion: This proof of concept for volumetric assessment of resection margins using MR-DSs, demonstrates promising potential for further development. Overall, sensitivity is higher than specificity for inadequate margin detection, because of the radiologist's tendency to overestimate tumor size.

Treatment of buccal mucosal carcinomas: A survey amongst head and neck surgeons in the Netherlands.

Objective: Currently, there is no up-to-date guideline for the treatment of buccal mucosal squamous cell carcinoma (BMSCC) in the Netherlands. A questionnaire was used to investigate the opinions of Dutch head and neck surgeons on BMSCC of the cheek treatment. Methods: A questionnaire was sent to all 91 head and neck surgeons in the Netherlands. Their opinions on surgical tumor-free margins, through-and-through defects, and indications for local adjuvant therapy were questioned. Results: The response rate was 51%. To prevent a through-and-through defect, 67% of the surgeons would accept a deep clinical (macroscopic) margin of ≤5 mm. The less adverse histological characteristics a tumor has, the less consensus there is amongst the surgeons for local adjuvant treatment in case of close margins. Conclusion: There is no consensus amongst Dutch head and neck surgeons about the optimal treatment for BMSCC of the cheek. There are different opinions on acceptable resection margins, indications for a through-and-through defect, and indications for adjuvant treatment. BMSCC of the cheek treatment should be more uniform and less surgeon dependent. Level of evidence: N/A.

A closer look at the resection margins of buccal mucosa cancer: Their influence on local recurrence free survival.

BACKGROUND: The adequate surgical margin for local control of buccal mucosa squamous cell carcinoma (BMSCC) is under debate. This study investigates surgical margins and other factors associated with local recurrence free survival (LRFS) in a large cohort of BMSCC patients. METHODS: Multiple factors were evaluated retrospectively in 97 patients with BMSCC. Cox-regression and Kaplan-Meier curves were used for analysis. RESULTS: The local recurrence rate was 23%. The tumor-free margin was <5.0 mm in 89% of the patients and the deep margin was significantly more often inadequate. Multivariate analysis associated pT3-classification, former smokers, tumor-free margin status, and postoperative (chemo)radiation (PO(ch)RT) with local recurrence. Re-resections did not improve LRFS in patients with <5.0 mm tumor-free margins. CONCLUSIONS: Adequate tumor-free margins are pivotal for LRFS of BMSCC. PO(ch)RT, not re-resection, can improve LRFS in patients with <5.0 mm tumor-free margins.

Ultrasound-guided resection for squamous cell carcinoma of the buccal mucosa: A feasibility study.

BACKGROUND: Image-guided surgery could help obtain clear (≥5.0 mm) resection margins. This feasibility study investigated ultrasound-guided resection accuracy of buccal mucosa squamous cell carcinoma (BMSCC). METHODS: MRI and ultrasound measurements of tumor thickness were compared to histology in 13 BMSCC-patients. Ultrasound measured margins (at five locations) on the specimen were compared to the corresponding histological margins. RESULTS: Accuracy of in- and ex-vivo ultrasound (mean deviation from histology: 1.6 mm) for measuring tumor thickness was comparable to MRI (mean deviation from histology: 2.6 mm). The sensitivity to detect clear margins using ex-vivo ultrasound was low (48%). If an ex-vivo ultrasound cutoff of ≥7.5 mm would be used, the sensitivity would increase to 86%. CONCLUSIONS: Ultrasound-guided resection of BMSCC's is feasible. In- and ex-vivo ultrasound measure tumor thickness in BMSCC accurately. We recommend ≥7.5 mm resection margins on ex-vivo ultrasound to obtain histological clear margins. Additional research is required to establish the effect of 7.5 mm ultrasound cutoff.

Application and accuracy of ultrasound-guided resections of tongue cancer.

OBJECTIVES: Surgical removal of squamous cell carcinoma of the tongue (SCCT) with tumour-free margin status (≥5 mm) is essential for loco-regional control. Inadequate margins (<5 mm) often indicate adjuvant treatment, which results in increased morbidity. Ultrasound (US)-guided SCCT resection may be a useful technique to achieve more adequate resection margins compared to conventional surgery. This study evaluates the application and accuracy of this technique. METHODS: Forty patients with SCCT were included in a consecutive US cohort. During surgery, the surgeon aimed for a 10-mm echographic resection margin, while the tumour border and resection plane were captured in one image. Ex-vivo US measurements of the resection specimen determined whether there was a need for an immediate re-resection. The margin status and the administration of adjuvant treatment were compared those of with a consecutive cohort of 96 tongue cancer patients who had undergone conventional surgery. A receiver operating characteristic analysis was done to assess the optimal margin of ex-vivo US measurements to detect histopathologically inadequate margins. RESULTS: In the US cohort, the frequency of free margin status was higher than in the conventional cohort (55% vs. 16%, p < 0.001), and the frequency of positive margins status (<1 mm) was lower (5% vs. 15%, respectively, p < 0.001). Adjuvant radiotherapy was halved (10% vs. 21%), and the need for re-resection was comparable (10% vs. 9%). A cut-off value of 8 mm for ex-vivo measurements prevented histopathologically inadequate margins in 76%. CONCLUSION: US-guided SCCT resections improve margin status and reduce the frequency of adjuvant radiotherapy.

Feasibility study of ultrasound-guided resection of tongue cancer with immediate specimen examination to improve margin control - Comparison with conventional treatment.

OBJECTIVES: Squamous cell carcinoma of the tongue (SCCT) is preferably treated by surgery. Free resection margins (≥5 mm) provide local control and disease-free survival. However, close (1-5 mm) and positive margins (<1 mm) are frequently encountered. We present our first experience of in-vivo ultrasound (US) guided SCCT resections followed by ex-vivo US control on the resection specimen to obtain free margins. We compare the results with those from a hisorical cohort of 91 conventionally treated SCCT patients. MATERIALS AND METHODS: Ten patients with SCCT were included in a consecutive US-cohort. We aimed for a 5-10 mm margin during surgery, while we visualized the resection plane on US. Ex-vivo US measurements on the resection specimen determined whether there was any need for an immediate re-resection. US measurements were then compared with histopathology. Histopathological margins were compared with a consecutive cohort of 91 patients who had undergone conventional surgery for a SCCT. RESULTS: In the US cohort, 70% of the margins were free. In the conventional cohort, this figure was 17% (P = 0.005). US predicted minimal histopathological margin distance with a mean ± SD error of 1.9 ± 1.8 mm. The mean ± SD of the histopathological overall submucosal/deep margin distance was 7.9 ± 2.1 mm in the US cohort and 7.0 ± 2.2 mm in the conventional cohort (P = 0.188). Ex-vivo examination through use of US indicated an immediate re-resection, which prevented local adjuvant treatment. CONCLUSION: Use of US-guided SCCT resection is feasible and improves margin control.

HIF-1a expression and differential effects on survival in patients with oral cavity, larynx, and oropharynx squamous cell carcinomas.

BACKGROUND: Hypoxia is a negative prognostic factor in head and neck squamous cell carcinomas. Under hypoxia, the hypoxia-inducible factor (HIF)-1a transcription factor is overexpressed. We investigated whether there were site differences in HIF-1a expression and its effect on patient outcomes per subsite. DESIGN/METHOD: A total of 941 patients with HNSCC in the squamous cell carcinoma of the oropharynx (OPSCC, n = 302), oral cavity (OSCC, n = 391), or larynx (LSCC, n = 248) were included. Expression of HIF-1a in tissue samples was investigated using immunohistochemistry. Overall survival (OS), disease-free survival (DFS), and locoregional control (LRC) were analyzed. RESULTS: HIF-1a expression was higher in OSCC than in LSCC and OPSCC. High HIF-1a expression led to worse prognosis in OPSCC (OS P = .029, DFS P = .085) and LSCC (OS P = .041, DFS P = .011) and better in OSCC (OS P = .055, DFS P = .012). There was no association between HIF-1a and LRC. CONCLUSIONS: High HIF-1a expression is related to poor outcome in OPSCC and LSCC and better outcome in OSCC.

Assessment of tumour depth in early tongue cancer: Accuracy of MRI and intraoral ultrasound.

OBJECTIVES: Complete resection of tongue cancer is necessary to achieve local control. Unfortunately, deep resection margins are frequently inadequate. To improve deep margin control, accurate knowledge of tumour thickness is pivotal. Magnetic resonance imaging (MRI) and intraoral ultrasound (ioUS) are frequently applied for tumour staging. This study explores the accuracy of these techniques to estimate depth of invasion. MATERIALS AND METHODS: The data of patients with a T1-2 tongue cancer that had been treated surgically between 2014 and 2018 were retrospectively analysed. Measurements that had been taken by either MRI or ioUS were compared with those taken during histopathology. RESULTS: A total of 83 patients with tongue cancer had undergone a pre-operative MRI and 107 had been studied through an ioUS. Tumour thickness measured by MRI (r = 0.72) and ioUS (r = 0.78) correlated significantly (p < 0.001) with histopathological depth of invasion (DOI). In tumours with a DOI of 0-10 mm, MRI has a mean absolute difference with histopathology of 3.1 mm (SD 3.2 mm) and ioUS of 1.6 mm (SD 1.3 mm). In tumours with a DOI greater than 10 mm, MRI has a mean absolute difference of 3.5 mm (SD 3.0 mm) and ioUS of 4.7 mm (SD 3.5 mm). CONCLUSION: Estimation of histopathological DOI in tongue cancers with DOI till 10 mm is very accurate through use of ioUS. ioUS tends to underestimate DOI in tumors exceeding 10 mm DOI. MRI tends to overestimate DOI in both thin and thick tumours. Since ultrasound measurements can be performed during surgery, ioUS could potentially guide the surgeon in the achievement of adequate resection margins.

Intraoral ultrasonography to measure tumor thickness of oral cancer: A systematic review and meta-analysis.

Early oral cancer is preferably treated by surgery. Its complete removal is essential for locoregional control and disease-free survival. Inadequate resection margins require adjuvant therapy such as re-resection or (chemo)radiation, that causes extra morbidity and oral discomfort. Intraoral ultrasonography (US) is reported to be of value in determining tumor thickness. Intraoperative visualization of the tumor may facilitate the resection and ensure adequate surgical margins. Furthermore, accurate prediction of tumor thickness could help determine the treatment strategy of the clinically node-negative neck, as thickness and depth of invasion are predictors of cervical metastasis as well as prognosticators of survival. The 8th edition of the American Joint Committee on Cancer staging system for oral squamous cell carcinoma has included depth of invasion as parameter for cT-stage. The aim of this review is to analyze the accuracy of intraoral US in determining tumor thickness in oral cancer. A systematic search was conducted, and the quality of the included papers was assessed using the QUADAS-2 tool for diagnostic accuracy studies. Subsequently, a meta-analysis was performed on the available individual participant data of 240 patients. Most of the twelve included studies focused on T1-2 tongue cancer (n = 129). Meta-analysis showed a high correlation in tumor thickness within this subgroup as measured by intraoral US and histopathology (r = 0.82, p < .001), with minor overestimation of 0.5 mm on US. It is concluded that intraoral US is very accurate in determining tumor thickness in early oral tongue cancer.

Cytokeratin 19 expression in early oral squamous cell carcinoma and their metastasis: Inadequate biomarker for one-step nucleic acid amplification implementation in sentinel lymph node biopsy procedure.

BACKGROUND: Intraoperative analysis of lymph nodes during a sentinel lymph node biopsy (SLNB) procedure could result in one-step surgery for early oral squamous cell carcinoma (OSCC) with an occult nodal metastasis. One-step nucleic acid amplification rapidly detects cytokeratin 19 (CK19) RNA with high accuracy. Sensitivity and specificity of CK19 expression in OSCC was evaluated. METHODS: Immunohistochemical CK19 expression was done in 207 patients with OSCC with 65 cases of paired nodal metastases. RESULTS: CK19 was expressed in 65% of all OSCC and even less in early OSCC (56%), with poor correlation between primary tumor and (occult) nodal metastasis. Furthermore, ectopic glandular tissue in close proximity of lymph nodes showed strong CK19 expression. CONCLUSION: CK19 lacks both sensitivity and specificity as a biomarker for nodal metastasis in OSCC, which questions the suitability for CK19-based one-step nucleic acid amplification in SLNB procedures. Therefore, future studies should focus on other biomarkers, because the concept of fast intraoperative diagnostics during SLNB remains attractive.