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The spatial organization of the tumor microenvironment has a profound impact on biology and therapy response. Here, we perform an integrative single-cell and spatial transcriptomic analysis on HPV-negative oral squamous cell carcinoma (OSCC) to comprehensively characterize malignant cells in tumor core (TC) and leading edge (LE) transcriptional architectures. We show that the TC and LE are characterized by unique transcriptional profiles, neighboring cellular compositions, and ligand-receptor interactions. We demonstrate that the gene expression profile associated with the LE is conserved across different cancers while the TC is tissue specific, highlighting common mechanisms underlying tumor progression and invasion. Additionally, we find our LE gene signature is associated with worse clinical outcomes while TC gene signature is associated with improved prognosis across multiple cancer types. Finally, using an in silico modeling approach, we describe spatially-regulated patterns of cell development in OSCC that are predictably associated with drug response. Our work provides pan-cancer insights into TC and LE biology and interactive spatial atlases ( http://www.pboselab.ca/spatial_OSCC/ ; http://www.pboselab.ca/dynamo_OSCC/ ) that can be foundational for developing novel targeted therapies., (© 2023. Springer Nature Limited.)

There are few prognostic biomarkers and targeted therapeutics currently in use for the clinical management of oral squamous cell carcinoma (OSCC) and patient outcomes remain poor in this disease. A majority of mutations in OSCC are loss-of-function events in tumour suppressor genes that are refractory to conventional modes of targeting. Interestingly, the chromosomal segment 3q22-3q29 is amplified in many epithelial cancers, including OSCC. We hypothesized that some of the 468 genes located on 3q22-3q29 might be drivers of oral carcinogenesis and could be exploited as potential prognostic biomarkers and therapeutic targets. Our integrative analysis of copy number variation (CNV), gene expression and clinical data from The Cancer Genome Atlas (TCGA), identified two candidate genes: NCBP2, TFRC, whose expression positively correlates with worse overall survival (OS) in HPV-negative OSCC patients. Expression of NCBP2 and TFRC is significantly higher in tumour cells compared to most normal human tissues. High NCBP2 and TFRC protein abundance is associated with worse overall, disease-specific survival, and progression-free interval in an in-house cohort of HPV-negative OSCC patients. Finally, due to a lack of evidence for the role of NCBP2 in carcinogenesis, we tested if modulating NCBP2 levels in human OSCC cell lines affected their carcinogenic behaviour. We found that NCBP2 depletion reduced OSCC cell proliferation, migration, and invasion. Differential expression analysis revealed the upregulation of several tumour-promoting genes in patients with high NCBP2 expression. We thus propose both NCBP2 and TFRC as novel prognostic and potentially therapeutic biomarkers for HPV-negative OSCC., (© 2023. The Author(s).)

Background: Close margins influence treatment and outcome in patients with oral squamous cell carcinoma (OSCC). This study evaluates 187 cases of surgically treated OSCC regarding the impact of close margins on recurrence-free survival (RFS) and disease-specific survival (DSS)., Methods: Predictors of worsened outcome were identified using Kaplan-Meier analysis and multivariate Cox regression analysis., Results: Tumour size [HR:1.70(0.95-3.08)], nodal status [HR:2.15(1.00-4.64)], presence of extracapsular spread (ECS) [HR:6.36(2.41-16.74)] and smoking history [HR:2.87(1.19-6.86)] were associated with worsened RFS. Similar factors were associated with worsened DSS. Close margins did not influence RFS or DSS., Conclusions: While most conventional risk factors for OSCC conferred a worsened outcome, close margins did not. One explanation for this would be that close margins (< 5 mm) are equivalent to clear margins and the cutoff definition for a close margin should be re-evaluated. Lack of standardized pathology could also reduce accuracy of reporting of close surgical margins.

Importance: High local recurrence rates with aggressive disease remain the main concern in oral cancer survival. Use of a translational device using fluorescence visualization (FV) approved by the US Food and Drug Administration and Health Canada, has shown a marked reduction in the 3-year local recurrence rate of high-grade oral lesions in a single-center observational study., Objective: To determine whether FV- guided surgery can improve local control rates in the treatment of in situ or T1 to T2 category oral squamous cell carcinoma (OSCC)., Design, Setting, and Participants: A multicenter randomized clinical trial was conducted in a surgical setting. A total of 457 patients were enrolled between January 18, 2010, and April 30, 2015. Data analysis of the intention-to-treat population was performed from April 3, 2019, to March 20, 2020. Patients with histologically confirmed high-grade dysplasia/carcinoma in situ or T1 to T2 category OSCC were randomized to receive traditional peroral surgery or FV-guided surgery., Intervention: Fluorescence visualization during surgery., Main Outcomes and Measures: The primary outcome was local recurrence of OSCC. Secondary outcomes were failure of the first-pass margin, defined as a histologically confirmed positive margin for severe dysplasia or greater histologic change of the main specimen (ie, not the margins taken from the resection bed), regional or distant metastasis, and death due to disease., Results: Of the 457 patients enrolled in the study, 443 patients (264 [59.6%] men; mean [SD] age, 61.5 [13.3] years) completed the randomized treatment: 227 FV-guided and 216 non-FV guided surgery. The median follow-up was 52 (range, 0.29-90.8) months. In total, 45 patients (10.2%) experienced local recurrence. The 3-year local recurrence rate was 9.4% in the FV-guided group and 7.2% in the non-FV group (difference, 2.2%; 95% CI, -3.2% to 7.4%). Other similarities between the FV vs non-FV groups included failure of first-pass margin (68/227 [30.0%]) vs 65/216 [30.1%]), regional failure (39/227 [17.2%] vs 37/216 [17.1%]), disease-specific survival (23/227 [10.1%] vs 19/26 [8.8%]), and overall survival (41/227 [18.1%] vs 38/216 [17.6%]) were also similar between groups. No adverse events were judged to be related to the intervention., Conclusions and Relevance: In this randomized clinical trial, FV-guided surgery did not improve local control rates in the treatment of patients with in situ or T1 to T2 category oral cancer. Under a controlled environment, FV-guided surgery did not have an evident effect in reduction of local recurrence for localized OSCC. This result suggests that attention be directed to strategies other than improving definitions of nonapparent disease at clinical margins to identify the sources of local recurrence., Trial Registration: ClinicalTrial.gov Identifier: NCT01039298.

Objectives: To test the performance of an oral cancer prognostic 13-gene signature for the prediction of survival of patients diagnosed with HPV-negative and p16-negative oral cavity cancer., Materials and Methods: Diagnostic formalin-fixed paraffin-embedded oral cavity cancer tumor samples were obtained from the Fred Hutchinson Cancer Research Center/University of Washington, University of Calgary, University of Michigan, University of Utah, and seven ARCAGE study centers coordinated by the International Agency of Research on Cancer. RNA from 638 Human Papillomavirus (HPV)-negative and p16-negative samples was analyzed for the 13 genes using a NanoString assay. Ridge-penalized Cox regressions were applied to samples randomly split into discovery and validation sets to build models and evaluate the performance of the 13-gene signature in predicting 2-year oral cavity cancer-specific survival overall and separately for patients with early and late stage disease., Results: Among AJCC stage I/II patients, including the 13-gene signature in the model resulted in substantial improvement in the prediction of 2-year oral cavity cancer-specific survival. For models containing age and sex with and without the 13-gene signature score, the areas under the Receiver Operating Characteristic Curve (AUC) and partial AUC were 0.700 vs. 0.537 (p < 0.001), and 0.046 vs. 0.018 (p < 0.001), respectively. Improvement in predicting prognosis for AJCC stage III/IV disease also was observed, but to a lesser extent., Conclusions: If confirmed using tumor samples from a larger number of early stage oral cavity cancer patients, the 13-gene signature may inform personalized treatment of early stage HPV-negative and p16-negative oral cavity cancer patients., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: In 2014, Alberta Health Services released guidelines for treating oral squamous cell carcinoma (OSCC)., Methods: A retrospective analysis was performed to assess the historical selection of patients with OSCC for postoperative radiotherapy (PORT) in a prospectively collected cohort of patients being treated with primary surgery. The primary outcome was compliance with the 2014 Alberta Health Services (AHS) guideline recommendations for PORT. The secondary outcome was the selection of PORT according to published pathological indications of high, intermediate, and low risk of recurrence. Reasons for discordance were analyzed., Result: Noncompliance with the new AHS guidelines and published indications for PORT was observed in 17% and 30% of cases, respectively. The reasons for discordance with published indications included: clinician decision (n = 41) and unmodifiable factors (n = 17)., Conclusion: The impact of noncompliance on patient outcomes is being studied. The effect of the publication of the guidelines on selection for PORT going forward will be monitored. © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1529, 2016., (© 2016 Wiley Periodicals, Inc.)

Background: The lack of prognostic biomarkers in oral squamous cell carcinoma (OSCC) has hampered treatment decision making and survival in OSCC remains poor. Histopathological features are used for prognostication in OSCC and, although useful for predicting risk, manual assessment of histopathology is subjective and labour intensive. In this study, we propose a method that integrates multiple histopathological features of the tumor microenvironment into a single, digital pathology-based biomarker using nuclear fractal dimension (nFD) analysis., Methods: One hundred and seven consecutive OSCC patients diagnosed between 1998 and 2006 in Calgary, Canada were included in the study. nFD scores were generated from DAPI-stained images of tissue microarray (TMA) cores. Ki67 protein expression was measured in the tumor using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA®). Lymphocytic infiltration (LI) was measured in the stroma from haematoxylin-eosin (H&E)-stained TMA slides by a pathologist., Results: Twenty-five (23.4%) and 82 (76.6%) patients were classified as high and low nFD, respectively. nFD was significantly associated with pathological tumor-stage (pT-stage; P = 0.01) and radiation treatment (RT; P = 0.01). High nFD of the total tumor microenvironment (stroma plus tumor) was significantly associated with improved disease-specific survival (DSS; P = 0.002). No association with DSS was observed when nFD of either the tumor or the stroma was measured separately. pT-stage (P = 0.01), pathological node status (pN-status; P = 0.02) and RT (P = 0.03) were also significantly associated with DSS. In multivariate analysis, nFD remained significantly associated with DSS [HR 0.12 (95% CI 0.02-0.89, P = 0.04)] in a model adjusted for pT-stage, pN-status and RT. We also found that high nFD was significantly associated with high tumor proliferation (P < 0.0001) and high LI (P < 0.0001), factors that we and others have shown to be associated with improved survival in OSCC., Conclusions: We provide evidence that nFD analysis integrates known prognostic factors from the tumor microenvironment, such as proliferation and immune infiltration, into a single digital pathology-based biomarker. Prospective validation of our results could establish nFD as a valuable tool for clinical decision making in OSCC.

Background: Oral cavity squamous cell carcinoma (SCC) represents the most common SCC affecting the head and neck region. Long-term survival of patients with oral cavity SCC is adversely affected by lymph node metastasis and further decreased by the presence of lymph node extracapsular spread (ECS)., Methods: Using a case-control design, preoperative CT scans from patients with oral cavity SCC and metastatic lymphadenopathy were evaluated by 2 independent neuroradiologists, blinded to the study, for a number of radiologic parameters, including central node necrosis. Multivariate logistic regression was used to identify parameters independently predicting pathologic ECS., Results: For both neuroradiologists, central node necrosis was a significant predictor of ECS, with high interrater agreement (kappa = 0.71). On multivariate analysis, only central node necrosis independently predicted ECS (odds ratio [OR] = 12.1; 95% confidence interval [CI] = 1.24-119). Central node necrosis predicted ECS with 91% sensitivity and 88% negative predictive values., Conclusion: Our findings suggest that central node necrosis on preoperative CT scans is strongly associated with the presence of ECS., (© 2014 Wiley Periodicals, Inc.)

The ING1 epigenetic regulator and tumor suppressor plays a central role in apoptosis. The Ing1 gene is functionally inactivated in many cancer types but is rarely mutated. Although most studies have implicated the major ING1 isoform, p33ING1b, in nuclear apoptotic signalling, we recently discovered a novel and potent apoptosis-inducing effect of p33ING1b translocation to the mitochondria in response to DNA damage. In the present study, we examined the impact of cytoplasmic/mitochondrial localization of p33ING1b in oral squamous cell carcinoma (OSCC) patient samples and explored the therapeutic potential of adenovirally-overexpressed p33ING1b in OSCC cell lines in combination with ionizing radiation (IR) treatment. In contrast with previous reports, we found that p33ING1b protein and mRNA levels are higher in OSCC compared to normal epithelial cells. In OSCC patient samples, higher levels of intra-tumoral cytoplasmic p33ING1b correlated with increased apoptotic markers and significantly better patient survival. This association was strongest in patients who received post-operative radiotherapy. IR treatment induced p33ING1b translocation to the mitochondria and adenoviral-p33ING1b synergized with IR to kill OSCC cells. Our results identify a novel functional relationship between cytoplasmic p33ING1b and patient survival and highlight the potential for the use of p33ING1b as a therapeutic agent in combination with adjuvant radiotherapy in OSCC.

Background: Tumour hypoxia is associated with impaired apoptosis, resistance to therapy and poor prognosis. We previously reported that high stromal expression of the endogenous marker of hypoxia, carbonic anhydrase IX (CAIX), is associated with significantly reduced survival in oral squamous cell carcinoma (OSCC). In addition to hypoxia, CAIX expression is regulated by proliferation-associated signalling. We hypothesised that incorporating Ki67, a proliferation marker, into our existing CAIX-based stratification of OSCC would identify patients with the least favourable prognosis., Methods: Surgically resected tumours from 60 OSCC patients were analysed for CAIX, Ki67 and BAX expression using fluorescence immunohistochemistry and automated quantitative analysis (AQUA)., Results: In patients expressing high stromal CAIX (sCAIX), stratification by tumour Ki67 expression revealed significantly distinct survival outcomes (P=0.005). In our OSCC cohort, below-median Ki67 and top-quartile sCAIX expression (Ki67(lo)sCAIX(hi)) were associated with significantly worse disease-specific survival in univariate (HR 7.2 (2.5-20.4), P=0.001) and multivariate (HR 4.2 (1.4-12.8), P=0.011) analyses. Hypoxia is associated with decreased BAX expression; the Ki67(lo)sCAIX(hi) group was more strongly associated with low BAX expression than high sCAIX alone., Conclusion: These data suggest that combined analysis of tumour Ki67 and sCAIX expression may provide a more clinically relevant assessment of tumour hypoxia in OSCC.

The risk model is a validated outcome predictor for patients with head and neck squamous cell carcinoma (Brandwein-Gensler et al. in Am j surg pathol 20:167-178, 2005; Am J Surg Pathol 34:676-688, 2010). This model may potentially shift treatment paradigms for patients with low-stage cancers, as current protocols dictate that they might receive only primary surgery. Here we test the hypothesis that the Risk Model has added prognostic value for low-stage oral cavity squamous cell carcinoma (OCSCC) patients. 299 patients with Stage I/II OCSCC were characterized according to the risk model (Brandwein-Gensler et al. in Am J Surg Pathol 20:167-178, 2005; Am J Surg Pathol 34:676-688, 2010). Cumulative incidence and competing risk analysis were performed for locoregional recurrence (LRR) and disease-specific survival (DSS). Receiver operating characteristic analyses were performed for worst pattern of invasion (WPOI) and the risk categories. 292 patients were analyzed; 30 T1N0 patients (17%) and 26 T2N0 patients (23%) developed LRR. Disease-specific mortality occurred in 9 T1N0 patients (6%) and 9 T2N0 patients (10%). On multivariable analysis, the risk model was significantly predictive of LRR (p = 0.0012, HR 2.41, 95% CI 1.42, 4.11) and DSS (p = 0.0005, HR 9.16, 95% CI 2.65, 31.66) adjusted for potential confounders. WPOI alone was also significantly predictive for LRR adjusted for potential confounders with a cut-point of either WPOI-4 (p = 0.0029, HR 3.63, 95% CI 1.56, 8.47) or WPOI-5 (p = 0.0008, HR 2.55, 95% CI 1.48, 4.41) and for DSS (cut point WPOI-5, p = 0.0001, HR 6.34, 95% CI 2.50, 16.09). Given a WPOI-5, the probability of developing locoregional recurrence is 42%. Given a high-risk classification for a combination of features other than WPOI-5, the probability of developing locoregional recurrence is 32%. The Risk Model is the first validated model that is significantly predictive for the important niche group of low-stage OCSCC patients.

Aim: The prognostic significance of Ki67 expression in cancers, including oral squamous cell carcinoma (OSCC), is unclear. This may be partly attributed to the lack of consensus surrounding the optimal approach for measuring tumour Ki67 expression. The aim of this study was to evaluate the association between different measures of Ki67 expression and disease-specific survival (DSS) in OSCC., Methods: Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded (FFPE) pre-treatment tumour tissue obtained from 121 OSCC patients diagnosed between 1998 and 2006. Ki67 expression was quantified using fluorescence immunohistochemistry (IHC) and AQUAnalysis® in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Intensity and percentage-based approaches for Ki67 scoring were tested for their association with survival., Results: Ki67 scores obtained from intensity and percentage-based approaches had similar associations with prognosis. We also found that high basal (lowest observed in triplicate cores) Ki67 expression was more strongly associated with improved 5-year disease-specific survival than hot-spot and average Ki67 measurements. The association of high basal Ki67 expression with improved prognosis was most pronounced in patients who received postoperative radiation. Cox proportional hazards analysis showed that the basal Ki67 expression is an independent prognostic marker in our OSCC cohort when adjusted for pathological T-stage, nodal status and treatment., Conclusions: Our study provides a framework for reaching a consensus on the optimal approach for measuring Ki67 expression in cancers. Our results suggest that rigorous comparisons of measurement approaches should be applied in a tumour-type and treatment-specific manner to enhance the clinical application of Ki67 assessment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Resistance to apoptosis is a hallmark of cancer and proteins regulating apoptosis have been proposed as prognostic markers in several malignancies. However, the prognostic impact of apoptotic markers has not been consistently demonstrated in oral squamous cell carcinoma (OSCC). This inconsistency in reported associations between apoptotic proteins and prognosis can be partly attributed to the intrinsic low resolution and misclassification associated with manual, semi-quantitative methods of biomarker expression measurement. The aim of this study was to examine the association between apoptosis-regulating proteins and clinical outcomes in oral squamous cell carcinoma (OSCC) using the quantitative fluorescence immunohistochemistry (IHC) based AQUAnalysis technique., Methods: Sixty-nine OSCC patients diagnosed between 1998-2005 in Calgary, Alberta, Canada were included in the study. Clinical data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of formalin-fixed paraffin embedded pre-treatment tumour tissue. Bax, Bcl-2 and Bcl-XL protein expression was quantified using fluorescent IHC and AQUA technology in normal oral cavity squamous epithelium (OCSE) and OSCC tumour samples. Survival was analyzed using Kaplan-Meier plots and the Cox proportional hazard model., Results: Bax expression was predominantly nuclear in OCSE and almost exclusively cytoplasmic in OSCC. No similar differences in localization were observed for Bcl-2 or Bcl-XL. Only Bax expression associated with disease-specific survival (DSS), with 5-year survival estimates of 85.7% for high Bax versus 50.3% for low Bax (p = 0.006), in univariate analysis. High Bax expression was also significantly associated with elevated Ki67 expression, indicating that increased proliferation might lead to an improved response to radiotherapy in patients with elevated Bax expression. In multivariate analyses, Bax protein expression remained an independent predictor of DSS in OSCC [HR 0.241 (0.078-0.745), p = 0.013]., Conclusions: The AQUA technique used in our study eliminates observer bias and provides reliable and reproducible estimates for biomarker expression. AQUA also provides essential measures of quality control that cannot be achieved with manual biomarker scoring techniques. Our results support the use of Bax protein expression as a prognostic marker in conjunction with other clinico-pathological variables when designing personalized treatment strategies for OSCC patients.

Every year, approximately 25,000 patients are diagnosed with oral cavity squamous cell carcinoma (OCSCC) in the USA. The 5-year survival rate for OCSCC is approximately 40%. Intratumoral hypoxia confers poor prognosis and treatment failure but direct tumor oxygen measurement is challenging. Carbonic anhydrase IX (CAIX) is a marker of tissue hypoxia and we have recently shown that stromal CAIX is associated with reduced survival in patients with HPV-negative head and neck cancer. We examined the importance of this observation in OCSCC patients. We identified patients diagnosed and treated with OCSCC in Calgary (Alberta, Canada) between 1998 and 2005. Clinical and pathologic data were obtained from the Alberta Cancer Registry and chart review. Tissue microarrays (TMAs) were assembled from triplicate cores of archived tumor tissue. Stromal CAIX expression was assessed by quantitative immunohistochemistry (AQUA-HistoRx). The primary endpoint was disease-specific survival. We identified 102 patients with OCSCC; 87 patients had surgery as their primary treatment and adequate tumor tissue for TMA construction was available for all patients. CAIX expression was evaluable for 61 patients. High (top quartile) stromal CAIX expression was associated with significantly reduced 5-year disease-specific survival compared to low stromal CAIX expression (p<0.006). This study confirms our previously reported association between high stromal CAIX expression and significantly reduced overall survival in an independent, predominantly p16-negative, cohort of surgically treated OCSCC. Assessment of stromal CAIX expression could identify patients with the least favorable prognosis and inform therapeutic strategies in OCSCC., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The tumor-specific EGFR deletion mutant, EGFRvIII, is characterised by ligand-independent constitutive signalling. Tumors expressing EGFRvIII are resistant to current EGFR-targeted therapy. The frequency of EGFRvIII in head and neck squamous cell carcinoma (HNSCC) is disputed and may vary by specific sub-site. The purpose of this study was to measure the occurrence of EGFRvIII mutations in a specific HNSCC subsite, oral squamous cell carcinoma (OSCC), using a novel real-time PCR assay., Methodology: Pre-treatment Formalin Fixed Paraffin Embedded (FFPE) cancer specimens from 50 OSCC patients were evaluated for the presence of EGFRvIII using a novel hydrolysis probe-based real-time PCR assay. EGFR protein expression in tumor samples was quantified using fluorescent immunohistochemistry (IHC) and AQUA® technology., Principal Findings: We detected EGFRvIII in a single OSCC patient in our cohort (2%). We confirmed the validity of our detection technique in an independent cohort of glioblastoma patients. We also compared the sensitivity and specificity of our novel real-time EGFRvIII detection assay to conventional RT-PCR and direct sequencing. Our assay can specifically detect EGFRvIII and can discriminate against wild-type EGFR in FFPE tumor samples. AQUAnalysis® revealed that the presence of EGFRvIII transcript is associated with very high EGFR protein expression (98(th) percentile). Contrary to previous reports, only 44% of OSCC over-expressed EGFR in our study., Conclusion and Significance: Our results suggest that the EGFRvIII mutation is rare in OSCC and corroborate previous reports of EGFRvIII expression only in tumors with extreme over-expression of EGFR. We conclude that EGFRvIII-specific therapies may not be ideally suited as first-line treatment in OSCC. Furthermore, highly specific and sensitive methods, such as the real-time RT-PCR assay and AQUAnalysis® described here, will provide accurate assessment of EGFR mutation frequency and EGFR expression, and will facilitate the selection of optimal tailored therapies for OSCC patients.