Recurrent Oral Squamous Cell Carcinoma Research Articles

Cholesterol Is a Regulator of CAV1 Localization and Cell Migration in Oral Squamous Cell Carcinoma.

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-β-cyclodextrin and increased using the methyl-β-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

Predictors of survival in advanced oral cancers after salvage surgery with free tissue flap reconstruction

ObjectiveTo identify prognostic factors for patients with advanced persistent, recurrent, or 2nd primary oral cavity squamous cell carcinoma (OCSCC) potentially unsuitable for salvage surgery with free tissue flap (FTF) reconstruction.Materials and methodsA population-based cohort of 83 consecutive patients with advanced OCSCC who underwent salvage surgery with FTF reconstruction at a tertiary referral centre between 1990 and 2017. Retrospective uni- and multivariable analyses were performed to identify factors affecting all-cause mortality (ACM), i.e., overall survival (OS), as well as disease-specific mortality (DSM), i.e., disease-specific survival (DSS) after salvage surgery.ResultsMedian disease-free interval until recurrence was 15 months with recurrent stage I/II in 31% and III/IV in 69%. Median age at salvage surgery was 67 years (range 31–87) and the median follow-up (alive patients) 126 months. At 2, 5, and 10 years after salvage surgery, respectively, DSS rates were 61%, 44%, and 37% and OS rates 52%, 30%, and 22%. Median DSS was 26 and OS 43 months. Multivariable analysis identified recurrent clinical regional (cN-plus) disease [HR 3.57; p < .001] and elevated gamma-glutamyl transferase (GGT) [HR 3.30; p = .003] as independent pre-salvage predictors for poor OS after salvage, whereas initial cN-plus [HR 2.07; p = .039] and recurrent cN-plus disease [HR 5.14; p < .001] predicted poor DSS. Among post-salvage factors, extranodal extension according to histopathology [HR ACM 6.11; HR DSM 9.99; p < .001] as well as positive [HR ACM 4.98; DSM 7.51; p < 0.001] and narrow surgical margins [HR ACM 2.12; DSM HR 2.80; p < 0.01] emerged as independent factors for poor survival.ConclusionWhile salvage surgery with FTF reconstruction is the primary curative option for patients with advanced recurrent OCSCC, the present findings may help guide discussions with patients who have advanced recurrent regional disease and high GGT preoperatively, especially if there is a small chance of reaching surgical radicality.

Histological risk score and its role in predicting recurrence in early-stage oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) comprises more than 90% of oral cancers and is the most common carcinoma affecting the oral cavity. Early stage T1/T2 OSCC have a heterogeneous prognosis and a significant number of patients develop loco regional recurrence (LRR) and have reduced disease free survival (DFS) with increased disease related mortality. To assess the impact of the three parameters used in Brandwein-Gensler risk model along with lympho-vascular invasion (LVI), depth of invasion (DOI) and lymph node metastases in predicting LRR in early stage OSCC. This was a retrospective study on early stage T1/2 OSCC patients over a period of 2 years who received treatment by surgical resection and had follow-up data. LRR was assessed based on recurrence of OSCC at the initial site or in regional lymph nodes. Out of 1135 OSCC cases during our study period a total of 207 cases befitted our inclusion criteria. Recurrence was noted in 113 (54.6%) cases. Univariate analysis identified LVI (P < 0.00001), DOI (P < 0.00001), nodal involvement (P < 0.00001), worst pattern of invasion (WPOI) (P < 0.00001), lymphocytic host response (LHR) (P = 0.004), perineural invasion (PNI) (P = 0.012) as strong statistically significant risk factors for LRR. Adequate assessment of simple parameters on routine H and E by incorporating Brandwein-Gensler histological risk scoring model at the initial presentation can help prognosticate and predict LRR and select patients for post-surgical adjuvant therapy.

Peritumoural and Intratumoural Distribution of Langerhan Cell in Oral Squamous Cell Carcinoma and its Association with Known Prognostic Factors

Introduction: Oral Squamous Cell Carcinoma (OSCC) accounts for about 2/3rd of the Head and Neck Squamous Cell Carcinomas (HNSCC). It is the sixth most common cancer worldwide and the third most common cancer in developing countries including India. More than half the HNSCC patients have advanced disease at the time of diagnosis and overall long-term survival is low (50%), despite aggressive therapeutic regimens. This has demanded the need for prognostic indicators that can be used to improve the ability to select a more individualised approach for treatment relative to the risks of recurrence, metastases and death. Aim: To study the intratumoural and peritumoural distribution and density of Langerhans Cells (LC) in OSCC using Immunohistochemistry (IHC) markers Cluster of Differentiation1a (CD1a) and Langerin and to study its association with known prognostic factors. Materials and Methods: This was a hospital-based crosssectional study conducted at the Department of Pathology, Amala institute of Medical Sciences, Kerala, India during a period of 18 months, from January 2019 to June 2020. In total, 28 specimens of wide local excision with cervical lymph node dissection done for OSCC were enrolled in the study. Small biopsy samples and cases who had undergone neoadjuvant treatment as well as cases of recurrent OSCC were excluded. Clinical details were obtained from the patients’ case files. The immunohistochemical staining for LCs using CD1a and Langerin was done. The distribution of LCs was compared to the tumour grade and lymph node status along with lymphovascular and perineural invasion. Statistical analysis was performed using sample t-test after identifying normality of distribution. The p-value of &lt;0.05 was taken as significant. Results: Fourteen cases of well differentiated and 14 cases of moderate to poorly differentiated OSCC were selected for the study. The LC distribution in these cases were compared to prognostic factors like histological grade, Lymphovascular Invasion (LVI), perineural invasion, tumour size, depth of invasion, T stage and lymph node status. A significant positive association was observed between the tumour differentiation and peritumoural CD1a positive cell density (p-value: 0.001). Further, a negative association was also established between the lymph node staging and peritumoural CD1a positive cell density (p-value: 0.004). There was no association between the LC density and presence of lymphovascular or perineural invasion in OSCC. Conclusion: A depleted peritumoural LC density in OSCC reflects poor tumour immunity, associated with higher grade and higher stage of lymph node metastasis. LC density may hence be of prognostic value in OSCC.

Immunoexpression of p53 mutant-type in Iranian patients with primary and recurrence oral squamous cell carcinoma.

Mutations in tumor suppressor p53 protein can occur at different phases of malignant transformation and affect the patient's prognosis. This study aimed to evaluate the expression of mutant p53 protein in Iranian patients with the primary and recurrence oral squamous cell carcinoma (OSCC). This retrospective cross-sectional study conducted on a group of patients with the primary OSCC (n=122) and the control subjects with oral noncancerous reactive lesions (n=80). Immunohistochemistry was performed with the DO-7 monoclonal antibody against p53 protein, and samples with ≥10% immunostaining were considered positive. Statistical analyses were carried out using SPSS. Positive staining for p53 was observed in none of the control subjects and 57.4% (70 of 122) of the primary OSCC patients (p<0.0001, OR=107.69, 95%CI=6.49-179.0). The p53 immunopositivity had no significant differences between males and females (54.2% vs. 62%, p=0.390), but significantly different between those aged below and over 50 years (p<0.0001, OR=4.52, 95%CI=1.07-12.05). During follow-up, OSCC recurrence occurred in 104 patients, but the phenotype of the mutant p53 protein in patients who relapsed was the same as in matched primary tumors (p=0.763). Risk of recurrence had no significant differences between p53-positive and p53-negative cases (p=0.953), males and females (p=0.263), and age below and over 50 years (p=0.223). Despite its confirmed diagnostic value, the immunoexpression of the p53 mutant protein in OSCC in cancer recurrence was the same as in the primary tumor. However, further studies with a larger sample size and longer follow-up are needed to confirm or change our conclusions.

Complete response of recurrent oral squamous cell carcinoma treated with cetuximab in combination with radiotherapy: A case series.

Salvage surgery for recurrent oral squamous cell carcinoma (OSCC) often leads to a poor quality of life (QOL). The present study described three cases that resulted in favorable locoregional control with cetuximab in combination with radiotherapy (Cmab + RT). Case 1 had regional recurrence of OSCC at the lower right mastoid area 4 months after primary surgery. Case 2 had regional recurrence of OSCC at the parotid area 7 months after primary surgery. Case 3 had local recurrence of OSCC at the masticatory muscle and Rouviere's lymph nodes 1 year and 3 months after primary surgery. In all cases, Cmab + RT was performed, and disease-free survival was confirmed 4 months, 2 years and 6 months, and 10 months after Cmab + RT, respectively. Immunohistochemically, all resected tumors had no expression of 110-kDa catalytic subunit of class IA phosphatidylinositol 3-kinase (PI3Kp110α). In conclusion, if salvage surgery for recurrent OSCC results in a significantly low QOL, then shifting to chemoradiotherapy may be appropriate as a treatment strategy. In addition, strong evidence indicated that PI3Kp110α expression is associated with Cmab therapy efficacy.

Identification of Biological Functions and Prognostic Value of NNMT in Oral Squamous Cell Carcinoma.

Background: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. Methods: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. Results: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial–mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. Conclusions: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy.

Outcomes for recurrent oral cavity squamous cell carcinoma

ObjectivesWe sought to determine overall survival (OS), prognostic factors, cost, and functional outcomes after surgery for locally recurrent oral cavity squamous cell carcinoma (OCSCC). Materials and MethodsWe retrospectively reviewed 399 cases of locally recurrent OCSCC from 1997 to 2011, of which 259 patients were treated with salvage surgery. Survival and prognostic factors were evaluated using univariable and multivariable Cox regression, the Kaplan-Meier method, and the log-rank test. ResultsThe 5-year OS for patients undergoing surgical salvage, nonsurgical therapy, or supportive care was 44.2%, 1.5%, and 0%, respectively. For patients who underwent surgical salvage, 133 (51%) patients experienced a second recurrence at a median of 17 months. Factors associated with OS included disease-free interval ≤ 6 months (P =.0001), recurrent stage III-IV disease (P <.0001), and prior radiation (P =.0001). Patients with both advanced stage and prior radiation had a 23% 5-year OS, compared with 70% for those with neither risk (P <.001). Functionally, 85% of patients had > 80% speech intelligibility and 81% were able to eat by mouth following salvage surgery. Of the patients who required tracheostomy, 78% were decannulated. The adjusted median hospital and professional charges for patients were $129,696 (range $9,238-$956,818). ConclusionsPatients with recurrent OCSCC who underwent salvage surgery have favorable functional outcomes with half of alive at 5 years but poorer OS for advanced disease, disease-free interval ≤ 6 months, and prior radiation. Additionally, treatment is associated with high cost, and about half of patients ultimately develop another recurrence.

Porphyromonas gingivalis Activation of Tumor-Associated Macrophages via DOK3 Promotes Recurrence of Oral Squamous Cell Carcinoma.

BackgroundOral squamous cell carcinoma (OSCC) is the most common head and neck malignancy, characterized by high recurrence rate resulting in poor prognosis. Porphyromonas gingivalis, most closely correlated with chronic periodontitis, is increasingly thought to play a significant role in OSCC development via influencing tumor-associated macrophages. However, its specific function remains unclear. In this study, we attempted to explore the mechanism of action of P. gingivalis in the recurrence of OSCC by bioinformatics analysis, to lay a foundation for subsequent basic experiments.Material/MethodsThe P. gingivalis-infected macrophage microarray dataset (GSE24897) and the OSCC advanced relapse patient microarray dataset (GSE87593) were retrieved from the Gene Expression Omnibus (GEO) database. Differentially-expressed genes (DEGs) were screened using R system, and the intersected DEGs were analyzed for functional enrichment, and protein–protein interaction (PPI) networks were constructed. The expression of significant DEG in GSE24897 microarray was assessed to determine its effect on macrophage immune infiltration in pan-cancer by applying the TIMER 2.0 repository. To detect the expression of DOK3 in OSCC specimens, immunohistochemical (ICH) assay was used.ResultsA total of 106 co-expressed DEGs were upregulated and 131 were downregulation. The biological processes were mainly enriched in DNA-templated transcription terms, the cellular component enrichment was mainly enriched in the nucleus terms, and the molecular function enrichment was mainly enriched in protein-binding function terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DEGs were mainly enriched in the MAPK signaling pathway. Overall analysis of the PPI network showed a significant aggregation, with the top 10 hub co-expressed genes (CASP3, FYN, HNRNPA2B1, NR3C1, RELA, REL, POLR2F, RAN, RHOA, and STAT5B). DOK3 is significantly upregulated in P. gingivalis-infected macrophages, which is associated with macrophage infiltration and differentiation. There was more positive DOK3 staining in the group with P. gingivalis infection.ConclusionsP. gingivalis can affect the recurrence of OSCC by increasing the expression of DOK3 in TAMs, which may be involved in activation of signaling pathways such as TNF and MAPK.

Combined evaluation of both WEE1 and phosphorylated cyclin dependent kinase 1 expressions in oral squamous cell carcinomas predicts cancer recurrence and progression

Background/purposeWEE1 is a mitotic inhibitor at G2 checkpoint of the cell cycle that negatively regulates cyclin-dependent kinase 1 (CDK1) through inhibitory phosphorylation. This study assessed whether the expressions of both WEE1 and phosphorylated CDK1 in specimens of oral squamous cell carcinoma (OSCC) might predict the OSCC recurrence and progression. Materials and methodsThis study used immunohistochemistry to examine the expressions of WEE1 and phosphorylated CDK1 proteins in 75 specimens of OSCC and 30 specimens of normal oral mucosa (NOM). ResultsThe mean WEE1 labeling index (LI) was significantly lower in 75 OSCC samples than in 30 NOM samples (P < 0.001), whereas the mean phosphorylated CDK1 LI was significantly higher in 75 OSCC samples than in 30 NOM samples (P < 0.001). We found a significant association of low WEE1 LI (<21%) with OSCC recurrence (P = 0.047) and a significant association of low phosphorylated CDK1 LI (<10%) with larger tumor size (P = 0.011) and more advanced clinical stages (P = 0.021) of OSCC. ConclusionCombined evaluation of WEE1 and phosphorylated CDK1 LI in specimens of OSCC may predict the OSCC recurrence and progression.

Novel Machine Learning Model to Predict Interval of Oral Cancer Recurrence for Surveillance Stratification.

We aimed to develop a machine learning (ML) model to accurately predict the timing of oral squamous cell carcinoma (OSCC) recurrence across four 1-year intervals. Patients with surgically treated OSCC between 2012-2018 were retrospectively identified from the Yale-New Haven Health system tumor registry. Patients with known recurrence or minimum follow-up of 24 months from surgery were included. Patients were classified into one of five levels: four 1-year intervals and one level for no recurrence (within 4 years of surgery). Three sets of data inputs (comprehensive, feature selection, nomogram) were combined with 4 ML architectures (logistic regression, decision tree (DT), support vector machine (SVM), artificial neural network classifiers) yielding 12 models in total. Models were primarily evaluated using mean absolute error (MAE), lower values indicating better prediction of 1-year interval recurrence. Secondary outcomes included accuracy, weighted precision, and weighted recall. 389 patients met inclusion criteria: 102 (26.2%) recurred within 48 months of surgery. Median follow-up time was 25 months (IQR: 15-37.5) for patients with recurrence and 44 months (IQR: 32-57) for patients without recurrence. MAE of 0.654% and 80.8% accuracy were achieved on a 15-variable feature selection input by 2 ML models: DT and SVM classifiers. To our knowledge, this is the first study to leverage multiclass ML models to predict time to OSCC recurrence. We developed a model using feature selection data input that reliably predicted recurrence within 1-year intervals. Precise modeling of recurrence timing has the potential to personalize surveillance protocols in the future to enhance early detection and reduce extraneous healthcare costs. 3 Laryngoscope, 133:1652-1659, 2023.

Feasibility of oral microbiome profiles associated with oral squamous cell carcinoma

ABSTRACT Objective Recently, the possibility that oral microbiomes is associated with oral squamous cell carcinoma (OSCC) initiation and progression has attracted attention; however, this association is still unclear. Here, we comprehensively analyze the microbiome profiles of saliva samples using next-generation sequencing followed by determining the association between oral microbiome profiles and OSCC. Materials and Methods Microbiome profiles in saliva samples from patients with OSCC, oral leukoplakia (OLK), and postoperative OSCC (Post) were analyzed. Candidate OSCC-associated bacteria were identified by comparing the bacterial diversity and relative abundance of each group based on these microbiome profiles, and their applicability as OSCC detection tools were evaluated. Results There were significant differences in genus abundances (Streptococcus, Aggregatibacter, and Alloprevotella) among the groups from saliva samples. In the OSCC group, compared with the OLK and Post groups, abundances of the genus Fusobacterium, phylum Fusobacteria and phylum Bacteroidetes were markedly increased and that of the genus Streptococcus and phylum Firmicutes were decreased. Conclusion The results suggested a strong association of these bacteria with OSCC. Especially, phylum Fusobacterium was significantly associated with early recurrence of OSCC. Thus, oral microbiome analysis may have a potential of novel OSCC detection and prognostic tool.

Observation of dynamic changes in neutrophil-to-lymphocyte ratio is useful for evaluating treatment response to nivolumab in PD-L1-negative advanced oral cancer

ObjectiveNivolumab is widely used for treatment of recurrent or metastatic oral squamous cell carcinoma (OSCC), but few patients benefit from this therapy. The aim of this study was to evaluate useful predictive biomarkers in OSCC patients. MethodsThis study included seven patients who were diagnosed with OSCC and received nivolumab treatment more than twice at Yamanashi Central Hospital. We analyzed correlations of treatment efficacy with PD-L1 expression in the tumor microenvironment by immunohistochemistry, gene mutation profile of tumor, and neutrophil to lymphocyte ratio (NLR) in peripheral blood. Gene mutation profile was analyzed by PCR of microsatellite instability and whole-exome sequencing. NLR was calculated by division of absolute neutrophil and lymphocyte counts. ResultsTwo patients showed complete response (CR), while five showed no therapeutic effect. PD-L1 expression was correlated with treatment efficacy in most patients, but PD-L1 expression was absent in one patient with CR. Gene mutation analysis of tumor cells did not contribute to prediction of the treatment effect in the patient, either. Pre-treatment NLR was not correlated with treatment outcome, but NLR in responders declined gradually to < 4 after the third nivolumab treatment; this markedly differed from the findings in non-responders, who exhibited increased NLR during therapy. ConclusionsNLR changes reflect the early treatment response in OSCC patients treated with nivolumab, including patients whose immune and gene mutation profiles does not correlate with treatment efficacy.

Risk Factors of Microscopically Tumor-Free Surgical Margins for Recurrence and Survival of Oral Squamous Cell Carcinoma Patients.

ObjectivesThe concept of adequate surgical margins remains controversial in oral squamous cell carcinoma (OSCC) surgery. This study aimed to identify surgical margin-related indicators that might impact recurrence and survival of OSCC patients.Materials and MethodsHistopathological examination was performed using hematoxylin-eosin-stained surgical margin tissue sections in 235 OSCC patients. Axin2 and Snail expression at the surgical margin was detected by immunohistochemistry. The impact of the Axin2-Snail cascade on tumorigenesis of the immortalized human oral keratinocyte (IHOK) line was investigated in vivo.ResultsThe width and dysplasia of surgical margins were not significantly associated with the outcome of OSCC patients. In a multivariate analysis using variable clinicopathologic factors and with Axin2 and Snail expression as cofactors, higher age (hazard ratio [HR]:1.050; P=0.047), Axin2 (HR:6.883; P=0.014), and Snail abundance (HR:5.663; P=0.009) had independent impacts on worsened overall survival. Similarly, lesion site in retromolar trigone (HR:4.077; P=0.010), upper (HR:4.332; P=0.005) and lower gingiva (HR:3.545; P=0.012), presence of extranodal extension (HR:9.967; P<0.001), perineural invasion (HR:3.627; P=0.024), and Snail abundance (HR:3.587; P<0.001) had independent impacts on worsened recurrence-free survival. Furthermore, Axin2 knockdown induced decreased Snail expression and attenuated tumorigenesis in the IHOK line.ConclusionHistopathological examination of surgical margins may not be reliable to predict OSCC patient outcome. Molecular analysis may provide a more accurate risk assessment of surgical margins in OSCC. In particular, Axin2 and Snail are potential predictive biomarkers for the risk assessment of surgical margins in OSCC.

Pharmacological inhibition of HDAC6 overcomes cisplatin chemoresistance by targeting cancer stem cells in oral squamous cell carcinoma.

Chemoresistance is associated with recurrence and metastasis in oral squamous cell carcinoma (OSCC). The cancer stem cell (CSC) subpopulation is highly resistant to therapy, and they are regulated by epigenetic mechanisms. HDACs are histone deacetylase enzymes that epigenetically regulate gene expression. HDAC6 acts on several physiological processes, including oxidative stress, autophagy and DNA damage response, and its accumulation is associated with cancer. Here, we investigate the role of HDAC6 in CSC-mediated chemoresistance in oral carcinoma in addition to its application as a therapeutic target to reverse chemoresistance. Wild-type oral carcinoma cell lines (CAL27 WT and SCC9 WT), cisplatin-resistant (CAL27 CisR and SCC9 CisR), and the subpopulations of cancer stem cells (CSC+) and non-stem (CSC-) derived from CisR cells were investigated. HDAC6 accumulation was analyzed by Western blot and immunofluorescence; DNA damage was evaluated by immunofluorescence of phospho-H2A.X; the qPCR for PRDX2, PRDX6, SOD2, and TXN and ROS assay assessed oxidative stress. Apoptosis and CSC accumulation were investigated by flow cytometry. We identified the accumulation of HDAC6 in CisR cell lines and CSC. Cisplatin-resistant cell lines and CSC demonstrated a reduction in DNA damage and ROS and elevated expression of PRDX2. The administration of tubastatin A (a specific HDAC6 inhibitor) increased oxidative stress and DNA damage and decreased PRDX2. Tubastatin A as a monotherapy induced apoptosis in CisR and CSC and reduced the stemness phenotype. High levels of HDAC6 sustain CSC subpopulation and chemoresistance in OSCC, suggesting HDAC6 as a pharmacological target to overcome resistance and perhaps prevent recurrence in OSCC.

Preoperative prediction model to evaluate salvage surgery in patients with recurrent or second primary oral cavity squamous cell carcinoma

ObjectiveIn approximately 50% of patients with oral cavity squamous cell carcinoma (OCSCC), the disease progresses after curative surgery. However, the role of salvage surgery (SS) is controversial, and life expectancy after SS is unknown. MethodsIn this study, 262 patients with OCSCC with locoregional recurrence and second primary OCSCC were retrospectively enrolled and divided into a resectable (55.0%, 144/262) and unresectable (45.0%, 118/262) groups. After excluding neck recurrence only, SS had been performed 195 times in the resectable group. The corresponding preoperative clinicopathologic factors and postsurgery survival (PSS) of each SS were pooled for analysis. ResultsMedian survival after disease progression was 64.2 and 10.4 months for the resectable and unresectable groups, respectively. In the resectable group, one-fifth (19.5%, 37/190) of the patients died within 1 year of SS (PSS < 1 year), and one-third (32.8%, 64/195) of the patients had undergone SS two or more times. The interval from the last surgery ≤ 12 months, depth of invasion of the last surgery > 1 cm, and clinical evidence of nodal disease at the preoperative evaluation were independent predictors of poor PSS. A scoring prediction model was established with 1 point for each factor. The results revealed 1-year postsurgery death rates of 10.3% in the low-risk group (score: 0–1) and 48.6% in the high-risk group (score: 2 or 3) (P < 0.001). ConclusionsIn conclusion, an effective scoring model predicting life expectancy after SS for patients with OCSCC was established.

Differences in PD-L1 Expression between oral and oropharyngeal squamous cell carcinoma.

Treatment of metastasized or recurrent oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma remains challenging. Targeted antibody-based therapy inter alia for PD-1 / PD-L1 axis shows promising results, but whether PD-L1 expression varies between the subentities remains unclear. The expression pattern of PD-L1 (EPR19759 antibody, Abcam, Berlin, Germany) and p16 (CINtech® Histology Kit, Ventana, Oro Valley, USA) was determined immunohistochemically and analyzed by HALO™ Image Analysis Software (Indica Lab, Albuquerque, USA). For PD-L1, combined positivity score (CPS), tumor proportion score (TPS) and histoscore, were assessed and results correlated with epidemiological data. In total, 161 patients (OSCC: n = 78, OPSCC: n = 83) were included. A mean of 43.6% (±34.0%) of the specimen showed increased PD-L1 expression that did not differ quantitatively between subentities (TPS: p = 0.159, CPS: p = 0.078), but qualitatively (histoscore: p = 0.003). In the mean follow-up period (45.6 months), contrary to age (p = 0.006) and advanced T-Status (p = 0.018), PD-L1 expression did not correlate with overall (OS, p = 0.191) and recurrence free survival (RFS: p = 0.193) in both subentities. No correlation of p16 and PD-L1 expression was found (p = 0.844). PD-L1 is differentially expressed between OSCC and OPSCC, however without influence on OS. Furthermore, p16 status was not related to PD-L1 expression. This may have implications for future (immune) therapeutical approaches for oral cancer.

Application of near infrared fluorescence imaging in detection of residual cancer in oral squamous cell carcinoma

Objective: Local recurrence is the main cause of treatment failure in patients with oral squamous cell carcinoma (OSCC). This study was proposed to investigate the feasibility of near infrared fluorescence (NIF) via indocyanine green (ICG) for monitoring surgical marginal in operation for OSCC patients. Methods: In 35 patients with OSCC treated surgically in the Department of Oral and Maxillofacial Surgery, Nanjing University School of Medicine, from January 2019 to June 2020, ICG (0.75 mg/kg) was administered intravenously via elbow vein at (12±1) hours before surgery, and NIF was performed intraoperatively on the surgical field and the cut edge of the surgically excised specimen, and fluorescence intensity was measured for OSCC tissue and normal oral mucosa, abnormal fluorescence signals were taken and subjected to rapid cryopathological examination. Correlation between NIF tumor boundary grading and pathological tumor boundary grading was analyzed by Spearman correlation analysis. Results: Clear ICG NIF was obtained for tumor lesions in all 35 patients, with a positive rate of 100%. The fluorescence intensity of OSCC tissue was (412.73±146.56) au, which was higher than that of normal oral mucosa tissue [(279.38±82.56) au, P<0.01]. Abnormal fluorescence signals were detected at the tumor bed and the cut edge of the surgical resection specimen in 4 patients, of which 2 cases were pathologically confirmed as cancer cell residue and 2 cases as inflammatory cell infiltration. The rate of positive detection of cut margins using ICG NIF technique in OSCC was 5.7% (2/35). Twenty of the 35 OSCC patients had grade 1, 11 of grade 2, and 4 of grade 3 tumor borders revealed by NIF of surgical resection specimens, which was positively correlated with pathological tumor border (r=0.809, P<0.001). Conclusions: ICG NIF technique can effectively detect the residual cancer cells at the incision margin, which is of great clinical value in reducing local recurrence of OSCC after surgery due to intraoperative cancer residue.

Reappraisal of multimodality imaging for improved Radiation Therapy (RT) target volume determination of recurrent Oral Squamous Cell Carcinoma (OSCC): An original article

Objective: Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most frequent cancer sites around the globe. Within the heterogeneous group of HNSCC, Oral Squamous Cell Carcinoma (OSCC) deserves the utmost attention as an important subsite. Despite multimodality management, recurrence of OSCC is not uncommon. RT may play an integral role as part of initial management or as adjunctive therapy, or for treatment of recurrent disease. In this study, we evaluate multimodality imaging-based RT treatment volume definition for irradiation of recurrent OSCC. Materials and methods: Multimodality imaging-based RT treatment volume definition for radiotherapeutic management of recurrent OSCC was assessed. RT target volume determination by incorporation of Magnetic Resonance Imaging (MRI) or by Computed Tomography (CT)-simulation images only has been evaluated and comparative analysis has been performed for patients receiving salvage RT for recurrent OSCC. Results: Ground truth target volume has been found to be identical with treatment volume definition by CT-MR fusion-based imaging. Conclusion: Incorporation of MRI in target and treatment volume definition may improve target and treatment volume definition for recurrent OSCC.

Impact of secondary tumor-free resection margins in soft tissue on local, regional, and distant recurrence in R0-resected oral squamous cell carcinoma.

The impact of additional soft tissue resection on recurrence of oral squamous cell carcinoma (OSCC) remains controversial. The study aim was to compare recurrence between patients with secondary tumor-free resection margins after intraoperative additional resection (STF-RM) and patients with primary tumor-free resection margins without additional resection (PTF-RM). Forty-five patients with STF-RM were matched with patients with PTF-RM according to Union for International Cancer Control stage, tumor location, and treatment modality and compared for local, regional, and distant recurrence. Patients with STF-RM showed lower local and distant control rates compared to patients with PTF-RM (66.2% vs. 82.8%; p=0.045 and 86.3% vs. 100.0%; p=0.021). STF-RM was the only predictor of local recurrence accounting for tumor (T) status, nodal (N) status, tumor grade, margin distance, and extracapsular extension (hazard ratio 4.21 [95% confidence interval 1.26-14.04]; p=0.019). STF-RM have an adverse impact on local and distant recurrence of OSCC.