Oral Squamous Carcinoma Patients Research Articles

Abstract PO-031: Integrated bioinformatics and functional spatial analysis platform for the discovery of predictive biomarkers in oral cancer

Abstract Background: Oral cavity squamous cell carcinoma (OCSCC) has an unpredictable response to immunotherapy, and standard biomarkers evaluating the tumor immune microenvironment (TIME) lack widespread translational applicability. Defining the TIME is critical to understanding mechanisms and allows the development of reproducible and accurate prognostic and predictive biomarkers based on immune profiles; however, to date, this remains elusive for clinical use. To further study and deconstruct the TIME within OCSCC, we integrated our previously developed computational spatial analysis (CSA) with in-silico deconvolution analyses to comprehensively study the TIME in OCSCC. Methods: We leveraged access to two independent OCSCC cohorts (UM and TCGA) with paired bulk RNASeq, WES, and CSA-immune score data. TMB, neoantigen load, and deconvolution analysis (CIBERSORTx) were performed to generate in silico immune profiles. A previously described CSA evaluates the density and distribution of tumor-infiltrating lymphocytes (TILs) relative to cancer cells to generate immune scores (GCross function [GFx]). GFx scores were then correlated with WES and RNASeq-derived cell-type deconvolution results, immune cell activation expression signatures, and gene expression-based radiosensitivity index scores. Pearson's correlation coefficient and associated p-value were calculated for each comparison. Cox proportional hazards multivariate regression was performed using the entire cohort for survival and recurrence analysis. Results: Here, we better characterize the immunologic tumor environment by objectively measuring the TIL density in spatial relationships with cancer cells. We found that immunoreactive tumors with high GFx scores are highly prognostic for overall survival, with the lowest tiers associated with poor survival. Combining the spatial relationship with immunogenomics (WES and RNASeq immune profiles) increased the prognostic capability of the integrated bioinformatics analysis model. In addition, we further revealed that lymphocyte-infiltrated regions, specifically CD8+ T-cells, are associated with TMB, neoantigen load, and improved overall survival. Conclusion: This study provides an integrated dataset from which to generate future insights and possible immunomodulatory therapies to counteract the poor immunogenicity of OCSCC. We show that integrating computational spatial analysis and in-silico deconvolution analysis is generalizable to multiple patient populations with different demographic compositions and pathology images collected from distinctive digitization methods. Our machine-learning approaches provide clinically actionable predictions that confirm the relevance of these biomarkers with immune cell infiltration and suggest the role of digital pathology profiling in predicting response to immunotherapy which could inform treatments for oral squamous carcinoma patients. Citation Format: Michael M. Allevato, Lisa Chionis, Joshua Smith, Santhoshi Krishnan, Leanne Henry, Tingting Qin, Janice Farlow, Laura Rozek, Jonathan Mchugh, Arvind Rao, Maureen Sartor, Steven B. Chinn. Integrated bioinformatics and functional spatial analysis platform for the discovery of predictive biomarkers in oral cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-031.

Identification of copy number alternation profiles in metastatic oral squamous carcinoma patients by using microarray-based comparative genomic hybridization: A study on Turkish patients

ObjectiveOral squamous cell carcinoma (OSCC) is a severe form of cancer affecting different anatomic sites of the oral cavity. OSCC ranks as the sixth most common cancer type with an increasing prevalence globally. However, the mechanisms of OSCC process at later stages are not well understood. In this study, we aimed to determine genetic alternations in metastatic OSCC patients to identify genomic changes occurred at metastatic phase of the disease. Material and MethodsThe Illumina CytoSNP-12 Array was used to determine copy number variations in OSCC cancer genome. Hybridization procedures were performed according to the manufacturer procedures (Illumina). Arrays were scanned on iScan System (Illumina). Data were analyzed using Illumina Genotyping module of Genome Studio software (version 1.2, Illumina). Multiple CNV algorithms and copy number alternations were accessed by Genome Studio. CNVs in whole genome were investigated by using a chromosomal heat map. ResultsWe reported that gains in 8q21.11-ter, 9p21.3, 13q14.11-ter, 13q13.3-ter and losses in 5q14.3-ter, 5q35 and 17p13.3–12 were associated with the development of OSCC. In addition, we also detected that deletion in 2q33.2-ter and 2q35–37.3 regions were also associated with OSCC metastasis process. ConclusionsOur results were also showed that gains in 11q13.3-q13.4 and 2q13.2 chromosomal regions could promote the metastatic OSCC process. We believe that results of the study will help to find new biomarkers for diagnosis at later stage of OSCC.

WPOI-5: Accurately Identified at Intraoperative Consultation and Predictive of Occult Cervical Metastases.

Frozen section analysis of oral cancer specimens is ideal for assessing margin distances and depth of invasion (DOI); the latter impacts intraoperative decisions regarding elective neck dissection (END). Here, we show that intraoperative determination of worst pattern of invasion (WPOI), specifically WPOI-5, has a high level of accuracy. This relates to our demonstration herein that WPOI-5 predicts occult cervical metastases (OCM) for pT1 oral squamous carcinoma (OSC). The presence of OCM was correlated with WPOI in 228 patients with primary T1/T2/cN0 OSC undergoing resection and END. Concordance between intraoperative and final pathology WPOI determination was assessed on 51 cases of OSC. WPOI-5 predicts OCM in pT1 patients, compared with WPOI-4/WPOI-3 (p < 0.0001). Most pT1 WPOI-5 tumors had DOI of 4-5mm (24/59 or 40.7%). Only two pT1 WPOI-5 tumors had DOI < 4mm (3.0 and 3.5mm). If END were performed in this pT1 cohort for all WPOI-5 OSC patients regardless of DOI, OR all OSC patients with DOI ≥ 4mm regardless of WPOI, then no OCM would be missed (p = 0.017, 100% sensitivity, 29% specificity, 77% positive predictive value, 23% negative predictive value). With respect to intraoperative WPOI-5 determination, the accuracy, sensitivity, and specificity was 92.16, 73.33, and 100.0%, respectively. DOI ≥ 4mm is the dominant predictor of OCM. For the rare WPOI-5 OSC with DOI < 4mm, it is reasonable to suggest that surgeons perform END. WPOI-5 may be accurately determined intraoperatively. As microscopic instruction is needed to accurately assess WPOI-5, a teaching link is included in this manuscript.

Incidence and impact of skip metastasis in the neck in early oral cancer: Reality or a myth?

ObjectivesVarying evidence exists surrounding skip metastasis in clinically node-negative oral cancer. We aim to report its incidence, identify factors predicting its presence, and determine survival outcomes to possibly decide “adequacy” of neck dissection for this select group. MethodsA review of audit records (clinical and pathologic) was undertaken to identify early stage (cT1/T2 cN0) oral squamous carcinoma patients operated between January 2016 and December 2020. Skip metastasis was defined as presence of nodal metastases distant from the primary tumour and at a lower level without metastases at levels above this. The factors predicting its presence were determined using chi-square and multivariate tests. Survival analysis was performed using Kaplan-Meier and regression hazard models. ResultsOccult metastasis rate was 17.9 % among 761 patients, with 47.1 % travelling to level I. Skip metastasis at level III was seen in 1.2 % and level IV was 0.3 %. Presence of PNI predicted the presence of skip metastasis at level III (p = 0.041, OR-0.241, 95 %CI 0.062–0.942) and no significant factors were identified for level IV. The 5-year disease-free survival was significantly reduced with isolated level IV metastasis (50 % vs 79.6 %, p = 0.017). Isolated occult metastasis at level Ia was in 0.3 %, all from tongue cancers. Their presence did not impact the 5-year overall survival (100 % vs 90.7 %, p = 0.628) and disease-specific survival (100 % vs 92.5 %, p = 0.675). ConclusionThe negligible incidence of skip metastasis to level IV in a clinically node negative neck suggests that clearance up to level III might be adequate in a large majority of patients.

DMA, a Small Molecule, Increases Median Survival and Reduces Radiation-Induced Xerostomia via the Activation of the ERK1/2 Pathway in Oral Squamous Cell Carcinoma.

Simple SummaryRadiotherapy is commonly used to treat the majority of patients with head and neck cancers. Salivary glands in the radiation field are affected by this procedure. The purpose of this study was to investigate the role of DMA as a radiomodulator to evaluate the real possibilities of reducing the incidence and severity of xerostomia in head and neck squamous cell carcinoma (HNSCC) patients. The effect of DMA on the response of head and neck cell lines in the presence of radiation was analyzed using clonogenic survival and xenograft tumor assays for cell line-derived xenografts as well as patient-derived xenografts. The combinatorial treatment of DMA along with radiation influenced the migration of cells. The changes associated with migration were observed through a microscope and wound healing assay. In addition, the stimulated saliva was measured. Tissue sections were analyzed using immunohistochemistry to stain for molecular markers of cell proliferation and porin channels.Survival, recurrence, and xerostomia are considerable problems in the treatment of oral squamous carcinoma patients. In this study, we investigated the role of DMA (5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxyphenyl)5″benzimidazoyl]benzimidazole) as a salivary gland cytoprotectant in a patient-derived xenograft mouse model. A significant increase in saliva secretion was observed in the DMA-treated xenograft compared to radiation alone. Repeated doses of DMA with a high dose of radiation showed a synergistic effect on mice survival and reduced tumor growth. The mean survival rate of tumor-bearing mice was significantly enhanced. The increased number of Ki-67-stained cells in the spleen, intestine, and lungs compared to the tumor suggests DMA ablates the tumor but protects other organs. The expression of aquaporin-5 was restored in tumor-bearing mice injected with DMA before irradiation. The reduced expression of αvβ3 integrin and CD44 in DMA alone and DMA with radiation-treated mice suggests a reduced migration of cells and stemness of cancer cells. DMA along with radiation treatment results in the activation of the Ras/Raf/MEK/ERK pathway in the tumor, leading to apoptosis through caspase upregulation. In conclusion, DMA has strong potential for use as an adjuvant in radiotherapy in OSCC patients.

Establishment and Identification of Patient-Derived Xenograft Model for Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is the most common head and neck malignancy with high morbidity and mortality. Currently, platinum-based chemotherapy is the conventional chemotherapy regimen for patients with oral squamous cell carcinoma. However, due to the heterogeneity of tumors and individual differences of patients, chemotherapy regimens lacking individualized evaluation of tumor patients are often less effective. Therefore, personalized tumor chemotherapy is one of the effective methods for the future treatment of malignant tumors. The patient-derived xenograft model is a relatively new tumor xenograft model that relies on immunodeficient mice. This model can better maintain various histological characteristics of primary tumor grafts, such as pathological structural features, molecular diversity, and gene expression profiles. Therefore, the patient-derived xenograft model combined with drug screening technology to explore new tumor chemotherapy is the critical research direction for future tumor treatment. This study successfully established the patient-derived xenograft model of oral squamous cell carcinoma. It was verified by hematoxylin-eosin staining and immunohistochemistry that the constructed patient-derived xenograft model retained the pathological and molecular biological characteristics of primary tumors. Our patient-derived xenograft model can be used further to study the oncological characteristics of oral squamous carcinoma and can also be applied to personalize the treatment of oral squamous carcinoma patients, providing a practical resource for screening chemotherapy drugs.

Deregulation of hsa_circ_0001971/miR-186 and hsa_circ_0001874/miR-296 signaling pathways promotes the proliferation of oral squamous carcinoma cells by synergistically activating SHP2/PLK1 signals

It has been demonstrated that circ_0001874 and circ_0001971 are potential biomarkers for the diagnosis of oral squamous carcinoma (OSCC). MiR-186 was reported to serve as a tumor suppressor in OSCC, and the down-regulation of miR-186 was reported to lead to higher expression of oncogenic factor SHP2 and the activation of growth promoting signaling. In this study, we aimed to explore the possible molecular role of circ_0001874 and circ_0001971 signaling in the pathogenesis of OSCC. RT-qPCR, Western blot, online bioinformatics tools and luciferase assay were utilized to study the molecular signaling pathways of circ_0001874 and circ_0001971. MTT assay and FCM assay were performed to investigate the synergistic effect of circ_0001971 and circ_0001874 on cell proliferation and apoptosis. By observing the effect of different miRNAs on the levels of circ_0001847 and circ_0001971, it was identified that circ_0001847 and circ_0001971 respectively sponged the expression of miR-296 and miR-186 via binding to these miRNAs. Also, SHP2 mRNA and PLK1 mRNA were respectively targeted by miR-186 and miR-296-5p. We also established two signaling pathways, i.e., circ_0001971/miR-186/SHP2 and circ_0001874/miR-296-5p/PLK1, and validated the synergistic effect of circ_0001971 and circ_0001874 via observing their positive effect on cell proliferation and negative effect on cell apoptosis. The expression of miR-186 and miR-296-5p was generally lower in saliva of OSCC patients compared with that in OLK patients, while the expression of miR-186 and miR-296-5p was specifically up-regulated in saliva of OSCC patients. In conclusion, the finding of this study demonstrated that the relative level of hsa_circ_0001971 and hsa_circ_0001874 were different in the saliva of OSCC patients and could be used as predictive biomarkers for the development of OSCC. Furthermore, oncogenic effects of hsa_circ_0001971 and hsa_circ_0001874 in the development of OSCC might be, at least partially, mediated by its downstream signaling pathways including hsa_circ_0001971/microRNA-186/SHP2 and hsa_circ_0001874/microRNA-297/PLK1.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Predictive modelling of level IIb lymph node metastasis in oral squamous cell carcinoma

The aim of the present study was to examine the conditions, characteristics, and risk factors of level IIb lymph node metastases in oral squamous cell carcinoma and to formulate surgical criteria for level IIb lymph node dissection. We analyzed clinical and pathological records for 541 oral squamous carcinoma patients in relation to level IIb metastasis. Univariate and multivariate analyses were performed to detect risk factors for level IIb lymph node metastasis; a predictive model was built based on multivariate analysis and tested in a validation group. Univariate and multivariate analyses using the training group indicated that level IIa metastasis and Lymphovascular permeation (LVP) were two independent risk factors for level IIb lymph node metastasis. This model was built and tested in a validation group, the area under the curve being 0.697 (P < .0.001). The model’s sensitivity was 66.7% and specificity was 77.4%. Nomogram incorporating validated variables was developed for level IIb metastasis prediction. Expected survival probabilites were analysed to specify significance of model's variable on patients’ overall survival and recurrence. Level IIb dissection should be performed in patients with level IIa metastasis and LVP. However, thorough consideration of the oncologic safety of omitting level IIb dissection is compulsory.

Salivary Cytokines as Biomarkers for Oral Squamous Cell Carcinoma: A Systematic Review.

The prognosis of patients with oral squamous carcinoma (OSCC) largely depends on the stage at diagnosis, the 5-year survival rate being approximately 30% for advanced tumors. Early diagnosis, including the detection of lesions at risk for malignant transformation, is crucial for limiting the need for extensive surgery and for improving disease-free survival. Saliva has gained popularity as a readily available source of biomarkers (including cytokines) useful for diagnosing specific oral and systemic conditions. Particularly, the close interaction between oral dysplastic/neoplastic cells and saliva makes such fluid an ideal candidate for the development of non-invasive and highly accurate diagnostic tests. The present review has been designed to answer the question: “Is there evidence to support the role of specific salivary cytokines in the diagnosis of OSCC?” We retrieved 27 observational studies satisfying the inclusion and exclusion criteria. Among the most frequent cytokines investigated as candidates for OSCC biomarkers, IL-6, IL-8, TNF-α are present at higher concentration in the saliva of OSCC patients than in healthy controls and may therefore serve as basis for the development of rapid tests for early diagnosis of oral cancer.

Incidence and impact of dysplasia at final resection margins in cancers of the oral cavity

Background The literature often cites margin status as being free, close or involved by tumor but there is very sparse evidence of the relevance of dysplasia at resection margin. Aims/Objectives: To compare the impact of dysplasia at final resection margin (D-FRM) and other margin statuses on overall survival (OS) and disease free survival (DFS). Methods This is a retrospective review of 1700 treatment naïve oral squamous carcinoma patients who underwent surgery between January 2012 and December 2015. The study arm consisted of patients with dysplasia at final resection margin (D-FRM). Each of these patients were double propensity matched to obtain positive (P-FRM), close (C-FRM) and free final resection margins (F-FRM). Results There was no hazard discrimination in survival among the D-FRM and C-FRM (vs F-FRM; p-0.597, HR-1.207 (0.621–2.346) and p-0.075, HR-1.594 (0.947–2.684), respectively). A decreasing survival trend was observed as the grade and number of D-FRM margins increased. Although not significant, the addition of adjuvant therapy for D-FRM showed a trend towards improved survival outcomes compared to C-FRM, especially with chemotherapy. Conclusion and relevance: The presence of D-FRM, irrespective of grade, had a similar impact on survival as C-FRM and underscores the possible need for treatment intensification.

Prognostic value of serum MIR4435-2HG in oral squamous cell carcinoma

Objective To investigate the value of serum MIR4435-2HG level in the diagnosis and prognosis of oral squamous cell carcinoma. Methods This study was a retrospective case-control study. Five hundred and eighteen samples of oral squamous carcinoma of patients with head and neck squamous cell carcinoma in the cancer genome atlas project (TCGA) database, with long noncoding RNA MIR4435-2HG expression. The median was the boundary, and the patients were divided into high expression group and low expression group, and the 5-year disease-free survival rate and overall survival rate of the two groups were compared. Serum samples from 82 patients with oral squamous cell carcinoma who were admitted to the Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Huzhou Univerisity from January 2012 to January 2015 were enrolled to verify the prognostic value of MIR4435-2HG. Bioinformatics is used to predict the biological processes involved in MIR4435-2HG. Use the SPSS 23.0 to set the optimal diagnostic and prognostic cutoff for the MIR4435-2HG. Results A total of 518 oral squamous carcinoma patients in the TCGA database showed that the 5-year overall survival rate of the MIR4435-2HG high expression group [43.2% (112/259)] was significanthy lower than that of the MIR4435-2HG low expression group [51.7% (134/259)] (P 0.05). The lymph node metastasis rate of the MIR4435-2HG high expression group was significantly higher than that of the low expression group [12.9% (8/62)] (P<0.05). The histological grade of the high expression group [80.0 % (16/20)] was significantly lower than that of the low expression group [24.2 % (15/62)] (χ2=20.030, P<0.05). The results of bioinformatics analysis indicated that the biological functions of MIR4435-2HG target gene were mainly enriched in protein metabolism, processing of rRNA in nucleolus and cytoplasm, SEMA4D induced cell migration process, and mitochondrial translation process. Conclusions Serum MIR4435-2HG can be used as a potential prognostic marker for oral squamous cell carcinoma. Key words: Oral sprays; Diagnosis; Prognosis; Biomarker

Worst pattern of invasion - type 4 (WPOI-4) and Lymphocyte host response should be mandatory reporting criteria for oral cavity squamous cell carcinoma: A re-look at the American Joint Committee of Cancer (AJCC) minimum dataset.

A proportion of early-stage node-negative oral squamous carcinoma patients fail despite complete surgical resection. Adjuvant treatment in early oral cancer is controversial and is often individualized based on stage, depth, and margin status. We reviewed various histological markers in pT1/T2N0 cases, resected upfront with elective nodal dissection, with an emphasis on tumor-tissue interface characteristics of the worst pattern of invasion (WPOI), tumor cell nest size (sCNS), budding and lymphocytic host response (LHR), to assess their prognostic significance. Archived blocks of 95 cases were reviewed. Tumor stage, grade, size, depth of invasion, lymphovascular, and perineural invasion, WPOI, LHR, sCNS, and tumor bud (single cells or <5 cell clusters) score were recorded. Prognostic significance was statistically analyzed using SPSS software version 20. Depth of invasion (P = 0.008), WPOI- 4 and 5 (P = 0.033), sCNS (<5 cells) at tumor interface (P = 0.010), high bud count (≥3 buds/40 × hpf) (P = 0.021) and poor LHR (P = 0.019) correlated significantly with poor disease-free survival on univariate analysis. However, on multivariate analysis only LHR and WPOI-4 (that is presence of small cell nests or buds) were significant, with high hazard ratio of 4.351 (95% CI 1.290-14.676, P = 0.018) and 5.019 (95% CI 1.212-20.789, P = 0.026), respectively. We propose mandatory reporting of WPOI-4 at the tumor interface and absence of LHR, as significant markers of poor prognosis in early-stage oral cavity squamous carcinoma.

The prevalence of oral high-risk HPV infection in Indonesian oral squamous cell carcinoma patients.

The objective of this study was to report the integrated observations of high-risk HPV-related oral squamous carcinoma (OSCC) at our national referral center for cancer, the Dharmais National Cancer Hospital (DNCH), Jakarta, from 2003 to 2013. Seventy-eight formalin-fixed paraffin-embedded specimens obtained from OSCC cases were collected from 2003 to 2013 DNCH archives and were included in this high-risk HPV (HR-HPV) study. Seventy-nine DNA samples from the normal oral mucosa of healthy individuals were obtained from the Oral Biology Laboratory DNA archives from 2001 to 2005. Glyceraldehyde 3-phosphate dehydrogenase was used as a control to ensure the DNA integrity for the subsequent HPV DNA PCR detection. High-risk HPV16/18 DNA amplification was conducted by nested PCR using two pairs of primers that were designed specifically to identify the region of gene L1 HPV16 and the HPV16/18 region. A high prevalence of HPV16/18 was detected in OSCC cases (17.9%). HPV18 occurred more often than HPV16 (86%) among OSCC patients who were HPV positive. This result supports high HPV18 prevalence among Indonesian cervical cancer patients studied in 1995 and 2006. The prevalence of high-risk HPV remains low in the normal Indonesian population (3.8%), but HPV16 is consistently more frequently detected in non-cancer populations.

The Impact of Peritumoral Retraction Clefting & Intratumoral Eosinophils on Overall Survival in Oral Squamous Carcinoma Patients.

This retrospective study aimed to investigate the impact of peritumoral retraction clefts (RC) and tumor-associated tissue eosinophilia (TATE) as predictors of overall survival (OS) in oral squamous cell carcinoma (OSCC) patients. Their relationships with tumor-factors were also examined. Eighty-seven OSCC cases (pTNM: I+II/III+IV; 32/55), post-curative surgery, comprised the study cohort. Three observers independently estimated the percent RC semi-quantitatively in the selected tumor sections. Additionally, stromal eosinophils were counted in ten consecutive high-power fields of intratumoral and peritumoral regions to evaluate the corresponding TATE. The percent RC ranged between 0% -90% (Mean±SD: 16±24%; Median: 5%). The stromal eosinophils were greater in peritumoral as compared to intratumoral region. The events of death and tumor recurrence were reached in 16 (18.4%) and 36 (41%) cases respectively. The 3-years OS was 69% [Median OS: 1880days; Mean follow up: 471(Range; 36-1880) days]. Increased percent RC exhibited relationship with pathologic stage (pTNM III&IV), primary tumor (pT III&IV), tumor depth>4mm and categorical tumor recurrence. Additionally, peritumoral eosinophilic infiltrates increased with increasing tumor depths and muscle invasion. Kaplan-Meier curves revealed significantly reduced OS in OSCC cases exhibiting: increased percent RC (>2.5%), mild -moderate/absent intratumoral TATE (versus intense TATE) or categorical tumor recurrence. In subsequent multivariate tests, all the three variables retained significance. Additionally, intraclass correlation coefficient demonstrated acceptable internal consistency for the observers who estimated percent RC. In conclusion, RC and intratumoral TATE proved to be independent predictors of OS in our OSCC cohort. Additionally, increased percent RC pointed towards aggressive tumor behaviour.

A semi-quantitative World Health Organization grading scheme evaluating worst tumor differentiation predicts disease-free survival in oral squamous carcinoma patients.

We investigated World Health Organization (WHO) grading and pattern of invasion based histological schemes as independent predictors of disease-free survival, in oral squamous carcinoma patients. Tumor resection slides of eighty-seven oral squamous carcinoma patients [pTNM: I&II/III&IV-32/55] were evaluated. Besides examining various patterns of invasion, invasive front grade, predominant and worst (highest) WHO grade were recorded. For worst WHO grading, poor-undifferentiated component was estimated semi-quantitatively at advancing tumor edge (invasive growth front) in histology sections. Tumor recurrence was observed in 31 (35.6%) cases. The 2-year disease-free survival was 47% [Median: 656; follow-up: 14-1450] days. Using receiver operating characteristic curves, we defined poor-undifferentiated component exceeding 5% of tumor as the cutoff to assign an oral squamous carcinoma as grade-3, when following worst WHO grading. Kaplan-Meier curves for disease-free survival revealed prognostic association with nodal involvement, tumor size, worst WHO grading; most common pattern of invasion and invasive pattern grading score (sum of two most predominant patterns of invasion). In further multivariate analysis, tumor size (>2.5cm) and worst WHO grading (grade-3 tumors) independently predicted reduced disease-free survival [HR, 2.85; P=0.028 and HR, 3.37; P=0.031 respectively]. The inter-observer agreement was moderate for observers who semi-quantitatively estimated percentage of poor-undifferentiated morphology in oral squamous carcinomas. Our results support the value of semi-quantitative method to assign tumors as grade-3 with worst WHO grading for predicting reduced disease-free survival. Despite limitations, of the various histological tumor stratification schemes, WHO grading holds adjunctive value for its prognostic role, ease and universal familiarity.

Induction chemotherapy for squamous cell carcinomas of the oral cavity: a cumulative meta-analysis

Abstract Induction chemotherapy (ICT) is a controversial treatment for head and neck squamous cell carcinomas (HNSCC). Despite numerous randomized controlled trials (RCTs), a majority do not have enough statistical power alone to conclude ICT’s treatment value among oral squamous carcinoma patients (OSCC) since many addressed HNSCC as one entity instead of by specific subtypes. By performing a systematic review and cumulative meta-analysis, we aim to determine the benefits of ICT in OSCC therapy. A literature search identified for RCTs comparing OSCC patients who received ICT against those without. Log-hazard ratio, and relative risk were used for comparison. Heterogeneity was determined using the I 2 statistic package. The primary endpoint was overall survival (OS), followed by disease-free survival (DFS), locoregional recurrence (LRR) and distant metastasis (DM) as secondary endpoints. RESULTS: 27 randomized trials were included for analysis (n = 2872 patients). The shortest median follow-up was 15 months whereas the longest was 11.5 years. ICT does not improve OS (HR = 0.947, 95% CI 0.85–1.05, p = 0.318), DFS (RR = 1.05, 95% CI 0.92–1.21, p = 0.462) and DM (RR = 0.626, CI 95% 0.361–1.086, p = 0.096) compared to locoregional treatment alone. However, there was a significant improvement to LRR (RR = 0.778, 95% CI 0.622–0.972, p = 0.027). There is no evidence ICT improves survival outcomes for OSCC patients. However, ICT reduces locoregional recurrence of OSCC, which may need further verification.

Skewed Distribution of IL-7 Receptor-α-Expressing Effector Memory CD8+ T Cells with Distinct Functional Characteristics in Oral Squamous Cell Carcinoma

CD8+ T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8+ T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7−CD45RA−CD8+ T cells and CCR7− CD45RA+CD8+ T cells negatively correlated with each other. A significantly higher frequency of CD127lo CCR7−CD45RA−CD8+ T cells or CCR7−CD45RA+CD8+ T cells among total CD8+ T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127hi CCR7−CD45RA−CD8+ T cells or CCR7−CD45RA+CD8+ T cells maintained significantly higher IFN-γ, IL-2 productivity and ex vivo proliferative capacity, while the CD127lo CCR7−CD45RA−CD8+ T cells or CCR7−CD45RA+CD8+ T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7−CD45RA+CD8+ T cells to CCR7−CD45RA−CD8+ T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127lo CCR7−CD45RA−CD8+ T cells and CCR7−CD45RA+CD8+ T cells are functionally similar CD8+ T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8+ effector memory balance toward CCR7−CD45RA+CD8+ T cells is associated with OSCC progression.

OP002: Identification of seven miRNAs as potential biomarkers for oral lichen planus and oral squamous cell carcinoma – A pilot study

Objective The aim of this pilot study was to identify miRNAs in the exfoliated cells of oral mucosa, tissues and peripheral blood as potential novel biomarkers for the detection of OLP and its malignant transformation. Background So far there have been no biomarkers with reasonable sensitivity and specificity for the early detection of oral lichen planus (OLP) and its malignant transformation, which is oral squamous carcinoma (OSCC). The transcriptome sequencing (RNA-seq) analysis provides better understanding of molecular pathways leading to cancer, which in turn allows designing the effective strategies for diagnosis, therapeutic intervention and prediction of therapeutic outcome. As part of the differential analysis results, microRNAs (miRNAs) as biomarkers has opened new opportunities for the diagnosis of OLP and OSCC, primarily because they exhibit tumor-specific expression profiles and have been commonly observed in OLP patients, OSCC patients and healthy controls. Methodology/principal findings To investigate the differential transcriptome, we have applied next generation RNA-Seq method to tumors, OLP tissues and matched normal tissues from two patients with both OLP and OSCC. Among the huge differential transcriptome analysis data, seven miRNAs were chosen for the further biomarker identification. Quantitative real-time PCR (qRT-PCR) was used for validation of them in 11 OSCC patients, 29 OLP patients and 20 controls from the general population. The samples were prepared into three major groups, which is exfoliated cells of oral mucosa, tissues and peripheral blood, each of these were subdivided, leading to eight groups in all. Comparison among these groups’ qPCR results reveals significantly different expression of certain miRNAs in different morbid states. Conclusions/significance The results of this pilot study show that these seven miRNAs might be potential minimally or even non-invasive biomarkers for the diagnosis of OLP and might have relationship with its malignant transformation. In addition, our results support the concept of using the exfoliated cells of oral mucosa for biomarker discovery. However, for early detection of malignant transformation the feasibility of these seven miRNAs alone or in combination with other biomarkers needs to be analyzed in a prospective study.

Arginine deprivation as a new treatment strategy for head and neck cancer

Arginine is a nonessential amino acid which can regulate tumor growth. Argininosuccinate synthetase (ASS) is the rate-limiting enzyme for de novo arginine production. The expression pattern of ASS and the feasibility of arginine deprivation therapy in head and neck cancer have not been investigated. The growth-inhibitory effect of arginine deprivation therapy was assessed either by proliferation assay with head and neck cancer cells cultured in arginine-free medium, or by tetrazolium/formazan dye assay with cells treated with an arginine-depleting drug (arginine deiminase, ADI). The tumor ASS status of 73 oral squamous carcinoma (OSCC) patients was then evaluated immunohistochemically and subsequently correlated with the corresponding clinicopathological parameters. Head and neck cancer cells cultured in arginine-free medium either completely stopped proliferating, or proliferated minimally. In addition, ADI treatment inhibited the growth of all 8 head and neck cancer cell lines to different degrees. Although cellular ASS level did not correlate well with ADI-sensitivity among these cell lines, knockdown of endogenous ASS potentiated the growth-inhibitory effect of ADI in each individual cell line (FaDu and OEC-M1). In multivariable analysis, high tumor ASS level independently predicted an unfavorable disease-free survival in OSCC patients. High tumor ASS status is an independent variable predicting a poor disease-free survival in OSCC patients. Arginine deprivation therapy may potentially be used as a new approach to treat head and neck cancer.