Preoperative Circulating Tumor HPV DNA and Oropharyngeal Squamous Cell Disease.

Importance: The utility of preoperative circulating tumor tissue-modified viral human papillomavirus DNA (TTMV-HPV DNA) levels in predicting human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) disease burden is unknown. Objective: To determine if preoperative circulating tumor HPV DNA (ctHPVDNA) is associated with disease burden in patients with HPV+ OPSCC who have undergone transoral robotic surgery (TORS). Design, Setting, and Participants: This cross-sectional study comprised patients with HPV+ OPSCC who underwent primary TORS between September 2021 and April 2023 at one tertiary academic institution. Patients with treatment-naive HPV+ OPSCC (p16-positive) and preoperative ctHPVDNA levels were included, and those who underwent neck mass excision before ctHPVDNA collection were excluded. Main Outcomes and Measures: The main outcome was the association of increasing preoperative ctHPVDNA levels with tumor size and lymph node involvement in surgical pathology. The secondary outcome was the association between preoperative ctHPVDNA levels and adverse pathology, which included lymphovascular invasion, perineural invasion, or extranodal extension. Results: A total of 70 patients were included in the study (65 men [93%]; mean [SD] age, 61 [8] years). Baseline ctHPVDNA levels ranged from 0 fragments/milliliter of plasma (frag/mL) to 49 452 frag/mL (median [IQR], 272 [30-811] frag/mL). Overall, 58 patients (83%) had positive results for ctHPVDNA, 1 (1.4%) had indeterminate results, and 11 (15.6%) had negative results. The sensitivity of detectable ctHPVDNA for identifying patients with pathology-confirmed HPV+ OPSCC was 84%. Twenty-seven patients (39%) had pathologic tumor (pT) staging of pT0 or pT1, 34 (49%) had pT2 staging, and 9 patients (13%) had pT3 or pT4 staging. No clinically meaningful difference between detectable and undetectable preoperative ctHPVDNA cohorts was found for tumor size or adverse pathology. Although the median preoperative ctHPVDNA appeared to be higher in pT2 through pT4 stages and pN1 or pN2 stages, effect sizes were small (pT stage: η2, 0.002 [95% CI, -1.188 to 0.827]; pN stage: η2, 0.043 [95% CI, -0.188 to 2.600]). Median preoperative log(TTMV-HPV DNA) was higher in active smokers (8.79 [95% CI, 3.55-5.76]), compared with never smokers (5.92 [95% CI, -0.97 to 1.81]) and former smokers (4.99 [95% CI, 0.92-6.23]). Regression analysis did not show an association between tumor dimension or metastatic lymph node deposit size and preoperative log(TTMV-HPV DNA). After univariate analysis, no association was found between higher log(TTMV-HPV DNA) levels and adverse pathology. Conclusions and Relevance: In this cross-sectional study, preoperative ctHPVDNA levels were not associated with disease burden in patients with HPV+ OPSCC who underwent TORS.

Development and validation of nomogram models for predicting postoperative prognosis of early-stage laryngeal squamous cell carcinoma.

BACKGROUND: We aimed to investigate the postoperative prognosis in patients with early-stage laryngeal squamous cell carcinoma (LSCC) in association with the preoperative blood markers and clinicopathological characteristics and to develop nomograms for individual risk prediction. METHODS: The clinical data of 353 patients with confirmed early-stage LSCC between 2009 and 2018 were retrospectively retrieved from the First Affiliated Hospital with Nanjing Medical University. All patients were randomly divided into the training and testing groups in a 7:3 ratio. Univariate and multivariate analyses were performed, followed by the construction of nomograms to predict recurrence-free survival (RFS) and overall survival (OS). Finally, the nomograms were verified internally, and the predictive capability of the nomograms was evaluated and compared with that of tumour T staging. RESULTS: Univariate and multivariate analyses identified platelet counts (PLT), fibrinogen (FIB), and platelet to lymphocyte ratio (PLR) were independent factors for RFS, and FIB, systemic immune-inflammation index (SII), and haemoglobin (HGB) were independent prognostic factors for OS. The nomograms showed higher predictive C-indexes than T staging. Furthermore, decision curve analysis (DCA) revealed that the net benefit of the nomograms' calculation model was superior to that of T staging. CONCLUSIONS: We established and validated nomograms to predict postoperative 1-, 3- and 5-year RFS and OS in patients with early-stage LSCC based on significant blood markers and clinicopathological characteristics. These models might help clinicians make personalized treatment decisions.

Unveiling the Potential of Serum MiR-483-5p: A Promising Diagnostic and Prognostic Biomarker in OLP and OSCC Patients by In silico Analysis of Differential Gene Expression.

BACKGROUND: Oral squamous cell carcinoma (OSCC) and oral lichen planus (OLP) are two separate conditions affecting the mouth and result in varying clinical outcomes and levels of malignancy. Achieving early diagnosis and effective therapy planning requires the identification of reliable diagnostic biomarkers for these disorders. MicroRNAs (miRNAs) have recently received attention as powerful biomarkers for various illnesses, including cancer. In particular, miR-483-5p is a promising diagnostic and prognostic biomarker in various cancers. Therefore, this study aimed to investigate the role of serum miR-483-5p in the diagnosis and prognosis of OLP and OSCC patients by in silico analysis of differential gene expression. METHODS: GSE23558 and GSE52130 data sets were selected, and differential gene expression analysis was performed using microarray data from GSE52130 and GSE23558. The analysis focused on comparing OLP and OSCC samples with normal samples. The genes intersected through the differential gene expression analysis were then extracted to determine the overlapping genes among the upregulated or downregulated DEGs. The downregulated genes among the DEGs were subsequently imported into the miRWalk database to search for potential target genes of miRNA 483-5p that lacked validation. To gain insight into the biological pathways associated with the DEGs, we conducted pathway analysis utilizing tools, such as Enrichr. Additionally, the cellular components associated with these DEGs were investigated by analyzing the String database. On the other hand, blood serum samples were collected from 35 OSCC patients, 34 OLP patients, and 34 healthy volunteers. The expression level of miR-483-5p was determined using quantitative reverse transcription polymerase chain reaction (RT-qPCR). The Kruskal-Wallis test was utilized to investigate the considerable correlation. Moreover, this study explored the prognostic value of miR-483-5p through its association with clinicopathological parameters in OSCC patients. RESULTS: The results showed that serum expression of miR-483-5p was considerably higher in OSCC patients compared to OLP patients and healthy controls (p 0.0001) and that this difference was statistically significant. Furthermore, elevated miR-483-5p expression was associated with tumor size, lymph node metastasis, and stage of tumor nodal metastasis in OSCC patients (p 0.001, p 0.038, and p 0.0001, respectively). In silico analysis found 71 upregulated genes at the intersection of upregulated DEGs and 44 downregulated genes at the intersection of downregulated DEGs, offering insight into the potential underlying mechanisms of miR-483-5p's engagement in OSCC and OLP. The majority of these DEGs were found to be involved in autophagy pathways, but DEGs involved in the histidine metabolism pathway showed significant results. Most of these DEGs were located in the extracellular region. After screening for downregulated genes that were invalidated, miRNA 483-5p had 7 target genes. CONCLUSION: This study demonstrates the potential of serum miR-483-5p as a promising diagnostic and prognostic biomarker in OSCC and OLP patients. Its upregulation in OSCC patients and its association with advanced tumor stage and potential metastasis suggest the involvement of miR-483-5p in critical signaling pathways involved in cell proliferation, apoptosis, and cell cycle regulation, making it a reliable indicator of disease progression. Nevertheless, additional experimental studies are essential to validate these findings and establish a foundation for the advancement of targeted therapies and personalized treatment approaches.

An Insight into Survivin in Relevance to Hematological, Biochemical and Genetic Characteristics in Tobacco Chewers with Oral Squamous Cell Carcinoma.

BACKGROUND: Survivin is an inhibitor of apoptosis protein (IAP), encoded by the Baculoviral IAP Repeat Containing 5 (BIRC5) gene located on q arm (25.3) on chromosome 17. It is expressed in various human cancers and involved in tumor resistance to radiation and chemotherapy. The genetic analysis of the BIRC5 gene and its protein survivin levels in buccal tissue related to oral squamous cell carcinoma (OSCC) in South Indian tobacco chewers has not been studied. Hence, the study was designed to quantify survivin in buccal tissue and its association with pretreatment hematological parameters and to analyze the BIRC5 gene sequence. METHOD: In a single centric case control study, buccal tissue survivin levels were measured by ELISA. A total of 189 study subjects were categorized into Group 1 (n = 63) habitual tobacco chewers with OSCC, Group 2 (n = 63) habitual tobacco chewers without OSCC, and Group 3 (n = 63) healthy subjects as control. Retrospective hematological data were collected from Group 1 subjects and statistically analyzed. The BIRC5 gene was sequenced and data were analyzed using a bioinformatics tool. RESULTS: Survivin protein mean ± SD in Group 1 was (1670.9 ± 796.21 pg/mL), in Group 2 it was (1096.02 ± 346.17 pg/mL), and in Group 3 it was (397.5 ± 96.1 pg/mL) with significance (p < 0.001). Survivin levels showed significance with cut-off levels of absolute monocyte count (AMC), neutrophil/lymphocyte ratio (NLR), and lymphocyte/monocyte ratio (LMR) at (p = 0.001). The unique variants found only in OSCC patients were T → G in the promoter region, G → C in exon 3, C → A, A → G, G → T, T → G, A → C, G → A in exon 4, C → A, G → T, G → C in the exon 5 region. CONCLUSIONS: The tissue survivin level increased in OSCC patients compared to controls; pretreatment AMC, LMR, and NLR may serve as add-on markers along with survivin to measure the progression of OSCC. Unique mutations in the promoter and exons 3-5 were observed in sequence analysis and were associated with survivin concentrations.

Different inflammatory blood markers correlate with specific outcomes in incident HPV-negative head and neck squamous cell carcinoma: a retrospective cohort study.

BACKGROUND: Inflammatory blood markers have been associated with oncological outcomes in several cancers, but evidence for head and neck squamous cell carcinoma (HNSCC) is scanty. Therefore, this study aims at investigating the association between five different inflammatory blood markers and several oncological outcomes. METHODS: This multi-centre retrospective analysis included 925 consecutive patients with primary HPV-negative HNSCC (median age: 68 years) diagnosed between April 2004 and June 2018, whose pre-treatment blood parameters were available. Neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), lymphocyte to monocyte ratio (LMR), systemic inflammatory marker (SIM), and systemic immune-inflammation index (SII) were calculated; their associations with local, regional, and distant failure, disease-free survival (DFS), and overall survival (OS) was calculated. RESULTS: The median follow-up was 53 months. All five indexes were significantly associated with OS; the highest accuracy in predicting patients' survival was found for SIM (10-year OS = 53.2% for SIM < 1.40 and 40.9% for SIM ≥ 2.46; c-index = 0.569) and LMR (10-year OS = 60.4% for LMR ≥ 3.76 and 40.5% for LMR < 2.92; c-index = 0.568). While LMR showed the strongest association with local failure (HR = 2.16; 95% CI:1.22-3.84), PLR showed the strongest association with regional (HR = 1.98; 95% CI:1.24-3.15) and distant failure (HR = 1.67; 95% CI:1.08-2.58). CONCLUSION: Different inflammatory blood markers may be useful to identify patients at risk of local, regional, or distant recurrences who may benefit from treatment intensification or intensive surveillance programs.

Sphingolipids signature in plasma and tissue as diagnostic and prognostic tools in oral squamous cell carcinoma.

Enzymes related to sphingolipids metabolism has been suggested as altered in oral squamous cell carcinoma (OSCC). However, clinical relevance of diverse sphingolipids in OSCC is not fully known. Here, we evaluated sphingolipidomics in plasma and tumor tissues as a tool for diagnosis/prognosis in OSCC patients. Plasma was obtained from 58 controls and 56 OSCC patients, and paired tumor and surgical margin tissues (n = 42). The levels of 28 sphingolipids molecules were obtained by mass spectrometry. Furthermore, sphingolipids were analyzed with clinical and pathological characteristics to search the potential for diagnosis and prognosis. Lower levels of 17 sphingolipids was found in the plasma of OSCC patients compared to controls while four were elevated in tumor tissues. C18:0 dyhidroceramide and C24:0 lactosylceramide in plasma were associated with perineural invasion, while tissue levels of ceramide and dyhidroceramide were associated with advanced tumor stage and perineural invasion. High plasma levels of C24:0 ceramide (HR = 0.10, p = 0.0036) and C24:1 glucosylceramide (HR = 6.62, p = 0.0023), and tissue levels of C24:0 dyhidroceramide (HR = 3.95, p = 0.032) were identified as independent prognostic factors. Moreover, we identified signatures composed by i) sphinganine-1-phosphate and C16 ceramide-1-phosphate in plasma with significant diagnostic accuracy, while ii) C24:0 ceramide, C24:0 dyhidroceramide, and C24:1 glucosylceramide plasma levels, and iii) C24:0 dyhidrosphingomyelin and C24:0 ceramide tissue levels showed value to predict survival in patients aged 60 years or older. We proposed the sphingolipids signatures in plasma and tumor tissues as biomarkers candidates to diagnosis and prognosis in OSCC.

Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing.

Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV- HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.

A Prognostic Model Based on Clinicopathological Features and Inflammation- and Nutrition-Related Indicators Predicts Overall Survival in Surgical Patients With Tongue Squamous Cell Carcinoma.

Objectives: It is reported that inflammation- and nutrition-related indicators have a prognostic impact on multiple cancers. Here we aimed to identify a prognostic nomogram model for prediction of overall survival (OS) in surgical patients with tongue squamous cell carcinoma (TSCC). Methods: The retrospective data of 172 TSCC patients were charted from the Cancer Hospital of Shantou University Medical College between 2008 and 2019. A Cox regression analysis was performed to determine prognostic factors to establish a nomogram and predict OS. The predictive accuracy of the model was analyzed by the calibration curves and the concordance index (C-index). The difference of OS was analyzed by Kaplan-Meier survival analysis. Results: Multivariate analysis showed age, tumor node metastasis (TNM) stage, red blood cell, platelets, and platelet-to-lymphocyte ratio were independent prognostic factors for OS, which were used to build the prognostic nomogram model. The C-index of the model for OS was 0.794 (95% CI = 0.729-0.860), which was higher than that of TNM stage 0.685 (95% CI = 0.605-0.765). In addition, decision curve analysis also showed the nomogram model had improved predictive accuracy and discriminatory performance for OS, compared to the TNM stage. According to the prognostic model risk score, patients in the high-risk subgroup had a lower 5-year OS rate than that in a low-risk subgroup (23% vs 49%, P < .0001). Conclusions: The nomogram model based on clinicopathological features inflammation- and nutrition-related indicators represents a promising tool that might complement the TNM stage in the prognosis of TSCC.

The neuropeptide calcitonin gene-related peptide links perineural invasion with lymph node metastasis in oral squamous cell carcinoma.

OBJECTIVE: Although perineural invasion (PNI) is well-known to be correlated with and able to predict lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC), the clinical and molecular correlation between PNI and LNM has not been elucidated, and preoperative biomarkers for LNM prediction in OSCC are urgently needed. MATERIALS AND METHODS: The correlation between PNI and LNM was retrospectively evaluated using a cohort of 218 patients diagnosed with OSCC. Candidate neuropeptides were screened based on TCGA database and verified via immunohistochemistry and Western blot analyses. ELISA was used to detect calcitonin gene-related peptide (CGRP) in patient plasma. In vitro assays were used to explore the effects of CGRP on OSCC cells. RESULTS: OSCC patients with PNI had a higher incidence of LNM (69.86% vs. 26.2%, P < 0.0001, n = 218). CGRP expression was upregulated in the PNI niche and in metastatic lymph nodes, and was correlated with poor overall survival of OSCC patients. Preoperative plasma CGRP levels were higher in OSCC patients (n = 70) compared to healthy donors (n = 60) (48.59 vs. 14.58 pg/ml, P < 0.0001), and were correlated with LNM (P < 0.0001) and PNI (P = 0.0002). Preoperative plasma CGRP levels alone yielded an AUC value of 0.8088 to predict LNM, and CGRP levels combined with preoperative T stage reached an AUC value of 0.8590. CGRP promoted proliferation and migration abilities of OSCC cells, which could be antagonized by either pharmacological or genetic blockade of the CGRP receptor. CONCLUSIONS: The neuropeptide CGRP links PNI and LNM in OSCC, and preoperative plasma CGRP levels can be used to predict LNM in OSCC.

Serum exosome-derived biomarkers for the early detection of oral squamous cell carcinoma.

Blood exosomes help regulate communication between tumour cells, moderating their behaviour. We sought to determine the protein content in serum exosomes (SEs), to characterise SEs, and to discover novel clinical biomarkers of oral squamous cell carcinoma (OSCC). Differentially expressed proteins (DEPs) of OSCC were identified using proteomics and then analysed using bioinformatics, before validation using ELISA, IHC, and RT-PCR. The influence of SEs on oral cancer cells was detected using CCK-8 and migration assays. Twelve DEPs were found in SEs from OSCC. Four proteins were targeted for further verification. New biomarkers exhibiting high sensitivity and specificity in diagnosing OSCC comprised C-reactive protein (CRP), von willebrand factor (VWF), and leucine-rich alpha-2-glycoprotein (LRG). Combined biomarkers outperformed any single protein. We also demonstrated that tumour-derived exosomes promoted tumour cell migration, but not proliferation and apoptosis. Our study indicates that CRP, VWF, and LRG are potential clinically relevant OSCC biomarkers. OSCC-related SEs may help promote migration of oral cells.

Plasma Metabolic Phenotypes of HPV-Associated versus Smoking-Associated Head and Neck Cancer and Patient Survival.

BACKGROUND: Metabolic differences between human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and smoking-associated HNSCC may partially explain differences in prognosis. The former relies on mitochondrial oxidative phosphorylation (OXPHOS) while the latter relies on glycolysis. These differences have not been studied in blood. METHODS: We extracted metabolites using untargeted liquid chromatography high-resolution mass spectrometry from pretreatment plasma in a cohort of 55 HPV-associated and 82 smoking-associated HNSCC subjects. Metabolic pathway enrichment analysis of differentially expressed metabolites produced pathway-based signatures. Significant pathways (P < 0.05) were reduced via principal component analysis and assessed with overall survival via Cox models. We classified each subject as glycolytic or OXPHOS phenotype and assessed it with survival. RESULTS: Of 2,410 analyzed metabolites, 191 were differentially expressed. Relative to smoking-associated HNSCC, bile acid biosynthesis (P < 0.0001) and octadecatrienoic acid beta-oxidation (P = 0.01), were upregulated in HPV-associated HNSCC, while galactose metabolism (P = 0.001) and vitamin B6 metabolism (P = 0.01) were downregulated; the first two suggest an OXPHOS phenotype while the latter two suggest glycolytic. First principal components of bile acid biosynthesis [HR = 0.52 per SD; 95% confidence interval (CI), 0.38-0.72; P < 0.001] and octadecatrienoic acid beta-oxidation (HR = 0.54 per SD; 95% CI, 0.38-0.78; P < 0.001) were significantly associated with overall survival independent of HPV and smoking. The glycolytic versus OXPHOS phenotype was also independently associated with survival (HR = 3.17; 95% CI, 1.07-9.35; P = 0.04). CONCLUSIONS: Plasma metabolites related to glycolysis and mitochondrial OXPHOS may be biomarkers of HNSCC patient prognosis independent of HPV or smoking. Future investigations should determine whether they predict treatment efficacy. IMPACT: Blood metabolomics may be a useful marker to aid HNSCC patient prognosis.

Plasma extracellular vesicle microRNA-491-5p as diagnostic and prognostic marker for head and neck squamous cell carcinoma.

Poor survival of patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) is partly due to early diagnosis difficulties and the lack of reliable biomarkers for predicting treatment outcomes. In the discovery cohort, plasma-derived extracellular vesicles (EVs) from LA-HNSCC patients (n = 48) and healthy volunteers (n = 12) were used for profiling for microRNA (miRNA) expression by NanoString analysis. Ten EV-associated miRNAs were differentially expressed between LA-HNSCC patients and healthy volunteers. Subsequently, the results were validated in the individual discovery and additional cases (HNSCC, n = 73; control, n = 20) by quantitative RT-PCR. Among 10 EV-miRNAs, four (miR-27b-3p, miR-491-5p, miR-1910-5p, and miR-630) were significantly dysregulated in LA-HNSCC patients (n = 73) compared with healthy volunteers (n = 20). The miRNA prediction models were developed to discriminate HNSCC patients from healthy volunteers. The model using miR-491-5p was selected as a diagnostic biomarker for LA-HNSCC with a sensitivity and specificity of 46.6% and 100%, respectively (P < .001). The dynamic changes of miRNA model score (ΔmiRNAs) were determined using scores pre- and postdefinitive treatment to further investigate the prognostic value of miRNA prediction models. The univariate and multivariate analyses indicated that ΔmiR-491-5p was the most powerful and independent prognostic indicator for overall survival (hazard ratio [HR] 5.66, 95% confidence interval, 1.77-18.01; P = .003) and disease-free survival (HR 2.82, 95% CI, 1.13-7.05; P = .027) of HNSCC patients. In summary, the miR-491-5p prediction model could serve as a blood-based diagnostic marker for LA-HNSCC. Moreover, ΔmiR-491-5p could be a potential monitoring prognostic marker to reflect the survival of HNSCC patients.

FCGR2A Could Function as a Prognostic Marker and Correlate with Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: We aim to investigate the correlation between FCGR2A mRNA level and prognosis of head and neck squamous cancer (HNSC) in public databases. In addition, we investigated the correlation between FCGR2A expression and clinicopathological characteristics and tumor-infiltrating immune cells in HNSC patients. METHODS: FCGR2A mRNA expression in multiple cancers was analyzed based on Gene Expression Profiling Interactive Analysis. A protein-protein interaction network was obtained based on the STRING database. The 10 proteins most closely related to FCGR2A (i.e., CD3G, PLCG2, LAT, LYN, SYK, FCGR3A, PIK3R1, HCK, ITGAM, and ITGB2) were screened, followed by establishing the protein-protein interaction network. The correlation between FCGR2A expression and immunocytes was investigated, together with the effects of FCGR2A on the metastasis, recurrence, and survival of HNSC. RESULTS: FCGR2A expression in several carcinoma tissues was significantly higher than that of adjacent tissues. Significant differences were noticed in the HNSC samples and the adjacent tissue samples except the seven samples of grade 4. There were statistical differences between the FCGR2A expression in tissues of grade 1, grade 2, and grade 3 (P < 0.05). In the tissues of grade 4, the expression of FCGR2A was the lowest. The FCGR2A protein was a type of II-a receptor in γFc of the low-affinity immunoglobulin, which could bind with the Fc region of the immunoglobulin γ. There was a correlation between the FCGR2A gene and the distal HNSC metastasis. FCGR2A gene expression was correlated with the survival and prognosis. The GSE65858 dataset was selected for the validation. The FCGR2A expression was significantly correlated with total survival (P = 0.0107) and progression-free survival (P = 0.0362). CONCLUSIONS: Our findings shed light on the importance of FCGR2A in HNSC and illustrated a potential relationship between FCGR2A and tumor-immune interactions.

Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation. EXPERIMENTAL DESIGN: We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I-IVA human papillomavirus-negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls. RESULTS: CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples. CONCLUSIONS: Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Characterization and ex vivo expansion of rare in situ cytokine secreting T cell populations from tumor tissue and blood of oral squamous cell carcinoma patients.

Rare subpopulations of tumor antigen-reactive memory T cells, which actively secrete type-1 effector cytokines, particularly TNF-α in situ, possess anti-tumor activity and prognostic relevance. These cells are relevant for cancer immunotherapy; however, their low frequencies make them difficult to study and novel protocols for their culture and expansion ex vivo are needed. Here, we studied the presence of T cells secreting type-1 cytokines (Cy+T cells) in the blood and tumors of 24 patients with oral squamous cell carcinomas (OSCC) and explored possibilities for their isolation and expansion. More than 90% of OSCC patients contained enriched numbers Cy+T cells in the blood and tumors compared to healthy donors in which these were hardly detectable. The majority of TNF-α+T cells were CD4+ T helper cells while IFN-γ+TIL were predominantly CD8+. Cy+T helper cells in the blood were early-differentiated memory T cells while Cy+TIL and Cy+CD8+T cells showed advanced-differentiated memory T cell phenotypes. We explored different conditions for their in vitro culture and found that Cy+T cells can be efficiently expanded in vitro to similar levels as Cy-T cells and after expansion maintained their TNF-α secreting capacity. However, for optimal expansion they required specific culture conditions to support the maintenance of stem-like and central memory T cell phenotype. In conclusion, we show that Cy+T cells are enriched in OSCC patients and report a novel cell culture protocol optimized to specifically expand and functionally maintain these cells for further functional characterization or for their exploitation in immunotherapy of OSCC.

Prediction of lymph node metastasis in oral tongue squamous cell carcinoma using the neutrophil-to-lymphocyte ratio and platelet-to-neutrophil ratio.

BACKGROUND: Lymph node metastasis in a variety of tumors is associated with systemic inflammatory markers. However, this association has not been reported in oral tongue squamous cell carcinoma (OTSCC). This study aimed to investigate how the preoperative neutrophil-to-lymphocyte ratio (NLR) and platelet-to-neutrophil ratio (PNR) in OTSCC patients correlated with the occurrence of OTSCC and lymph node metastasis. METHODS: The data of 73 patients with primary OTSCC who underwent surgical resection were retrospectively analyzed. Patients with other malignant tumors, patients who had received radiotherapy or chemotherapy before surgery, and patients with active inflammation were excluded. The enrolled patients were divided into groups N0 (no early-stage lymph node metastasis) and N1 (early-stage lymph node metastasis). Venous blood samples were collected before surgery and at the third week after surgery and subjected to complete blood counting in a blood analyzer. Eighty-seven healthy people were included as a control group. In addition, the NLR and PNR in OTSCC patients were compared with those in the controls, and the postoperative NLR and PNR of group N0 were compared with those of group N1 . RESULTS: The NLR was significantly higher in the OTSCC patients than the controls (p < 0.05). The area under the receiver operating characteristic curve was 0.595. Further comparison of the NLR and PLR between group N0 and group N1 showed that when NLR was ≤1.622, and the probability of early-stage lymph node metastasis in OTSCC patients was 73.3%, and when PNR was >60.889, the probability was 86.7%. In re-examination 3 weeks postoperatively, the NLR and PNR were not significantly different between groups. CONCLUSION: The NLR has certain reference value for the diagnosis of OTSCC. The preoperative NLR and PNR can be used to predict early-stage lymph node metastasis in patients with histopathologically confirmed OTSCC.

MiR-3168, miR-6125, and miR-4718 as potential predictors of cisplatin-induced nephrotoxicity in patients with head and neck cancer.

BACKGROUND: No biomarker is available for identifying cancer patients at risk of developing nephrotoxicity when treated with cisplatin. METHODS: We performed microRNA (miRNA) sequencing using plasma collected 5 days after cisplatin treatment (D5) from twelve patients with head and neck cancer with and without nephrotoxicity (grade ≥ 2 increased serum creatinine). The most differentially expressed miRNAs between the two groups were selected for quantification at baseline and D5 in a larger cohort of patients. The association between miRNAs and nephrotoxicity was evaluated by calculating the odds ratio (OR) from univariate logistic regression. Receiver operating characteristic curves (ROC) were used to estimate the area under the curve (AUC), sensitivity, and specificity. RESULTS: MiR-3168 (p = 1.98 × 10- 8), miR-4718 (p = 4.24 × 10- 5), and miR-6125 (p = 6.60 × 10- 5) were the most differentially expressed miRNAs and were further quantified in 43, 48, and 53 patients, respectively. The baseline expression of miR-3168 (p = 0.0456, OR = 1.03, 95% CI: 1.00-1.06) and miR-4718 (p = 0.0388, OR = 1.56, 95% CI: 1.03-2.46) were associated with an increased risk of nephrotoxicity, whereas miR-6125 showed a trend (p = 0.0618, OR = 1.73, 95% CI: 0.98-3.29). MiR-4718 showed the highest AUC (0.77, 95% CI: 0.61-0.93) with sensitivity of 66.76 and specificity of 79.49. CONCLUSIONS: We have provided evidence of baseline plasmatic expression of miR-3168, miR-6125, and miR-4718 as potential predictors of cisplatin-induced nephrotoxicity.

Sarcopenia with inflammation as a predictor of survival in patients with head and neck cancer.

OBJECTIVE: Although both sarcopenia and systemic inflammation affect the outcomes of head and neck cancer (HNC) patients, the association between sarcopenia and systemic inflammation and the combined prognostic effect of these factors in HNC patients remain unknown. This study aimed to evaluate the effect of sarcopenia with systemic inflammation on survival and disease control in HNC patients. METHODS: We retrospectively reviewed medical records of HNC patients treated between 2009 and 2016. The skeletal muscle area was measured using a single computed tomography image slice at the level of the third cervical vertebra. A prognostic score (SPLR) was developed based on sarcopenia and the platelet-lymphocyte ratio (PLR), and its prognostic value was evaluated. RESULTS: Overall, 164 patients were enrolled. In the multivariate analysis, sarcopenia was significantly associated with poor overall survival (OS) (p < 0.01). However, neither sarcopenia nor a high PLR was an independent prognostic factor for disease-free survival (DFS) or locoregional recurrence-free survival (LRFS). A high PLR was an independent predictor for sarcopenia (p < 0.01). A high SPLR was associated with older age, lower serum hemoglobin, and lower body mass index (all p < 0.05). Multivariate analysis revealed that SPLR was a significant independent predictor of OS, DFS, and LRFS (all p < 0.05). CONCLUSIONS: Systemic inflammation is significantly associated with sarcopenia. The survival and oncological effects of sarcopenia were enhanced when PLR was high. Thus, the combination of these two parameters may be useful for identifying HNC patients at a risk of poor survival outcomes.

Presurgical platelet-lymphocyte ratio for prognosis in advanced hypopharyngeal squamous cell carcinoma in individuals undergoing radical resection.

BACKGROUND: Patient prognosis in hypopharyngeal carcinoma remains difficult to predict, necessitating new, readily available biomarkers. OBJECTIVE: Platelet-lymphocyte ratio (PLR)'s effects on recurrence-free survival (RFS) and overall survival (OS) were evaluated in individuals undergoing radical resection for advanced hypopharyngeal squamous cell carcinoma (HSCC). METHODS: A total of 89 patients were retrospectively assessed. PLR, and derived neutrophil-lymphocyte (dNLR) and neutrophil-lymphocyte (NLR) ratios were determined based on complete blood count. Then, the prognostic values of PLR, dNLR and NLR were assessed by univariate and multivariate Cox regression analyses adjusted for disease-specific prognostic factors. Endpoints of interest were RFS and OS. RESULTS: The optimal cutoff of PLR was 98.815, based on which individuals were categorized into the high- (PLR ≥98.815) and low- (PLR <98.815) PLR groups. High PLR (p = .022) had a significant association with reduced RFS, which still showed significance in multivariable analysis (HR = 2.020, 95%CI: 1.076-3.794, p = .029). In univariate analysis, PLR (p = .046) and positive surgical margin (p = .021) also had significant associations with OS. CONCLUSION: Elevated PLR has associations with increased risk of recurrence and reduced survival in advanced HSCC cases undergoing radical resection. High presurgical PLR may independently predict RFS. Therefore, further multi-institutional prospective studies are needed to better characterize the role of pre-operative blood PLR as prognostic factors in HSCC.

Pre-treatment absolute lymphocyte count predicts for improved survival in human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma.

BACKGROUND: The prognostic value of pretreatment complete blood count (CBC) data, including absolute lymphocyte count (ALC) and the neutrophil-to-lymphocyte ratio (NLR), has been reported for many diseases with decreased ALC and increased absolute neutrophil count (ANC) and NLR values correlating with worse outcomes. There is minimal data relating these hematologic parameters to oropharyngeal squamous cell carcinoma (OPSCC) prognosis. This study evaluates the prognostic value of pretreatment CBC data in OPSCC on overall survival (OS) and progression-free survival (PFS) in relation to HPV status. METHODS: A single-institutional retrospective review of patients with pretreatment hematologic data who received radiation for OPSCC was performed. Univariate and multivariate (UVA/MVA) Cox proportional hazard regression analyses were performed to identify prognostic variables. Translational studies related outcomes to the degree of tumor-infiltrating lymphocytes (TILs) in histologic specimens. RESULTS: From 2007 to 2018, 201 patients were treated for OPSCC. Median follow-up was 40 months. 3-year OS was 86.2% in the HPV-positive cohort, 46.3% for HPV-negative. Median NLR was 3.04. NLR ≥ 3 was associated with worse PFS (HR 1.67, p = 0.044. In the subset of 158 HPV + patients, MVA revealed increasing ALC to be associated with improved OS (HR 0.53; p = 0.040) and PFS (HR = 0.48; p = 0.0075). On UVA, high-TIL infiltration at diagnosis was associated with improved OS. CONCLUSION: In a cohort of HPV + OPSCC patients, increasing ALC is associated with improved OS and PFS. Our study is the first to identify pre-treatment ALC as an independent prognostic factor in HPV-associated OPSCC.