Integrative genomics in combination with RNA interference identifies prognostic and functionally relevant gene targets for oral squamous cell carcinoma.

In oral squamous cell carcinoma (OSCC), metastasis to lymph nodes is associated with a 50% reduction in 5-year survival. To identify a metastatic gene set based on DNA copy number abnormalities (CNAs) of differentially expressed genes, we compared DNA and RNA of OSCC cells laser-microdissected from non-metastatic primary tumors (n = 17) with those from lymph node metastases (n = 20), using Affymetrix 250K Nsp single-nucleotide polymorphism (SNP) arrays and U133 Plus 2.0 arrays, respectively. With a false discovery rate (FDR)<5%, 1988 transcripts were found to be differentially expressed between primary and metastatic OSCC. Of these, 114 were found to have a significant correlation between DNA copy number and gene expression (FDR<0.01). Among these 114 correlated transcripts, the corresponding genomic regions of each of 95 transcripts had CNAs differences between primary and metastatic OSCC (FDR<0.01). Using an independent dataset of 133 patients, multivariable analysis showed that the OSCC-specific and overall mortality hazards ratio (HR) for patients carrying the 95-transcript signature were 4.75 (95% CI: 2.03-11.11) and 3.45 (95% CI: 1.84-6.50), respectively. To determine the degree by which these genes impact cell survival, we compared the growth of five OSCC cell lines before and after knockdown of over-amplified transcripts via a high-throughput siRNA-mediated screen. The expression-knockdown of 18 of the 26 genes tested showed a growth suppression ≥ 30% in at least one cell line (P<0.01). In particular, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC, and the growth suppression was likely caused by increase in apoptosis. Further investigation is warranted to examine the biological role of these genes in OSCC progression and their therapeutic potentials.

Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival.

BACKGROUND: Lymphotropism in oral squamous cell carcinoma (OSCC) is one of the most important prognostic factors of 5-year survival. In an effort to identify genes that may be responsible for the initiation of OSCC lymphotropism, we examined DNA copy number gains and losses and corresponding gene expression changes from tumor cells in metastatic lymph nodes of patients with OSCC. RESULTS: We performed integrative analysis of DNA copy number alterations (CNA) and corresponding mRNA expression from OSCC cells isolated from metastatic lymph nodes of 20 patients using Affymetrix 250 K Nsp I SNP and U133 Plus 2.0 arrays, respectively. Overall, genome CNA accounted for expression changes in 31% of the transcripts studied. Genome region 11q13.2-11q13.3 shows the highest correlation between DNA CNA and expression. With a false discovery rate < 1%, 530 transcripts (461 genes) demonstrated a correlation between CNA and expression. Among these, we found two subsets that were significantly associated with OSCC (n = 122) when compared to controls, and with survival (n = 27), as tested using an independent dataset with genome-wide expression profiles for 148 primary OSCC and 45 normal oral mucosa. We fit Cox models to calculate a principal component analysis-derived risk-score for these two gene sets ('122-' or '27-transcript PC'). The models combining the 122- or 27-transcript PC with stage outperformed the model using stage alone in terms of the Area Under the Curve (AUC = 0.82 or 0.86 vs. 0.72, with p = 0.044 or 0.011, respectively). CONCLUSIONS: Genes exhibiting CNA-correlated expression may have biological impact on carcinogenesis and cancer progression in OSCC. Determination of copy number-associated transcripts associated with clinical outcomes in tumor cells with an aggressive phenotype (i.e., cells metastasized to the lymph nodes) can help prioritize candidate transcripts from high-throughput data for further studies.

A genetic expression profile associated with oral cancer identifies a group of patients at high risk of poor survival.

PURPOSE: To determine if gene expression signature of invasive oral squamous cell carcinoma (OSCC) can subclassify OSCC based on survival. EXPERIMENTAL DESIGN: We analyzed the expression of 131 genes in 119 OSCC, 35 normal, and 17 dysplastic mucosa to identify cluster-defined subgroups. Multivariate Cox regression was used to estimate the association between gene expression and survival. By stepwise Cox regression, the top predictive models of OSCC-specific survival were determined and compared by receiver operating characteristic analysis. RESULTS: The 3-year overall mean+/-SE survival for a cluster of 45 OSCC patients was 38.7+/-0.09% compared with 69.1+/-0.08% for the remaining patients. Multivariate analysis adjusted for age, sex, and stage showed that the 45 OSCC patient cluster had worse overall and OSCC-specific survival (hazard ratio, 3.31; 95% confidence interval, 1.66-6.58 and hazard ratio, 5.43; 95% confidence interval, 2.32-12.73, respectively). Stepwise Cox regression on the 131 probe sets revealed that a model with a term for LAMC2 (laminin gamma2) gene expression best identified patients with worst OSCC-specific survival. We fit a Cox model with a term for a principal component analysis-derived risk score marker and two other models that combined stage with either LAMC2 or PCA. The area under the curve for models combining stage with either LAMC2 or PCA was 0.80 or 0.82, respectively, compared with 0.70 for stage alone (P=0.013 and 0.008, respectively). CONCLUSIONS: Gene expression and stage combined predict survival of OSCC patients better than stage alone.

Prediction of survival of HPV16-negative, p16-negative oral cavity cancer patients using a 13-gene signature: A multicenter study using FFPE samples.

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

Gene expression profiling identifies genes predictive of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is associated with substantial mortality and morbidity. To identify potential biomarkers for the early detection of invasive OSCC, we compared the gene expressions of incident primary OSCC, oral dysplasia, and clinically normal oral tissue from surgical patients without head and neck cancer or preneoplastic oral lesions (controls), using Affymetrix U133 2.0 Plus arrays. We identified 131 differentially expressed probe sets using a training set of 119 OSCC patients and 35 controls. Forward and stepwise logistic regression analyses identified 10 successive combinations of genes which expression differentiated OSCC from controls. The best model included LAMC2, encoding laminin-gamma2 chain, and COL4A1, encoding collagen, type IV alpha1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I alpha1 chain, and PADI1, encoding peptidyl arginine deiminase, type 1, could also distinguish OSCC from controls. We validated these two models using an internal independent testing set of 48 invasive OSCC and 10 controls and an external testing set of 42 head and neck squamous cell carcinoma cases and 14 controls (GEO GSE6791), with sensitivity and specificity above 95%. These two models were also able to distinguish dysplasia (n = 17) from control (n = 35) tissue. Differential expression of these four genes was confirmed by quantitative reverse transcription-PCR. If confirmed in larger studies, the proposed models may hold promise for monitoring local recurrence at surgical margins and the development of second primary oral cancer in patients with OSCC.

Salivary metabolite profiling distinguishes patients with oral cavity squamous cell carcinoma from normal controls.

Oral cavity squamous cell carcinoma (OCC) and oropharyngeal squamous cell carcinoma (OPC) are among the most common cancers worldwide and are associated with high mortality and morbidity. The purpose of this study is to identify potential biomarkers to distinguish OCC/OPC from normal controls and to distinguish OCC patients with and without nodal metastasis. We tested saliva samples from 101 OCC, 58 OPC, and 35 normal controls using four analytical platforms (NMR, targeted aqueous by LC-MS/MS, global aqueous and global lipidomics by LC-Q-TOF). Samples from OCC and normal controls were divided into discovery and validation sets. Using linear regression adjusting for age, sex, race and experimental batches, we found the levels of two metabolites (glycine and proline) to be significantly different between OCC and controls (FDR < 0.1 for both discovery and validation sets) but did not find any appreciable differences in metabolite levels between OPC and controls or between OCC with and without nodal metastasis. Four metabolites, including glycine, proline, citrulline, and ornithine were associated with early stage OCC in both discovery and validation sets. Further study is warranted to confirm these results in the development of salivary metabolites as diagnostic markers.

Transcriptome analysis reveals differentially expressed lncRNAs between oral squamous cell carcinoma and healthy oral mucosa.

Oral cavity and oropharyngeal squamous cell carcinoma (OSCC) is a major cancer type in the head and neck region. To better understand the roles long non-coding RNA (lncRNA) play in OSCC carcinogenesis, we compared the expression levels of 3,054 probe sets for lncRNAs between 167 OSCCs and 45 healthy oral mucosa using an Affymetrix HG U133 plus 2.0 array dataset. We found 658 lncRNA transcripts (790 probe sets) to be significantly differentially expressed using a criteria of FDR < 0.01, with 36 of them (39 probe sets) showing more than a 2-fold change. We further validated the top differentially expressed lncRNAs in three independent datasets from Gene Expression Omnibus (GEO) repository: GSE42743, GSE9844, and GSE6791. Fourteen lncRNAs (15 probe sets) were validated in all three datasets using the criteria FDR < 0.01: LOC441178, C5orf66-AS1, HCG22, FLG-AS1, CCL14/CCL15-CCL14, LOC100506990, TRIP10, PCBP1-AS1, LINC01315, LINC00478, COX10-AS1/LOC100506974, MLLT4-AS1, MIR31HG, and DUXAP10/LINC01296. Three lncRNAs in the validated list which showed the highest fold change (LOC441178, HCG22 and C5orf66-AS1) were verified by quantitative RT-PCR in a subset of 20 OSCCs and 10 control samples. In silico prediction of their functional role has given us directions for further investigation.

Impact of tumoral carbonic anhydrase IX and Ki-67 expression on survival in oral squamous cell carcinoma patients.

Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed type of head and neck cancer, accounting for ~300,000 new cases worldwide annually. Carbonic anhydrase IX (CAIX) and Ki-67 have been associated with reduced disease-specific survival (DSS) in patients with OSCC. We previously proposed a combined CAIX and Ki-67 signature of 'functional hypoxia' and sought to replicate this association in a larger independent cohort of patients with OSCC at the Fred Hutchinson Cancer Research Center (FHCRC) in Seattle. The study population included patients with incident primary OSCC treated at the University of Washington Medical Center and the Harborview Medical Center in Seattle between December 2003 and February 2012. Archived tumor blocks were obtained with tissue samples from 189 patients, and triplicate 0.6 mm cores were assembled into tissue microarrays (TMAs). Fluorescence immunohistochemistry and AQUAnalysis® were used to quantify the expression of tumoral CAIX (tCAIX) and stromal CAIX (sCAIX) and tumoral Ki-67 for each TMA core. Hazard ratios for DSS were calculated using Cox proportional hazards analysis. High tCAIX and sCAIX expression levels were associated with reduced DSS (aHR=1.003, 95% CI:1.00-1.01 and aHR=1.010, 95% CI:1.001-1.019, per AQUA score unit, respectively). Ki-67 expression was not associated with survival (aHR=1.01, 95% CI:0.99-1.02) in the FHCRC cohort. DSS for patients with high sCAIX and low Ki-67 did not differ from that of other patient groups. Elevated tCAIX was associated with reduced DSS as a continuous and as a dichotomized (75%) variable. sCAIX was associated with DSS as a continuous variable but not when dichotomized (75%). However, the previously proposed 'functional hypoxia' signature was not replicated in the current FHCRC study. The failure to replicate our prior observation of poorer survival in patients with combined high sCAIX and low tumoral Ki-67 was likely due to the absence of an association between tumoral Ki-67 and DSS in this cohort. However, the association between DSS and tCAIX and sCAIX supports a role for CAIX in OSCC clinical outcomes.

Common TDP1 Polymorphisms in Relation to Survival among Small Cell Lung Cancer Patients: A Multicenter Study from the International Lung Cancer Consortium.

Purpose: DNA topoisomerase inhibitors are commonly used for treating small-cell lung cancer (SCLC). Tyrosyl-DNA phosphodiesterase (TDP1) repairs DNA damage caused by this class of drugs and may therefore influence treatment outcome. In this study, we investigated whether common TDP1 single-nucleotide polymorphisms (SNP) are associated with overall survival among SCLC patients.Experimental Design: Two TDP1 SNPs (rs942190 and rs2401863) were analyzed in 890 patients from 10 studies in the International Lung Cancer Consortium (ILCCO). The Kaplan-Meier method and Cox regression analyses were used to evaluate genotype associations with overall mortality at 36 months postdiagnosis, adjusting for age, sex, race, and tumor stage.Results: Patients homozygous for the minor allele (GG) of rs942190 had poorer survival compared with those carrying AA alleles, with a HR of 1.36 [95% confidence interval (CI): 1.08-1.72, P = 0.01), but no association with survival was observed for patients carrying the AG genotype (HR = 1.04, 95% CI, 0.84-1.29, P = 0.72). For rs2401863, patients homozygous for the minor allele (CC) tended to have better survival than patients carrying AA alleles (HR = 0.79; 95% CI, 0.61-1.02, P = 0.07). Results from the Genotype Tissue Expression (GTEx) Project, the Encyclopedia of DNA Elements (ENCODE), and the ePOSSUM web application support the potential function of rs942190.Conclusions: We found the rs942190 GG genotype to be associated with relatively poor survival among SCLC patients. Further investigation is needed to confirm the result and to determine whether this genotype may be a predictive marker for treatment efficacy of DNA topoisomerase inhibitors. Clin Cancer Res; 23(24); 7550-7. ©2017 AACR.

Genome-Wide Loss of Heterozygosity and DNA Copy Number Aberration in HPV-Negative Oral Squamous Cell Carcinoma and Their Associations with Disease-Specific Survival.

Oral squamous cell cancer of the oral cavity and oropharynx (OSCC) is associated with high case-fatality. For reasons that are largely unknown, patients with the same clinical and pathologic staging have heterogeneous response to treatment and different probability of recurrence and survival, with patients with Human Papillomavirus (HPV)-positive oropharyngeal tumors having the most favorable survival. To gain insight into the complexity of OSCC and to identify potential chromosomal changes that may be associated with OSCC mortality, we used Affymtrix 6.0 SNP arrays to examine paired DNA from peripheral blood and tumor cell populations isolated by laser capture microdissection to assess genome-wide loss of heterozygosity (LOH) and DNA copy number aberration (CNA) and their associations with risk factors, tumor characteristics, and oral cancer-specific mortality among 75 patients with HPV-negative OSCC. We found a highly heterogeneous and complex genomic landscape of HPV-negative tumors, and identified regions in 4q, 8p, 9p and 11q that seem to play an important role in oral cancer biology and survival from this disease. If confirmed, these findings could assist in designing personalized treatment or in the creation of models to predict survival in patients with HPV-negative OSCC.

A 13-gene signature prognostic of HPV-negative OSCC: discovery and external validation.

PURPOSE: To identify a prognostic gene signature for patients with human papilloma virus (HPV)-negative oral squamous cell carcinomas (OSCC). EXPERIMENTAL DESIGN: Two gene expression datasets were used: a training dataset from the Fred Hutchinson Cancer Research Center (FHCRC, Seattle, WA; n = 97) and a validation dataset from the MD Anderson Cancer Center (MDACC, Houston, TX; n = 71). We applied L1/L2-penalized Cox regression models to the FHCRC data on the 131-gene signature previously identified to be prognostic in patients with OSCCs to identify a prognostic model specific for patients with high-risk HPV-negative OSCCs. The models were tested with the MDACC dataset using a receiver operating characteristic (ROC) analysis. RESULTS: A 13-gene model was identified as the best predictor of HPV-negative OSCC-specific survival in the training dataset. The risk score for each patient in the validation dataset was calculated from this model and dichotomized at the median. The estimated 2-year mortality (± SE) of patients with high-risk scores was 47.1% (± 9.24%) compared with 6.35% (± 4.42) for patients with low-risk scores. ROC analyses showed that the areas under the curve for the age, gender, and treatment modality-adjusted models with risk score [0.78; 95% confidence interval (CI), 0.74-0.86] and risk score plus tumor stage (0.79; 95% CI, 0.75-0.87) were substantially higher than for the model with tumor stage (0.54; 95% CI, 0.48-0.62). CONCLUSIONS: We identified and validated a 13-gene signature that is considerably better than tumor stage in predicting survival of patients with HPV-negative OSCCs. Further evaluation of this gene signature as a prognostic marker in other populations of patients with HPV-negative OSCC is warranted.

Human papillomavirus-positive oral cavity and oropharyngeal cancer patients do not have better quality-of-life trajectories.

OBJECTIVE: To determine the role of human papillomavirus (HPV) status on quality of life (QOL) in patients with oral cavity and oropharyngeal squamous cell carcinoma (OSCC). Since OSCC that are associated with high-risk HPV have an improved response to treatment and survival, we hypothesized that patients with these tumors would have better QOL trajectories. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary care academic medical center and 2 affiliated hospitals. SUBJECTS AND METHODS: Head and neck-specific QOL was determined using the University of Washington Quality of Life scale version 4 in patients with newly diagnosed invasive OSSC (N = 228). RESULTS: Pretreatment QOL was higher in patients with high-risk HPV-associated tumors compared with patients with HPV-negative or low-risk HPV-associated tumors (P = .015). Patients with high-risk HPV-associated tumors had larger decreases in QOL from pretreatment to immediate posttreatment compared with patients with HPV-negative or low-risk HPV-associated tumors (P = .041). There was no association between HPV status and 1-year posttreatment QOL. CONCLUSION: Among OSCC patients, high-risk HPV-associated tumors were associated with higher pretreatment QOL and a larger decrease in QOL from pretreatment to immediate posttreatment, suggesting that treatment intensity in this unique population may adversely affect QOL.

Gene expression in uninvolved oral mucosa of OSCC patients facilitates identification of markers predictive of OSCC outcomes.

Oral and oropharyngeal squamous cell carcinomas (OSCC) are among the most common cancers worldwide, with approximately 60% 5-yr survival rate. To identify potential markers for disease progression, we used Affymetrix U133 plus 2.0 arrays to examine the gene expression profiles of 167 primary tumor samples from OSCC patients, 58 uninvolved oral mucosae from OSCC patients and 45 normal oral mucosae from patients without oral cancer, all enrolled at one of the three University of Washington-affiliated medical centers between 2003 to 2008. We found 2,596 probe sets differentially expressed between 167 tumor samples and 45 normal samples. Among 2,596 probe sets, 71 were significantly and consistently up- or down-regulated in the comparison between normal samples and uninvolved oral samples and between uninvolved oral samples and tumor samples. Cox regression analyses showed that 20 of the 71 probe sets were significantly associated with progression-free survival. The risk score for each patient was calculated from coefficients of a Cox model incorporating these 20 probe sets. The hazard ratio (HR) associated with each unit change in the risk score adjusting for age, gender, tumor stage, and high-risk HPV status was 2.7 (95% CI: 2.0-3.8, p = 8.8E-10). The risk scores in an independent dataset of 74 OSCC patients from the MD Anderson Cancer Center was also significantly associated with progression-free survival independent of age, gender, and tumor stage (HR 1.6, 95% CI: 1.1-2.2, p = 0.008). Gene Set Enrichment Analysis showed that the most prominent biological pathway represented by the 71 probe sets was the Integrin cell surface interactions pathway. In conclusion, we identified 71 probe sets in which dysregulation occurred in both uninvolved oral mucosal and cancer samples. Dysregulation of 20 of the 71 probe sets was associated with progression-free survival and was validated in an independent dataset.

Integrative analysis in oral squamous cell carcinoma reveals DNA copy number-associated miRNAs dysregulating target genes.

OBJECTIVE: To better understand possible mechanisms involved in the dysregulation of gene expression unique to oral squamous cell carcinoma (OSCC) metastasis, the investigators examined the differential expression of microRNAs (miRNAs) in OSCC metastasis and their functional impact on target gene expression. STUDY DESIGN: Observational assessment of DNA copy number, miRNA, and RNA expression in primary and metastatic OSCC. SETTING: University of Washington Medical Center and affiliated hospitals. SUBJECTS: Tumor samples were taken from patients with primary incident OSCC; cells were laser-capture microdissected from 17 nonmetastatic primary tumors and 20 metastatic lymph nodes. METHODS: DNA copy number aberrations and gene expression profiles were previously determined using Affymetrix 250K Nsp I SNP arrays and HU133 plus 2.0 expression arrays. miRNAs were interrogated with Exiqon's Ready-to-Use PCR Panels assessing the expression of 368 human miRNAs. RESULTS: Investigators found 31 miRNAs differentially expressed between metastatic and nonmetastatic samples (false discovery rate <0.4; 26 overexpressed and 5 underexpressed in metastatic samples). Expression of 7 of these miRNAs was significantly associated with their DNA copy numbers, and expressions of 8 of these miRNAs were significantly associated with their target genes. Among these unique miRNAs, miR-140-3p, miR-29c, and miR-29a were differentially expressed in metastasis versus nonmetastatic samples and had a strong positive correlation with their DNA copy numbers and a negative correlation with the expression of their target genes. CONCLUSION: Results suggest that DNA copy number aberration may play a role in the dysregulation of some differentially expressed miRNAs in OSCC metastasis, warranting further investigation.

Tumor and salivary matrix metalloproteinase levels are strong diagnostic markers of oral squamous cell carcinoma.

BACKGROUND: The matrix metalloproteinases (MMP) cause degradation of the extracellular matrix and basement membranes, and thus may play a key role in cancer development. METHODS: In our search for biomarkers for oral squamous cell carcinomas (OSCC), we compared primary OSCC, oral dysplasia and control subjects with respect to: (i) expression of MMP1, MMP3, MMP10, and MMP12 in oral epithelial tissue using Affymetrix U133 2.0 Plus GeneChip arrays, followed by quantitative reverse transcription-PCR (qRT-PCR) for MMP1, and (ii) determination of MMP1 and MMP3 concentrations in saliva. RESULTS: MMP1 expression in primary OSCC (n = 119) was >200-fold higher (P = 7.16 × 10(-40)) compared with expression levels in nonneoplastic oral epithelium from controls (n = 35). qRT-PCR results on 30 cases and 22 controls confirmed this substantial differential expression. The exceptional discriminatory power to separate OSCC from controls was validated in two independent testing sets (AUC% = 100; 95% CI: 100-100 and AUC% = 98.4; 95% CI: 95.6-100). Salivary concentrations of MMP1 and MMP3 in OSCC patients (33 stage I/II, 26 stage III/IV) were 6.2 times (95% CI: 3.32-11.73) and 14.8 times (95% CI: 6.75-32.56) higher, respectively, than in controls, and displayed an increasing trend with higher stage disease. CONCLUSION: Tumor and salivary MMPs are robust diagnostic biomarkers of OSCC. IMPACT: The capacity of MMP gene expression to identify OSCC provides support for further investigation into MMPs as potential markers for OSCC development. Detection of MMP proteins in saliva in particular may provide a promising means to detect and monitor OSCC noninvasively.

Can a metastatic gene expression profile outperform tumor size as a predictor of occult lymph node metastasis in oral cancer patients?

PURPOSE: To determine the differential gene expression between oral squamous cell carcinoma (OSCC) with and without metastasis to cervical lymph nodes and to assess prediction of nodal metastasis by using molecular features. EXPERIMENTAL DESIGN: We used Affymetrix U133 2.0 plus arrays to compare the tumor genome-wide gene expression of 73 node-positive OSCCs with 40 node-negative OSCCs (≥ 18 months). Multivariate linear regression was used to estimate the association between gene expression and nodal metastasis. Stepwise logistic regression and receiver operating characteristics (ROC) analysis were used to generate predictive models and to compare these with models by using tumor size alone. RESULTS: We identified five genes differentially expressed between node-positive and node-negative OSCCs after adjusting for tumor size and human papillomavirus status: REEP1, RNF145, CTONG2002744, MYO5A, and FBXO32. Stepwise regression identified a four-gene model (MYO5A, RFN145, FBXO32, and CTONG2002744) as the most predictive of nodal metastasis. A leave-one-out ROC analysis revealed that our model had a higher area under the curve (AUC) for identifying occult nodal metastasis compared with that of a model by tumor size alone (respective AUC: 0.85 and 0.61; P = 0.011). A model combining tumor size and gene expression did not further improve the prediction of occult metastasis. Independent validation using 31 metastatic and 13 nonmetastatic cases revealed a significant underexpression of CTONG2002744 (P = 0.0004). CONCLUSIONS: These results suggest that our gene expression markers of OSCC metastasis hold promise for improving current clinical practice. Confirmation by others and functional studies of CTONG2002744 is warranted.

Genomewide gene expression profiles of HPV-positive and HPV-negative oropharyngeal cancer: potential implications for treatment choices.

OBJECTIVE: To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral cavity and oropharyngeal squamous cell carcinoma (OSCC). DESIGN: We used Affymetrix U133 plus 2.0 arrays to examine gene expression profiles of OSCC and normal oral tissue. The HPV DNA was detected using polymerase chain reaction followed by the Roche LINEAR ARRAY HPV Genotyping Test, and the differentially expressed genes were analyzed to examine their potential biological roles using the Ingenuity Pathway Analysis Software, version 5.0. SETTING: Three medical centers affiliated with the University of Washington. PATIENTS: A total of 119 patients with primary OSCC and 35 patients without cancer, all of whom were treated at the setting institutions, provided tissues samples for the study. RESULTS: Human papillomavirus DNA was found in 41 of 119 tumors (34.5%) and 2 of 35 normal tissue samples (5.7%); 39 of the 43 HPV specimens were HPV-16. A higher prevalence of HPV DNA was found in oropharyngeal cancer (23 of 31) than in oral cavity cancer (18 of 88). We found no significant difference in gene expression between HPV-positive and HPV-negative oral cavity cancer but found 446 probe sets (347 known genes) differentially expressed in HPV-positive oropharyngeal cancer than in HPV-negative oropharyngeal cancer. The most prominent functions of these genes are DNA replication, DNA repair, and cell cycling. Some genes differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer (eg, TYMS, STMN1, CCND1, and RBBP4) are involved in chemotherapy or radiation sensitivity. CONCLUSION: These results suggest that differences in the biology of HPV-positive and HPV-negative oropharyngeal cancer may have implications for the management of patients with these different tumors.