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1.  Genome-Wide Loss of Heterozygosity and DNA Copy Number Aberration in HPV-Negative Oral Squamous Cell Carcinoma and Their Associations with Disease-Specific Survival 
PLoS ONE  2015;10(8):e0135074.
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.
PMCID: PMC4527746  PMID: 26247464
2.  A 13-gene signature prognostic of HPV-negative OSCC: discovery and external validation 
To identify a prognostic gene signature for HPV-negative OSCC patients.
Experimental Design
Two gene expression datasets were used; a training dataset from the Fred Hutchinson Cancer Research Center (FHCRC) (n=97), and a validation dataset from the MD Anderson Cancer Center (MDACC) (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 OSCC patients to identify a prognostic model specific for high-risk HPV-negative OSCC patients. The models were tested with the MDACC dataset using a receiver operating characteristic analysis.
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%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).
We identified and validated a 13-gene signature that is considerably better than tumor stage in predicting survival of HPV-negative OSCC patients. Further evaluation of this gene signature as a prognostic marker in other populations of patients with HPV-negative OSCC is warranted.
PMCID: PMC3593802  PMID: 23319825
gene signature; prognosis; HPV-negative; OSCC
3.  HPV-positive oral cavity and oropharyngeal cancer patients do not have better QOL trajectories 
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.
Tertiary care academic medical center and two affiliated hospitals.
Subjects and Methods
Head and neck-specific QOL was determined using the University of Washington Quality of Life (UW-QOL) scale version 4 in patients with newly diagnosed invasive OSSC (n=228).
Pre-treatment QOL was higher in patients with high-risk HPV-associated tumors compared to patients with HPV-negative or low-risk HPV-associated tumors (p=0.015). Patients with high-risk HPV-associated tumors had larger decreases in QOL from pre-treatment to immediate post-treatment compared to patients with HPV-negative or low-risk HPV-associated tumors (p=0.041). There was no association between HPV status and one year post-treatment QOL.
Among OSCC patients, high-risk HPV-associated tumors were associated with higher pre-treatment QOL and a larger decrease in QOL from pre-treatment to immediate post-treatment, suggesting that treatment intensity in this unique population may adversely affect QOL.
PMCID: PMC3535430  PMID: 22275190
head and neck cancer; quality of life; epidemiology/outcomes research; head and neck surgery; human papillomavirus; oropharyngeal squamous cell carcinoma
4.  Integrative Genomics in Combination with RNA Interference Identifies Prognostic and Functionally Relevant Gene Targets for Oral Squamous Cell Carcinoma 
PLoS Genetics  2013;9(1):e1003169.
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.
Author Summary
Neck lymph node metastasis is the most important prognostic factor in oral squamous cell carcinoma (OSCC). To identify genes associated with this critical step of OSCC progression, we compared DNA copy number aberrations and gene expression differences between tumor cells found in metastatic lymph nodes versus those in non-metastatic primary tumors. We identified 95 transcripts (87 genes) with metastasis-specific genome abnormalities and gene expression. Tested in an independent cohort of 133 OSCC patients, the 95 gene signature was an independent risk factor of disease-specific and overall death, suggesting a disease progression phenotype. We knocked down the expression of over-amplified genes in five OSCC cell lines. Knockdown of 18 of the 26 tested genes suppressed the cell growth in at least one cell line. Interestingly, cell lines derived from late-stage OSCC were more sensitive to the knockdown of G3BP1 than cell lines derived from early-stage OSCC. The knockdown of G3BP1 increased programmed cell death in the p53-mutant but not wild-type OSCC cell lines. Taken together, we demonstrate that CNA–associated transcripts differentially expressed in carcinoma cells with an aggressive phenotype (i.e., metastatic to lymph nodes) can be biomarkers with both prognostic information and functional relevance. Moreover, results suggest that G3BP1 is a potential therapeutic target against late-stage p53-negative OSCC.
PMCID: PMC3547824  PMID: 23341773
5.  Tumor and salivary matrix metalloproteinase levels are strong diagnostic markers of oral squamous cell carcinoma 
The matrix metalloproteinases (MMPs) cause degradation of the extracellular matrix and basement membranes, and thus may play a key role in cancer development.
In our search for biomarkers for oral squamous cell carcinomas (OSCC), we compared primary OSCC, oral dysplasia and control subjects with respect to: (1) expression of MMP1, MMP3, MMP10 and MMP12 in oral epithelial tissue using Affymetrix U133 2.0 Plus GeneChip arrays, followed by qRT-PCR for MMP1, and (2) determination of MMP1 and MMP3 concentrations in saliva.
MMP1 expression in primary OSCC (n=119) was >200-fold higher (p=7.16×10−40) compared with expression levels in non-neoplastic 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.
Tumor and salivary MMPs are robust diagnostic biomarkers of OSCC.
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 non-invasively.
PMCID: PMC3237810  PMID: 21960692
oral squamous cell carcinoma; matrix metalloproteinase; MMP; saliva; gene expression
6.  Gene Expression in Uninvolved Oral Mucosa of OSCC Patients Facilitates Identification of Markers Predictive of OSCC Outcomes 
PLoS ONE  2012;7(9):e46575.
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.
PMCID: PMC3460916  PMID: 23029552
7.  Can a Metastatic Gene Expression Profile Outperform Tumor Size as a Predictor of Occult Lymph Node Metastasis in Oral Cancer Patients? 
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 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 OSCC with 40 node-negative (≥18 months) OSCC. 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 using tumor size alone.
We identified five genes differentially expressed between node-positive and node-negative OSCC 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 to that of a model using 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 prediction of occult metastasis. Independent validation using 31 metastatic and 13 non-metastatic cases revealed a significant under-expression of CTONG2002744 (p = 0.0004).
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 are warranted.
PMCID: PMC3078996  PMID: 21300763
oral squamous cell carcinoma; oral cancer; genetic expression profiles; gene expression profiling; metastasis; microarrays
8.  Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival 
Molecular Cancer  2010;9:143.
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.
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).
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.
PMCID: PMC2893102  PMID: 20537188
9.  A Genetic Expression Profile Associated With Oral Cancer Identifies A Group of Patients at High-Risk of Poor Survival 
To determine if gene expression signature of invasive oral squamous cell carcinoma (OSCC) can sub-classify OSCC on the basis of survival.
Experimental Design
We analyzed the expression of 131 genes in 119 OSCC, 35 normal and 17 dysplastic mucosae to identify cluster-defined sub-groups. 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 Characteristics (ROC) analysis.
The 3-year overall mean survival (± SE) for a cluster of 45 OSCC patients was 38.7 ± 0.09%, compared to 69.1 ± 0.08% for the remaining patients. Multivariate analysis adjusted for age, sex and stage showed that the 45 OSCC cluster patients had worse overall and OSCC-specific survival (HR=3.31, 95% CI: 1.66, 6.58; HR=5.43, 95% CI: 2.32, 12.73, respectively). Stepwise Cox regression on the 131 probe sets revealed that a model with a term for LAMC2 (laminin, gamma 2) 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 (‘PCA’) 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 to 0.70 for stage alone (p=0.013 and 0.008, respectively).
Gene expression and stage combined predict survival of OSCC patients better than stage alone.
PMCID: PMC2683360  PMID: 19228736
10.  Genome-wide gene expression profiles of HPV-positive and HPV-negative oropharyngeal cancer: potential implications for treatment choices 
To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral squamous cell carcinoma (OSCC).
We used Affymetrix U133 plus 2.0 arrays to examine gene expression profiles of OSCC and normal oral tissue. HPV DNA was detected using PCR 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 (IPA 5.0).
Tumor tissue from 119 primary OSCC patients and normal oral tissue from 35 patients without cancer, all of whom were treated at three University of Washington-affiliated medical centers.
HPV DNA was found in 41 of 119 (34.5%) tumors and 2 of 35 (5.7%) normal tissue samples, with 39 of 43 HPV being HPV type 16; there was a higher prevalence of HPV DNA 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 between HPV-positive and HPV-negative oropharyngeal cancer. The most prominent functions of these genes are DNA replication, DNA repair, and cell cycle. Some genes differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer (e.g., TYMS, STMN1, CCND1 and RBBP4) are involved in chemotherapy or radiation sensitivity.
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.
PMCID: PMC2761829  PMID: 19221247
Human papillomavirus; oral cancer; gene expression
11.  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 early detection of invasive OSCC, we compared gene expression of incident primary OSCC, oral dysplasia, and clinically normal oral tissue from surgical patients without head and neck cancer or pre-neoplastic 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 gamma 2 chain, and COL4A1, encoding collagen, type IV, alpha 1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I, alpha 1 chain, and PADI1, encoding peptidyl arginine deiminase, type 1, also can 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 (HNSCC) 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 qRT-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 OSCC patients.
PMCID: PMC2575803  PMID: 18669583
oral squamous cell carcinoma; oral cancer; genetic expression profiles; microarrays

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