John R. Houck

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-γ2 chain, and COL4A1, encoding collagen, type IV α1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I α1 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. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2152–62)

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.

Genetic Factors in Catechol Estrogen Metabolism in Relation to the Risk of Endometrial Cancer

2-Hydroxylated metabolites of estrogen have been shown to have antiangiogenic effects and inhibit tumor cell proliferation, whereas 4-hydroxylated metabolites have been implicated in carcinogenesis. We examined whether polymorphisms in certain genes involved in estrogen metabolism are associated with endometrial cancer risk in a population-based case-control study with 371 cases and 420 controls. Based on previously published genotype-phenotype correlation studies, we defined variant alleles thought to increase estrogen 2-hydroxylation as presumptively low-risk (CYP1A1 m1 T6235C and m2 Ile462Val) and those thought to increase estrogen 4-hydroxylation as high-risk (CYP1A1 m4 Thr461Asn, CYP1A2 A734C, and CYP1B1 Leu432Val). Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression. Carrying at least one CYP1A1 m1 or m2 variant allele was associated with a decreased risk of endometrial cancer [ORs (95% CIs), 0.64 (0.44-0.93) and 0.54 (0.30-0.99), respectively]. No strong alteration in risk was observed among women with any of the putative high-risk alleles. When CYP1A1, CYP1A2, and CYP1B1 genotypes were combined and ranked by the number of putative low-risk genotypes carried, women with four or five low-risk genotypes had a reduced risk of endometrial cancer (OR, 0.29; 95% CI, 0.15-0.56) compared with women with one or none. No appreciable alteration in risk was observed among women carrying two or three low-risk genotypes. Some of our findings are consistent with the hypothesis that increased estrogen 2-hydroxylation is associated with decreased endometrial cancer risk, but replication of these results is required before any firm conclusions can be reached.

Evaluation of locus heterogeneity and EXT1 mutations in 34 families with hereditary multiple exostoses

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by growth of benign bone tumors. Three chromosomal loci have been implicated in this genetically heterogeneous disease: EXT1 at 8q24, EXT2 at 11p13, and EXT3 on 19p. EXT1 and EXT2 were recently cloned. We evaluated 34 families with EXT to estimate the proportion of disease attributable to EXT1, EXT2, and EXT3 and to investigate the spectrum of EXT1 mutations. Linkage analyses combined with heterogeneity testing provides strong evidence in favor of linkage of disease to both chromosomes 8 and 11, but does not support evidence of linkage to chromosome 19 in this data set. The 11 EXT1 exons were PCR‐amplified and sequenced in all 11 isolated cases and in 20 of the 23 familial cases. Twelve different novel EXT1 mutations were detected, including 5 frame‐shift deletions or insertions, 1 codon deletion, and 6 single base‐pair substitutions distributed across 8 of the exons. Only 2 of the mutations were detected in more than one family. Three mutations affect sites in which alterations were previously reported. Nonchain‐terminating missense mutations were identified in codons 280 and 340, both coding for conserved arginine residues. These residues may be crucial to the function of this protein. Although the prevalence of EXT has been estimated to be approximately 1/50,000 individuals, the disease has been reported to occur much more frequently in the Chamorro natives on Guam. Our detection of an EXT1 mutation in one Chamorro subject will allow investigation of a possible founder effect in this population. Combined mutational and heterogeneity analyses in this set of families with multiple exostoses suggest that 66% of our total sample, including 45% of isolated and 77% of familial cases, are attributable to abnormalities in EXT1. Hum Mutat 11:231–239, 1998.

Screening for Deleterious Nonsynonymous Single-Nucleotide Polymorphisms in Genes Involved in Steroid Hormone Metabolism and Response

To facilitate selection of single-nucleotide polymorphisms (SNP) for molecular epidemiologic studies investigating the hormonal carcinogenesis hypothesis, we used two sequence homology–based tools [Sort Intolerant from Tolerant (SIFT) and Polymorphism Phenotype (PolyPhen)] to predict the potential impact a nonsynonymous SNP (nsSNP), which results in an amino acid substitution, may have on the activity of proteins encoded by genes involved in the steroid hormone metabolism and response pathway. We screened 137 variants. Of these, 28% were predicted by SIFT and PolyPhen as having a potentially damaging effect on protein function. Investigation into the association of these variant alleles with hormone-related cancers may prove to be fruitful.

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.

Simultaneous Isolation of DNA and RNA from the Same Cell Population Obtained by Laser Capture Microdissection for Genome and Transcriptome Profiling

Laser capture microdissection (LCM) is used extensively for genome and transcriptome profiling. Traditionally, however, DNA and RNA are purified from separate populations of LCM-harvested cells, limiting the strength of inferences about the relationship between gene expression and gene sequence variation. There have been no published protocols for the simultaneous isolation of DNA and RNA from the same cells that are obtained by LCM of patient tissue specimens. Here we report an adaptation of the Qiagen AllPrep method that allows the purification of DNA and RNA from the same LCM-harvested cells. We compared DNA and RNA purified by the QIAamp DNA Micro kit and the PicoPure RNA Isolation kit, respectively, from LCM-collected cells from adjacent tissue sections of the same specimen. The adapted method yields 90% of DNA and 38% of RNA compared with the individual methods. When tested with the GeneChip 250K Nsp Array, the concordance rate of the single nucleotide polymorphism heterozygosity calls was 98%. When tested with the GeneChip U133 Plus 2.0 Array, the correlation coefficient of the raw gene expression was 97%. Thus, we developed a method to obtain both DNA and RNA material from a single population of LCM-harvested cells and herein discuss the strengths and limitations of this methodology.

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 γ2) 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.

Examination of Oral Cancer Biomarkers by Tissue Microarray Analysis

OBJECTIVE To validate the DNA microarray results on a subset of genes that could potentially serve as biomarkers of oral squamous cell carcinoma (OSCC) by examining their expression with an alternate quantitative method and by assessing their protein levels. DESIGN Based on DNA microarray data from our laboratory and data reported in the literature, we identified 6 potential biomarkers of OSCC to investigate further. We used quantitative real-time polymerase chain reaction to examine expression changes of CDH11, MMP3, SPARC, POSTN, TNC, and TGM3 in OSCC and histologically normal control tissues. We further examined validated markers at the protein level by immunohistochemical analysis of OSCC tissue microarray sections. RESULTS Quantitative real-time polymerase chain reaction analysis revealed upregulation of CDH11, SPARC, POSTN, and TNC gene expression and decreased TGM3 expression in OSCC tissue compared with control tissue; MMP3 was not found to be differentially expressed. In tissue microarray immunohistochemical analyses, SPARC (secreted protein, acidic, rich in cysteine), periostin, and tenascin C exhibited increased protein expression in tumor tissue compared with control tissue, and their expression was primarily localized within tumor-associated stroma rather than tumor epithelium. Conversely, transglutaminase 3 protein expression was found only within keratinocytes in control tissue and was significantly downregulated in cancer cells. CONCLUSIONS Of 6 potential gene markers of OSCC, initially identified by DNA microarray analyses, differential expression of CDH11, SPARC, POSTN, TNC, and TGM3 were validated by quantitative real-time polymerase chain reaction. Differential expression and localization of proteins encoded by SPARC, POSTN, TNC, and TGM3 were clearly shown by tissue microarray immunohistochemical analysis.

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. Cancer Epidemiol Biomarkers Prev; 20(12); 2628–36. ©2011 AACR.