[Show abstract][Hide abstract] ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) is characterized by significant genomic instability that could lead to clonal diversity. Intratumor clonal heterogeneity has been proposed as a major attribute underlying tumor evolution, progression, and resistance to chemotherapy and radiation. Understanding genetic heterogeneity could lead to treatments specific to resistant and metastatic tumor cells. To characterize the degree of intratumor genetic heterogeneity within a single tumor, we performed whole-genome sequencing on three separate regions of an human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma and two separate regions from one corresponding cervical lymph node metastasis. This approach achieved coverage of approximately 97.9% of the genome across all samples. In total, 5701 somatic point mutations (SPMs) and 4347 small somatic insertions and deletions (indels)were detected in at least one sample. Ninety-two percent of SPMs and 77% of indels were validated in a second set of samples adjacent to the discovery set. All five tumor samples shared 41% of SPMs, 57% of the 1805 genes with SPMs, and 34 of 55 cancer genes. The distribution of SPMs allowed phylogenetic reconstruction of this tumor's evolutionary pathway and showed that the metastatic samples arose as a late event. The degree of intratumor heterogeneity showed that a single biopsy may not represent the entire mutational landscape of HNSCC tumors. This approach may be used to further characterize intratumor heterogeneity in more patients, and their sample-to-sample variations could reveal the evolutionary process of cancer cells, facilitate our understanding of tumorigenesis, and enable the development of novel targeted therapies.
Neoplasia (New York, N.Y.) 12/2013; 15(12):1371-1378. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The outcome of allogeneic hematopoietic cell transplantation is influenced by donor/recipient genetic disparity at loci both inside and outside the MHC on chromosome 6p. Although disparity at loci within the MHC is the most important risk factor for the development of severe GVHD, disparity at loci outside the MHC that encode minor histocompatibility (H) antigens can elicit GVHD and GVL activity in donor/recipient pairs who are otherwise genetically identical across the MHC. Minor H antigens are created by sequence and structural variations within the genome. The enormous variation that characterizes the human genome suggests that the total number of minor H loci is probably large and ensures that all donor/recipient pairs, despite selection for identity at the MHC, will be mismatched for many minor H antigens. In addition to mismatch at minor H loci, unrelated donor/recipient pairs exhibit genetic disparity at numerous loci within the MHC, particularly HLA-DP, despite selection for identity at HLA-A, -B, -C, and -DRB1. Disparity at HLA-DP exists in 80% of unrelated pairs and clearly influences the outcome of unrelated hematopoietic cell transplantation; the magnitude of this effect probably exceeds that associated with disparity at any locus outside the MHC.
[Show abstract][Hide abstract] ABSTRACT: 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.
Observational assessment of DNA copy number, miRNA, and RNA expression in primary and metastatic OSCC.
University of Washington Medical Center and affiliated hospitals.
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.
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.
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.
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.
Otolaryngology Head and Neck Surgery 04/2012; 147(3):501-8. · 1.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Candidate genetic associations with acute GVHD (aGVHD) were evaluated with the use of genotyped and imputed single-nucleotide polymorphism data from genome-wide scans of 1298 allogeneic hematopoietic cell transplantation (HCT) donors and recipients. Of 40 previously reported candidate SNPs, 6 were successfully genotyped, and 10 were imputed and passed criteria for analysis. Patient and donor genotypes were assessed for association with grades IIb-IV and III-IV aGVHD, stratified by donor type, in univariate and multivariate allelic, recessive and dominant models. Use of imputed genotypes to replicate previous IL10 associations was validated. Similar to previous publications, the IL6 donor genotype for rs1800795 was associated with a 20%-50% increased risk for grade IIb-IV aGVHD after unrelated HCT in the allelic (adjusted P = .011) and recessive (adjusted P = .0013) models. The donor genotype was associated with a 60% increase in risk for grade III-IV aGVHD after related HCT (adjusted P = .028). Other associations were found for IL2, CTLA4, HPSE, and MTHFR but were inconsistent with original publications. These results illustrate the advantages of using imputed single-nucleotide polymorphism data in genetic analyses and demonstrate the importance of validation in genetic association studies.
[Show abstract][Hide abstract] ABSTRACT: Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a major complication of cancer treatment. We compared gene expression in CD34+ cells from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for lymphoma with controls who did not develop t-MDS/AML. We observed altered gene expression related to mitochondrial function, metabolism, and hematopoietic regulation in pre-aHCT samples from patients who subsequently developed t-MDS/AML. Progression to overt t-MDS/AML was associated with additional alterations in cell-cycle regulatory genes. An optimal 38-gene PBSC classifier accurately distinguished patients who did or did not develop t-MDS/AML in an independent group of patients. We conclude that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and accurately identify patients at risk for this complication.
Cancer cell 11/2011; 20(5):591-605. · 25.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Affymetrix GeneChip Exon Array can be used to detect alternative splice variants. Microarray Detection of Alternative Splicing (MIDAS) and Partek(®) Genomics Suite (Partek(®) GS) are among the most popular analytical methods used to analyze exon array data. While both methods utilize statistical significance for testing, MIDAS and Partek(®) GS could produce somewhat different results due to different underlying assumptions. Comparing MIDAS and Partek(®) GS is quite difficult due to their substantially different mathematical formulations and assumptions regarding alternative splice variants. For meaningful comparison, we have used the previously published generalized probe model (GPM) which encompasses both MIDAS and Partek(®) GS under different assumptions. We analyzed a colon cancer exon array data set using MIDAS, Partek(®) GS and GPM. MIDAS and Partek(®) GS produced quite different sets of genes that are considered to have alternative splice variants. Further, we found that GPM produced results similar to MIDAS as well as to Partek(®) GS under their respective assumptions. Within the GPM, we show how discoveries relating to alternative variants can be quite different due to different assumptions. MIDAS focuses on relative changes in expression values across different exons within genes and tends to be robust but less efficient. Partek(®) GS, however, uses absolute expression values of individual exons within genes and tends to be more efficient but more sensitive to the presence of outliers. From our observations, we conclude that MIDAS and Partek(®) GS produce complementary results, and discoveries from both analyses should be considered.
International journal of biomedical science : IJBS. 09/2011; 7(3):172-180.
[Show abstract][Hide abstract] ABSTRACT: Exposure to estrogens increases the risk of endometrial cancer. Certain estrogen metabolites can form bulky DNA adducts, which are removed via nucleotide excision repair (NER), and the ability to carry out this repair might be related to endometrial cancer risk.
We examined 64 tag and functional single-nucleotide polymorphisms (SNPs) in the NER genes ERCC1, ERCC2 (XPD), ERCC3 (XPB), ERCC4 (XPF), ERCC5 (XPG), LIG1, XPA, and XPC in a population-based case-control study in Washington state, with 783 endometrial cancer cases and 795 controls.
The presence of ERCC5 rs4150386 C, LIG1 rs3730865 C, XPA rs2808667 T, or XPC rs3731127 T alleles was associated with risk of endometrial cancer, with respective age-, county-, and reference year-adjusted per-allele ORs and 95% CIs of 0.68 (0.53-0.87, P = 0.002), 1.46 (1.02-2.10, P = 0.04), 0.71 (0.52-0.97, P = 0.03), and 1.57 (1.13-2.17, P = 0.007), respectively.
Certain ERCC5, LIG1, XPA, and XPC genotypes might influence endometrial cancer risk.
Because of multiple redundancies in DNA repair pathways (and therefore a low prior probability) and the large number of associations examined, false-positive findings are likely. Further characterization of the relation between variation in NER genes and endometrial cancer risk is warranted.
[Show abstract][Hide abstract] ABSTRACT: In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15-12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.
Radiation Research 02/2011; 175(2):172-84. · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
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.
Clinical Cancer Research 02/2011; 17(8):2466-73. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Molecular Cancer 01/2010; 9:143. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral cavity and oropharyngeal squamous cell carcinoma (OSCC).
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.
Three medical centers affiliated with the University of Washington.
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.
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.
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.
Archives of otolaryngology--head & neck surgery 03/2009; 135(2):180-8. · 1.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine if gene expression signature of invasive oral squamous cell carcinoma (OSCC) can subclassify OSCC based on survival.
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.
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).
Gene expression and stage combined predict survival of OSCC patients better than stage alone.
Clinical Cancer Research 03/2009; 15(4):1353-61. · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CYP19A1 encodes for aromatase, which irreversibly converts androgens to estrogens; variation in this gene may affect individual susceptibility to breast cancer and other sex hormone-dependent outcomes. In a case-control study nested within a breast self-examination trial conducted in China, we examined whether CYP19A1 polymorphisms (rs1870049, rs1004982, rs28566535, rs936306, rs11636639, rs767199, rs4775936, rs11575899, rs10046, and rs4646) were associated with risk of breast cancer and fibrocystic breast conditions. Cases were diagnosed with breast cancer (n = 614) or fibrocystic breast conditions (n = 465) during 1989 to 2000. Controls were free of breast disease during the same period (n = 879). Presence of proliferative changes within the extratumoral tissue of women with breast cancer and the lesions of women with fibrocystic conditions only was assessed. None of the polymorphisms were associated with overall risk of breast cancer or fibrocystic breast conditions. Differences in breast cancer risk, however, were observed by proliferation status. The risk of breast cancer with (but not without) proliferative fibrocystic conditions was increased among women homozygous for the minor allele of rs1004982 (C), rs28566535 (C), rs936306 (T), and rs4775936 (C) relative to those homozygous for the major allele [age-adjusted odds ratios (95% confidence intervals), 2.19 (1.24-3.85), 2.20 (1.27-3.82), 1.94 (1.13-3.30), and 1.95 (1.07-3.58), respectively]. Also, haplotypes inferred using all polymorphisms were not associated with overall risk of either outcome, although some block-specific haplotypes were associated with an increased risk of breast cancer with concurrent proliferative fibrocystic conditions. Our findings suggest that CYP19A1 variation may enhance breast cancer development in some women, but further confirmation is warranted.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Diagnostics 04/2008; 10(2):129-34. · 3.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is one of the most common and deadly forms of hematopoietic malignancies. We hypothesized that microarray studies could identify previously unrecognized expression changes that occur only in AML blasts. We were particularly interested in those genes with increased expression in AML, believing that these genes may be potential therapeutic targets. To test this hypothesis, we compared gene expression profiles between normal hematopoietic cells from 38 healthy donors and leukemic blasts from 26 AML patients. Normal hematopoietic samples included CD34+ selected cells (N = 18), unselected bone marrows (N = 10), and unselected peripheral bloods (N = 10). Twenty genes displayed AML-specific expression changes that were not found in the normal hematopoietic cells. Subsequent analyses using microarray data from 285 additional AML patients confirmed expression changes for 13 of the 20 genes. Seven genes (BIK, CCNA1, FUT4, IL3RA, HOMER3, JAG1, WT1) displayed increased expression in AML, while 6 genes (ALDHA1A, PELO, PLXNC1, PRUNE, SERPINB9, TRIB2) displayed decreased expression. Quantitative RT/PCR studies for the 7 over-expressed genes were performed in an independent set of 9 normal and 21 pediatric AML samples. All 7 over-expressed genes displayed an increased expression in the AML samples compared to normals. Three of the 7 over-expressed genes (WT1, CCNA1, and IL3RA) have already been linked to leukemogenesis and/or AML prognosis, while little is known about the role of the other 4 over-expressed genes in AML. Future studies will determine their potential role in leukemogenesis and their clinical significance.
Genes Chromosomes and Cancer 02/2008; 47(1):8-20. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metastasis is the most important predictor of survival in patients with oral squamous cell carcinoma (OSCC). We tested the hypothesis that there is a genetic expression profile associated with OSCC metastasis.
We obtained samples from 6 OSCC node-positive primary tumors and their matched metastatic lymph nodes, and 5 OSCC node-negative primary tumors. Using laser capture microdissection, we isolated OSCC cells from metastatic lymph nodes and compared them with those from matched primary tumors and unmatched node-negative primary tumors using Affymetrix Human Genome Focus arrays.
Comparison of tumor cells from the lymph nodes with those from the unmatched, node-negative primary tumors revealed differential expression of 160 genes. Hierarchical clustering and principal component analysis using this 160-gene set showed that the node-negative samples were distinguishable from both, node-positive primary tumors and tumors in the lymph nodes. Many of the expression changes found in the metastatic cells from the lymph nodes were also found in the node-positive primary tumors. Immunohistochemical analysis for transglutaminase-3 and keratin 16 confirmed the differential genetic expression for these genes.
These preliminary results suggest that there may be a metastatic gene expression profile present in node-positive primary OSCC.
Head & Neck 10/2007; 29(9):803-14. · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip that uses multiple oligonucleotide probes (i.e. probe set), since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip gene expression array data.
We developed a two-step approach to predict alternative splicing from GeneChip data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic) samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic medulloblastomas to non-metastatic ones. We checked the consistency of some of our findings with information in UCSC Human Genome Browser.
The two-step approach described in this paper is capable of predicting some alternative splicing from multiple oligonucleotide-based gene expression array data with GeneChip technology. Our method employs the extensive repositories of gene expression array data available and generates alternative splicing hypotheses, which can be further validated by experimental studies.
Theoretical Biology and Medical Modelling 02/2006; 3:19. · 1.27 Impact Factor