Cell wall protein variation, break-induced replication, and subtelomere dynamics in Candida glabrata.

Candida glabrata is an opportunistic pathogen of humans, responsible for up to 30% of disseminated candidiasis. Adherence of C. glabrata to host cells is mediated by adhesin-like proteins (ALPs), about half of which are encoded in the subtelomeres. We performed a de novo assembly of two C. glabrata strains, BG2 and BG3993, using long single-molecule real-time (SMRT) reads, and constructed high-quality telomere-to-telomere assemblies of all 13 chromosomes to assess differences between C. glabrata strains. We documented variation between strains, and in agreement with earlier studies, found high (~0.5%-1%) frequencies of SNVs across the genome, including within subtelomeric regions. We documented changes in ALP gene structure and complement: there are large length differences in ALP genes in different strains, resulting from copy number variation in tandem repeats. We compared strains to characterize chromosome rearrangement events including within the poorly characterized subtelomeric regions. We show that rearrangements within the subtelomere regions all affect ALP-encoding genes, and 14/16 involve just the most terminal ALP gene. We present evidence that these rearrangements are mediated by break-induced replication. This study highlights the constrained nature of subtelomeric changes impacting ALP gene complement and subtelomere structure.

De novo genome assembly of Candida glabrata reveals cell wall protein complement and structure of dispersed tandem repeat arrays.

Candida glabratais an opportunistic pathogen in humans, responsible for approximately 20% of disseminated candidiasis. Candida glabrata's ability to adhere to host tissue is mediated by GPI-anchored cell wall proteins (GPI-CWPs); the corresponding genes contain long tandem repeat regions. These repeat regions resulted in assembly errors in the reference genome. Here, we performed a de novo assembly of the C. glabrata type strain CBS138 using long single-molecule real-time reads, with short read sequences (Illumina) for refinement, and constructed telomere-to-telomere assemblies of all 13 chromosomes. Our assembly has excellent agreement overall with the current reference genome, but we made substantial corrections within tandem repeat regions. Specifically, we removed 62 genes of which 45 were scrambled due to misassembly in the reference. We annotated 31 novel ORFs of which 24 ORFs are GPI-CWPs. In addition, we corrected the tandem repeat structure of an additional 21 genes. Our corrections to the genome were substantial, with the length of new genes and tandem repeat corrections amounting to approximately 3.8% of the ORFeome length. As most corrections were within the coding regions of GPI-CWP genes, our genome assembly establishes a high-quality reference set of genes and repeat structures for the functional analysis of these cell surface proteins.

Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity.

Inflammatory processes can reliably be assessed by (19)F MRI using perfluorocarbons (PFCs), which is primarily based on the efficient uptake of emulsified PFCs by circulating cells of the monocyte-macrophage system and subsequent infiltration of the (19)F-labeled cells into affected tissue. An ideal candidate for the sensitive detection of fluorine-loaded cells is the biochemically inert perfluoro-15-crown-5 ether (PFCE), as it contains 20 magnetically equivalent (19)F atoms. However, the biological half-life of PFCE in the liver and spleen is extremely long, and so this substance is not suitable for future clinical applications. In the present study, we investigated alternative, nontoxic PFCs with predicted short biological half-lives and high fluorine content: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD) and trans-bis-perfluorobutyl ethylene (F-44E). Despite the complex spectra of these compounds, we obtained artifact-free images using sine-squared acquisition-weighted three-dimensional chemical shift imaging and dedicated reconstruction accomplished with in-house-developed software. The signal-to-noise ratio of the images was maximized using a Nutall window with only moderate localization error. Using this approach, the retention times of the different PFCs in murine liver and spleen were determined at 9.4 T. The biological half-lives were estimated to be 9 days (PFD), 12 days (PFOB) and 28 days (F-44E), compared with more than 250 days for PFCE. In vivo sensitivity for inflammation imaging was assessed using an ear clip injury model. The alternative PFCs PFOB and F-44E provided 37% and 43%, respectively, of the PFCE intensities, whereas PFD did not show any signal in the ear model. Thus, for in vivo monitoring of inflammatory processes, PFOB emerges as the most promising candidate for possible future translation of (19)F MR inflammation imaging to human applications.

Vascular phenotyping of brain tumors using magnetic resonance microscopy (µMRI).

Abnormal vascular phenotypes have been implicated in neuropathologies ranging from Alzheimer's disease to brain tumors. The development of transgenic mouse models of such diseases has created a crucial need for characterizing the murine neurovasculature. Although histologic techniques are excellent for imaging the microvasculature at submicron resolutions, they offer only limited coverage. It is also challenging to reconstruct the three-dimensional (3D) vasculature and other structures, such as white matter tracts, after tissue sectioning. Here, we describe a novel method for 3D whole-brain mapping of the murine vasculature using magnetic resonance microscopy (µMRI), and its application to a preclinical brain tumor model. The 3D vascular architecture was characterized by six morphologic parameters: vessel length, vessel radius, microvessel density, length per unit volume, fractional blood volume, and tortuosity. Region-of-interest analysis showed significant differences in the vascular phenotype between the tumor and the contralateral brain, as well as between postinoculation day 12 and day 17 tumors. These results unequivocally show the feasibility of using µMRI to characterize the vascular phenotype of brain tumors. Finally, we show that combining these vascular data with coregistered images acquired with diffusion-weighted MRI provides a new tool for investigating the relationship between angiogenesis and concomitant changes in the brain tumor microenvironment.

Presence of 5-methylcytosine in CpNpG trinucleotides in the human genome.

While the methylation machinery of mammalian cells has been shown to be capable of both maintenance and de novo methylation at CpNpG sites, CpNpG methylation in the human genome has not been demonstrated. Here, we report the first observation of 5-methylcytosines in CpNpG triplets in the human genome. We identify the existence of CpNpG methylation in a number of genes which contain trinucleotide repeat regions, including the androgen receptor (AR). We further analyzed DNA extracted from primary tissue samples and found the same pattern of CpNpG methylation. To confirm our results, we performed Southern blot analysis by analyzing the cleavage sites of restriction enzymes within exon 1 of the AR gene and found direct evidence of the presence of 5mCs in CpNpG triplets in the human genome. Our results also suggest that this methylation pattern may be due to the human DNA methyltransferases DNMT1 and DNMT3A. Although the functional significance needs to be tested further, the discovery of inheritable CpNpG methylation in the human genome may have important implications in our understanding of gene regulation and of the development of various diseases, including cancer.

TP53 mutations and survival in squamous-cell carcinoma of the head and neck.

BACKGROUND: The abrogation of function of the tumor-suppressor protein p53 as a result of mutation of its gene, TP53, is one of the most common genetic alterations in cancer cells. We evaluated TP53 mutations and survival in patients with squamous-cell carcinoma of the head and neck. METHODS: A total of 560 patients with squamous-cell carcinoma of the head and neck who were treated surgically with curative intent were enrolled in our prospective multicenter, 7-year study. TP53 mutations were analyzed in DNA from the tumor specimens with the use of the Affymetrix p53 chip and the Surveyor DNA endonuclease and denaturing high-performance liquid chromatography. Mutations were classified into two groups, disruptive and nondisruptive, according to the degree of disturbance of protein structure predicted from the crystal structure of the p53-DNA complexes. TP53 mutational status was compared with clinical outcome. RESULTS: TP53 mutations were found in tumors from 224 of 420 patients (53.3%). As compared with wild-type TP53, the presence of any TP53 mutation was associated with decreased overall survival (hazard ratio for death, 1.4; 95% confidence interval [CI], 1.1 to 1.8; P=0.009), with an even stronger association with disruptive mutations (hazard ratio, 1.7; 95% CI, 1.3 to 2.4; P<0.001) and no significant association with nondisruptive mutations (hazard ratio, 1.2; 95% CI, 0.9 to 1.7; P=0.16). In multivariate analyses a disruptive TP53 alteration, as compared with the absence of a TP53 mutation, had an independent, significant association with decreased survival (hazard ratio, 1.7; 95% CI, 1.2 to 2.4; P=0.003). CONCLUSIONS: Disruptive TP53 mutations in tumor DNA are associated with reduced survival after surgical treatment of squamous-cell carcinoma of the head and neck.

Occurrence, shape, and dimensions of large surface hemimicelles made of semifluorinated alkanes. Elongated versus circular Hemimicelles. Pit- and tip-centered hemimicelles.

The formation of large (approximately 20-35 nm) surface hemimicelles in monolayers of semifluorinated alkanes, C(n)F(2)(n)(+1)C(m)H(2)(m)(+1) (FnHm), observed after transfer onto silicon wafers, is a general phenomenon. F6H16 and F8H14 exclusively form highly monodisperse circular hemimicelles, organized in a hexagonal array. The other FnHm investigated form both circular and elongated hemimicelles. The longer FnHm is, the larger the area fraction of elongated micelles; both the hydrocarbon block (H-block) and the fluorocarbon block (F-block) affect this area fraction. The length of the elongated micelles increases with the total length of the diblocks. The diameter of the circular micelles increases with the length of the H-block but, unexpectedly, not with that of the F-block. Model calculations account for these observations. Close examination of the circular micelles showed that they generally present a pit or a tip at their center. The width of the elongated micelles is comparable to the radius of the circular micelles, suggesting that the latter arise from a partition of elongated micelles, followed by coalescence of the edges of the resulting fragments. The elongated micelles become shorter and fewer when surface pressure increases, further suggesting a conversion of elongated into circular micelles. This conversion is reversible. The surface pressure-molecular area isotherms do not present any feature that forebears the existence of hemimicelles. The obtaining of stable surface patterns from simple, "nonpolar" molecular fluorocarbon/hydrocarbon diblocks opens a new approach for producing featured nanostructures from organic templates.

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus.

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

Colorimetric approach to high-throughput mutation analysis.

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.

Increased mitochondrial DNA content in saliva associated with head and neck cancer.

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

Epstein-Barr virus in head and neck cancer assessed by quantitative polymerase chain reaction.

OBJECTIVES/HYPOTHESIS: Epstein-Barr virus (EBV) has classically been associated with nasopharyngeal carcinoma and Burkitt's lymphoma. Recently, multiple studies have been published linking EBV with oral squamous cell carcinoma and, to a lesser extent, hypopharyngeal and laryngeal tumors. Using a sensitive method of detection, the authors sought to analyze the presence and quantity of EBV DNA in a large cohort of head and neck cancers. STUDY DESIGN: : Retrospective cohort study. METHODS: Three hundred head and neck cancer samples exclusive of nasopharyngeal carcinoma were examined for the presence of EBV using quantitative polymerase chain reaction. Eighty-four tumor samples from the larynx, 30 from the hypopharynx, 73 from the oropharynx, and 113 from the oral cavity were analyzed for EBV quantity, which was expressed as the number of viral copies per cell genome. Representative samples, which contained the highest EBV DNA levels, were examined using in situ hybridization. Results were correlated with tumor grade and site and tobacco and alcohol exposure. RESULTS: Three of 300 (1%) tumor samples were overtly positive for EBV DNA (defined as >0.1 copies of viral DNA/cell genome). Five of 300 (2%) tumor samples showed low levels (defined as >0.01 and <0.1 copies of viral DNA/cell genome), and 68 of 300 tumor samples (23%) showed trace levels (defined as < 0.01 copies of viral DNA/cell genome) of EBV DNA. No correlation was found between EBV positivity and tobacco exposure, alcohol exposure, or tumor grade. CONCLUSION: In the overwhelming majority of head and neck cancers in this North American cohort, EBV did not appear to contribute to growth of a dominant clonal population with integrated EBV genome and was unlikely to be a genetic etiological agent in tumor development. The low quantities of EBV detected in a minority of head and neck cancers may be related to the presence of EBV genome in rare lymphoid or epithelial cells adjacent to the primary head and neck cancer.

Gene expression alterations over large chromosomal regions in cancers include multiple genes unrelated to malignant progression.

In solid tumors, the relationship between DNA copy number and global expression over large chromosomal regions has not been systematically explored. We used a 12,626-gene expression array analysis of head and neck squamous cell carcinoma and normal oral mucosa and annotated gene expression levels to specific chromosomal loci. Expression alterations correlated with reported data using comparative genomic hybridization. When genes with significant differences in expression between normal and malignant lesions, as defined by significance analysis of microarrays (SAM), were compared to nonsignificant genes, similar chromosomal patterns of alteration in expression were noted. Individual tumors underwent microsatellite analysis and chi(2) analysis of expression at 3p and 22q. Significant 3p underexpression and 22q overexpression were found in all primary tumors with 3p and 22q allelic imbalance, respectively, whereas no tumor without allelic imbalance on these chromosomal arms demonstrated expression differences. Loss and gain of chromosomal material in solid cancers can alter gene expression over large chromosomal regions, including multiple genes unrelated to malignant progression.

Real-time gap ligase chain reaction: a rapid semiquantitative assay for detecting p53 mutation at low levels in surgical margins and lymph nodes from resected lung and head and neck tumors.

PURPOSE: We have developed a real-time semiquantitative gap ligase chain reaction for detecting p53 point mutations at low level in a background of excess of wild-type DNA. EXPERIMENTAL DESIGN: This method was validated by direct comparison to a previously validated but cumbersome phage plaque hybridization assay. Forty-one surgical margins and lymph nodes from 10 cases of head and neck squamous cell carcinoma and lung carcinoma were tested for p53 mutant clones. RESULTS: Both methods detected p53 mutants in margins from 8 of the 10 cases, whereas standard pathology detected cancer cells in only 3 cases. Positive margins included tissue samples with a tumor/normal DNA ratio of up to 1:1000. CONCLUSIONS: This novel molecular approach can be performed in <5 h facilitating intraoperative use for real-time surgical resection.

Intraoperative molecular margin analysis in head and neck cancer.

BACKGROUND: Tumor-specific molecular alterations in surgical margins have been shown to predict risk of local recurrence. However, assays used for these analyses are time-consuming and therefore cannot be used in the intraoperative setting. OBJECTIVE: To detect and quantify tumor-specific methylated promoter sequences in surgical margins in a time frame suitable for intraoperative use. DESIGN: A novel quantitative methylation-specific polymerase chain reaction (QMSP) protocol. METHODS: A total of 13 patients with head and neck squamous cell carcinoma (HNSCC) were initially characterized for molecular alterations in their tumor at the time of biopsy. Six primary tumors were found to harbor promoter hypermethylation for p16 and O6-methylguanine-DNA-methyltransferase (MGMT) genes. Rapid QMSP was then used to identify promoter hypermethylation of these genes in the surgical margins. Results were compared with standard intraoperative histologic frozen section analysis and with conventional QMSP. RESULTS: Using our rapid QMSP assay, we found that 3 patients had methylation-positive margins. Tumor margins from 2 patients were methylated for p16 alone, and margins from 1 patient were methylated for p16 and MGMT simultaneously. Molecular margin analysis was completed in less than 5 hours, a time frame appropriate for selected major HNSCC resections that require combined primary tumor resection, cervical lymphadenectomy, and complex reconstruction. This technique was comparable in sensitivity to conventional QMSP. CONCLUSION: Rapid molecular margin analysis using QMSP is feasible and may be performed intraoperatively in selected patients with HNSCC that requires extensive resection.

Serum protein MALDI profiling to distinguish upper aerodigestive tract cancer patients from control subjects.

BACKGROUND: There are no reliable blood markers for the early detection and monitoring of aerodigestive tract tumors. Recent studies have suggested that serum protein patterns may be able to distinguish cancer patients from control subjects. METHODS: We used matrix-assisted laser desorption and ionization (MALDI) mass spectroscopy to obtain serum protein patterns from patients with head and neck cancer (n = 99) or lung cancer (n = 92) and from control subjects (n = 143) at risk for the development of these cancers. From the mass spectra, we predicted the cancer status of patients using a simple classification procedure based on a t test feature selection and linear discriminant analysis (LDA). We cross-validated the data with 200 random data simulations to establish a range of the LDA tuning parameter, which was used to construct receiver operating characteristic (ROC) curves. RESULTS: Average total protein levels were higher in case patients than in control subjects, although the differences were not statistically significant. Ten individual m/z peaks, from 5 to 111 kd, appeared frequently in head and neck cancer patients but not in control subjects. Using the 45 top predictors, selected by spectral mass and LDA, we observed that ROC curves differed from those expected under the null hypothesis, suggesting that spectral profiles from the sera of patients with head and neck cancer statistically significantly differed from the sera of control subjects. The model developed on head and neck cancer patients could also be used to identify patients with lung cancer. CONCLUSIONS: The pattern of protein spectra in total serum reliably distinguished cancer case patients from control subjects. Incorporation of MALDI assays into prospective longitudinal trials to assess the true predictive values of protein spectra in cancer detection is needed.

Death-associated protein kinase promoter hypermethylation in normal human lymphocytes.

A high frequency of death-associated protein kinase (DAPK) promoter hypermethylation has been noted in B-cell malignancies, head and neck cancers, and other solid tumors, and it has been used as a tumor marker in molecular detection strategies. Low levels of DAPK promoter hypermethylation, ranging from 0.003 to 1.181%, were detected in peripheral blood cells from 75 of 143 (52%) normal subjects by quantitative methylation-specific PCR (Q-MSP). In 10 of 10 selected patients, MSP amplification of a portion of the DAPK promoter followed by PCR product sequencing confirmed dense hypermethylation of the CpG island in their peripheral blood cells. Q-MSP analysis of fluorescence-activated cell-sorted peripheral blood cells from three of these patients demonstrated that a significantly greater proportion of B cells (1.074-6.026%) were DAPK hypermethylated than were T cells, monocytes, or neutrophils, which were <0.06% hypermethylated. Further analysis after sorting of one subject's B cells into IgM+, IgM-, IgG+, and IgG- subpopulations demonstrated that DAPK hypermethylation was predominantly present in the IgM- compared with IgM+ B cells (3.338% versus 0.436%). DAPK promoter hypermethylation was found in IgM- B cells in normal individuals. The same hypermethylation identified in B-cell malignancies may reflect a clonal outgrowth of B cells arising from this compartment and may indicate a susceptibility to neoplastic transformation in a subset of B cells. Normal circulating lymphocytes with DAPK promoter hypermethylation may act as confounding factors in tumor detection based on DAPK hypermethylation.

Epigenetic inactivation of RASSF1A in head and neck cancer.

PURPOSE: RASSF1A, a recently identified candidate tumor suppressor gene, was found to be inactivated in lung cancer and other tumor types. We sought to understand the role of RASSF1A in head and neck cancer. EXPERIMENTAL DESIGN: We analyzed the status of RASSF1A and presence of high-risk human papilloma virus (HPV) in head and neck cancer squamous cell carcinoma (HNSCC) cell lines and primary tumors. We used methylation-specific PCR to detect promoter hypermethylation and direct sequence analysis to detect point mutations in primary tumors and cell lines. 5-aza-2-deoxycytidine was used to demethylate the RASSF1A promoter in cell lines. RESULTS: Promoter methylation of RASSF1A was detected in 42.9% (3 of 7) cell lines and 15% (7 of 46) primary tumors but not in the normal control DNA. Direct sequence analysis revealed a point mutation in a cell line and another in a primary HNSCC. After treatment with 5-aza-2-deoxycytidine, re-expression and demethylation of RASSF1A gene were detected in cell lines with promoter hypermethylation. HPV DNA was detected in 34.7% (16 of 46) primary HNSCC. We found a significant inverse correlation between RASSF1A promoter methylation and HPV infection (P = 0.038). CONCLUSIONS: Our results suggest that RASSF1A is inactivated in a subset of HNSCC primary tumors. Moreover, an inverse correlation between RASSF1A and HPV supports a biological mechanism in which both RASSF1A promoter methylation and HPV infection abrogate the same pathway in tumorigenesis.

A transcriptional progression model for head and neck cancer.

PURPOSE: A genetic progression model for head and neck squamous cell carcinoma (HNSC) has been established and implies the presence of transcriptional dysregulation as a consequence of accumulation of genetic alterations. Although expression array data have been provided for HNSC, the timing of transcriptional dysregulation in the progression from normal mucosa to dyplastic epithelium to invasive HNSC has not been described. Here, we describe a transcriptional progression model of HNSC. EXPERIMENTAL DESIGN: Expression arrays representing >12,000 genes and expressed sequence tags were used to examine malignant lesions (M), premalignant lesions (PM), distant, histopathologically normal mucosa from patients with premalignant or malignant lesions (MN), and normal mucosa from the upper aerodigestive tract of patients with noncancer diagnoses (N). Significance analysis of microarrays, hierarchical clustering, and principal components analysis was used to identify genes with differential expression patterns. RESULTS: Using a false discovery rate of <5% for significance analysis of microarray, the M group revealed 965 up-regulated and 1106 down-regulated genes relative to the N group. The PM group demonstrated 108 up-regulated and 226 down-regulated genes relative to the N group, whereas the M group demonstrated only 5 up-regulated and 13 down-regulated genes relative to the PM group. Both hierarchical cluster analysis and principal components analysis revealed a consistent separation between the N, PM, and M groups, with a closer association between the PM and M groups. To provide independent validation of the microarray data, quantitative reverse transcription-PCR was performed for a significantly up-regulated gene, integrin alpha 6, correlating well with microarray data (linear regression analysis, P < 0.0001). CONCLUSIONS: Similarly to the genetic progression model of HNSC, this transcriptional model shows that the majority of alterations occurs before the development of malignancy and identifies key targets of transcriptional dysregulation during progression from a normal to a premalignant state and from a premalignant to a malignant state.

p53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study.

BACKGROUND: The p53 gene is frequently mutated in non-small-cell lung cancer (NSCLC); however, the effect of p53 gene mutations on patient prognosis remains unclear. Therefore, we initiated a prospective study to determine the association of p53 gene mutations with survival in patients with stage I NSCLC. METHODS: Tumor samples were collected prospectively from 188 patients with operable NSCLC (stages I, II, and IIIA). p53 mutations were detected by direct dideoxynucleotide sequencing and p53 GeneChip analysis. Association of clinical and pathologic variables (e.g., alcohol consumption, sex, age, pathologic stage) with mutation of the p53 gene was determined by logistic regression. Associations between p53 mutation status, clinical and pathologic variables, and survival were assessed using a Cox proportional hazards regression model. All statistical tests were two-sided. RESULTS: p53 mutations were detected in 55% (104/188) of tumors. These mutations were associated with non-bronchoalveolar tumors, a history of alcohol consumption, and younger patient age. The risk of death was statistically significantly higher in patients with p53 mutations in their tumors (hazard ratio [HR] = 1.6, 95% confidence interval [CI] = 1.0 to 2.4; P =.049) than in patients with wild-type p53 in their tumors. Tumor stage, the presence of a p53 mutation, and increasing patient age were statistically significant predictors of patient death in the entire patient group; however, the statistically significant prognostic effect of p53 mutation was limited to patients with stage I NSCLC (stage I HR = 2.8, 95% CI = 1.4 to 5.6; stage II HR = 1.8, 95% CI = 0.74 to 4.4; and stage III HR = 0.70, 95% CI = 0.32 to 1.5). Among patients with stage I NSCLC, actuarial 4-year survival was statistically significantly higher in those with wild-type p53 than in those with mutant p53 (78% versus 52%, respectively; difference in 4-year survival = 26%, 95% CI = 6% to 46%; P =.009, log-rank test). CONCLUSION: Tumor p53 mutations are a statistically significant predictor of poor outcome in patients with stage I NSCLC.

DeltaNp63alpha and TAp63alpha regulate transcription of genes with distinct biological functions in cancer and development.

The p63 gene shows remarkable structural similarity to the p53 and p73 genes. Because of two promoters, the p63 gene generates two types of protein isoforms, TAp63 and DeltaNp63. Each type yields three isotypes (alpha, beta, gamma) because of differential splicing of the p63 COOH terminus. The purpose of this study was to determine whether there is a functional link between the distinct p63 isotypes in their transcriptional regulation of downstream targets and their role in various cellular functions. TAp63alpha and DeltaNp63alpha adenovirus expression vectors were introduced into Saos2 cells for 4 and 24 h, and then gene profiling was performed using a DNA microarray chip analysis. Seventy-four genes (>2-fold change in expression) were identified that overlapped between two independent studies. Thirty-five genes were selected for direct expression testing of which 27 were confirmed by reverse transcription-PCR or Northern blot analysis. A survey of these genes shows that p63 can regulate a wide range of downstream gene targets with various cellular functions, including cell cycle control, stress, and signal transduction. Our study thus revealed p63 transcriptional regulation of many genes in cancer and development while often demonstrating opposing regulatory functions for TAp63alpha and DeltaNp63alpha.