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TP53 mutations are analyzed in DNA from tumor specimens of patients with squamous-cell carcinoma of the head and neck, as classified into two groups, disruptive and nondisruptive, to compare the mutational status with clinical outcome. Disruptive TP53 mutations in tumor DNA are associated with reduced survival after surgical treatment of squamous-cell carcinoma of the head and neck.

An investigation of the surface morphology of Langmuir monolayers of a series of C(sub n)F(sub 2n+1)C(sub m)H(sub 2m+1) (FnHm) molecular diblocks after transfer in silicon wafers is presented. Langmuir monolayers of semifluorinated alkanes FnHm consist of surface hemimicelles rather than of classical, laterally featureless monolayers.

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. [ABSTRACT FROM AUTHOR]

Inflammatory processes can reliably be assessed by 19F 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 19F-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 19F 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 19F MR inflammation imaging to human applications. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2013 John Wiley & Sons, Ltd.)

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.

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.

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.

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.

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.