In-Depth Look at the Anatomical Relationship of the Lesser Occipital Nerve, Great Auricular Nerve, and Spinal Accessory Nerve and Their Implication in Safety of Operations in the Posterior Triangle of the Neck.

BACKGROUND: Migraine surgery is an increasingly popular treatment option for migraine patients. The lesser occipital nerve is a common trigger point for headache abnormalities, but there is a paucity of research regarding the lesser occipital nerve and its intimate association with the spinal accessory nerve. METHODS: Six cadaver necks were dissected. The lesser occipital, great auricular, and spinal accessory nerves were identified and systematically measured and recorded. These landmarks included the longitudinal axis (vertical line drawn in the posterior), the horizontal axis (defined as a line between the most anterosuperior points of the external auditory canals) and the earlobe. Mean distances and standard deviations were calculated to delineate the relationship between the spinal accessory, lesser occipital, and great auricular nerves. RESULTS: The point of emergence of the spinal accessory nerve was determined to be 7.17 ± 1.15 cm lateral to the y axis and 7.77 ± 1.10 caudal to the x axis. The lesser occipital nerve emerges 7.5 ± 1.31 cm lateral to the y axis and 8.47 ± 1.11 cm caudal to the x axis. The great auricular nerve emerges 8.33 ± 1.31 cm lateral to the y axis and 9.4 ±1.07 cm caudal to the x axis. The decussation of the spinal accessory and the lesser occipital nerves was found to be 7.70 ± 1.16 cm caudal to the x axis and 7.17 ± 1.15 lateral to the y axis. CONCLUSION: Understanding the close relationship between the lesser occipital nerve and spinal accessory nerve in the posterior, lateral neck area is crucial for a safer approach to occipital migraine headaches, occipital neuralgia, and new daily persistent headaches and other reconstructive or cosmetic operations.

Impact of drug therapy, radiation dose, and dose rate on renal toxicity following bone marrow transplantation.

PURPOSE: To demonstrate a radiation dose response and to determine the dosimetric and chemotherapeutic factors that influence the incidence of late renal toxicity following total body irradiation (TBI). METHODS AND MATERIALS: A comprehensive retrospective review was performed of articles reporting late renal toxicity, along with renal dose, fractionation, dose rate, chemotherapy regimens, and potential nephrotoxic agents. In the final analysis, 12 articles (n = 1,108 patients), consisting of 24 distinct TBI/chemotherapy conditioning regimens were included. Regimens were divided into three subgroups: adults (age > or =18 years), children (age <18 years), and mixed population (both adults and children). Multivariate logistic regression was performed to identify dosimetric and chemotherapeutic factors significantly associated with late renal complications. RESULTS: Individual analysis was performed on each population subgroup. For the purely adult population, the only significant variable was total dose. For the mixed population, the significant variables included total dose, dose rate, and the use of fludarabine. For the pediatric population, only the use of cyclosporin or teniposide was significant; no dose response was noted. A logistic model was generated with the exclusion of the pediatric population because of its lack of dose response. This model yielded the following significant variables: total dose, dose rate, and number of fractions. CONCLUSION: A dose response for renal damage after TBI was identified. Fractionation and low dose rates are factors to consider when delivering TBI to patients undergoing bone marrow transplantation. Drug therapy also has a major impact on kidney function and can modify the dose-response function.

Acute toxicity in definitive versus postprostatectomy image-guided radiotherapy for prostate cancer.

PURPOSE: To assess the incidence of acute gastrointestinal (GI) and genitourinary (GU) injury and the dose-volume response in patients with clinically localized prostate cancer treated with image-guided radiotherapy using helical tomotherapy. METHODS AND MATERIALS: Between November 2004 and March 2007, 146 consecutive patients with localized prostate cancer were treated with helical tomotherapy at the City of Hope Medical Center. Of the 146 patients, 70 had undergone prostatectomy. Acute GI and GU toxicities were evaluated using the Radiation Therapy Oncology Group/European Organization for Research and Cancer of Medical scoring system. Events were scored for patients developing Grade 2 or greater morbidity within 90 days after the end of radiotherapy (RT). The dosimetric parameters included the minimal dose received by the highest 10%, 20%, 50%, 80%, and 90% of the target volume, the mean rectal dose, minimal rectal dose, maximal rectal dose, and the volume receiving > or =45, > or =65, and > or =70 Gy. These variables, plus the status of radical prostatectomy, hormonal therapy, RT techniques, and medical conditions, were included in a multivariate logistic regression analysis. A goodness-of-fit evaluation was done using the Hosmer-Lemeshow statistic. RESULTS: A dose-response function for acute GI toxicity was elicited. The acute GI Grade 2 or greater toxicity was lower in the definitive RT group than in the postoperative RT group (25% vs. 41%, p <0.05). Acute GU Grade 2 or greater toxicity was comparable between the two groups. No grade 3 or greater complications were observed. No dosimetric variable was significant for GU toxicity. For acute GI toxicity, the significant dosimetric parameters were the minimal dose received by 10%, 20%, and 50% of the target volume and the mean rectal dose; the most predictive parameter was the minimal dose received by 10% of the target volume. The dose-modifying factor was 1.2 for radical prostatectomy. CONCLUSION: The results of our study have shown that acute rectal symptoms are dose-volume related. Postprostatectomy RT resulted in a greater incidence of acute GI toxicity than did definitive RT. For postoperative RT, it would be prudent to use different dose-volume limits.

Prognostic relevance of methylation markers in patients with non-muscle invasive bladder carcinoma.

There is increasing evidence for the role of epigenetic gene silencing in superficial bladder cancer. The aim of the current study was to investigate the prognostic value of epigenetic alterations in patients with non-muscle invasive bladder carcinoma. We checked the methylation status of 20 cancer associated genes (p14ARF, p16 CDKN2A, STAT-1, SOCS-1, DR-3, DR-6, PIG-7, BCL-2, H-TERT, BAX, EDNRB, DAPK, RASSF-1A, FADD, TMS-1, E-Cadherin, ICAM-1, TIMP-3, MLH-1, COX-2) for DNA methylation. We analysed microdissected tumour samples from 105 consecutive patients with primary non-muscle invasive bladder carcinoma. Quantitative methylation analysis of CpG sites in the promoter region of the genes was performed with methylation sensitive quantitative real time PCR ('Methylight'). Univariate analysis for association with tumour recurrence was carried out with the Kaplan-Meier analysis and the log-rank test. Follow-up data were available in 95/105 patients (91.4%). A tumour recurrence was observed in 26 patients (27.3%). We could identify six genes (SOCS-1, STAT-1, BCL-2, DAPK, TIMP-3, E-Cadherin), where methylation was associated with tumour recurrence. In Kaplan-Meier analysis, TIMP-3 showed a significant association with recurrence free survival. Methylation of TIMP-3 predicted prolonged disease free interval. In this study, we report a comprehensive analysis on prognostic relevance of gene methylation in non-muscle invasive bladder cancer. We identified one gene (TIMP-3) where methylation was associated with a more favourable outcome. Our data strongly support the usefulness of gene methylation as a prognostic marker in patients with non-muscle invasive bladder cancer.

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology.

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy.

1-(Beta-d-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) corresponds structurally to cytidine minus the exocyclic 4-amino group. The increased electrophilic character of its simple aglycon endows the molecule with unique biologic properties as a potent inhibitor of both cytidine deaminase and DNA cytosine methyltransferase. The latter activity makes zebularine a promising antitumor agent that is hydrolytically stable, preferentially targets cancer cells, and shows activity both in vitro and in experimental animals, even after oral administration.

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

PURPOSE: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. EXPERIMENTAL DESIGN: We analyzed the methylation status of the 5' regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. RESULTS: The 5' regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P < or = 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P < or = 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P < or = 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P < or = 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. CONCLUSIONS: Our results indicate that methylation of the 5' region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

Hepatic pseudotumor due to nodular fatty sparing: the diagnostic role of opposed-phase MRI.

OBJECTIVE: We sought to describe the role of opposed-phase MRI in the diagnosis of nodular fatty sparing in the liver. CONCLUSION: Nodular fatty sparing of the liver displays characteristic findings on opposed-phase MRI. Because the lesion may otherwise mimic a hepatic tumor, knowledge of these findings may prevent unnecessary biopsy or inappropriate treatment.

Preferential response of cancer cells to zebularine.

The frequent silencing of tumor suppressor genes by altered cytosine methylation and chromatin structural changes makes this process an attractive target for epigenetic therapy. Here we show that zebularine, a stable DNA cytosine methylation inhibitor, is preferentially incorporated into DNA and exhibits greater cell growth inhibition and gene expression in cancer cell lines compared to normal fibroblasts. In addition, zebularine preferentially depleted DNA methyltransferase 1 (DNMT1) and induced expression of cancer-related antigen genes in cancer cells relative to normal fibroblasts. Our results demonstrate that zebularine can be selective toward cancer cells and may hold clinical promise as an anticancer therapy.

Expression of Maspin in non-muscle invasive bladder carcinoma: correlation with tumor angiogenesis and prognosis.

OBJECTIVES: Maspin is a member of the serpin (serine protease inhibitor) family and has been shown to be a suppressor of tumor growth and an inhibitor of angiogenesis as well as metastasis in several types of tumors. We studied expression patterns of Maspin in pTa/pT1 urothelial carcinoma of the bladder and compared them with microvessel density (MVD) for two vascular markers (CD34 and CD105) and correlated the findings with clinical outcome. MATERIAL AND METHODS: We investigated tumor samples of 110 patients undergoing transurethral resection for pTa/pT1 bladder carcinoma (pTa, n=84; pT1, n=26; grade 1, n= 22; grade 2, n= 81; grade 3, n=7). Immunohistochemical studies were performed using the monoclonal antibodies, anti-human Maspin (NCL Maspin), anti-CD34 Class II and anti-CD105. Maspin expression level was classified according to the staining intensity (- to +++). The blood vessels (CD34) and specifically proliferating blood vessels (CD105) were counted as vessels per field (microvessel density, MVD). RESULTS: Of the 110 tumors, 27 showed a negative immunostaining for Maspin, 46 tumors stained +, 29 stained ++, and 8 stained +++. Maspin expression correlated inversely with CD34 reactivity. In tumors with loss of or only weak Maspin expression, the MVD for CD34 was 21.7 vessels per field, and 4.2 vessels per field for proliferating vessels (CD105), whereas Maspin-positive tumors had an MVD of 17.7 vessels per field (CD34), and of 6.0 vessels per field (CD105). Complete follow-up data are available in 92 patients. After a median follow-up of 25 months, 18 of the 92 patients (19.6%) had tumor recurrences. Tumors with decreased Maspin expression (-/+) had a shorter disease-free interval (23 months) than patients with stronger Maspin (++/+++) expression (29 months), whereas a Kaplan-Meier analysis and the log-rank test showed no significant difference in disease-free survival between the patients. CONCLUSION: The clinical importance of Maspin has been mainly investigated regarding tumor progression or metastasis. We found a decreased Maspin expression in a large portion of pTa/pT1 bladder tumors. Even if patients with decreased Maspin expression have a slightly shorter disease-free survival Maspin does not appear to be a promising prognostic marker.

Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome.

Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (approximately 25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5' regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.

Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation.

Several alternatively spliced variants of DNA methyltransferase (DNMT) 3b have been described. Here, we identified new murine Dnmt3b mRNA isoforms and found that mouse embryonic stem (ES) cells expressed only Dnmt3b transcripts that contained exons 10 and 11, whereas the Dnmt3b transcripts in somatic cells lacked these exons, suggesting that this region is important for embryonic development. DNMT3b2 and 3b3 were the major isoforms expressed in human cell lines and the mRNA levels of these isoforms closely correlated with their protein levels. Although DNMT3b3 may be catalytically inactive, it still may be biologically important because D4Z4 and satellites 2 and 3 repeat sequences, all known DNMT3b target sequences, were methylated in cells that predominantly expressed DNMT3b3. Treatment of cells with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) caused a complete depletion of DNMT1, 3a, 3b1, and 3b2 proteins. Human DNMT3b3 and the murine Dnmt3b3-like isoform, Dnmt3b6, were also depleted although less efficiently, suggesting that DNMT3b3 also may be capable of DNA binding. Moreover, de novo methylation of D4Z4 in T24 cancer cells after 5-Aza-CdR treatment only occurred when DNMT3b3 was expressed, reinforcing its role as a contributing factor of DNA methylation. The expression of either DNMT3b2 or 3b3, however, was not sufficient to explain the abnormal methylation of DNMT3b target sequences in human cancers, which may therefore be dependent on factors that affect DNMT3b targeting. Methylation analyses of immunodeficiency, chromosomal instabilities, and facial abnormalities cells revealed that an Alu repeat sequence was highly methylated, suggesting that Alu sequences are not DNMT3b targets.

Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells.

During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2'-deoxycytidine followed by zebularine hindered the remethylation of the p16 5' region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.

Inhibition of DNA methylation and reactivation of silenced genes by zebularine.

BACKGROUND: Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. METHODS: We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. RESULTS: In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). CONCLUSIONS: Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.