Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma is highly prevalent in Southern China and Southeast Asia. To unveil the molecular basis of this endemic disease, high-resolution comparative genomic hybridization arrays were used for systematic investigation of genomic abnormalities in 26 nasopharyngeal carcinoma samples. A comprehensive picture of genetic lesions associated with tumorigenesis of nasopharyngeal carcinoma was generated. Consistent chromosomal gains were frequently found on 1q, 3q, 8q, 11q, 12p, and 12q. High incidences of nonrandom losses were identified on chromosomes 3p, 9p, 11q, 14q, and 16q. In addition to previously characterized regions, we have identified several novel minimal regions of gains, including 3q27.3-28, 8q21-24, 11q13.1-13.3, and 12q13, which may harbor candidate nasopharyngeal carcinoma-associated oncogenes. In this study, gain of 11q13.1-13.3 was the most frequently detected chromosomal aberration and a 5.3-Mb amplicon was delineated at this region. Within this 11q13 amplicon, concordant amplification and overexpression of cyclin D1 (CCND1) oncogene was found in nasopharyngeal carcinoma cell lines, xenografts, and primary tumors. Knockdown of cyclin D1 by small interfering RNA in nasopharyngeal carcinoma cell lines led to significant decrease of cell proliferation. The findings suggest that cyclin D1 is a target oncogene at 11q13 in nasopharyngeal carcinoma and its activation plays a significant role in nasopharyngeal carcinoma tumorigenesis.

Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis.

Although the integration of hepatitis B virus (HBV) into human DNA has been found to be associated with the development of hepatocellular carcinoma (HCC), the molecular mechanism remains unclear. In order to obtain additional insight into the correlation of HBV integration and HCC development, integrated HBV in 14 primary HCC cases was isolated and characterized by sequencing analysis. Our findings in this study showed that: (1) none of the known cellular oncogene or tumor suppressor gene was affected by the HBV integration; (2) although the integration of HBV is random, the integration site was often within or close to human repetitive sequences; (3) integrated HBV may possess the capacity to transpose to another chromosome region through reintegration; (4) rearrangements of HBV sequence were observed in all the 14 integrants, involving (most frequently) X (12/14 integrants), P (8/14), S (7/14), and C (7/14) genes; and (5) 3'-deleted X gene and consequent C-terminal truncated X protein caused by HBV integration was observed in 10 cases. These deletions cause the losses of p53-dependent transcriptional repression binding site, transcription factor Sp1 binding site, and growth-suppressive effect domain, leading to cell proliferation and transformation. This finding suggests that 3'-deleted X gene caused by the HBV integration may play an important role in the HCC development.

Establishment and characterization of a human non-small cell lung cancer cell line.

Lung cancer is one of the most common causes of cancer death worldwide. Although many efforts have been made to explore the mechanisms involved in the development of lung cancer, the genetic events involved in the pathogenesis of lung cancer are still unclear. For a better mechanistic scope of study, a well-established cellular model is essential. We report the establishment of a squamous cell carcinoma (SCC) cell line of human lung, SCC-37. Chromosomal abnormalities and global genomic alterations of SCC-37 were studied by spectral karyotyping (SKY) and comparative genomic hybridization (CGH), respectively. Results showed that SCC-37 was a hypodiploid with complex chromosomal rearrangements. Some of the alterations, such as the gain of 1q25-qter in SCC-37, have been correlated to the tumor recurrence of non-small cell lung cancer (NSCLC). Other interesting findings include the amplification of 3q25-qter and 12q13, suggesting the existence of important oncogenes in the amplicons. This cell line may thus provide a useful cellular resource for studying the pathogenesis of SCC of the lung in the future.

Detection of hypermethylated RIZ1 gene in primary tumor, mouth, and throat rinsing fluid, nasopharyngeal swab, and peripheral blood of nasopharyngeal carcinoma patient.

PURPOSE: The aims of this study are to examine the methylation status of the Fn2 RIZ1 gene in nasopharyngeal (NP) tumors and nasopharyngeal carcinoma (NPC) cell lines and to evaluate the clinical value of methylated RIZ1 gene in body fluid samples of NPC patients. EXPERIMENTAL DESIGN: Methylation status of RIZ1 was evaluated by MSP on CNE-2 and M1 cell lines, 30 tumor biopsies, and their matched body fluid samples, including mouth and throat (M & T) rinsing fluid, NP swabs, plasma, and buffy coat. Normal controls included 8 normal NP biopsies and body fluid samples from 29 healthy volunteers. Sequencing was performed on MSP products from one NP tumor and one M & T rinsing fluid. Transcription of the RIZ1 gene before and after 5-aza-2'-deoxycytidine treatment was examined on CNE-2. RESULTS: The methylated RIZ1 gene was detected in both CNE-2 and M1, 18 (60%) NP tumors, but not in any of the normal controls. Of 30 matched body fluid samples, methylated RIZ1 DNA was found in 11 (37%) NP swabs, 9 (30%) M & T rinsing fluid, 7 (23%) plasma, and 3 (10%) buffy coat samples. Sequencing analysis confirmed all cytosines to uracils conversion, excluding cytosines in CpG dinucleotides in methylated PCR products. Promoter methylation correlated with loss of RIZ1 mRNA expression, and 5-aza-2'-deoxycytidine treatment restored its expression in CNE-2. CONCLUSION: Our results suggest that promoter hypermethylation of the RIZ1 gene is commonly found in NPC. Its detection in body fluid samples of NPC patients but not in normal controls indicates that it is worth to further evaluate its clinical application in assisting screening of NPC and monitoring recurrence after treatment.

Quantitative plasma hypermethylated DNA markers of undifferentiated nasopharyngeal carcinoma.

PURPOSE: Gene-specific methylation is common in primary undifferentiated nasopharyngeal carcinoma (NPC). DNA released from apoptotic or necrotic cell death including those aberrantly methylated promoter DNA of cancer cells is absorbed into the circulation as cell-free plasma DNA of the patient. This study aims at evaluation of the potential use of methylated gene promoter DNA as a serological tumor marker of primary and potentially salvageable local or nodal recurrent NPC. EXPERIMENTAL DESIGN: The quantity of plasma hypermethylated gene promoters of CDH1, DAPK1, p15, p16, RASSF1A, and MLH1 of 41 NPC patients before treatment and 43 normal individuals were studied using real-time quantitative PCR. The post-treatment plasma hypermethylated CDH1, DAPK1,and p16 were also measured in 13 NPC patients with locoregional recurrence and 17 patients in remission. RESULTS: Concentrations of cell-free circulating DNA were significantly higher in NPC patients than normal controls (28.79 ng/ml versus 16.57 ng/ml, respectively). There was no significant difference in plasma DNA concentration of EBV-positive and -negative normal individuals. Methylated DNA was detectable in plasma of NPC patients before treatment including 46% for CDH1,42% for p16,20% for DAPK1,20% for p15,and 5% for RASSF1A. Hypermethylated MLH1 was not detected in plasma of all of the NPC patients and normal individuals. Aberrantly hypermethylated promoter DNA of at least one of the five genes was detectable in 29 of 41 (71%) plasma of NPC patients before treatment. Hypermethylated promoter DNA of at least one of the three genes (CDH1, DAPK1, and p16) was detectable in post-treatment plasma of 5 of 13 (38%) recurrent NPC patients and none of the patients in remission. CONCLUSIONS: Our results suggested that cell-free circulating methylated gene promoter DNA is a possibly useful serological marker in assisting in screening of primary and potentially salvageable local or regional recurrent NPC.

Promoter hypermethylation of high-in-normal 1 gene in primary nasopharyngeal carcinoma.

PURPOSE: The methylation of high-in-normal-1 (HIN-1) gene promoter in undifferentiated nasopharyngeal carcinoma (NPC) is studied. EXPERIMENTAL DESIGN: The methylation status of HIN-1 in NPC cell lines, primary NPC, paired nasopharyngeal swabs, paired throat-rinsing fluid, and paired peripheral blood was assessed by methylation-specific PCR assay. The relationship between HIN-1 promoter methylation and transcription in NPC cell lines was evaluated by reverse transcription-PCR and demethylation agent treatment (5-aza-2-deoxycytidine). RESULTS: Hypermethylated promoter was observed in five of five (100%) NPC cell lines and not found in three normal nasopharyngeal outgrowths, two tonsil epithelial cell cultures, and two skin fibroblast cultures. Reverse transcription-PCR assay indicated that HIN-1 transcription was significantly down-regulated in the NPC cell line with promoter methylation. Treatment with demethylation agent, 5-aza-2-deoxycytidine, restored HIN-1 transcription in the NPC cell line. Methylated HIN-1 promoter was found in 36 of 47 (77%) primary NPC tumors and not found in the normal nasopharyngeal biopsies. Methylated HIN-1 promoter was detected in 12 of 26 (46%) nasopharyngeal swabs, 5 of 26 (19%) throat-rinsing fluids, 2 of 11 (18%) plasmas, and 5 of 11 (46%) buffy coats of peripheral blood of the NPC patients but was not detectable in all normal controls. CONCLUSION: HIN-1 promoter hypermethylation is common in NPC. Methylated promoter DNA in nasopharyngeal swab, throat-rinsing fluid, and peripheral blood might be potentially useful as tumor marker for screening of NPC.

High frequency of promoter hypermethylation of the death-associated protein-kinase gene in nasopharyngeal carcinoma and its detection in the peripheral blood of patients.

PURPOSE: Death-associated protein (DAP)-kinase gene is frequently inactivated by promoter hypermethylation in cancer. The aim of this study was to evaluate the promoter methylation status of the DAP-kinase gene in nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN: The methylation status was evaluated by methylation-specific PCR (MSP). Thirty-two NPC biopsy specimens, plasma and buffy coat of 12 patients, 5 NPC cell lines, 3 normal nasopharyngeal biopsy tissues, and 2 normal nasopharyngeal epithelial primary cultures were included in this study. RESULTS: There was no promoter hypermethylation in all 3 normal nasopharyngeal tissues and 2 normal nasopharyngeal primary cultures. Hypermethylation was found in 24 (75%) NPC primary tumor biopsies and 4 (80%) NPC cell lines. Of the 24 patients with hypermethylation of DAP-kinase promoter in the primary tumors, 12 patients had their plasma and buffy coat DNA available for MSP study. Hypermethylated DAP-kinase promoter was detectable in 5 patients in the plasma but not in the buffy coat, 2 patients in the buffy coat but not in the plasma, and 1 patient in both plasma and buffy coat. Four patients had no detectable hypermethylated DAP-kinase promoter in both plasma and buffy coat. Hypermethylation of DAP-kinase promoter was found in both early- and late-stage NPC. CONCLUSIONS: Our results show that hypermethylation of the DAP-kinase promoter is a common early event in NPC. The high frequency of identification of hypermethylated DAP-kinase promoter in plasma and buffy coat of NPC patients illustrates its potential clinical application as tumor marker for the diagnosis and monitoring of treatment result.

Differential gene methylation in undifferentiated nasopharyngeal carcinoma.

Differential gene methylation is observed in a variety of human malignancies. The study of the pattern of methylated genes helps to understand carcinogenesis and to identify potential marker tumor genes for clinical use. The differential methylated genes in undifferentiated nasopharyngeal carcinoma (NPC) of Chinese were studied by methylation-specific polymerase chain reaction (MSP). Methylation status of 11 tumor-associated genes (ARF, caspase-8, CDH1, CDKN2A, CDKN2B, MGMT, MLH1, RASSF1A, THBS1, TP73 and VHL) was studied in 30 primary undifferentiated NPC and paired peripheral blood of 12 patients. The methylation profile of NPC in order of frequency was CDH1 (50%), CDKN2B (50%), THBS1 (50%), RASSF1A (46%), MLH1 (40%), MGMT (28%), CDKN2A (23%), TP73 (20%), caspase-8 (7%), ARF (3%) and VHL (0%). Methylation of at least 1 gene was observed in 93% of primary NPC. Of the 12 patients with at least 1 methylated gene in the primary tumor, all 12 (100%) patients had at least 1 of the methylated gene promoter detectable in their peripheral blood. The results show high frequency of methylation in NPC and the potential of using methylation as peripheral blood tumor marker in screening NPC.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry serum protein profiling to identify nasopharyngeal carcinoma.

BACKGROUND: Diagnosis of nasopharyngeal carcinoma (NPC) at an early disease stage is important for successful treatment and improving the outcome of patients. The use of serum protein profiles and a classification tree algorithm were explored to distinguish NPC from noncancer. METHODS: Serum samples were applied to metal affinity protein chips to generate mass spectra by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Protein peak identification and clustering were performed using the Biomarker Wizard software. Proteomic spectra of serum samples from 50 NPC patients and 54 noncancer controls were used as a training set and a classification tree with 6 distinct protein masses was generated by using Biomarker Pattern software. The validity of the classification tree was then challenged with a blind test set including another 20 NPC patients and 25 noncancer controls. RESULTS: The software identified an average of 93 mass peaks/spectrum and 6 of the identified peaks were used to construct the classification tree. The classification tree correctly determined 83% (123 of 149) of the test samples with 83% (58 of 70) of the NPC samples and 82% (65 of 79) of the noncancer samples. In a combination of the serum protein profiles with Epstein-Barr (EBV) nuclear antigen 1 (EBNA1 IgA) test, the diagnostic sensitivity and specificity were increased to 99% and 96%, respectively. CONCLUSIONS: The results suggest that SELDI-TOF-MS serum protein profiles could discriminate NPC from noncancer. The combination of serum protein profiles with an EBV antibody serology test could further improve the accuracy of NPC screening.

Functional studies of the chromosome 3p21.3 candidate tumor suppressor gene BLU/ZMYND10 in nasopharyngeal carcinoma.

Chromosome 3p plays an important role in tumorigenesis in many cancers, including nasopharyngeal carcinoma (NPC). We have previously shown chromosome 3p can suppress tumor growth in vivo by using the monochromosome transfer approach, which indicated the chromosome 3p21.3 region was critical for tumor suppression. BLU/ZMYND10 is one of the candidate tumor suppressor genes mapping in the 3p21.3 critical region and is a candidate TSG for NPC. By quantitative RT-PCR, it is frequently downregulated in NPC cell lines (83%) and NPC biopsies (80%). However, no functional studies have yet verified the functional role of BLU/ZMYND10 as a tumor suppressor gene. In the current study, a gene inactivation test (GIT) utilizing a tetracycline regulation system was used to study the functional role of BLU/ZMYND10. When BLU/ZMYND10 is expressed in the absence of doxycycline, the stable transfectants were able to induce tumor suppression in nude mice. In contrast, downregulation of BLU/ZMYND10 in these tumor suppressive clones by doxycycline treatment restored the tumor formation ability. This study provides the first significant evidence to demonstrate BLU/ZMYND10 can functionally suppress tumor formation in vivo and is, therefore, likely to be one of the candidate tumor suppressor genes involved in NPC.

Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid, nasopharyngeal swab and peripheral blood of nasopharygeal carcinoma patient.

The purpose of our study was to evaluate the frequency of hypermethylated tumor suppressor genes (TSGs) in peripheral blood, mouth and throat (M&T) rinsing fluid and nasopharyngeal (NP) swabs of nasopharyngeal carcinoma (NPC) patients. Six normal NP tissues, 43 M&T rinsing fluid, 37 NP swabs and 43 peripheral blood from healthy non-smokers and non-drinkers without a family history of NPC, and 30 NPC tumors and their matched body fluid were analyzed for the presence of hypermethylated p15, p16, Ras association domain family 1 (RASSF1A), E-cadherin, and death-associated protein kinase (DAPK) by methylation-specific PCR. Sequencing analysis was carried out on selected NPC tumors and body fluid samples. Twenty-nine (97%) tumors displayed methylation in at least 1 of the 5 genes. The methylation frequencies were 80% for p15, 77% for DAPK, 67% for RASSF1A, 53% for E-cadherin and 33% for p16. The frequency range of aberrant methylated genes in the body fluids were NP swabs (17-63%) and M&T rinsing fluid (17-50%). Methylation was found in <20% of peripheral blood for each respective gene. Methylation was, however, detected in 1 M&T rinsing fluid in which the primary tumor showed methylation free for RASSF1A. Five healthy individuals exhibited methylation for DAPK, or RASSF1A, or p15 in their body fluid samples. All body fluid samples of healthy controls showed methylation free for E-cadherin and p16. Epigenetic change is found frequently in NPC and the high detection rate in body fluids suggest its potential application in non-invasive screening of NPC or detection of residual carcinoma after treatment.