Wichai Pornthanakasem

Human papillomavirus DNA in plasma of patients with cervical cancer

BackgroundHuman papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients.ResultsWhereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA.ConclusionsThe plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

Genomic alterations in nasopharyngeal carcinoma: loss of heterozygosity and Epstein-Barr virus infection

Nasopharyngeal carcinoma is a subset of head and neck squamous cell cancers with unique endemic distribution and aetiological co-factors. Epstein-Barr virus has been revealed to be an important aetiological factor for most nasopharyngeal carcinomas. Nevertheless, additional genetic alterations may be involved in their development and progression. The aim of this study was to determine the likely chromosomal locations of tumour-suppressor genes related to Epstein-Barr virus-associated nasopharyngeal carcinoma. Fifty-six microsatellite polymorphic markers located on every autosomal arm were used to estimate the incidence of loss of heterozygosity in 27 Epstein-Barr virus-associated nasopharyngeal carcinomas. High frequencies of allelic loss were observed on chromosome 3p (75.0%) and 9p (87.0%). Chromosome 9q, 11q, 13q and 14q displayed loss in over 50%, while chromosome 3q, 6p, 16q, 19q and 22q exhibited loss in 35-50%. Furthermore, several other chromosomal arms demonstrated allelic loss in 20-35%. Additionally, 1 of the 27 cases showed microsatellite instability at multiple loci. These findings provide evidence of multiple genetic alterations during cancer development and clues for further studies of tumour-suppressor genes in Epstein-Barr virus-associated nasopharyngeal carcinoma.

Loss of heterozygosity on chromosome 14 in nasopharyngeal carcinoma.

The main objective of this study was to determine the precise frequency of chromosome 14q loss of heterozygosity in nasopharyngeal carcinomas and to define its minimal deletion regions. Thirty‐nine tumors were selected for PCR‐based deletion mapping using 19 microsatellite polymorphic markers spanning the long arm of this chromosome. Loss of heterozygosity for at least one marker was observed in 29 (74.4%) tumors, while 24 of these tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated, the first defined by markers D14S278 and D14S288, the second being between D14S51 and the telomere. These data confirmed 2 potential tumor‐suppressor‐gene loci at 14q12‐13 and 14 q32. Interestingly, the third region of loss was located at the T‐cell‐receptor delta‐chain locus. This may reflect another tumor‐suppressor‐gene locus at 14q11.2, or may be the consequence of a specific genomic rearrangement of this region. In addition, these allelic losses occurred with high frequency in all tumor grades and stages and in all histological sub‐types. These findings suggest that the genetic alteration of chromosome 14 is common and crucial during nasopharyngeal‐carcinoma development. Int. J. Cancer 78:153–156, 1998.© 1998 Wiley‐Liss, Inc.

Replication independent DNA double-strand break retention may prevent genomic instability

BackgroundGlobal hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs), and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs) located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ), mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM)-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability.ResultsWe observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA) and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51.ConclusionsMethylated RIND-EDSBs are retained in non-acetylated heterochromatin because there is a prolonged time lag between RIND-EDSB production and repair. The rapid cellular responses to DSBs may be blocked by compact heterochromatin structure which then allows these breaks to be repaired by a more precise ATM-dependent pathway. In contrast, Ku-mediated NHEJ can repair euchromatin-associated EDSBs. Consequently, spontaneous mutations in hypomethylated genome are produced at faster rates because unmethylated EDSBs are unable to avoid the more error-prone NHEJ mechanisms.

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

Astroblastoma: Report of a case with microsatellite analysis

A 5‐year‐old girl who developed progressive headache, vomiting, and left hemiparesis was found to have a cystic tumor with an enhanced mural nodule in the right frontoparietal region on a computed tomography examination. The lesion was histologically and ultrastructurally verified as an astroblastoma, an uncommon neuroepithelial tumor of uncertain origin. Molecular analysis using 17 microsatellite markers on chromosomes 9, 10, 11, 17, 19, and 22 showed loss of heterozygosity at the D19S412 locus on the long arm of chromsome 19. This observation suggests that there is a tumor suppressor gene in this chromosomal region, which plays a role in the pathogenesis of astroblastoma.

Identification of distinct regions of allelic loss on chromosome 13q in nasopharyngeal cancer from paraffin embedded tissues

Our main purpose was to identify tumor suppressor gene loci on chromosome 13 responsible for nasopharyngeal cancer (NPC) development by analyzing loss of heterozygosity (LOH) and RB protein expression in paraffin embedded tissues. Normal and tumor DNA were extracted from microdissected samples, and their whole genomes were amplified using degenerate oligonucleotide primers. The polymerase chain reaction (PCR) products were analyzed by repeated amplification using primers derived from 16 microsatellite regions spanning the long arm of this chromosome. Among 50 informative cases, LOH was observed in 44 tumors. Thirty‐one tumors displayed partial loss and provided an informative basis for detailed deletion mapping. Three minimal regions of loss were delineated; the first flanked by D13S120 and D13S219, the second by D13S126 and D13S119, and the third by D13S137 and 13qter. These 3 regions were linked to BRCA2 on 13q12, RB1 on 13q14, and 13q14.3‐ter, respectively. Seven and 4 cases showed LOH either on 13q12 or 13q14, respectively. Nineteen cases showed LOH of both loci separately. One NPC displayed 13q12 and 13q14.3‐ter LOH. RB protein expression was detectable in 76% of the cases. Ten out of 15 cases with the allelic losses limited to 13q14 showed RB protein expression. Contrasting that, 6 out of 7 cases devoid of RB protein expressions showed 13q14LOH. In conclusion, 13qLOH, involving 3 tumor suppressor gene loci, appears to be a frequent genetic event occurring during NPC development. However, other tumor suppressor genes besides RB1, may be responsible for the majority of 13q14LOH. Int. J. Cancer 83:210–214, 1999.

LINE-1 insertion dimorphisms identification by PCR

Differentiating between recent LINE-1 (L1) insertion dimorphisms (LIDs) is predominantly useful for studying not only the transposition mechanism but also population genet-ics or evolution based on polymorphic markers where the ancestral state is known (i.e., absence of the insertion) (1–5). Previous techniques such as L1 display (1) or ATLAS (2) have identi-fied new LIDs from distinct popula-tions. To improve both efficiency and simplicity, we designed a PCR-based approach, LID identification by PCR (LIDSIP), by combining and modify-ing ligation-mediated PCR (LMPCR) (6) and interspersed repetitive sequence PCR (IRSPCR) (7). This method requires only a small amount of DNA, a limited and specific number of PCRs to provide a genome-wide scan within a PCR range of an appropriate restriction site, conventional molecular genetic techniques such as agarose gel elec-trophoresis devoid of radioactive label, and in addition, this method yields clear, easily distinguishable, specific results. The aim of applying LIDSIP was to globally map active L1 in the human genome by amplifying the 3′ untrans-lated region (UTR) end of the L1-Ta subset up to its next specific restric-tion enzyme recognition sequences. First, 500 ng human genomic DNA were digested with BstYI (New Eng-land Biolabs, Beverly, MA, USA) and purified with phenol-chloroform extraction and ethanol precipitation. Second, the DNA was ligated to 20 pmol each of LIDSIP-LINK (5′-AGG-TAACGAGTCAGACCACCGACTC-GTGGACGT-3′) and BstYI-LINK (5′-GATCACGTCCACGAG-3′) using T4 DNA ligase (New England Biolabs) at 16°C overnight. Finally 50 ng of the purified ligated DNA were subjected to nested PCR. The first PCR (50 µL total volume) contained 200 µM each of the four dNTPs, 1× PCR buffer (contains 1.5 mM MgCl

Central nervous system lymphoma and Epstein-Barr virus in immunocompetent patients in Thailand

A series of 11 apparently immunocompetent patients with primary non‐Hodgkins lymphoma of the central nervous system (NHL‐CNS) together with six patients with systemic lymphoma involving the spinal cord and/or brain were studied for immunophenotyping and the presence of Epstein‐Barr virus (EBV). Immunohistochemistry and polymerase chain reaction (PCR) were performed on paraffin‐embedded tissues. Nine of 11 primary NHL‐CNS and all six secondary CNS lesions were of B cell origin. The EBV sequences were detected in six primary tumors and four systemic lesions by PCR while the immuno‐histochemical marker for the EBV‐latent membrane protein was positive in five primary lesions and three secondary neoplasms. Our results suggest that the association of EBV and NHL‐CNS is not only restricted to patients with immunosuppression but that it includes a broad spectrum of conditions in which this relationship occurs in patients without immunodeficiency. The mortality rate is high particularly in patients with EBV‐associated NHL‐CNS.