Mutation analysis of IDH1 in paired gliomas revealed IDH1 mutation was not associated with malignant progression but predicted longer survival.

Recurrence and progression to higher grade lesions are characteristic behaviors of gliomas. Though IDH1 mutation frequently occurs and is considered as an early event in gliomagenesis, little is known about its role in the recurrence and progression of gliomas. We therefore analysed IDH1 and IDH2 status at codon 132 of IDH1 and codon 172 of IDH2 by direct sequencing and anti-IDH1-R132H immunohistochemistry in 53 paired samples and their recurrences, including 29 low-grade gliomas, 16 anaplastic gliomas and 8 Glioblastomas. IDH1/IDH2 mutation was detected in 32 primary tumors, with 25 low-grade gliomas and 6 anaplastic gliomas harboring IDH1 mutation and 1 low-grade glioma harboring IDH2 mutation. All of the paired tumors showed consistent IDH1 and IDH2 status. Patients were analyzed according to IDH1 status and tumor-related factors. Malignant progression at recurrence was noted in 22 gliomas and was not associated with IDH1 mutation. Survival analysis revealed patients with IDH1 mutated gliomas had a significantly longer progression-free survival (PFS) and overall survival (OS). In conclusion, this study demonstrated a strong tendency of IDH1/IDH2 status being consistent during progression of glioma. IDH1 mutation was not a predictive marker for malignant progression and it was a potential prognostic marker for gliomas of Chinese patients.

Loss of heterozygosity and methylation of multiple tumor suppressor genes on chromosome 3 in hepatocellular carcinoma.

BACKGROUND: Genetic and epigenetic alterations are the two key mechanisms in the development of hepatocellular carcinoma (HCC). However, how they contribute to hepatocarcinogenesis and the correlation between them has not been fully elucidated. METHODS: A total of 48 paired HCCs and noncancerous tissues were used to detect loss of heterozygosity (LOH) and the methylation profiles of five tumor suppressor genes (RASSF1A, BLU, FHIT, CRBP1, and HLTF) on chromosome 3 by using polymerase chain reaction (PCR) and methylation-specific PCR. Gene expression was analyzed by immunohistochemistry and reverse transcription (RT)-PCR. RESULTS: Sixteen of 48 (33.3 %) HCCs had LOH on at least one locus on chromosome 3, and two smallest common deleted regions (3p22.3-24.3 and 3p12.3-14.2) were identified. RASSF1A, BLU, and FHIT showed very high frequencies of methylation in HCCs (100, 81.3, and 64.6 %, respectively) and noncancerous tissues, but not in liver tissues from control patients. Well-differentiated HCCs showed high methylation frequencies of these genes but very low frequencies of LOH. Furthermore, BLU methylation was associated with an increased level of alpha-fetoprotein, and FHIT methylation was inversely correlated with HCC recurrence. In comparison, CRBP1 showed moderate frequencies of methylation, while HLTF showed low frequencies of methylation, and CRBP1 methylation occurred mainly in elderly patients. Treatment with 5-aza-2'-deoxycytidine demethylated at least one of these genes and restored their expression in a DNA methylation-dependent or -independent manner. CONCLUSIONS: Hypermethylation of RASSF1A, BLU, and FHIT is a common and very early event in hepatocarcinogenesis; CRBP1 methylation may also be involved in the later stage. Although LOH was not too frequent on chromosome 3, it may play a role as another mechanism in hepatocarcinogenesis.

Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma.

Overexpression of high mobility group AT-hook 1 (HMGA1) is common in human cancers. Little is known about the mechanisms underlying its deregulation and downstream targets, and information about its clinical and biological significance in medulloblastoma (MB) is lacking. Here, we demonstrated frequent genomic gain at 6p21.33-6p21.31 with copy number increase leading to overexpression of HMGA1 in MB. The overexpression correlated with a high proliferation index and poor prognosis. Moreover, we found that hsa-miR-124a targeted 3'UTR of HMGA1 and negatively modulated the expression in MB cells, indicating that loss/downregulation of hsa-miR-124a reported in our previous study could contribute to the overexpression. Regarding the biological significance of HMGA1, siRNA knockdown and ectopic expression studies revealed the crucial roles of HMGA1 in controlling MB cell growth and migration/invasion through modulation of apoptosis and formation of filopodia and stress fibers, respectively. Furthermore, we identified cdc25A as a target of HMGA1 and showed that physical interaction between HMGA1 and the cdc25A promoter is required for transcriptional upregulation. In clinical samples, HMGA1 and cdc25A were concordantly overexpressed. Functionally, cdc25A is involved in the HMGA1-mediated control of MB cell growth. Finally, netropsin, which competes with HMGA1 in DNA binding, reduced the expression of cdc25A by suppression of its promoter activity and inhibited in vitro and in vivo intracranial MB cell growth. In conclusion, our results delineate the mechanisms underlying the deregulation and reveal the functional significance of HMGA1 in controlling MB cell growth and migration/invasion. Importantly, the results highlight the therapeutic potential of targeting HMGA1 in MB patients.

Dyslexia-associated kiaa0319-like protein interacts with axon guidance receptor nogo receptor 1.

To identify the putative interacting partners for Kiaa0319-like protein. KIAA0319-like, located near the dyslexia susceptibility locus, DYX8 in chromosome 1p34.3, has been suggested as a positional candidate for developmental dyslexia due to its homology with another gene, KIAA0319 which has been strongly established as a candidate gene for developmental dyslexia. Previous research has shown that a single marker, rs7523017 (P = 0.042) has been associated with developmental dyslexia by a Canadian group. There is little functional information about this gene and protein. In this article, we put forward further evidence that support Kiaa0319-like is a candidate for this disorder. A yeast-2-hybrid screen and co-immunopreciptiation assays were performed to find protein interacting partners of KIAA0319L. A human cortex immunohistochemistry assay was performed to show the colocalization of Kiaa0319-like and its specific interacting partner in cells. Nogo Receptor 1 (NgR1), an axon guidance receptor, was identified to have physical interactions with Kiaa0319-like protein. These two proteins interact predominantly in the cytoplasmic granules of cortical neurons in the human brain cortex. Based on this data, it can be concluded that Kiaa0319-like protein interacts with Nogo Receptor 1, supporting the idea that Kiaa0319-like protein participates in axon guidance.

KIAA0495/PDAM is frequently downregulated in oligodendroglial tumors and its knockdown by siRNA induces cisplatin resistance in glioma cells.

Co-deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT-related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31-p36.32 that might be involved in OTs. A novel gene KIAA0495/p53-dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild-type p53. B-cell CCL/lymphoma 2 (BCL2)-like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM-silenced cells after cisplatin treatment, suggesting that alteration of anti-apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin-resistant phenotype. Moreover, our data suggested that PDAM might function as a non-protein-coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53-dependent anti-apoptotic genes.

Association between temporal lobe P-glycoprotein expression and seizure recurrence after surgery for pharmacoresistant temporal lobe epilepsy.

Surgery is recommended for pharmacoresistant temporal lobe epilepsy (TLE), but seizures recur in approximately one third of patients postsurgery. P-glycoprotein is an efflux multidrug transporter that is overexpressed in a range of epileptogenic pathologies. We hypothesized that increased expression of P-glycoprotein in the epileptogenic temporal lobe might be a marker for recurrence of pharmacoresistant seizures postsurgery. We performed immunohistochemistry on temporal lobe tissues resected from 69 patients who underwent anterior temporal lobectomy for pharmacoresistant TLE with histopathological proven hippocampal sclerosis. P-glycoprotein expression was rated by three pathologists independently. Patients with seizure recurrence (n=22) had greater number of positively stained capillaries (p=0.001) and higher P-glycoprotein immunoreactive score in capillaries (p=0.002) in the white matter of resected temporal lobe. The differences remained significant in multivariate analysis (p=0.002 and 0.006, respectively). The results suggest that P-glycoprotein expression in temporal lobe may be associated with seizure recurrence after surgery for pharmacoresistant TLE.

Increased expression of centrosomal alpha, gamma-tubulin in atypical ductal hyperplasia and carcinoma of the breast.

Centrosomal abnormalities have been found in various cancer types. We sought to determine whether centrosomal dysfunctions occur in the atypical ductal hyperplasia (ADH)-carcinoma sequence of breast cancer. As alpha and gamma-tubulins are the structural components of centrosomes, we performed real time quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH) and immunnohistochemistry (IHC) to determine the DNA copy levels, messenger RNA (mRNA) expression, and protein expression of alpha and gamma-tubulins respectively. Gamma-tubulin staining was used for the localization and quantification of centrosomes. We found that alpha-tubulin or gamma-tubulin mRNA was increasingly expressed from normal breast tissue (NBT) to ADH, ductal carcinoma in situ (DCIS), and infiltrative ductal carcinoma (IDC), respectively, with the highest expressions being found in DCIS. The expression profiles of alpha, gamma-tubulin proteins were concordant with that of mRNA, except that the highest expression was found in IDC. Similarly, DNA copies of alpha, gamma-tubulins showed a rising tendency, with the highest level for gamma-tubulin attained in IDC and that for alpha-tubulin was found in DCIS. However, there was no significant difference of alpha, gamma-tubulin DNA copy levels, mRNA expression, and protein expression between DCIS and IDC. Our results demonstrate that centrosomal aberrations may play key roles in the early stage of breast tumorogenesis. The malignant transformation sequence is probably attributable to the amplification of centrosomal DNA leading to mRNA and protein over-expression of these centrosomal proteins. Furthermore, determination of alpha, gamma-tubulins using combined qPCR with ISH may be useful in assisting the diagnosis of premalignant lesions of the breast.

Clinicopathologic significance of immunophenotypic profiles related to germinal center and activation B-cell differentiation in diffuse large B-cell lymphoma from Chinese patients.

Diffuse large B-cell lymphoma (DLBCL) can be subdivided into prognostically significant groups with germinal center B-cell-like (GCB), activated B-cell-like (ABC), and type 3 groups. In this study, tissue microarray slides composed of 163 de novo DLBCLs from Chinese patients were immunostained for CD20, CD10, Bcl-6, MUM1, CD138, Bcl-2, Ki-67, cyclin D3, geminin, and P27(Kip1). One hundred forty-nine of 163 DLBCLs could then be classified into GCB group (pattern A), activated GCB group (pattern B) and activated non-GCB group (pattern C) according to the expression of CD10, Bcl-6, MUM1, and CD138. Of the 149 cases, 40 (26%) showed pattern A expression and were grouped as GCB group, lower than reported frequency of the studies involving mostly Western population. Compared with cases with pattern A, those with pattern B (activated GCB group) and C (activated non-GCB group) more often presented with more aggressive tumors and a shorter survival time. These results indicate that most of DLBCLs from Chinese patients can be classified into prognostically different groups based on the antigenic expression models using a panel of GCB- and ABC-associated markers. Polymerase chain reaction analysis of t(14;18) showed that 11 of 64 cases were t(14;18)-positive, and most (10 of 11) of it occurred in the group with pattern A. The translocation was significantly associated with expression of Bcl-2 protein. The group with pattern B demonstrated more frequent expression of Ki-67, cyclin D3, geminin, and showed higher proliferative activity than the group with pattern A. These findings suggest that high proliferative activity of tumors with pattern B may be associated with aggressive tumor behavior and poor clinical outcome in patients with DLBCL.

Novel blood-based, five-gene biomarker set for the detection of colorectal cancer.

PURPOSE: We applied a unique method to identify genes expressed in whole blood that can serve as biomarkers to detect colorectal cancer (CRC). EXPERIMENTAL DESIGN: Total RNA was isolated from 211 blood samples (110 non-CRC, 101 CRC). Microarray and quantitative real-time PCR were used for biomarker screening and validation, respectively. RESULTS: From a set of 31 RNA samples (16 CRC, 15 controls), we selected 37 genes from analyzed microarray data that differed significantly between CRC samples and controls (P < 0.05). We tested these genes with a second set of 115 samples (58 CRC, 57 controls) using quantitative real-time PCR, validating 17 genes as differentially expressed. Five of these genes were selected for logistic regression analysis, of which two were the most up-regulated (CDA and MGC20553) and three were the most down-regulated (BANK1, BCNP1, and MS4A1) in CRC patients. Logit (P) of the five-gene panel had an area under the curve of 0.88 (95% confidence interval, 0.81-0.94). At a cutoff of logit (P) >+0.5 as disease (high risk), <-0.5 as control (low risk), and in between as an intermediate zone, the five-gene biomarker combination yielded a sensitivity of 94% (47 of 50) and a specificity of 77% (33 of 43). The intermediate zone contained 22 samples. We validated the predictive power of these five genes with a novel third set of 92 samples, correctly identifying 88% (30 of 34) of CRC samples and 64% (27 of 42) of non-CRC samples. The intermediate zone contained 16 samples. CONCLUSION: Our results indicate that the five-gene biomarker panel can be used as a novel blood-based test for CRC.

[Hypermethylation of fragile histidine triad gene and 3p14 allelic deletion in ovarian carcinomas].

OBJECTIVE: The FHIT (fragile histidine triad) is a candidate tumor suppressor gene (TSG) located on chromosome 3p14.2. Hypermethylation and loss of heterozygosity (LOH) are major mechanisms in the inactivation of tumor suppressor genes. In this study, the methylation status of FHIT and LOH of 3p14 in 61 cases of human sporadic ovary carcinomas were investigated. METHODS: Sixty-one primary ovary carcinomas and 10 borderline ovarian tumors were analyzed with methylation specific PCR (MSP) to detect the CpG island methylation status in the FHIT promoter region. In addition, 45 cases of ovary carcinomas and their corresponding non-tumor ovary tissues were investigated with D3S1287 microsatellite polymorphic marker for LOH. RESULTS: Hypermethylation of FHIT gene was observed in 39.3% (24/61) of ovarian carcinomas. The frequencies of hypermethylation in serous ovarian carcinoma, mucinous ovarian carcinoma, endometrioid ovarian carcinoma and ovary borderline tumor were 45.2% (19/42), 14.3% (1/7), 33.3% (4/12) and 60.0% (6/10), respectively. Ten of twenty-three (43.5%) informative tumors showed LOH and 6 of 18 (33.3%) informative cases showed homozygous deletions. The status of FHIT methylation was not associated with clinical stage and differentiation grade, there was no significant difference between the malignant and borderline tumors. CONCLUSION: Hypermethylation and allelic deletion of FHIT are frequent events in ovarian carcinomas and are important mechanisms for the loss of expression of this gene.

[Hypermethylation of Ras association domain family protein 1A, hypermethylated in cancer 1 and p73 genes in hepatocellular carcinoma].

OBJECTIVE: To evaluate the status of promoter hypermethylation of Ras association domain family protein 1A (RASSF1A), hypermethylated in cancer 1 (HIC1) and p73 genes in hepatocellular carcinoma (HCC) and to explore the correlation with clinicopathological features. METHODS: Forty cases of HCC and their corresponding non-tumor liver tissues, other 2 cases of healthy donor livers were detected using methylation specific polymorphism chain reaction (MSP) method. RESULTS: The frequency of promoter hypermethylation of RASSF1A showed 90.0% and 72.5% in tumor and corresponding non-tumor tissues respectively, and there was significant difference between them (P < 0.05). The frequency of promoter hypermethylation of HIC1 showed 77.5% and 70.0% in tumor and corresponding non-tumor tissues respectively. The frequency of hypermethylation of HIC1 in non-tumor liver tissues showed significant correlation between younger and older patients. The frequency of promoter hypermethylation of p73 showed 5.0% in tumor tissues. However, none of hypermethylation of the gene was detected in corresponding non-tumor liver tissues. There was none of hypermethylation of the three genes showed in two cases of healthy donor livers. CONCLUSION: Promoter hypermethylation of RASSF1A and HIC1 genes are common event in HCC and play an important role in the pathogenesis and may be used to develop novel diagnostic and therapeutic approaches for HCC in the future.

[Methylation of mismatch repair gene (MMR) in primary hepatocellular carcinoma].

OBJECTIVE: To assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC). METHODS: Samples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR. RESULTS: CpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines. CONCLUSIONS: Our results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.