Frequent Allelic Loss Research Articles

Allelic loss of HLA class I facilitates evasion from immune surveillance in cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) has been reported to occur in HLA regions in cervical intraepithelial neoplasia (CIN) and cervical cancer. However, the details of how this is related to the progression of CIN have been unclear. In this study, we examined the human papillomavirus (HPV) antigen-presenting capacity of people with CIN and the significance of LOH of HLA class I in the progression of CIN. It was shown that differences in antigen-presenting capacity among each case depended on HLA types, not HPV genotypes. Focusing on the HLA type, there was a positive correlation between antigen-presenting capacity against HPV and the frequency of allelic loss. Furthermore, the lost HLA-B alleles had a higher HPV antigen-presenting capacity than intact alleles. In addition, frequency of LOH of HLA class I was significantly higher in advanced CIN (CIN2-3) than in cervicitis or early-stage CIN (CIN1): around half of CIN2-3 had LOH of any HLA class I. Moreover, the antigen-presenting capacity against E5, which is the HPV proteins that facilitate viral escape from this immune surveillance by suppressing HLA class I expression, had the most significant impact on the LOH in HLA-B. This study suggests that HPV evades immune surveillance mechanisms when host cells lose the capacity for antigen presentation by HLA class I molecules, resulting in long-term infection and progression to advanced lesions.

Clinicopathological Significance of Overall Frequency of Allelic Loss (OFAL) in Lesions Derived from Thyroid Follicular Cell.

BackgroundLoss of heterozygosity (LOH) and microsatellite instability (MSI) are frequent molecular events in thyroid tumor etiopathogenesis occurring in several chromosomal critical areas, including 3p12–25.3, 7q21–31, 10q22–24, and 15q11–13, with loci of tumor suppressor genes.ObjectiveWe evaluated the usefulness of LOH/MSI as a diagnostic/prognostic biomarker in lesions derived from thyroid follicular cells: follicular thyroid carcinoma (FTC); follicular adenoma (FA), papillary thyroid carcinoma (PTC), and nodular goiter (NG).MethodsWe performed allelotyping (GeneMapper Software v. 4.0.) of ten microsatellite markers linked to the 1p31.2, 3p21.3, 3p24.2, 9p21.3, 11p15.5, and 16q22.1 region on DNA from 93 primary thyroid lesions then evaluated the LOH/MSI frequency and overall frequency of allelic loss (OFAL).ResultsWe found regions with significantly increased frequency of LOH/MSI for specific histotypes: the 3p24.2 region for FA and 1p31.2 for FTC. LOH/MSI in 3p21.3 was significantly elevated in PTC and FTC. LOH/MSI in 3p21.3 was increased for small size tumors (T1a + T1b), tumors with no regional lymph node involvement (N0 + Nx), American Joint Committee on Cancer (AJCC) stage I tumors, and tumor diameter (Td) < 10 mm; in 1p31.2 for T2–3, N1, stage II–IV, and Td 10–30 mm; in 11p15.5 for T2–3, N1, stage II–IV, and Td > 30 mm. OFAL values were significantly higher in younger patients (< 40 years), in men, in those with T2–3 stage tumors, in those with increased Td, and in FA and FTC compared with NG and PTC.ConclusionsWe confirmed the occurrence of LOH/MSI in 3p21.3 at an early stage of tumorigenesis and mapped 1p31.2 and 11p15.5 as characteristic for advanced-stage tumors. The results of our study may enable consideration of OFAL, defined as LOH/MSI coincidence in various chromosomal regions, as a tumor progression marker. OFAL values were significantly higher in follicular neoplasms (FA and FTC) than in PTC or NG; hence, increased OFAL values can be regarded as a characteristic feature of the follicular phenotype.

Mutational and copy number asset of primary sporadic neuroendocrine tumors of the small intestine

Small intestine neuroendocrine tumors (SI-NETs) represent the most common histotype among small intestine neoplasms, and metastatic disease is usually present at diagnosis. A retrospective series of 52 sporadic primary surgically resected SI-NETs, which were metastatic at diagnosis, was analyzed by high-coverage target sequencing (HCTS) for the mutational status of 57 genes and copy number status of 40 genes selected from recently published genome sequencing data. Seven genes were found to be recurrently mutated: CDKN1B (9.6%), APC and CDKN2C (each 7.7%), BRAF, KRAS, PIK3CA, and TP53 (each 3.8%). Copy number analysis showed frequent allelic loss of 4 genes located on chromosome 18 (BCL2, CDH19, DCC, and SMAD4) in 23/52 (44.2%) and losses on chromosomes 11 (38%) and 16 (15%). Other recurrent copy number variations were gains for genes located on chromosomes 4 (31%), 5 (27%), 14 (36%), and 20 (20%). Univariate survival analysis showed that SRC gene copy number gains were associated with a poorer prognosis (p = 0.047). Recurrent copy number variations are important events in SI-NET and SRC may represent a novel prognostic biomarker for this tumor type.

Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia

AbstractTo identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[−]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002−) using a new monoclonal antibody specific to this allele, B4002− granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(−) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002− granulocytes that retained the HLA-A allele on the same haplotype (B4002−A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002−A−) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002−A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002− granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Association between histopathological features of dysplasia in oral leukoplakia and loss of heterozygosity

Oral leukoplakia (OL) dysplasia is graded on the basis of architectural and cytological features, and grade does not correlate well with malignant transformation. Loss of heterozygosity (LOH) profiles have been validated as risk predictors of OL malignant transformation. We aimed to assess whether the histological parameters used to grade dysplasia show different LOH profiles. Areas of epithelial dysplasia of 29 OL samples were microdissected, and LOH was assessed by use of a panel of 11 microsatellite markers located on chromosomes 3, 9, 11, and 17. Dysplasia was graded, and the cytological and architectural parameters were scored. Dysplasia was graded as mild in 18 samples, moderate in nine, and severe in two. The moderate/severe dysplasias and the mild dysplasias did not show different frequencies of allelic loss. Irregular epithelial stratification was associated with LOH at marker D3S1234 (3p14.2). In addition, the presence of drop-shaped rete ridges and premature keratinization in single cells showed associations with LOH at D9S162 (9p22) and P53 (17p13.1), respectively. We provide evidence that architectural and cellular changes in OL have different LOH patterns.

Lip cancer and pre-cancerous lesions harbor TP53 mutations, exhibit allelic loss at 9p, 9q, and 17p, but no BRAFV600E mutations.

Molecular mechanisms of lip squamous cell carcinoma (LSCC) and actinic cheilitis (AC) are unclear. We aimed at assessing loss of heterozygosity (LOH) and TP53 and BRAF V600E mutations in these lesions. Formalin-fixed paraffin-embedded (FFPE) samples of 17 LSCC and 16 AC were included, with additional 5 fresh LSCC genotyped for TP53 mutations. LOH was assessed by six polymorphic markers located at 9p22, 9q22, and 17p13 and correlated with cell proliferation (Ki-67) and P53 immunostaining. Direct sequencing of TP53 exons 2-11 (fresh samples), and exons 5-9 (FFPE samples) was carried out. BRAF V600E mutation was genotyped in eight LSCC. LOH occurred in at least one marker in 15/17 LSCC and in 9/16 AC. The marker exhibiting the highest frequency of allelic loss (FAL) in LSCC was D9S157 (8/12 informative cases) and D9S287 in AC (4/11 informative cases). Cell proliferation was not correlated with LOH or with the FAL and no correlation between P53 IHC and 17p LOHwas observed. We found TP53 missense mutations in both lesions and nonsense in LSCC, including CC>TT transition, which is a marker of UV damage. BRAF V600E mutation was not detected. LOH and TP53 mutations detected in LSCC and AC may be associated with tumorigenesis, whereas BRAF V600E mutation does not seem to significantly contribute to LSCC pathogenesis.

Sa1990 Frequent Allelic Loss of HIF3A Region in Liver Metastasis From Primary Colorectal Cancers

Sa1990 Frequent Allelic Loss of HIF3A Region in Liver Metastasis From Primary Colorectal Cancers

Evidence That HLA-B*40:02 and HLA-Α*31:01 Are Strongly Involved in the Presentation of Autoantigens to CTLs Responsible for the Development of Acquired Aplastic Anemia

The presence of leukocytes lacking one haplotype of the human leukocyte antigen (HLA) gene as a result of copy-neutral loss of heterozygosity of chromosome 6p (6pLOH) is compelling evidence that cytotoxic T cells (CTLs) have a key role in the development of acquired aplastic anemia (AA). Pathogenic auto-antigens are presumed to be presented by particular HLAs, such as HLA-B*40:02, HLA-A*31:01, HLA-A*02:01 and HLA-A*02:06, all of which are frequently lost due to 6pLOH (Katarigi, Blood 2011). However, this presumption may be incorrect, because the frequency of lacking haplotypes that contain KIR-ligands (KIR-Ls), such as HLA-A*24:02 and HLA-B*52:01, the most frequent alleles of HLA-A and HLA-B, may be underestimated as a result of the killing of 6pLOH(+) leukocytes by NK cells. Indeed, all four frequently missing HLA alleles are non-KIR-Ls. To address these issues, we conducted a mass screening of 6pLOH(+) leukocytes in newly diagnosed AA patients using an assay that allows us to detect 6pLOH(+) cells within one day, and reanalyzed haplotypes that are likely to be lost. The HLA-B and –C allele-specific duplex real time PCR (2qPCR) that we established compared the copy number of heterozygous HLA alleles in a single reaction mixture using two allele-specific TaqMan probes labeled with two different fluorochromes (VIC and FAM) and one primer pair complementary to consensus sequences, which allowed us to detect as small as 5% 6pLOH(+) leukocytes in the total leukocytes. The HLA haplotypes of 6pLOH(+) patients were determined based on the HLA-A antigen expression as demonstrated by flow cytometry and the HLA-B and -C allele data from the 2qPCR, in combination with the haplotype database of the Japanese population available in the HLA laboratory website. A total of 498 patients with AA were subjected to this analysis and 60 6pLOH(+) patients, including 39 6pLOH(+) patients that had been identified by our previous study were used in this analysis. The allelic loss frequencies of HLA-B*40:02 (25%, 33/132) and HLA-A*31:01 (19%, 14/75) were markedly higher than those of the other HLA-B (2.9%, 24/814, P = 7x10-16) and HLA-A (4.9%, 36/731, P = 0.002) alleles, while the frequencies of HLA-A*02:01 (11%, 11/99) and HLA-A*02:06 (11%, 11/99) were similar to those of the other HLA-A alleles. In 41 6pLOH(+) patients possessing either HLA-B*40:02 or HLA-A*31:01, these alleles were all contained in the missing haplotype. On the other hand, four of the 15 patients with HLA-A*02:01(+) haplotypes and two of the 13 patients with HLA-A*02:06(+) haplotypes had these alleles in the retained haplotype. The 6pLOH(+) patients could be divided into three groups according to the status of KIR-Ls (HLA-Bw4 and HLA-C1/C2) of their haplotype; A, the lack of the KIR-L does not occur when either of the two haplotypes is lost; B, the lack of KIR-L occurs when one specific haplotypes is lost; C, the lack of KIR-L occurs when either haplotype is lost. These groups comprised 42%, 53% and 5% of the total. The proportion of group C was much lower than that expected in the general population (16%) and only five (16%) of the 31 group B patients lacked KIR-Ls, thus suggesting that NK cells had an effect on the appearance of 6pLOH(+) leukocytes in AA patients. However, the very high frequency of HLA-loss in the HLA-B*40:02 and HLA-A*31:01 alleles could not be explained by the absence of KIR-Ls in the missing haplotype. Of particular note, the lack of KIR-Ls occurred in eight patients as a result of 6pLOH; six of the eight lost a haplotype containing HLA-B*40:02 and one lost an A*31:01-containing haplotype, suggesting that CTLs specific for autoantigens presented by these class I alleles more dominantly inhibit HSCs than NK cells. Together, these results indicate that HLA-B*40:02 and HLA-A*31:01 have particularly important roles in the presentation of autoantigens to T cells in AA. Studies of T-cell responses to autoantigens restricted by these class I alleles are thus warranted. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

BackgroundGenetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer.MethodsMicroarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction.ResultsOverall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro.ConclusionsThe relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence.

Thymidylate synthase (TS) expression as a prognostic molecular marker in stage II/III colon cancer.

3577 Background: Studies on the association between colorectal cancer (CC) outcome and thymidylate synthase expression have provided inconsistent results. In this study we attempted to resolve the issue by assessing the associations between TS expression and outcome in a population of primary CC patients (pts), who after resection were randomized to 5-FU/FA vs. FOLFIRI adjuvant therapy. Methods: Immunohistochemical staining for TS protein was successfully performed for 1211 pts in the PETACC3 trial. TS immunoreactivity was scored as high expression (≥75% positive) and low expression (&lt;75% positive). Gene expression respectively copy number data were available for 853 respectively 306 of these samples. Twelve single nucleotide polymorphisms (SNPs) close to the TYMS gene were assessed in 923 pts. Association of variables with relapse-free (RFS) and overall survival (OS) was assessed using Cox regression models. Results: High TS expression and RNA level were strongly associated (log fold change 0.65, p&lt;0.001). Both were significantly higher in proximal CC. As expected, both were associated with other characteristics of proximal CC: MSI, BRAF mutation, high tumor grade. RNA was significantly correlated with gene copy number, distal CC showing more frequent allelic loss. Three SNPs were associated with gene expression which was validated in data from the 1000 genomes project, but none with survival. High TS expression was more strongly associated with better OS in pts receiving FOLFIRI (HR 0.4, 95% CI 0.3–0.6, p&lt;0.001), than 5-FU/FA (HR 0.8, 95% CI 0.5–1.1, p=0.13), with a significant interaction (p=0.05). Similar results were observed for RFS (HR 0.5, p&lt;0.001 vs. HR 0.7, p=0.07; interaction p=0.11). TS expression is still highly prognostic in multivariate models adjusting for factors associated with risk or proximal tumors in FOLFIRI treated pts (OS: HR 0.5, p=0.008; RFS: HR 0.6, p=0.02), but not in F-FU/FA treated pts (OS and RFS: HR=1, p=1). Conclusions: TS expression is lower in distal CC, partly due to deletion of the TYMS locus. Pts with high TS expression have longer RFS and OS, notably when treated with FOLFIRI. For these pts addition of irinotecan to 5-FU/FA adjuvant chemotherapy might be considered. Clinical trial information: NCT00026273.

Expression Profiles in Stage II Colon Cancer According to APC Gene Status

Colorectal cancer is one of the most common cancers in the world. Histoclinical staging is efficient, but combination with molecular markers may improve the classification of stage II cancers. Several tumor-suppressor genes have been associated with colorectal cancer, and the most frequent allelic losses have been extensively studied for their prognosis effect, but the results remain controversial. In a previous study, we found a possible influence of the chromosome 5 status in the development of liver metastases in stage II colon cancers. We have here investigated the role of the APC gene, located in chromosome arm 5q, in a series of 183 colon adenocarcinomas through a combined analysis of gene expression, mutation, allelic loss and promoter methylation, and metastasis occurrence. Point mutations were found in 73% of cases and allelic losses were found in 39%; 59% of tumors presented with a biallelic inactivation, with a very strong interdependence of the two APC hits (P=2.1×10-9). No associationwas found between expression, number and type of APC alterations, and metastatic evolution. Our results show that the determination of APC status cannot help in the prediction of metastasis and cannot be used to subclassify stage II colon cancers.

High frequency of allelic loss at the BRCA1 locus in ovarian cancers: clinicopathologic and molecular associations

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR(qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P<0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations.

Aberrant protein expression and frequent allelic loss of MSH3 in colorectal cancer with low-level microsatellite instability

High level of microsatellite instability (MSI-H) in colorectal cancer (CRC) is caused by the inactivation of mismatch repair (MMR) genes; however, it is unknown for tumors with low level MSI (MSI-L). The protein complex involving MSH3 preferentially recognizes insertion/deletion loops (IDLs) of two to eight bases and di- and tetranucleotide repeats are affected in the majority of MSI-L CRC. We selected 10 and eight MSI-L CRCs from 228 and 204 patients with sporadic and hereditary disease, respectively. The tumors were analyzed for protein expression of MSH3, MSH2, MSH6, MLH1, and PMS2, and for mutations and loss of heterozygosity (LOH) in MSH3. Four tumors showed a markedly reduced MSH3 expression, whereas all 18 tumors had normal expression of the remaining MMR proteins. Twenty-five different sequence variants were identified. None of these results in a truncated protein, though L902W represents the first constitutional missense mutation in MSH3 predicted to be functional based on conservation among mutS homologues. All variants have also been found in normal DNA of the patients and in controls. LOH intragenic to MSH3 was evident for 12 of 16 (75%) informative tumors. Occurrence of sequence variants in normal DNA of the patients and in controls excludes somatic mutations and mutations specific to the CRC patient population, respectively. In contrast, the high frequency of LOH as well as the aberrant protein expression in some tumors indicates an involvement of MSH3 impairment in MSI-L CRC.

Inactivation of the hypermethylated in cancer 1 tumour suppressor - not just a question of promoter hypermethylation?

The chromosomal region 17p13.3 is frequently deleted or epigenetically silenced in a variety of human cancers. It includes the hypermethylated in cancer 1 (HIC1) gene placed telomerically to the p53 tumour suppressor gene. HIC1 encodes a transcriptional repressor, and its targets identified to date are genes involved in proliferation, tumour growth and angiogenesis. In addition, HIC1 functionally cooperates with p53 to suppress cancer development. Frequent allelic loss at position 17p13.1 in human cancers often points to mutations of the tumour suppressor p53. However, in a variety of cancer types, allelic loss of the short arm of chromosome 17 may hit regions distal to p53 and, interestingly, without leading to p53 mutations. Furthermore, the neighbouring region 17p13.3 often shows loss of heterozygosity or DNA hypermethylation in various types of solid tumours and leukaemias. In line with this concept, Wales et al. described a new potential tumour suppressor in this region and named it hypermethylated in cancer 1 (HIC1). Further, it was shown that in the majority of cases hypermethylation of this chromosomal region leads to epigenetic inactivation of HIC1. A role for HIC1 in tumour development is further supported by a mouse model, since various spontaneous, age- and gender-specific malignant tumours occur in heterozygous Hic1+/- knockout mice. Furthermore, exogenously delivered HIC1 leads to a significant decrease in clonogenic survival in cancer cell lines. This review highlights the role of HIC1 inactivation in solid tumours and particularly in leukaemia development.

The association of frequent allelic loss on 17p13.1 with early metastastic recurrence of hepatocellular carcinoma after liver transplantation

Identification and characterization of loss of heterozygosity (LOH) can determine putative tumor suppressor genes (TSGs) and provide a variety of molecular markers for hepatocellular carcinoma (HCC). This study aimed to investigate LOH status on chromosomes 4q, 6q, 8p, 9p, and 17p, and to explore their clinical significances in HCC post-liver transplantation. A total of 37 patients with HCC who underwent liver transplantation were enrolled. LOH was examined using 34 microsatellite markers located on 4q13-3q5, 6q27, 8p22-p23, 9p21-p22, and 17p12-p13. The frequency of LOH at each microsatellite locus ranged from 23% to 75%, with a mean value of 53.1%. Frequencies of LOH on 4q, 6q, 8p, 9p, and 17p were 62% (23 of 37), 30% (11 of 37), 49% (18 of 37), 46% (16 of 35), and 68% (25 of 37), respectively. LOHs on certain chromosomal regions were significantly associated with age, AFP level, tumor size, tumor multiplicity, histological grade, and metastatic recurrence. LOH on 17p13.1 correlated to metastatic HCC recurrence, while LOH on 4q and 8p was found to be associated with progression of HCC. Thus, potential novel biomarkers or TSGs for prognosis and treatment of HCC may harbor on these regions.

Tumor‐specific mutation and downregulation of ING5 detected in oral squamous cell carcinoma

Our previous study showed high frequency of allelic loss at chromosome 2q37 region in oral cancer. This location contains several candidate tumor suppressor genes such as PPP1R7, ILKAP, DTYMK and ING5. We previously showed 3 members of inhibitor of growth (ING) family, ING1, ING3 and ING4 as tumor suppressor gene in head and neck cancer. As ING5 shows high homology with other members of ING genes including highly conserved carboxy-terminal plant homeodomain and nuclear localization signal, we first picked up ING5 and examined it as a possible tumor suppressor in oral cancer. For this aim, mutation and mRNA expression status of ING5 in paired normal and oral squamous cell carcinoma samples were examined by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. Three missense mutations located within leucine zipper like (LZL) finger and novel conserved region (NCR) domains in ING5 protein were detected, probably abrogating its normal function. We also found 5 different alternative splicing variants of ING5. Then, we examined mRNA level of ING5 by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis, which demonstrated decreased expression of ING5 mRNA in 61% of the primary tumors as compared to the matched normal samples. In conclusion, tumor-specific mutation and downregulation of ING5 mRNA suggested it as a tumor suppressor gene in oral squamous cell carcinoma.

Genetic Alterations and Expression Pattern of CEACAM1 in Colorectal Adenomas and Cancers

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelial cells throughout the intestinal tract and is a negative regulator of tumor cell growth, suggesting that it may function as a tumor suppressor. In this study, to determine whether the CEACAM1 is involved in colorectal tumorigenesis, we have investigated the genetic alterations, including mutations and allelic loss, of the CEACAM1 gene in 17 colonic adenomas and 123 sporadic colorectal cancers. In addition, the expression pattern of the CEACAM1 protein was examined in 60 colonic adenomas and 123 sporadic colorectal adenocarcinomas. No mutation was found in colonic adenomas, but four somatic missense mutations, L36F, T312I, V398I and A445V, were detected in colorectal cancers. Interestingly, all of the mutations were found in left-side colon cancers of the patients with clinical stage III. In LOH analysis, nine adenomas were informative for at least one of the markers and five (55.6%) showed allelic loss. Thirty-eight cancers were informative at D19S211 and D19S872 markers and 21 (56.3%) showed LOH at these markers. Statistically, the frequency of allelic loss at the CEACAM1 locus was not associated with clinicopathologic parameters (P > 0.05). In immunohistochemical analysis, loss of expression of CEACAM1 protein was detected in nine (15.0%) and 30 (24.4%) of 60 colorectal adenomas and 123 colorectal cancers. Statistically, there was no significant relationship between loss of CEACAM1 expression and clinicopathologic parameters, including clinical stage, tumor location, tumor size, lymph node metastasis and 5-year survival (P > 0.05). These data suggest that genetic alteration and loss of expression of the CEACAM1 may contribute to the development of colorectal cancers, as an early event.

Reduced expression of EphB2 is significantly associated with nodal metastasis in Chinese patients with gastric cancer

EphB2 is a member of the Eph receptor tyrosine kinase family that has been involved in the regulation of cytoskeleton organization and cell migration in various cell types. Its role and regulation in carcinogenesis is controversial, especially in gastric cancer. We detected EphB2 expression and determined its clinical significance and explored its underlying molecular mechanism in gastric cancers. Tissue microarray blocks containing primary gastric cancer, lymph node metastases, and adjacent normal mucosa specimens obtained from 337 Chinese patients were constructed. Expression of EphB2 in these specimens was analyzed using immunohistochemistry. Mutation analysis at the A9 tract in exon 17 and loss of heterozygosity analysis at the EphB2 gene locus were carried out in 13 sporadic EphB2-negative gastric cancers. Complete loss of EphB2 expression was observed in 177 (52.5%) of the 337 primary tumor and 41 (82%) of the 50 nodal metastases. Loss of EphB2 expression was significantly associated with advanced T stage, nodal metastasis, advanced disease stage, and poor histological differentiation. Loss of EphB2 expression correlated significantly with poor survival rates in both univariate and multivariate analysis. No frameshift mutation, but a higher frequency of allelic loss, was found in EphB2-negative primary and metastatic tumor samples. Frequent deletion and decreased expression of EphB2 protein suggested it as a negative biomarker for gastric carcinogenesis and a potential predictor of the outcome of patients with gastric cancer.

Integrated genomic profiling identifies candidate genes implicated in glioma‐genesis and a novel LEO1‐SLC12A1 fusion gene

We performed genotyping and exon-level expression profiling on 21 glioblastomas (GBMs) and 19 oligodendrogliomas (ODs) to identify genes involved in glioma initiation and/or progression. Low-copy number amplifications (2.5 < n < 7) and high-copy number amplifications (n > 7) were more frequently observed in GBMs; ODs generally have more heterozygous deletions per tumor. Four high-copy amplicons were identified in more than one sample and resulted in overexpression of the known oncogenes EGFR, MDM2, and CDK4. In the fourth amplicon, RBBP5, a member of the RB pathway, may act as a novel oncogene in GBMs. Not all hCNAs contain known genes, which may suggest that other transcriptional and/or regulatory elements are the target for amplification. Regions with most frequent allelic loss, both in ODs and GBMs, resulted in a reduced expression of known tumor suppressor genes. We identified a homozygous deletion spanning the Pragmin gene in one sample, but direct sequencing of all coding exons in 20 other glioma samples failed to detect additional genetic changes. Finally, we screened for fusion genes by identifying aberrant 5'-3' expression of genes that lie over regions of a copy number change. A fusion gene between exon 11 of LEO1 and exon 10 of SLC12A1 was identified. Our data show that integrated genomic profiling can identify genes involved in tumor initiation, and/or progression and can be used as an approach to identify novel fusion genes.

Epigenetic Inactivation of the Thyroid Hormone Receptor β1 Gene at 3p24.2 in Lung Cancer

Frequent allelic loss on chromosome 3p in various human cancers suggests the presence of tumor suppressor genes in this region. The thyroid hormone receptor beta1 (TRbeta1) gene is located at 3p24.2, where allelic loss frequently occurs in lung cancer, and aberrant TRbeta1 methylation was observed in several human cancers. We examined the expression, mutation, and promoter methylation of TRbeta1 in 18 small cell lung cancer (SCLC) and 29 non-small cell lung cancer (NSCLC) cell lines by reverse-transcription polymerase chain reaction (RT-PCR), direct sequencing, or methylation-specific PCR. Four lung cancer cell lines lacking TRbeta1 expression were treated with 5-aza-2-deoxy-cytidine and/or trichostatin-A, and the TRbeta1 expression was determined by RT-PCR. We also examined the TRbeta1 methylation in 116 NSCLC surgical specimens and analyzed the correlation between methylation status and clinicopathological parameters or mutations of KRAS and EGFR. TRbeta1 expression was absent in 61% of SCLCs and 48% of NSCLCs, and 67% of SCLCs and 45% of NSCLCs carried TRbeta1 promoter methylation, while no somatic mutation was found in all cell lines. TRbeta1 methylation status was significantly associated with loss of TRbeta1 expression. TRbeta1 expression was restored by treatment with 5-aza-2-deoxy-cytidine and/or trichostatin-A in four cell lines. TRbeta1 methylation was found in 47% of NSCLC surgical specimens; however, the methylation was not significantly associated with any clinicopathological parameters or mutations of KRAS and EGFR. This is the first study to demonstrate epigenetic inactivation of TRbeta1 through aberrant methylation in lung cancer, while TRbeta1 mutations are not common in lung cancer.