Hee Eun Lee

Promoter CpG island hypermethylation during breast cancer progression

This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15xa0CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (nu2009=u200910), ADH/FEA (nu2009=u200930), DCIS (nu2009=u200935), and IDC (nu2009=u200930) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.

Characteristics of KIT-negative gastrointestinal stromal tumours and diagnostic utility of protein kinase C theta immunostaining

Aims: To characterise KIT-negative gastrointestinal stromal tumours (GISTs) clinically, pathologically, immunohistochemically and genetically, and to establish the usefulness of protein kinase C theta (PKCθ) as a diagnostic marker in KIT-negative GIST. Methods: 252 consecutive cases of GIST were evaluated for clinicopathological characteristics and immunostained for various antibodies. Mutational analyses of KIT and platelet-derived growth factor receptor α (PDGFRA) were also performed in 62 cases. Results: 20 (7.9%) GISTs showed negative immunostaining for KIT. KIT-negative GISTs were more likely to originate from omentum or peritoneum, have an epithelioid histology, and be classified as high risk. The overall survival rate of patients with KIT-negative GISTs (5-year survival rate 68.7% (SD 10.7%)) was lower than that of patients with KIT-positive GISTs (5-year survival rate, 79.9% (3.0%)) (pu200a=u200a0.042, log-rank test). Negative KIT expression was an independent prognostic factor in multivariate Cox regression analysis when the risk of aggressive behaviour and the status of imatinib treatment were adopted as covariates. KIT-negative GISTs also showed lower expression rates of CD34, Bcl-2, and PKCθ than KIT-positive GISTs; mutational analysis revealed that 30% of KIT-negative GISTs harboured a PDGFRA exon 18 mutation. Immunostaining on PKCθ showed that 93.9% of all GISTs expressed PKCθ protein. However, 21.9% of 64 mesenchymal tumours other than GIST also showed positivity on PKCθ. Conclusions: KIT-negative GISTs had characteristics that differ from those of KIT-positive GISTs, and negative KIT expression was an independent prognostic indicator for overall survival of patients. Although PKCθ is a sensitive diagnostic marker for GIST, its usefulness is limited because of low sensitivity and low specificity in KIT-negative GISTs.

Epithelial-mesenchymal transition increases during the progression of in situ to invasive basal-like breast cancer

Epithelial-mesenchymal transition (EMT) is known to play an important role in breast cancer invasion and metastatic progression. However, the pattern of expression of EMT markers in the progression from in situ to invasive breast carcinoma is not clear. To investigate this, we performed immunohistochemical analyses of EMT markers (expression of vimentin, smooth muscle actin, osteonectin, and N-cadherin; loss of E-cadherin; alteration of β-catenin), breast cancer stem cell (CSC) markers (CD44(+)/CD24(-), ALDH1), and CD146, an EMT inducer, in invasive carcinomas and ductal carcinoma in situ (DCIS) of the breast. Expression of EMT markers was closely associated with the basal-like subtype and CSC phenotype in invasive carcinoma but not in pure DCIS, except for vimentin. The expression of smooth muscle actin and N-cadherin, loss of E-cadherin, and alteration of β-catenin were significantly higher in invasive carcinomas than in pure DCIS (P = .015, P = .029, P = .001, and P = .007, respectively). Subgroup analyses revealed greater loss of E-cadherin and alteration of β-catenin in invasive carcinoma than in pure DCIS in basal-like subtype (P = .001) but not in non-basal-like subtypes. Moreover, expression of EMT markers and CD146 was higher in the invasive than in the DCIS component of basal-like cancers. Our study confirmed that EMT is an intrinsic characteristic of basal-like subtype and is associated with CSC phenotype. Furthermore, we showed higher expression of EMT markers in invasive carcinomas than in pure DCIS, especially in basal-like subtype, and in the invasive component of basal-like breast cancers, suggesting that EMT may be involved in the progression from in situ to invasive basal-like breast cancers.

Distinct patterns of promoter CpG island methylation of breast cancer subtypes are associated with stem cell phenotypes

Although DNA methylation profiles in breast cancer have been connected to breast cancer molecular subtype, there have been no studies of the association of DNA methylation with stem cell phenotype. This study was designed to evaluate the promoter CpG island methylation of 15 genes in relation to breast cancer subtype, and to investigate whether the patterns of CpG island methylation in each subtype are associated with their cancer stem cell phenotype represented by CD44+/CD24− and ALDH1 expression. We performed MethyLight analysis of the methylation status of 15 promoter CpG island loci involved in breast cancer progression (APC, DLEC1, GRIN2B, GSTP1, HOXA1, HOXA10, IGF2, MT1G, RARB, RASSF1A, RUNX3, SCGB3A1, SFRP1, SFRP4, and TMEFF2) and determined cancer stem cell phenotype by CD44/CD24 and ALDH1 immunohistochemistry in 36 luminal A, 33 luminal B, 30 luminal–HER2, 40 HER2 enriched, and 40 basal-like subtypes of breast cancer. The number of CpG island loci methylated differed significantly between subtypes, and was highest in the luminal–HER2 subtype and lowest in the basal-like subtype. Methylation frequencies and levels in 12 of the 15 genes differed significantly between subtypes, and the basal-like subtype had significantly lower methylation frequencies and levels in nine of the genes than the other subtypes. CD44+/CD24− and ALDH1+ putative stem cell populations were most enriched in the basal-like subtype. Methylation of promoter CpG islands was significantly lower in CD44+/CD24-cell (+) tumors than in CD44+/CD24-cell (−) tumors, even within the basal-like subtype. ALDH1 (+) tumors were also less methylated than ALDH1 (−) tumors. Our findings showed that promoter CpG island methylation was different in relation to breast cancer subtype and stem cell phenotype of tumor, suggesting that breast cancers have distinct patterns of CpG island methylation according to molecular subtypes and these are associated with different stem cell phenotypes of the tumor.

Constitutive phosphorylation of the FOXO1A transcription factor as a prognostic variable in gastric cancer.

Increased phosphorylation of FOXO1A, a FOXO transcription factor, has been implicated in several human cancers; however, it has not been studied in the gastric cancer to date. To determine the status of pFOXO1A expression in human gastric cancers and to determine its relationship with other tumor-associated proteins, we performed immunohistochemical staining on tissue array slides containing 272 human gastric carcinoma specimens. In non-neoplastic gastric mucosa, the expression of pFOXO1A was observed primarily in cells in the proliferative zone and in areas of intestinal metaplasia. In gastric carcinomas, the expression of pFOXO1A was observed in 230 (84.6%) out of 272 cases examined, and was positively correlated with the Ki-67-labeling index (P=0.026). The expression of pFOXO1A was higher in the early stages of pTNM (P<0.001), and was inversely correlated with the intestinal type by Laurens classification (P=0.001), lymphatic invasion (P=0.017) and lymph node metastasis (P<0.001). Moreover, the expression of pFOXO1A was correlated with a longer patient survival (P=0.004). In addition, the expression of pFOXO1A was correlated with that of pAKT1 (P<0.001), PTEN (P=0.009), CDKN2A (P=0.012), APC (P=0.048), SMAD4 (P<0.001), CD82 (P=0.011), and BCL2 (P=0.011). In conclusion, our results showed that the expression of pFOXO1A is a frequent and early event in gastric tumorigenesis and that there is a significant correlation between pFOXO1A and better prognosis. Thus, our data suggest that the expression of pFOXO1A may serve as a valuable prognostic variable in gastric carcinoma and have significant implications for the development and application of targeted therapy.

In situ analysis of HER2 mRNA in gastric carcinoma: comparison with fluorescence in situ hybridization, dual-color silver in situ hybridization, and immunohistochemistry.

The importance of anti-HER2 therapy has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. In the present study, we evaluated HER2 mRNA expression using a new mRNA in situ detection technique called RNAscope in 211 cases of formalin-fixed, paraffin-embedded gastric carcinoma. In addition, we compared the results with the conventional methods of immunohistochemistry, fluorescence in situ hybridization, and dual-color silver in situ hybridization. RNA in situ hybridization (in situ hybridization) showed that 162 cases (76.8%) were score 0, 5 cases (2.4%) were score 1, 10 cases (4.7%) were score 2, 13 cases (6.2%) were score 3, and 21 cases (10.0%) were score 4. HER2 transcription levels were found to be significantly related to pT class, pN class, and tumor recurrence. mRNA expression was well correlated with protein overexpression and gene amplification; 20 cases out of 23 with DNA amplification showed a score of 4 in RNA in situ hybridization (P < .001). Three cases showed false negative and one case showed false positive results by in situ hybridization. More studies are needed to determine whether the in situ hybridization method can identify additional patients that may benefit from anti-HER2 therapy or exclude those who may be resistant to anti-HER2 therapy.

Constitutive phosphorylation of the FOXO1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis-related molecules.

BackgroundAlthough FOXO transcription factors may have an anti-angiogenic role, little is known about their role in tumor angiogenesis. The present study was performed to investigate the correlation between the constitutive expression of phosphorylated FOXO1 (pFOXO1) and angiogenesis in gastric cancer.MethodsImmunohistochemistry was performed on tissue array slides containing 272 gastric carcinoma specimens, and the correlations between the cytoplasmic pFOXO1 expression in gastric cancer cells and CD34-immunopositive microvessel area (MVA) or the expressions of angiogenesis-related molecules were analyzed. In vitro analyses with Western blotting and semiquantitative reverse transcription-polymerase chain reaction were performed using the stable SNU-638 gastric cancer cell line transfected with lentivirus-delivered FOXO1 short hairpin RNA.ResultsThe cytoplasmic expression of pFOXO1 in tumor cells was observed in 85% of gastric carcinoma cases, and was found to be positively associated with higher MVA (P = 0.048). Moreover, pFOXO1 expression was positively correlated with the expressions of several angiogenesis-related proteins, including hypoxia inducible factor-1α (HIF-1α, P = 0.003), vessel endothelial growth factor (P = 0.004), phosphorylated protein kinase B (P < 0.001), and nuclear factor-κB (P = 0.040). In contrast, the expression of pFOXO1 was not correlated with that of phosphorylated signal transducer and activator of transcription 3 or β-catenin. In addition, cell culture experiments showed that FOXO1 suppression increased the mRNA and protein expressions of HIF-1α.ConclusionOur results suggest that pFOXO1 expression in cancer cells plays a role in gastric cancer angiogenesis via mechanisms involving various angiogenesis-related molecules. Animal experiments are needed to confirm the anti-angiogenic role of FOXO1 in human gastric cancer.

Low Ki‐67 proliferation index is an indicator of poor prognosis in gastric cancer

We designed this study to assess the biologic significance of Ki‐67 proliferation index (PI) in gastric cancer.

Expression of apoptosis-related proteins and its clinical implication in surgically resected gastric carcinoma.

Apoptosis, via caspase cascade, is involved in tumorigenesis and progression, and thus, altered apoptosis-related protein expressions have clinical and prognostic significance. Moreover, the apoptosis pathway is highlighted due to the recent introduction of apoptosis-targeted therapy for several genes such as the X-linked inhibitor of apoptosis protein (XIAP). XIAP is the most potent direct inhibitor of caspase, and XIAP-associated factor 1 (XAF1) and secondary mitochondrial activator of caspase/direct IAP-binding protein with low PI (Smac/DIABLO) are negative regulators of XIAP. In this study, we evaluated the expression of these proteins and investigated their clinical and prognostic significance in gastric carcinomas. Immunohistochemical analysis by using the tissue array method was performed for XIAP, survivin, Bcl-2, XAF1, Smac/DIABLO, and cleaved caspase-3 proteins in 1,162 surgically resected gastric carcinoma cases. XIAP expression was related to the advanced stage. The expression of XIAP showed negative relationship with XAF1 and Smac/DIABLO expressions. In addition, XIAP expression was associated with a poor prognosis and was also proved to be an independent prognostic factor. Cleaved caspase-3 expression was related to the early stage. In addition, cleaved caspase-3 expression was associated with a favorable prognosis and was also proved to be an independent prognostic factor. The expression of XIAP showed an inverse relationship with cleaved caspase-3. In addition, the expression of XAF1 and Smac/DIABLO had a positive relationship with cleaved caspase-3. These findings are consistent with their known functions, and they may help to identify individuals best suited for apoptosis-targeted therapy as a baseline data in gastric carcinoma.

Downregulation of methylthioadenosin phosphorylase by homozygous deletion in gastric carcinoma

The methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 telomeric to the CDKN2A tumor suppressor gene. Loss of MTAP gene is frequently associated with CDKN2A homozygous deletion. Although the homozygous deletion of MTAP has been reported in various human cancers, its function in gastric carcinogenesis is unknown. Here, we determined the status of the MTAP gene by using a combination of array‐based comparative genomic hybridization and oligonucleotide microarray. It was found that MTAP was deleted and downregulated in 2 of 10 gastric cancer cell lines. Of the 494 primary gastric carcinomas examined, MTAP expression at the protein level was reduced in 59 (11.9%). Furthermore, a lack of MTAP expression was found to be associated with poor survival (P = 0.038). The genomic loss of MTAP and CDKN2A in gastric carcinomas was investigated by quantitative real‐time PCR. Among 20 gastric carcinomas, two cases showed deletion of both MTAP and CDKN2A, and three samples showed homozygous deletion of MTAP, but not of CDKN2A. An analysis of gastric carcinomas revealed that reduced MTAP expression correlated significantly with a genomic deletion. Furthermore, functional assays by transfecting the siRNA or the expressional cDNA into gastric cancer cell lines demonstrated that MTAP regulates cell growth and invasion. The present study suggests that MTAP plays an important role in the regulation of gastric carcinogenesis and, in particular, that MTAP loss is implicated in some way with tumor growth via the modulation of cellular properties, which, in turn, suggests that MTAP has therapeutic applications.