Showing papers by "Nam Jin Yoo published in 2011"

A Randomized Phase II Study of Gefitinib Plus Simvastatin Versus Gefitinib Alone in Previously Treated Patients with Advanced Non–Small Cell Lung Cancer

Abstract: Purpose: To evaluate the efficacy and safety of gefitinib plus simvastatin (GS) versus gefitinib alone (G) in previously treated patients with advanced non–small cell lung cancer (NSCLC). Experimental Design: Between May 2006 and September 2008, 106 patients (51% men, 75% adenocarcinoma, 50% never smoker) were randomly assigned to G alone (250 mg/d, n = 54) or GS (250 and 40 mg/d, respectively, n = 52). One cycle was 4 weeks of treatment. Therapy was continued until disease progression or intolerable toxicity was observed. The primary endpoint was response rate (RR). Secondary endpoints included toxicity, progression-free survival (PFS), and overall survival (OS). Results: The RR was 38.5% (95% CI, 25.3–51.7) for GS and 31.5% (95% CI, 19.1–43.9) for G. The median PFS was 3.3 months [M] (95% CI, 1.4–5.2M) for GS and 1.9M (95% CI, 1.0–2.8M) for G. The median OS was 13.6M (95% CI, 7.1–20.1M) for GS and 12.0M (95% CI, 7.8–16.2M) for G. In exploratory subgroup analysis, GS showed higher RR (40% vs. 0%, P = 0.043) and longer PFS (3.6M vs. 1.7M, P = 0.027) compared with G alone in patients with wild-type epidermal growth factor receptor ( EGFR ) nonadenocarcinomas. Adverse events in both arms were generally mild and mainly consisted of skin rashes. Conclusions: Although no superiority of GS to G was demonstrated in this unselected NSCLC population, GS showed higher RR and longer PFS compared with G alone in patients with wild-type EGFR nonadenocarcinomas. Simvastatin may improve the efficacy of gefitinib in that subgroup of gefitinib-resistant NSCLC patients. Clin Cancer Res; 17(6); 1553–60. ©2011 AACR.

Genetic and expressional alterations of CHD genes in gastric and colorectal cancers

Abstract: Kim M S, Chung N G, Kang M R, Yoo N J & Lee S H (2011) Histopathology58, 660–668 Genetic and expressional alterations of CHD genes in gastric and colorectal cancers Aims: Chromodomain helicase DNA-binding protein (CHD) is a regulator of the chromatin remodelling process The aim was to determine the CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9mutational status of mononucleotide repeats in gastric and colorectal cancers with microsatellite instability (MSI) Methods and Results: The repeats were determined in 28 gastric cancers (GCs) with high MSI (MSI-H), 45 GCs with low MSI (MSI-L)/stable MSI (MSS), 35 colorectal cancers (CRCs) with MSI-H and 45 CRCs with MSI-L/MSS by single-strand conformation polymorphism analysis CHD4 and CHD8 expressionwas also examined in GCs and CRCs by immunohistochemistry CHD1, CHD2, CHD3, CHD4, CHD7, CHD8 and CHD9 mutations were found in five, 19, three, five, seven, 10 and seven cancers, respectively They were detected in MSI-H cancers, but not in MSI-L/MSS cancers Loss of CHD4 expression was observed in 564% of the GCs and 557% of the CRCs, and loss of CHD8 was observed in 357% of the GCs and 286% of the CRCs The cancers with CHD4 and CHD8 mutations showed loss of CHD4 and CHD8 expression, respectively Conclusions: Frameshift mutation and loss of expression of CHD genes are common in GCs and CRCs with MSI-HThese alterations might contribute to cancer pathogenesis by deregulating CHD-mediated chromatin remodelling

Mutational and expressional analyses of ATG5, an autophagy-related gene, in gastrointestinal cancers.

Abstract: There is mounting evidence that alterations of cell death processes are involved in cancer pathogenesis. ATG5 is a key regulator of autophagic and apoptotic cell death. The aim of this study was to see whether alterations of ATG5 protein expression and somatic mutation of ATG5 gene are features of human gastrointestinal cancers. In this study, we analyzed ATG5 somatic mutation in 45 gastric, 45 colorectal, and 45 hepatocellular carcinomas by single-strand conformation polymorphism (SSCP). Also, we analyzed ATG5 protein expression in 100 gastric, as well as in 95 colorectal and hepatocellular carcinomas using immunohistochemistry. Overall, we detected two somatic missense mutations of ATG5 gene in the coding sequences p.Leu112Phe and p.His41Tyr. The mutations were observed in one gastric and one hepatocellular carcinoma. Immunohistochemically, ATG5 protein was well expressed in normal stomach, colon, and liver epithelial cells, while it was lost in 21 (21%) of the gastric carcinomas, in 22 (23%) of the colorectal carcinomas, and in 5 (10%) of the hepatocellular carcinomas. Our data suggest that ATG5 gene could be altered in gastrointestinal cancers at the mutational or expressional level. Despite the low incidences of the alterations, our data led us to conclude that somatic mutation and loss of expression of ATG5 gene might play a role in gastrointestinal cancer pathogenesis by altering autophagic and apoptotic cell death.

Expressional and mutational analyses of ATG5 gene in prostate cancers

Abstract: Autophagy is an evolutionarily conserved mechanism that plays important roles in both cell death and cell survival. ATG5 is an essential constituent for autophagosome formation, which sequesters cytoplasmic materials before lysosomal delivery. Although both cell death and survival are important in cancer development, the role of autophagy in prostate cancer development remains unclear. The aim of this study was to see whether alterations of ATG5 protein expression and somatic mutations of the ATG5 gene are found in prostate cancers. In the present study, we analyzed ATG5 protein expression in 107 prostate carcinomas by immunohistochemistry; additionally, we assayed the presence of ATG5 somatic mutations in 45 prostate carcinomas by single-strand conformation polymorphism. Immunostaining of ATG5 in normal prostate cells was observed in 44.9% of the cases, whereas in prostate intraepithelial neoplasm (PIN) and prostate cancer cells, ATG5 was observed in 100% and 89.7% of the cases, respectively. Cytoplasmic expression of ATG5 that might be related to autophagy was seen in PIN (100%) and cancers (83.2%), but not in normal cells (0%). ATG5 expression was not associated with any of the pathologic characteristics, including size of the cancers, age, Gleason score, and stage. As for the ATG5 gene, we found no somatic mutations in the prostate cancers. In this study, we analyzed ATG5 expression and mutation in prostate cancers, and found that ATG5 expression was altered in prostate cancers. The expression of ATG5, especially in the cytoplasm, in the prostate cancers compared with normal prostate cells suggested that overexpression of this protein may be related to autophagy and might play a role in prostate tumorigenesis.

Detection of low-level EGFR T790M mutation in lung cancer tissues.

Abstract: Epidermal growth factor receptor (EGFR) gene mutation status is critical to predicting responsiveness to EGFR tyrosine kinase inhibitor (TKI) therapies in non-small cell lung cancer (NSCLC) patients. However, a vast majority of the patients experience recurrence of the cancers by a secondary mutation of EGFR (T790M). Earlier studies suggested evidence that subclones bearing EGFR T790M mutation pre-exist in NSCLCs even prior to the therapies. However, to date, the status of T790M mutation in primary NSCLC is largely known. In this study, we developed an assay using peptide nucleic acid (PNA)-clamping PCR for detection of low-level EGFR T790M mutation. We found that the assay showed the highest sensitivity (0.01% mutation detection) in the clamping condition. We analyzed 147 NSCLC tissues [70 adenocarcinomas (AD), 62 squamous cell carcinomas (SQ), 12 large cell carcinomas (LC), and three adenosquamous carcinomas] that had not been exposed to the TKI therapies, and found 12 (8.2%; 12/147) EGFR T790M mutation in eight AD (11.4%), three SQ (4.8%), and one LC (8.3%) by the PNA-clamping PCR. However, this mutation was not detected by conventional DNA sequencing. Our data indicate that EGFR T790M exists in pretreatment NSCLC at low levels irrespective of histologic types. This study provides a basis for developing an applicable protocol for detecting low-level EGFR T790M mutation in primary NSCLC, which might contribute to predicting recurrence of the tumor in response to the TKI therapies.

Expression of AIMP1, 2 and 3, the scaffolds for the multi-tRNA synthetase complex, is downregulated in gastric and colorectal cancer.

Abstract: Aminoacyl -tRNA synthetase -interacting multifunctional proteins (AIMPs) form a protein complex with aminoacyl -tRNA synthetases In addition to protein transla tion, AIMPs play a role in diverse biological processes Earlier studies suggested that AIMPs may act as tumor suppressors However, the expression status of the AIMP proteins in human cancer tissues is largely unknown In this study, we analyzed the expression of AIMP members (AIMP1, AIMP2 and AIMP3) in gastric cancer (GC) and colorectal cancer (CRC) tissues We analyzed the expression of these proteins in 100 GC and 103CRC tissues by immunohistochemistry using atissue microarray method Normal gastric and colon mucosa expressed AIMP1, AIMP2 and AIMP3 in nearly all of the cases (95-100%)However, the expression of AIMP1, AIMP2 and AIMP3 wassig nificantly decreased in the GC samples (60%, 52% and 70% of the cases , respectively) and in the CRC samples (66%, 53% and 81% of the cases , respectively) (P <001) Ex pression of AIMP1 , AIMP2 or AIMP3 was not associated with clinicopathological pa rameters including differentiation, depth of invasion and TNM stage The decreased expression of AIMP1, AIMP2 and AIMP3 in the GC and CRC tissues compared to the corresponding normal tissues suggested that downregulation of these proteins may be related to inactivation of the tumor suppressor functions of AIMP proteins and might play a role in the development of GC and CRC

Somatic mutations of caspase-2 gene in gastric and colorectal cancers

Abstract: There is mounting evidence that evasion of apoptosis is a hallmark of cancer. Caspase-2, which plays roles in both extrinsic and intrinsic apoptosis pathways, is considered a candidate tumor suppressor. The aim of this study was to explore the possibility that genetic alterations of caspase-2 gene are present in human cancers. In this study, we analyzed the entire coding sequences of human caspase-2 gene for the detection of somatic point mutations in 90 gastric carcinomas and 100 colorectal carcinomas by polymerase chain reaction (PCR)–single strand conformation polymorphism (SSCP). Of the cancers analyzed, two gastric cancers (2/90; 2.2%) and two colorectal cancers (2/100; 2.0%) harbored somatic missense mutations of caspase-2. The mutations consisted of p.V46M (at prodomain), p.S157L (at prodomain), p.R357K (at p13 subunit), and p.R397L (at p13 subunit). We expressed these tumor-derived mutants in 293 T cells and found that three of the mutants decreased cell death activity of caspase-2. Our data indicate that somatic mutation of caspase-2 is rare in gastric and colorectal carcinomas. However, functional data of the caspase-2 mutations also suggest that caspase-2 gene mutation might affect the pathogenesis of some gastric and colorectal cancers by inactivating cell death function of caspase-2.

Mutational and expressional analysis of a haploinsufficient tumor suppressor gene DOK2 in gastric and colorectal cancers.

Abstract: The docking proteins (DOKs) are tyrosinephosphorylated adaptor proteins that are substrates for several protein kinases (1). DOK proteins negatively regulate tyrosine kinase activation by feedback signaling mechanisms (1–3). Berger et al. (4) analyzed roles of Dok genes by knocking out one or more Dok genes heterozygously in mice and found that the mice without any of DOK1 or DOK2 or DOK3 gene developed lung adenocarcinomas. In human lung adenocarcinoma, DOK2 was a target of copy-number loss and mRNA downregulation (4). They also observed that DOK2 suppressed lung cancer cell proliferation in vitro and in vivo (4). These studies suggest that DOK2 may be a haploinsufficient tumor suppressor gene. However, currently, the data on DOK2 point mutation and expression are lacking in human cancers. In this study, we analyzed somatic DOK2 mutation in five types of common solid cancers, and DOK2 protein expression in gastric (GC) and colorectal cancers (CRC). For the mutation analysis of DOK2, 235 methacarn-fixed tissues from 47 CRC, 47 GC, 47 prostate carcinomas, 47 breast carcinomas and 47 hepatocellular carcinomas were randomly selected for the study. We analyzed protein expression of DOK2 in the 47 GC and 47 CRC by immunohistochemistry. Approval was obtained from the institutional review board of Catholic University of Korea, College of Medicine. For mutation detection of DOK2, genomic DNA each from tumor and corresponding normal cells was amplified with 10 primer pairs that encompass entire coding exons (exons 1–5) of DOK2. Radioisotope was incorporated into the PCR products for detection by autoradiogram. After single-strand conformation polymorphism (SSCP), migration of the PCR products on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. For immunohistochemistry, we used DAKO REAL EnVision System (DAKO, Glostrup, Denmark) with a rabbit polyclonal antibody against human DOK2 (dilution 1 ⁄100; Abcam, Cambridge, UK). Other procedures for mutation and immunohistochemistry were described in our previous reports (5, 6). We analyzed allelic status of DOK2 in the 47 GC and 47 CRC. The microsatellite markers and the intragenic polymorphism markers for the LOH analysis are shown in Table 1. Loss of heterozygosity (LOH) was defined as a significant reduction (>50%) in the intensity of one of the two alleles in tumor DNA compared with two alleles (informative cases) in normal DNA. Single-strand conformation polymorphism analysis showed aberrantly migrating bands in one CRC (1 ⁄47; 2.1%) compared to wild-type bands from its normal tissue. There was no aberrantly migrating band of DOK2 in the other cancer types. The mutation was a missense mutation in the exon 4 (c.518G>A), which would result in substitution of Arg by Gln at amino acid residue 173 (p.Arg173Gln). The SSCP and DNA sequencing patterns of the CRC with the mutation showed both wild-type and mutant signals, indicating that the mutation was a heterozygous one. The CRC with the

Somatic mutation of proapoptotic caspase-2 gene is rare in acute leukemias and common solid cancers.

Abstract: To the Editor: Apoptosis is essential for the development and homeostasis of organisms and is a critical component in cellular response to injury (1). Caspases play a central role in the activation and propagation of apoptosis signaling. Caspase-2 is cleaved into p18 and p13 subunits and activated during apoptosis (1). During the intrinsic apoptosis, caspase-2 contributes to release of cytochrome c and Smac from mitochondria and to translocation of Bax from cytoplasm to mitochondria (1). Also during the extrinsic apoptosis, caspase-2 primes cells for apoptosis by processing caspase-8 (1). There is evidence that activation of caspase-2 is related to TNFa-induced cell death (1). In addition, caspase-2 plays roles as a regulator of cell cycle and DNA damage response (2). Inactivation of apoptosis is considered a ‘cancer hallmark’. Previously, we reported that caspase genes were inactivated by somatic mutations in hematologic malignancies and solid cancers (3, 4). However, data on somatic mutation of caspase-2 in most human cancers are lacking. In this study, to explore the possibility that the alterations in caspase-2 gene might play a role in human cancer development, we analyzed cancer tissues from acute leukemias, breast cancers, lung cancers, and liver cancers for the detection of caspase-2 somatic mutations. For this, we analyzed somatic mutations of caspase-2 gene in fresh bone marrow aspirates of 94 leukemias (60 acute myelogenous leukemias, 20 B-cell acute lymphoblastic leukemias, and 14 T-cell acute lymphoblastic leukemias) by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. Also, we analyzed 82 breast invasive ducal cell carcinomas, 47 hepatocellular carcinomas, and 47 non-small-cell lung cancers. Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Genomic DNA each from tumor cells and normal cells (remission bone marrow cells in the cases of leukemias) from the same patients was amplified with 11 primer pairs covering entire coding region (11 exons) of human caspase-2 gene. Radioisotope ([P]dCTP) was incorporated into PCR products for detection by autoradiography. After SSCP, mobility shifts on the SSCP were determined by visual inspection. Other procedures of the PCR–SSCP and DNA sequencing were described in our previous studies (3, 4). SSCP and DNA sequencing analysis of aberrantly migrating bands on the SSCP led to the identification of one caspase-2 mutation in a breast carcinoma, but no caspase-2 mutation was detected in the other solid cancers and leukemias. However, the caspase-2 mutation detected was a silent mutation, which showed a C-to-T transition at a nucleotide [c.336C>T (p.Gly112Gly)]. To confirm the SSCP results, we repeated our experiments twice, including PCR and SSCP and to ensure the specificity of the results, and found that the data were consistent. Roles of apoptosis deregulation in cancer development led us to analyze caspase-2 somatic mutation in this study. For example, in El-Myc lymphoma model of mice, loss of caspase-2 alleles resulted in an accelerated tumorigenesis (5). However, we detected no caspase-2 mutation that might possibly inactivate caspase-2 tumor suppressor function in acute leukemias, breast cancers, lung cancers, and liver cancers. Our data indicate that somatic mutation of caspase-2 gene in acute leukemias, breast cancers, lung cancers, and liver cancers may be a very rare event. The data also suggest that the caspase-2 mutation may not be a direct target of inactivation in tumorigenesis of these malignancies. Our data suggest a possibility that apoptosis inactivation by caspase-2, if any, may be driven by other mechanisms besides somatic mutation.

Absence of paxillin gene mutation in lung cancer and other common solid cancers.

Abstract: Aims and background Mounting evidence indicates that deregulated cell adhesion is involved in the mechanisms of cancer pathogenesis. A recent study showed that the paxillin gene (PXN) encoding a focal adhesion protein was somatically mutated in lung cancers. The aim of this study was to confirm the presence of PXN mutations in lung cancers as well as in other common solid cancers. Methods We analyzed somatic PXN mutations in 45 lung, 45 gastric, 45 colorectal, 45 breast, 45 liver and 45 prostate cancers by polymerase chain reaction and single-strand conformation polymorphism assay. Results Neither lung nor other cancers were found to be associated with somatic mutations of PXN. Conclusions In contrast to the previous report, our study revealed that PXN mutation was absent in lung cancers and other common solid cancers, suggesting that PXN mutation may not play a principal role in solid cancer development.

A Frameshift Mutation of the Pro-Apoptotic VDAC1 Gene in Cancers with Microsatellite Instability.

Abstract: Dear editor, Apoptosis is a fundamental biochemical cell-death pathway that plays important roles in various physiological and pathological processes during fetal development and in adult tissues.1 In the intrinsic pathway, a number of death stimuli engage the apoptotic machinery by causing release of cytochrome c from mitochondria, which in turn induces recruitment of caspase-9 into an apoptosome.1 Activated caspase-9 initiates a proteolytic cascade by activating effector caspases that cleave key molecules to induce apoptosis. As inactivation of pro-apoptotic genes through genetic alterations have been reported in many cancers, they are considered tumor suppressor genes.2,3 Voltage-dependent anion channel 1 (VDAC1) is located in the outer mitochondrial membrane (OMM), and is involved in controlling metabolic cross-talk between mitochondria and the cytosol.4 In addition, VDAC1 plays a key role during intrinsic apoptosis. VDAC1 constitutes an OMM channel that mediates the release of a number of apoptogenic molecules, including cytochrome c, second mitochondria-derived activator of caspase (Smac), HTRA serine peptidase 2 (HTRA2), apoptosis-inducing factor (AIF), and endonuclease G (EndoG) from mitochondria to the cytosol by OMM permeabilization.4 VDAC1-deficient mitochondria from a mutant cell did not exhibit a Bax/Bak-induced loss in membrane potential of OMM and in cytochrome c release.5 Since VDAC1 plays an important role in apoptosis signaling, it could be hypothesized that VDAC1 gene is inactivated through somatic mutation in human cancers. To date, however, the data on the mutation status of VDAC1 in human cancers is lacking. By analyzing the public database (http://genome.cse.ucsc.edu/), we found an A8 repeat in exon 6 of VDAC1 gene (nucleotides 325-332) that had not been analyzed for mutations in cancers. Microsatellite instability (MSI) is defined by length alterations in repeated DNA sequences, and 10% to 30% of colorectal cancer (CRC) and gastric cancer (GC) are classified as MSI-positive cancers.6 To see whether the A8 repeat is mutated in GC and CRC with MSI, we performed polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP). We analyzed 30 GC with high-MSI (MSI-H) and 10 GC with stable MSI (MSS), 37 CRC with MSI-H, and 14 CRC with MSS according to the NCI criteria.7 The GC consisted of 21 diffuse-type and 19 intestinal-type adenocarcinomas by Lauren's classification. The TNM stages of the GC were 14 stage I, 16 stage II, 8 stage III, and 2 stage IV, while those of the CRC were 8 stage I, 18 stage II, 22 stage III, and 3 stage IV. Male to female ratios of gastric and colorectal cancers were 23:17 and 30:21, respectively. All of the specimens were surgically removed by gastrectomy and colectomy. Approval for this study was obtained from The Catholic University of Korea, College of Medicine's Institutional Review Board. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection as described previously.2,3 This microdissection method was proven to procure tumor cells with nearly being devoid of normal cell contamination.2,3 DNA from tumor and normal cells were amplified by PCR using a primer pair that could amplify the A8 (product size 128 bps). Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. After SSCP, mobility shifts compared to wild-type bands were analyzed by visual inspection. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. We repeated the experiments twice to ensure the specificity of the results. PCR-SSCP analysis identified aberrant bands in one of the GC with MSI-H (1/30, 3.3%) and one of the CRC with MSI-H (1/37, 2.7%), but not in those with MSS. DNA from normal tissue showed no shifts in SSCP, indicating the mutations had risen somatically. Direct DNA sequencing of the cancers with the aberrant bands led to identification of a recurrent VDAC1 mutation in the A8 repeat sequence (Fig. 1). This mutation, c.332delA, leads to a premature stop codon, resulting in truncation of the amino acid synthesis (p.Asn111MetfsX34). The SSCP and DNA sequencing patterns indicate that the mutation was heterozygous (Fig. 1). There was no significant difference of the mutations with respect to the clinicopathologic features of the GC and CRC (Fisher's exact test, p>0.05). Fig. 1 Frameshift mutations of VDAC1 in a gastric cancer and a colonic cancer with microsatellite instability. Direct DNA sequencing analyses of VDAC1 exon 6 from a gastric adenocarcinoma (A) and a colonic adenocarcinoma (B) with MSI-H show heterozygous A deletion ... Earlier studies showed that VDAC1 exists as oligomers of varying sizes in the cells.4,8 Mader et al.8 demonstrated that a VDAC1 mutant bound with wild-type VDAC1 displayed a dominant-negative effect in the intrinsic apoptosis pathway. The frameshift mutation of VDAC1 identified in this study would lead to a premature stop of amino acid synthesis in VDAC1 protein (p.Asn111MetfsX34) and would remove about 60% length of C-terminal VDAC1 protein. In this case, it is possible that the hemizygously mutated VDAC1 may bind with normal VDAC1 to construct a structurally abnormal oligomer. As a possible mechanism of apoptosis inactivation in human cancers, we analyzed VDAC1 mutation in GC and CRC with MSI, and we identified for the first time a frameshift mutation in human cancer. To our knowledge, there has been no report on functions of VDAC1 protein related to MSI. To address consequences of such down-regulations in cancer development (especially related to cancers with MSI-H), additional functional studies on the mutated gene should be performed. Although we identified somatic mutations of VDAC1 in both GC and CRC, the incidence was not high. To see whether other mechanisms of VDAC1 inactivation are present in these cancers, characterization of additional somatic alterations in VDAC1 should be undertaken. Antibody activity against frameshift neopeptides provides a promising tool for diagnostic application in MSI cancer patients. It might be clinically significant to analyze the immunogenic behavior of the VDAC1 neopeptides in future studies.

Rare somatic mutation and loss of expression of EMX2 gene in common solid cancers.

Abstract: Alteration of tumor suppressor genes plays important roles in cancer development (1). EMX2 is a human homolog ofDrosophila empty spiracles gene (ems) that is a homeodomain-containing transcription factor with important functions in the development (2). In addition, recent studies revealed that the EMX2 gene is involved in cancer development. EMX2 suppressed cell proliferation and invasive phenotypes in lung and endometrial cancers, and down-regulation of EMX2 expression was observed in these cancers (3, 4). Together, these data suggest that EMX2 may be a tumor suppressor gene and inactivation of EMX2 might contribute to tumorigenesis. Somatic mutation is the main mechanism by which functions of tumor suppressor gene are inactivated in cancers (1). Somatic mutation of EMX2 gene has been studied in a small number (N = 20) of endometrial cancers, and there were two EMX2 mutations in them (4). Although EMX2 is considered a candidate tumor suppressor gene, it remains unknown whether mutational and expressional status ofEMX2 gene is common in other cancers. In this study, we analyzed somatic mutations of EMX2 gene in methacarn-fixed tissues of 45 prostate carcinomas, 51 non-small cell lung cancers (25 squamous cell carcinomas and 26 adenocarcinomas), 43 gastric carcinomas, 44 colorectal carcinomas, and 43 breast carcinomas by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. In the tumors, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1 ⁄2 hypodermic needle affixed to a micromanipulator, as described previously (5). Genomic DNA each from tumor cells and normal cells from the same patients were amplified with three primer pairs covering entire coding region of human EMX2 gene. Radioisotope ([P]dCTP) was incorporated into PCR products for detection by autoradiography. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. With immunohistochemistry, we analyzed tissue expression of EMX2 protein in prostate (N = 107), gastric (N = 60) and colorectal (N = 60) cancers using DAKO REAL EnVision System (DAKO, Glostrup, Denmark) and a mouse monoclonal antibody against human EMX2 (Abgent, Taipei, Taiwan; dilution 1 ⁄200). Other procedures for mutation and expression were described in our previous reports (5, 6). On the SSCP autoradiograms, all the PCR products were clearly seen. However, none of the SSCP from prostate, gastric, colorectal, breast and lung carcinomas showed aberrantly migrating bands compared to wild-type bands from normal tissues, indicating that there was no evidence of EMX2 somatic mutation in the cancers analyzed. Normal epithelial cells from prostate, stomach, and colon were positive for EMX2 immunostaining. Positive immunostaining for EMX2 was observed in 103 (96%) of the 107 prostate cancers, 57 (95%) of the 60 gastric cancers and 59 (98%) of the 60 colorectal cancers (Fig. 1). Statistically, there was no significant difference of the EMX2 immunostaining between normal and the cancers (Fisher’s exact test, p > 0.05). The aim of this study was to determine whether somatic mutation of EMX2 is present and whether EMX2 expression is altered in the common solid cancers. In the present study, we detected no mutation in the cancers, indicating that somatic mutation of EMX2 is rare in prostate, gastric, colorectal, breast and lung carcinomas. In addition, we found that EMX2 expression was lost only in 2–5% of prostate, gastric, and colorectal cancers. These results indicate that EMX2 gene is not altered in the cancers either by somatic mutation or loss of expression. To our knowledge, this is the first report on EMX2 mutation and expression in human tissues besides lung and endometrial cancers. In contrast to the earlier data from lung and endometrial cancers that showed somatic mutation and loss of expression of EMX2, our data show that other common solid cancers rarely harbor such alterations, suggesting that EMX2 might not act as a tumor suppressor gene in the common solid cancers.

Mutational analysis of NCOA2 gene in prostate cancer and other common cancers.

Abstract: Dear Editor, One of the aims in cancer research is to identify mutated genes that are causally involved in cancer development. In an effort to discover cancerrelated genes in prostate cancer, Taylor et al. analyzed DNA copy number, mRNA expression, and somatic mutation in prostate cancers (1). They found that 6.2% of prostate cancers harbored amplification of locus-spanning NCOA2 gene and these were significantly correlated with elevated NCOA2 transcript levels. In addition, two somatic mutations of NCOA2 gene (p.G435S and p.S1042N) were found in prostate cancers (2 ⁄91; 2.2%). The NCOA2 gene encodes nuclear receptor coactivator 2, which aids in functions of nuclear hormone receptors that are conditional transcription factors important in various aspects of cell growth, development, and homeostasis by controlling expression of specific genes (2). Functionally, increased NCOA2 dosages amplified androgen receptor pathway transcriptional output in cancer, suggesting a mechanism for its role as an oncogene. Of note, somatic mutations of NCOA2 gene have been reported in malignant melanoma and lung cancer (p.G439D, p.P470S, and p.Q1032E) by COSMIC database (http:// www.sanger.ac.uk/genetics/CGP/cosmic). Also, a fusion protein of ETV6 (a transcriptional repressor) and NCOA2 has been detected in acute leukemia (3). Together, these data suggest that NCOA2 may be a candidate oncogene and that its alterations might contribute to development not only of prostate cancer, but also of other cancers. To further characterize NCOA2 mutation in human cancers, we analyzed somatic mutation of NCOA2 gene in frozen tissues of 187 prostate cancers by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) assay. All of the prostate cancers were primary adenocarcinomas that had been removed by prostatectomy, and were graded as Gleason score 6 (n = 38), Gleason score 7 (n = 95), Gleason score 8 (n = 39), and Gleason score 9 (n = 15). In addition, we analyzed methacarn-fixed tissues of 45 gastric carcinomas, 45 colorectal carcinomas, 45 hepatocellular carcinomas, 45 breast carcinomas, and 45 non-small cell lung cancers (23 squamous cell carcinomas and 22 adenocarcinomas), and fresh bone marrow aspirates of 47 leukemia (32 acute myelogenous and 15 acute lymphoblastic leukemias). Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. In solid tumors, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a 30G1 ⁄2 hypodermic needle affixed to a micromanipulator, as described previously (4). NCOA2 somatic mutations were previously detected in exons 11 and 15 (1). Thus, we analyzed these exons in the present study. Radioisotope ([P]dCTP) was incorporated into PCR products for detection by autoradiography. After SSCP, mobility shifts on the SSCP as potential mutations were determined by visual inspection. On the SSCP autoradiograms, all of the PCR products were clearly seen. However, none of the SSCP from prostate cancers and other cancers revealed aberrantly migrating bands compared with wild-type bands from normal tissues, indicating that there was no evidence of NCOA2 mutation in the cancers analyzed (Fig. 1). To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR, and SSCP to ensure specificity of the results, and found that the data were consistent.

Mutational analysis of mononucleotide repeats in HDAC4, 5, 6, 7, 9 and 11 genes in gastric and colorectal carcinomas with microsatellite instability

Abstract: To the Editor,Histone acetylation in conjunction with deacetylation serves as a crucial modulation process of chromatin structure and is one of the main epigenetic mechanisms for gene regulation [1...

Copy Number Alterations of BCAS1 in Squamous Cell Carcinomas

Abstract: Cancer is a genetic disease. The beginning of cancer stems from genetic changes that are critical in the control of cell cy­ cles. For decades, researchers have investigated genes that pro­ mote or suppress the development of cancers. Alterations of many genes in cancers were reported and their functions in car­ cinogenesis were elucidated. Among the alterations, studies on copy number changes have turned out to be important in un­ derstanding pathogenesis and providing better patient care. Sometimes, copy number alterations cause changes in gene ac­ tivity. As a result, tumor suppressor genes can be inactivated and an oncogene can be activated. These changes may lead to the disruption of the normal cell cycle and a malignant tumor may develop as a result. Knowing the copy number of a gene in certain tumor types can be valuable in terms of diagnosis, treatment and predicting prognosis of a cancer patient. For example, a 1p deletion in neu­ roblastoma correlates with an unresectable and metastatic dis­ ease; N-MYC amplification of neuroblastoma is related to poor prognosis; and human epidermal growth factor receptor 2 (HER2) amplification of breast cancer correlates with an aggressive phenotype. Among the many copy number alterations reported, chromo­ some 20q13 is the commonly amplified region in breast can­ cers. Several genes, including amplified in breast cancer 1 (AIB1), breast tumor amplified kinase (BTAK), breast carcinoma amplified sequence 1 (BCAS1), CAS, and transcription factor AP­2 gamma (TFAP2C), are located in this region and report­ ed to be associated with various phenotypes. BCAS1 is one of genes located in the region and is considered to be an oncogene candidate. BCAS1 is amplified in several breast cancer cell lines and overexpressed in some of the cell lines with amplification. However, how BCAS1 promotes breast cancer development is not known. Inducing BCAS1 expression in NIH3T3 cell lines in vitro failed to induce cellular transformation. This is why BCAS1 is not considered a prototypical oncogene. Besides breast cancers, amplification of the 20q13 region is Copy Number Alterations of BCAS1 in Squamous Cell Carcinomas

Mutational and Expressional Analysis of DOK2 Gene in Non-small Cell Lung Cancers

Abstract: Purpose: Mounting evidence indicates that perturbation of tyrosine phosphorylation is implicated in the development of many human diseases, including cancers. Docking proteins (DOKs) are tyrosine-phosphorylated proteins that negatively regulate tyrosine kinase signaling and they are considered to be tumor suppressors. Deletion and the altered expression of the DOK2 gene have been studied in leukemias and lung cancers. However, the somatic mutation status of the DOK2 gene has not been studied in lung cancers. The aim of this study was to see whether alterations of DOK2 protein expression and somatic mutation of the DOK2 gene are present in human non-small cell lung cancer (NSCLC). Materials and Methods: We analyzed DOK2 somatic mutation in 45 NSCLCs (23 adenocarcinomas (AD) and 22 squamous cell carcinomas (SCC) by single-strand conformation polymorphism (SSCP). We examined the DOK2 protein expression in 45 NSCLCs by immunohistochemistry. Results: SSCP analysis revealed no evidence of somatic mutation in the DNA sequences encoding the DOK2 gene in the 45 NSCLCs. Among the informative cases, 27% and 21% of the ADs and SCCs showed allelic loss in the DOK2 locus, respectively. On the immunohistochemistry, DOK2 protein was expressed in the normal bronchial epithelial cells, while it was lost in 10 (22%) of the NSCLCs. Conclusion: Our data indicates that DOK2 is altered in NSCLC at the expressional level, but not at the mutational level. The data also suggests that loss of the expression of DOK2 might play roles in NSCLC development by possibly altering tyrosine kinase signaling. (J Lung Cancer 2011;10(1):26 �� 31)