Showing papers by "Nam Jin Yoo published in 2009"

Mutational analysis of IDH1 codon 132 in glioblastomas and other common cancers

Abstract: Missense somatic mutations in IDH1 gene affecting codon 132 have recently been reported in glioblastoma multiforme (GBM) and other gliomas. The recurrent nature of the IDH1 mutations in the same amino acid strongly suggests that the mutations may play important roles in the pathogenesis of glial tumors. The aim of this study was to see whether the IDH1 codon 132 mutations occur in other human cancers besides glial tumors. We also attempted to confirm the occurrence of the IDH1 mutations in GBM of Korean patients. We have analyzed 1,186 cancer tissues from various origins, including carcinomas from breast, colon, lung, stomach, esophagus, liver, prostate, urinary bladder, ovary, uterine cervix, skin and kidney, and malignant mesotheliomas, primary GBM, malignant meningiomas, multiple myelomas and acute leukemias by single-strand conformation polymorphism analysis. We found four IDH1 codon 132 mutations in the GBM (4/25; 16.0%), two in the prostate carcinomas (2/75; 2.7%) and one in the B-acute lymphoblastic leukemias (B-ALL) (1/60; 1.7%), but none in other cancers. The IDH1 mutations consisted of five p.R132H and two p.R132C mutations. The data indicate that IDH1 codon 132 mutations occur not only in GBM, but also in prostate cancers and B-ALL. This study suggests that despite the infrequent incidence of the IDH1 mutations in prostate cancers and B-ALL, mutated IDH1 could be therapeutically targeted in these cancers and in glial tumors with the IDH1 mutations.

Frameshift mutations of autophagy‐related genes ATG2B, ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability

Abstract: Mounting evidence indicates that alterations of autophagy processes are directly involved in the development of many human diseases, including cancers. Autophagy-related gene (ATG) products are main players in the autophagy process. In humans there are 16 known ATG genes, of which four (ATG2B, ATG5, ATG9B and ATG12) have mononucleotide repeats with seven or more nucleotides. Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). It is not known whether ATG genes with mononucleotide repeats are altered by frameshift mutations in gastric and colorectal carcinomas with MSI. For this, we analysed the mononecleotide repeats in ATG2B, ATG5, ATG9B and ATG12 in 32 gastric carcinomas with high MSI (MSI-H), 13 gastric carcinomas with low MSI (MSI-L), 43 colorectal carcinomas with MSI-H and 15 colorectal carcinomas with MSI-L by a single-strand conformation polymorphism (SSCP) analysis. We found ATG2B, ATG5, ATG9B and ATG12 mutations in 10, 2, 13 and 0 cancers, respectively. The mutations were detected in MSI-H cancers but not in MSI-L cancers. Gastric and colorectal cancers with MSI-H harboured one or more ATG mutations in 28.1% and 27.9%, respectively. Our data indicate that frameshift mutations in ATG genes with mononucleotide repeats are common in gastric and colorectal carcinomas with MSI-H, and suggest that these mutations may contribute to cancer development by deregulating the autophagy process.

Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers

Abstract: Activation of nuclear factor-kappa B (NF-kappaB) signaling is considered an important mechanism in the development of prostate cancers. A recent study revealed that IkappaB kinase epsilon (IKKepsilon), an activator of NF-kappaB, was overexpressed in breast cancers and acted as an oncogene. Expression of NF-kappaB members has been reported in prostate cancer tissues, but expression of IKKepsilon has not yet been studied in prostate cancers. In this study, we attempted to explore as to whether expressions of IKKepsilon and NF-kappaB members p50/105, p52/p100 and RelA are altered in prostate cancers. We analyzed the expression of IKKepsilon, p50/105, p52/p100 and RelA in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. In the TMA, IKKepsilon is expressed in basal cells, but not in alveolar cells in normal prostate glands. IKKepsilon is expressed in 60.0% of prostate intraepithelial neoplasm (PIN) and 70.1% of the prostate cancers in the cytoplasm. Nuclear immunostainings of NF-kappaB members p50/105, p52/p100 and RelA, which are considered activation of NF-kappaB signaling, were observed respectively in 28.0%, 18.7% and 37.4% of the cancers. Nuclear staining was detected neither in normal alveolar cells nor in PIN. However, none of the expression of p50/105 nor p52/p100 nor RelA nor IKKepsilon was associated with pathologic characteristics, including size of the cancers, age, Gleason score and stage. The increased cytoplasmic expression of IKKepsilon as well as the increased nuclear expressions of p50/105, p52/p100 and RelA in the prostate cancers compared to normal alveolar cells suggested that overexpression of these proteins may be related to activation of the NF-kappaB pathway and might play a role in tumorigenesis of prostate cancers.

Mutational Analysis of WTX Gene in Wnt/ β-Catenin Pathway in Gastric, Colorectal, and Hepatocellular Carcinomas

Abstract: A recent study of Wilms’ tumors discovered a new X chromosome gene, Wilms’ tumor gene on the X chromosome (WTX), which was found to harbor small deletions and point mutations. WTX protein negatively regulates Wnt/ β-catenin signaling, and is considered a tumor-suppressor gene. One of the questions about the WTX gene is whether the genetic alterations of the WTX gene are specific to only Wilms’ tumors. To see whether somatic point mutations of WTX occur in other malignancies, we analyzed the WTX gene for the detection of mutations in 141 cancer tissues by a single-strand conformation polymorphism assay. The cancer tissues consisted of 47 gastric adenocarcinomas, 47 colorectal adenocarcinomas, and 47 hepatocellular carcinomas. Overall, we detected one WTX mutation in the colorectal carcinomas (1/47; 2.1%), but there was no WTX mutation in other cancers analyzed. The detected mutation was a missense mutation (c. 1117G > A (p.Ala373Thr)). Although the WTX mutation is common in Wilms’ tumors, our data indicate that it is rare in colorectal, gastric, and hepatocellular carcinomas. The data also suggest that deregulation of Wnt/ β-catenin signaling by WTX gene mutation may be a rare event in the pathogenesis of colorectal, gastric, and hepatocellular carcinomas.

Mutational analysis of hypoxia-related genes HIF1alpha and CUL2 in common human cancers.

Abstract: Biochemistry, College of Medicine,The Catholic University of Korea, Seoul, KoreaPark SW, Chung NG, Hur SY, Kim HS, Yoo NJ, Lee SH. Mutational analysis of hypoxia-relatedgenes HIF1a and CUL2 in common human cancers. APMIS 2009; 117: 880–5.Hypoxia is a general feature of solid cancer tissues. Hypoxia upregulates hypoxia-inducible factor 1a(HIF1a) that transactivates downstream genes and contributes to cancer pathogenesis. HIF1a is upreg-ulated not only by hypoxia but also by genetic alterations in HIF1a-related genes, including VHL.Cullin 2 (CUL2) interacts with the trimeric VHL-elongin B-elongin C complex and plays an essentialrole in the ubiquitinated degradation of HIF1a. The aim of this study was to explore whether HIF1aand CUL2 genes are somatically mutated, and contribute to HIF1a activation in common human can-cers. For this, we have analyzed the coding region of oxygen-dependent degradation domain of HIF1ain 47 colon, 47 gastric, 47 breast, 47 lung, and 47 hepatocellular carcinomas, and 47 acute leukemias bya single-strand conformation polymorphism assay. In addition, we analyzed mononucleotide repeatsequences (A8) in CUL2 in 55 colorectal and 45 gastric carcinomas with microsatellite instability(MSI). We found one HIF1a mutation (p.Ala593Pro) in the hepatocellular carcinomas (1⁄47; 2.1%),but none in other cancers. We found two CUL2 frameshift mutations in colon cancers (p.Asn292MetfsX20), which were exclusively detected in high MSI cancers (4.9%; 2⁄41). Our data indicate thatsomatic mutation of HIF1a is rare in common cancers, and somatic mutation of CUL2 occurs in a frac-tion of colorectal cancers (colorectal cancers with high MSI). The data suggest that neither HIF1a norCUL2 mutation may play a central role in HIF1a activation in gastric, colorectal, breast, lung andhepatocellular carcinomas, and acute leukemias.Key words: HIF1a; CUL2; hypoxia; mutation; cancer; microsatellite instability.Sug Hyung Lee, Department of Pathology, College of Medicine, The Catholic University of Korea,505 Banpo-dong, Socho-gu, Seoul 137 701, Korea. e-mail: suhulee@catholic.ac.kr*

Mutational analysis of TTK gene in gastric and colorectal cancers with microsatellite instability.

Abstract: PURPOSE: The TTK gene plays a crucial role in regulation of the mitotic checkpoint. The TTK gene has an A9 mononucleotide repeat in the coding sequences, which harbors mutations in gastric (GC) and colorectal cancers (CRC) with microsatellite instability (MSI). However, there are three more repeats (the A7s) in the coding sequences that have not been analyzed. The aim of this study was to explore whether the three A7s as well as the A9 are altered in GC and CRC, and to find any association of TTK mutation with clinocopathologic characteristics of GC and CRC. MATERIALS AND METHODS: We analyzed exon 5 (A7 and A7) and exon 22 (A9 and A7) which have repeat sequences in 30 GC with high MSI (MSI-H), 15 GC with low MSI (MSI-L), 35 CRC with MSI-H, and 15 CRC with MSI-L, by single-strand conformation polymorphism (SSCP) and DNA sequencing assays. RESULTS: Overall, we detected 23 frameshift mutations in the repeat sequences of TTK in the GC with MSI-H (11/30; 36.7%) and the CRC with MSI-H (12/35; 34.3%), but not in the cancers with MSI-L. The mutations were observed in both A9 and A7 of exon 22, but in neither of the two A7s of exon 5. The mutations consisted of c.2560delA, c.2560dupA, c.2571delA and c.[2560delA(+)2571delA]. All of the mutations were frameshift mutations and would result in premature stops of TTK protein synthesis. There was no significant difference in clinopathologic parameters of the cancers with the mutations. CONCLUSION: Our data indicate that frameshift mutations of TTK are common in both GC and CRC with MSI-H, and that the mutations occur not only in the A9 repeat but also in the A7 repeat. The data suggest that frameshift mutations of TTK might alter cell cycle control in the affected cells and contribute to pathogenesis of cancers with MSI-H.

Absence of IDH2 codon 172 mutation in common human cancers.

Abstract: Sang Wook Park, Nak Gyun Chung, Ji Youn Han, Hyeon Seok Eom, Ji Youl Lee, Nam Jin Yoo and Sug Hyung Lee* Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea Department of Pediatrics, College of Medicine, The Catholic University of Korea, Seoul, Korea Center for Lung Cancer, National Cancer Center of Korea, Goyang, Korea Hematology-Oncology Clinic, National Cancer Center of Korea, Goyang, Korea Department of Urology, College of Medicine, The Catholic University of Korea, Seoul, Korea

Immunohistochemical analysis of Fas and FLIP in prostate cancers.

Abstract: Fas-mediated apoptosis is considered a principal pathway for apoptosis induction in normal and cancer cells. Expression of Fas has been reported in prostate tissues several times, but the data were not consistent. Expression of FLICE-like inhibitory protein (FLIP), an inhibitor of Fas-mediated apoptosis, has not been studied by immunohistochemistry in prostate tissues. The aim of this study is to explore whether alterations of Fas and FLIP expression occur in prostate cancer tissues. We analyzed the expression of Fas and FLIP in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray approach. Normal glandular cells of the prostates strongly expressed both Fas and FLIP proteins. Prostate intraepithelial neoplasm also showed a strong Fas immunoreactivity. Fas expression was strongly positive in 60 cancers (56.1%), but the remaining 47 cancers showed no (6.5%) or markedly decreased (37.4%) Fas immunostaining compared with the normal glandular cells of the same patients. By contrast, FLIP expression was strong in most (103/107; 96.3%) of the cancers, and only four cancers (3.7%) showed decreased immunoreactivities compared with the normal cells. The decreased expression of Fas was not associated with pathologic characteristics, including FLIP expression, size of the cancers, age, Gleason score and stage. The decreased expression of Fas in a large fraction of prostate cancers compared with their normal cells suggested that loss of Fas expression might play a role in tumorigenesis in some prostate cancers possibly by inhibiting apoptosis mediated by Fas.

Absence of oncogenic AKT1 E17K mutation in prostate, esophageal, laryngeal and urothelial carcinomas, hepatoblastomas, gastrointestinal stromal tumors and malignant meningiomas

Abstract: AKT1, which contains a pleckstrin homology (PH) domain, is a serine/threonine protein kinase. Mounting evidence indicates that activation of AKT1 is important in cancer development. An activating A...

Somatic mutation of PIK3R1 gene is rare in common human cancers

Abstract: The PI3K signaling pathway regulates many normal cellular processes, and perturbation of the PI3K signaling results in deregulated kinase activity and malignant transformation [1]. Phosphatidylinos...

Somatic mutation of GNAQ gene is rare in common solid cancers and leukemias.

Abstract: pathologic stage of IA. Her neuropsychiatric symptoms failed to abate despite surgery and a course of intravenous immunoglobulins. Fourteen days postoperatively, she was started on adjuvant chemotherapy with a standard chemotherapeutic regime of BEP, receiving a total of three cycles. Her dystonia became less prominent rapidly after initiation of her first cycle, but it was only after 20 days after commencement of chemotherapy that she regained orientation and was able to answer questions in a relevant and appropriate manner. She returned to work four months after completion of chemotherapy. These two cases highlight the role of chemotherapy as an adjunct to surgery in a multimodality approach to paraneoplastic encephalitis associated with teratoma. In the first patient, the temporal association is definitive where neuropsychiatric symptoms only developed after the teratomas were resected. These symptoms resolved completely following chemotherapy. Given that tumour foci were identified on the omentum and in the peritoneal fluid, it is conceivable there may have been residual disease after surgery resulting in a delayed onset of encephalitis. This would further support the role of chemotherapy as an adjunct to surgery. In the second patient, since both surgery and intravenous immunoglobulin was administered prior to chemotherapy, one cannot exclude the possibility that her improvement may have represented a delayed effect of treatment. However, a strong temporal association between initiation of chemotherapy and symptom improvement was noted. In a review of patients reported to have paraneoplastic encephalitis associated with immature ovarian teratomas in the English literature (Table I), only three of the ten patients received chemotherapy, with a fourth receiving cyclophosphamide only [1� 6]. The grade and stage of the disease as well as the details of treatment

Analysis of NOTCH1 extracellular juxtamembrane expansion mutations in acute leukemias and multiple myelomas

Abstract: NOTCH signaling plays crucial roles in embryonic development (1). Upon activation, NOTCH proteins (NOTCH1-4) translocate to the nuclei and regulate transcription of target genes. Several lines of evidence indicate that NOTCH proteins are implicated in tumorigenesis of haematopoietic malignancies (2). Several studies documented a high incidence of gain-of-function mutations of NOTCH1 in T-cell acute lymphoblastic leukemia (ALL) (3). The NOTCH1mutations were found in N-terminal heterodimerization domain, C-terminal heterodimerization domain and PEST domain. The NOTCH1 mutations in these domains activate NOTCH signaling (3). Recently, Sulis et al. (4) reported novelNOTCH1mutations in T-ALL. The mutations were ‘JuxtaMembrane Expansion (JME)’ alleles that consist of internal duplication insertions in the vicinity of exon 28 of NOTCH1. They detected the mutations in seven of 210 primary T-ALLs as well as in Jurkat T-ALL. Functionally, the JME mutants activate NOTCH signaling (4). To see whether the JMEmutations occur in other haematopoietic malignancies as well, we analyzed the mutations in 217 malignancies (106 acute myelogenous leukemias (AML), 80 ALLs (60 B-ALL and 20 T-ALL) and 31 multiple myelomas (MM)) from Koreans. Genomic DNA was isolated, and analyzed for the JME mutations by polymerase chain reaction (PCR) with two primer pairs. Primer A was the same primer pair that had been used by Sulis et al. (4). Primer B was designed by ourselves. We analyzed the mutations by both conventional and slowdown PCR. The PCR products were displayed on 2% agarose gels and analyzed by DNA sequencing. PCR products from the 217 haematopoietic malignancies by primer A and B were observed at 346 and 217 bps, respectively. These sizes were the expected size of the wild-type sequences. There were no additional bands besides them. By direct DNA sequencing, we found that the sequences were the same with the wild-type sequences. We changed PCR conditions, including concentration of DNA, annealing temperatures and PCR cycles, but could not detect any additional PCR band. By slowdown PCR, we did not find any mutant bands for the mutations, either. Because the NOTCH1 JME mutations had been detected in T-ALLs, we expected to detect the JME mutations in Korean T-ALLs. However, we detected no JME mutation in T-ALLs in this study. The earlier study detected the JME mutations in seven from 210 T-ALLs (3.3%), and our study detected none from the 14 T-ALLs (0.0%). However, there was no significant difference of the mutation incidence between the studies (Fisher’s exact test, p=0.632), suggesting that a racial difference between the two does not exist. Moreover, we detected no JME mutation in other haematopoietic (AML, B-ALL and MM) malignancies nor 463 solid cancers (unpublished data). This observation indicates that the NOTCH1 JME mutations may be rare in common human malignancies. In summary, we identified in this study that the NOTCH1 JME mutations are rare in common haematopoietic cancers, suggesting that the JME mutations may not play a crucial role in the development of haematopoietic cancers. Because the most impressive examples of recent cancer therapies target activated proteins by mutations, cancer researches have focused on finding activating mutations as promising molecular targets for cancer treatment (5). The discovery of the NOTCH1 JME mutations in cancers raised a possibility to treat cancers by targeting the mutated NOTCH1. However, our data in this study suggest that the possibility of the NOTCH1 JME mutations as therapeutic targets is low.

Somatic mutation of the death domain of TNF-R1 is rare in common cancers.

Abstract: Tumor necrosis factor (TNF)-a regulates various biological processes, including immune reaction, inflammation and apoptosis. Of two TNF receptors (TNF-R1 and R2), TNF-R1 is primarily responsible for the actions by TNF-a (1). TNF receptor family consists of more than 20 proteins, including TNF-R1, Fas, DR4 and DR5 (1). While Fas, DR4 and DR5 have main functions in cell death induction, TNF-R1 is involved in both NF-kB activation and apoptosis induction (1). Activation of TNF receptor family members is a major pathway for the induction of apoptosis in cells and tissues (1). Failure of apoptosis allows survival of transformed cells that are prone to undergo genetic damages and plays an important role in development of cancers. Somatic mutations of genes encoding TNF receptor family members, including Fas and Death Receptor 5 (DR5), have been reported in many human cancers, and many of the mutations were proven to inactivate cell death (2–6). Of note, most of the mutations were discovered in death domains (DD) of Fas andDR5. The DD of TNF receptor family members is an evolutionally conserved intracellular domain that transmits death signals from the ligands (1). Although the DD is crucial in apoptosis signaling and somatic mutations in the DD of Fas and DR5 are well known, currently the data on somatic mutations of TNF-R1 DD is lacking. In order to determine whether the DD of TNF-R1 is mutated in human cancers as well, we analyzed the exon 10 of TNF-R1gene by polymerase chain reaction (PCR) with three primer pairs and subsequent single-strand conformation polymorphism (SSCP) assay. The exon 10 contains DNA sequences that encode the DD of TNF-R1. The cancers analyzed consisted of methacarn-fixed tissues of 47 gastric adenocarcinomas, 47 colorectal adenocarcinomas, 47 breast invasive ductal carcinomas, 47 hepatocellular carcinomas and 47 non-small cell lung cancers, and non-fixed fresh tissues of 47 acute myelogenous leukemias (subgroup details available on request). Malignant cells and normal cells of the solid cancers were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle by microdissection (2). Genomic DNA was extracted by a modified single-step method using proteinase K. Genomic DNA each from tumor and normal cells were amplified by PCR, and the PCR products were subsequently displayed on SSCP gels. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. Other procedures of the PCR-SSCP were described in our previous studies (2). On the SSCP, all of the PCR products from the cancers were clearly seen, but they did not reveal any aberrantly migrating band compared with wild-type bands from the normal tissues, indicating there was no evidence of somatic mutations in the DD of TNF-R1 in the cancers. We directly sequenced the PCR products of normal and cancers in five cases, but there was no sequence alteration. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR and SSCP to ensure the specificity of the results, and found that the data were consistent. As a possible mechanism of apoptosis inactivation in common cancers, we analyzed TNF-R1 DD mutation in gastric, colorectal, breast, hepatocellular and lung carcinomas, and acute myelogenous leukemias. However, we detected no TNF-R1 mutation in the cancer tissues. This is in contrast to the high incidences of Fas DD mutations in lung cancers (7.7%), bladder cancers (23.3%) and multiple myelomas (10.4%), and DR5 DD mutations in lung cancers (8.7%) and breast cancers (10.5%) (2–6). Our data indicate that apoptosis resistance frequently observed in gastric, colorectal, lung, hepatocellular and breast cancers, and acute myelogenous leukemias may not be a result of TNF-R1 DD mutation in the cancer cells. The reason why we could not find any TNF-R1 DD mutation might be that the primary effect of the TNF-R1 receptor is activation of survival pathways through NF-kB activation (1), and not activation of apoptosis pathways. Apoptosis of cancer cells can be delayed or blocked by several APMIS 117: 547–548