Ji Youn Han

Alterations of Fas (Apo-1/CD95) gene in non-small cell lung cancer.

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many non-lymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas-resistance, may be involved in the pathogenesis of non-lymphoid malignancies as well. In this study, we have analysed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 65 human non-small cell lung cancers by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing. Overall, five tumors (7.7%) were found to have the Fas mutations, which were all missense mutations. Four of the five mutations identified were located in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal and one mutation was located in the transmembrane domain. This is the first report on the Fas gene mutations in non-lymphoid malignancies, and the data presented here suggests that alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human lung cancers.

Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin

Nuclear factor erythroid‐related factor 2 (NRF2) encodes a transcription factor that induces expression of cytoprotective proteins upon oxidative stress and oncogenic NRF2 mutations have been found in lung and head/neck cancers that inactivate KEAP1‐mediated degradation of NRF2. The aim of this study was to catalogue NRF2 mutations in other human cancers. For this, we analysed 1145 cancer tissues from carcinomas from oesophagus, skin, uterine cervix, lung, larynx, breast, colon, stomach, liver, prostate, urinary bladder, ovary, uterine cervix, and kidney, and meningiomas, multiple myelomas, and acute leukaemias by single‐strand conformation polymorphism (SSCP) assay. We detected NRF2 mutations in oesophagus (8/70; 11.4%), skin (1/17; 6.3%), lung (10/125; 8.0%), and larynx (3/23; 13.0%) cancers. Of note, all of the 22 mutations except one were found in squamous cell carcinomas (SCCs) (95.5%). The mutations were observed within or near DLG and ETGE motifs that are important in NRF2 and KEAP1 interaction. All of the oesophageal SCCs and skin SCCs with the NRF2 mutations showed increased NRF2 expression in the nuclei. However, none of the SCCs from oesophagus and skin harboured KEAP1 mutation. Our study demonstrated here that NRF2 mutation occurs not only in lung and head/neck cancers, but also in oesophageal and skin cancers. Our data suggest that the NRF2 mutation plays a role in the development of SCC and is a feature of SCC. Copyright

Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes

Detection of somatic mutations in clinical cancer specimens is often hampered by excess wild-type DNA. The aim of this study was to develop a simple and economical protocol without using fluorescent probes to detect low-level mutations. In this study, we combined peptide nucleic acid (PNA)-clamping PCR with asymmetric primers and a melting curve analysis using an unlabeled detection probe. PNA-clamping PCR, which suppressed amplification of the wild-type allele, was more sensitive for KRAS codon 12 mutation detection than nonclamping PCR in 5 different mutant cell lines. Three detection probes were tested (a perfectly matched antisense, a mismatched antisense, and a mismatched sense), and the mismatched sense detection probe showed the highest sensitivity (0.1% mutant detection) under clamping conditions. With this probe, we were able to detect not only the perfectly matched KRAS mutation, but also 4 other mismatched mutations of KRAS. We then applied this protocol to 10 human colon cancer tissues with KRAS codon 12 mutations, successfully detecting the mutations in all of them. Our data indicate that the combination of perfectly matched antisense PNA and a mismatched sense detection probe can detect KRAS mutations with a high sensitivity in both cell lines and human tissues. Moreover, this study might prove an easily applicable protocol for the detection of low-level mutations in other cancer genes.

Absence of IDH2 codon 172 mutation in common human cancers

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

Mutational analysis of CASP10 gene in colon, breast, lung and hepatocellular carcinomas

Aims: Evasion of apoptosis is a feature of cancer cells. As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro‐apoptotic genes have been reported in many cancers. Caspase‐10 is an initiation‐phase caspase, and somatic mutation of CASP10 that encodes caspase‐10 has been found in non‐Hodgkins lymphoma and gastric carcinoma. Methods: The aim of this study was to explore whether CASP10 gene is somatically mutated in colon, breast, lung, and hepatocellular carcinomas. We analysed the entire coding region and all splice sites of CASP10 in 47 colon, 47 breast, 47 lung, and 47 hepatocellular carcinomas by a single‐strand conformation polymorphism (SSCP) assay. Results: We found two CASP10 mutations in the colon cancers (2/47; 4.3%), but none in breast, lung or hepatocellular carcinomas. One mutation [c.41A > C (p.Lys14Thr)] was a missense mutation, while the other was a substitution mutation in a splice site (c.684 + 4G > A). The colon cancer with the CASP10 missense mutation harboured additional CASP gene mutations (CASP3, 7 and 8). Conclusion: Our data indicate that somatic mutation of CASP10 is rare in colon, breast, lung, and hepatocellular carcinomas. However, the data also suggest that CASP10 mutation might contribute to the pathogenesis of some colon carcinomas together with other CASP gene mutations.

Somatic mutations of EGFR, ERBB2, ERBB3 and ERBB4 in juxtamembrane activating domains are rare in non-small cell lung cancers

Receptor tyrosine kinases (RTK) regulate diverse cellular functions, including cell survival and growth. Epidermal growth factor receptor (EGFR) is a prototypical RTK, and the EGFR family consists of EGFR (ERBB1 ⁄ HER1), ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4) (1). The ERBB genes are mammalian equivalents of viral oncogenes, and are deregulated in many cancers (1). Lung adenocarcinomas harbor EGFR mutations, which predict clinical responses to the EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) (2). ERBB2 amplification is present in about 25% of breast cancers, and trastuzumab (Herceptin) against the ERBB2 is effective in treating breast cancers with ERBB2 amplification (3). Kinase domain mutations of ERBB2 and ERBB4 mutations have also been detected in several types of human cancers (4, 5). Intracellular kinase domain is crucial in activation of ERBB proteins (1). However, in addition to the kinase domain, alterations in other domains such as deletions of extracellular domain of EGFR are known to activate ERBB signaling (1). Recently, Red Brewer et al. (6) discovered that a region in the juxtamembrane domain (JMD) of EGFR functioned as an activating domain of EGFR. The JMD of EGFR is located between the transmembrane domain and the kinase domain. The C-terminal region of the EGFR JMD (amino acid residues 688– 706) contributes to EGFR activation, whereas the N-terminal region of the JMD (amino acid residues 669–687) does not (6). The active JMD region contributes to EGFR dimerization and stabilizes the dimers (6). They also found that point mutations in the JMD (V689A and L703A) in non-small cell lung cancers (NSCLC) were activating mutations. The V689A-mutated EGFR promotes cellular transformation and tumorigenesis in mice (6). These data indicate that mutation of the EGFR JMD may play an important role in NSCLC development. Like EGFR, ERBB2, ERBB3 and ERBB4 possess a JMD and most of the amino acids in each JMD region are conserved (6). It could be hypothesized that the active regions in JMD of ERBB2, ERBB3 and ERBB4 could also be activated by somatic mutations in NSCLC. For this, we have analyzed 200 surgically resected NSCLC tissues, including 120 adenocarcinomas and 80 squamous cell carcinomas from Korean patients. Male to female ratios of the adenocarcinoma patients and the squamous cell carcinoma patients were 58:62 and 62:18, respectively. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides using a hypodermic needle affixed to a micromanipulator. DNA extraction was performed by a modified single-step DNA extraction method. Genomic DNA each from tumor cells and corresponding normal cells was amplified by polymerase chain reaction (PCR) with four primer pairs covering the C-terminal active JMD region of EGFR, ERBB2, ERBB3 and ERBB4 in their exon 18 (Table 1). Radioisotope was incorporated into the PCR products for detection by autoradiogram. After singlestrand 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. On the SSCP autoradiograms, all of the PCR products from the cancers were clearly seen. However, the SSCPs from them did not reveal any aberrantly migrating band compared with wild-type bands from the normal tissues, indicating that there was no evidence of the somatic mutation of EGFR or ERBB2 or ERBB3 or ERBB4 in the NSCLCs. 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. Most of the EGFR mutations detected in NSCLC were located in either exon 19 or exon 21 in the kinase domain. However, several

Somatic mutation of PIK3R1 gene is rare in common human cancers

1Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea, 2Hematology-Oncology Clinic, National Cancer Center of Korea, Goyang, Korea, 3Center for Lung Cancer, National Cancer Center of Korea, Goyang, Korea, 4Department of General Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea and 5Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea