Mi Ran Kang

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

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.

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

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

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. Copyright

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

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

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

Seo SI, Song SY, Kang MR, Kim MS, Oh JE, Kim YR, Lee JY, Yoo NJ, Lee SH. Immunohistochemical analysis of NF‐κB signaling proteins IKKε, p50/p105, p52/p100 and RelA in prostate cancers. APMIS 2009; 117:623–8.

Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability

Song SY, Kang MR, Yoo NJ, Lee SH. Mutational analysis of mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability. APMIS 2010; 118: 389–93.

Novel somatic frameshift mutations of genes related to cell cycle and DNA damage response in gastric and colorectal cancers with microsatellite instability

Aims and background Microsatellite instability (MSI) in sporadic gastric cancer (GC) and colorectal cancer (CRC) causes frameshift mutations in gene sequences that contribute to cancer pathogenesis. Many mutations have already been identified in these two cancer types, but some are still undiscovered. Methods We analyzed seven genes (cell cycle control and DNA damage signaling/repair-related genes) with seven or more mononucleotide repeats in 30 GC samples with high MSI (MSI-H), 15 GC samples with low MSI (MSI-L), 45 GC samples that were microsatellite stable (MSS), 33 CRC samples with MSI-H, 15 CRC samples with MSI-L, and 45 CRC samples that were MSS. Single-strand conformation polymorphism (SSCP) and DNA sequencing were used for the analysis. Results We found somatic frameshit mutations of the KNTC1 (6.7% GC, 12.1% CRC), ZC3H13 (3.3% GC, 15.2% CRC), CENPH (6.7% GC), TOPBP1 (3.0% CRC), NDC80 (3.0% CRC), RIF1 (6.7% GC), and NBS1 (3.3% GC, 3.0% CRC) genes in the cancers with MSI-H. Mutations were detected in MSI-H, but not in MSI-L or MSS samples. Conclusions Novel frameshift mutations occurred in seven genes in GC and CRC with MSI-H. The results of our study suggest that the mutations might contribute to the development of GC and CRC with MSI by deregulation of the cell cycle and DNA damage signaling/repair. Free full text available at www.tumorionline.it

Frameshift mutations of ATBF1, WNT9A, CYLD and PARK2 in gastric and colorectal carcinomas with high microsatellite instability

implication on the treatment. In all of the subsequent cases reported after the original report, the short term prognosis has been good with no recurrence or metastasis reported. The long term prognosis is unclear at this stage but will become clearer with further patient monitoring and follow-up. In this study, we described an additional case of PAMT of the stomach. Our current case shares the clinical, histological, immunophenotypical and ultrastructural features of the previously described cases (Table 1).

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

Expression of apoptosis-related proteins caspase-6, caspase-9, FLIP and BNIP3 in oesophageal squamous cell carcinomas

Sir, The Fas-Fas ligand system is recognised as a major pathway for the induction of apoptosis in cells and tissues. Fas-mediated apoptosis can be blocked by several mechanisms, including overexpression of FLICE-like inhibitory protein (FLIP). Among caspases, the initiator caspases would appear to be caspases 8, 9 and 10, and the effector caspases would appear to be caspases 3, 6 and 7. Bcl-2/ adenovirus E1B-19kDa-interacting protein3 (BNIP3) was identified as a pro-apoptotic member of Bcl-2 family and plays an important role in apoptosis, necrosis and autophagy. Apoptosis of cancer cells can be altered by several mechanisms, including somatic mutations of apoptosisrelated genes and aberrant expressions of apoptosis-related proteins. Oesophageal squamous cell carcinoma (ESCC) is known to be resistant to various apoptosis stimuli, including chemotherapies. Earlier studies found alterations of apoptosis-related genes and proteins in ESCC. However, expression status of pro-apoptotic caspase-6, caspase-9 and BNIP3, and anti-apoptotic FLIP in ESCC has not been reported. In the present study, we analysed the expression of caspase-6, caspase-9, FLIP, and BNIP3 in a series of ESCC tissues by immunohistochemistry using a tissue microarray (TMA) approach. Four TMA recipient blocks were constructed containing paraffin-embedded primary ESCC tissues from 58 archival patient specimens. Ages of the patients ranged from 47 to 79 years with an average of 63 years. The TNM stages of the ESCC were 33IIB and 25III. From every archival paraffin block, five cylinders (two from normal and three from cancer tissues of each patient: total 290 cylinders) of 1.0 mm diameter tissue were taken from representative areas. Using sections from the TMA sections, immunohistochemistries for caspase-6 (Cell Signaling, USA; dilution 1/ 100), caspase-9 (Santa Cruz Biotechnology, USA; dilution 1/100), FLIPL (Sigma, USA; dilution 1/50), and BNIP3 (Abcam, USA; dilution 1/100) were performed using Dako Real EnVision System (Dako, Denmark). Reaction products were developed with diaminobenzidine and counterstained with haematoxylin. By visual inspection under microscope, tumours were interpreted as positive by immunohisto-chemistry when at least weak (þ) to intense (þþ) staining was seen in greater than 30% of the neoplastic cells. The data on the immunostainings are summarised in Table 1 and shown in Fig. 1. The normal squamous cells in the oesophageal mucosa displayed caspase-6, caspase-9, FLIP and BNIP3 immunoreactivities in two (3%), three (5%), three (5%), and six (10%) tissues, respectively (Fig. 1A,C,E,G). Even in the positive immunostainings of these proteins in the normal cells, all of them displayed a weak immunoreactivity (Table 1). In the ESCC, immunopositivity (defined as 30% of the neoplastic cells) was observed for caspase-6 in 24 (41%), caspase-9 in 32 (55%), FLI in 30 (52%), and BNIP3 in 36 (62%) of the 58 cancers (Fig. 1B,D,F,H). All of the immunostainings of caspase-6, caspase-9, FLIP, and BNIP3, when present, were cytoplasmic (Fig. 1A–H). A negative control using the blocking solution instead of the primary antibody showed no signal (Figure 1I). Statistically, there were differences in caspase-6, caspase-9, and BNIP3 immunostainings between normal and cancer cells (Fisher’s exact test, p5 0.01). We also analysed the relationship of each immunostaining with various pathological parameters (differentiation, depth of invasion, TNM stage and survival), but there was no significant association (w test, p4 0.05). In this study, both caspase-6 and -9 were highly expressed in the ESCC, irrespective of the pathological characteristics (Fig. 1). Also, a pro-apoptotic Bcl-2 protein BNIP3 was well expressed in the ESCC. Interestingly, however, an anti-apoptotic protein FLIP was expressed in the ESCC as well. These results suggest that ESCC cells may express both antiand pro-apoptotic proteins, and that there may be complicated regulatory pathways for the induction of apoptosis in the cells. While caspases 6 and 9, FLIP, and BNIP3 are expressed in ESCC cells, these proand anti-apoptotic proteins are negatively or weakly expressed in normal oesophageal squamous cells in the mucosa that may be a counterpart of the ESCC. These results suggest that changes of expression of these proteins might be involved in the development of ESCC. Apoptosis index and some apoptosis-related proteins have been reported to be associated with aggressiveness or prognosis or disease progression of ESCC. However, in our study we failed to find any relationship between clinicopathological parameters, including survival, with expression of caspase-6, caspase-9, FLIP or BNIP3. Apoptosis is a complicated pathway in which numerous molecules are involved, and it might not be easy to link the alterations in expression of several proteins to clinical characteristics. Also, using tiny tissue in the TMA might be a possible cause for the lack of clinical correlation of the proteins. Further studies are required for identifying the clinical