Young Hwa Soung

PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas

A recent report revealed that phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene is somatically mutated in several types of human cancer, suggesting the mutated PIK3CA gene as an oncogene in human cancers. However, because the previous report focused the mutational search primarily on colon cancers, the data on PIK3CA mutations in other types of human cancers have been largely unknown. Here, we performed mutational analysis of the PIK3CA gene by polymerase chain reaction-single-strand conformation polymorphism assay in 668 cases of common human cancers, including hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas, and non-small-cell lung cancers. We detected PIK3CA somatic mutations in 26 of 73 hepatocellular carcinomas (35.6%), 25 of 93 breast carcinomas (26.9%), 12 of 185 gastric carcinomas (6.5%), one of 88 acute leukemias (1.1%), and three of 229 non-small-cell lung cancers (1.3%). Some of the PIK3CA mutations were detected in the early lesions of breast cancer carcinoma, hepatocellular carcinoma, and gastric carcinomas, suggesting that PIK3CA mutation may occur independent of stage of the tumors. The high incidence and wide distribution of PIK3CA gene mutation in the common human cancers suggest that alterations of lipid kinase pathway by PIK3CA mutations contribute to the development of human cancers.

Somatic Mutations of EGFR Gene in Squamous Cell Carcinoma of the Head and Neck

Purpose: Recently, the kinase domain mutations of epidermal growth factor receptor (EGFR) gene have been identified in non–small-cell lung cancer, and these mutations have been related to the clinical response to the tyrosine kinase inhibitor gefitinib. Gefitinib treatment has also shown clinical benefits in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore the possibility that SCCHN harbored the EGFR mutations. Experimental Design: In this study, we analyzed EGFR gene in 41 SCCHN for the detection of the somatic mutations by PCR-single-strand conformational polymorphism analysis. Results: Overall, we detected three EGFR mutations (7.3%), and all of the mutations were the same in-frame deletion mutation in exon 19 (E746_A750del). Conclusion: These data indicated that in addition to non–small-cell lung cancer, SCCHN harbors the EGFR gene mutations, and suggested the rationale for the clinical applicability of gefinitib to SCCHN patients.

Inactivating mutations of caspase-8 gene in colorectal carcinomas

BACKGROUND & AIMS There has been evidence that dysregulation of apoptosis is involved in the pathogenesis of cancer development. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis. The aim of this study was to explore the possibility that mutation of the caspase-8 gene might be involved in the development of colorectal cancer. METHODS We analyzed the entire coding region of the caspase-8 gene for the detection of somatic mutations in 180 colorectal tumors (98 invasive carcinomas and 82 adenomas) by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. RESULTS Overall, we detected a total of 5 somatic mutations in 98 invasive carcinomas (5.1%), but no mutations were detected in 82 adenomas (0%). The frequency of caspase-8 mutation in the carcinomas was significantly higher than that in adenomas (P < 0.05). The 5 mutations consisted of 1 frameshift, 1 nonsense mutation, and 3 missense mutations. We expressed the 5 tumor-derived caspase-8 mutants and found that 3 of the 5 mutations markedly decreased apoptosis activity of caspase-8. Furthermore, expression of the inactivating caspase-8 mutants interfered with apoptosis by death receptor overexpression, indicating that these mutants have dominant-negative inhibition of the death receptor-induced apoptosis. CONCLUSIONS The presence of caspase-8 mutation in colon carcinomas suggests that caspase-8 gene mutation might lead to the loss of its apoptotic function and contribute to the pathogenesis of colorectal carcinomas, especially at the late stage of colorectal carcinogenesis.

Somatic Mutations of ERBB2 Kinase Domain in Gastric, Colorectal, and Breast Carcinomas

Purpose: Recent reports revealed that the kinase domain of the ERBB2 gene is somatically mutated in lung adenocarcinoma, suggesting the mutated ERBB2 gene as an oncogene in human cancers. However, because previous reports focused the mutational search of ERBB2 primarily on lung cancers, the data on ERBB2 mutations in other types of human cancers have been largely unknown. Experimental Design: Here, we did a mutational analysis of the ERBB2 kinase domain by PCR single-strand conformational polymorphism assay in gastric, colorectal, and breast carcinoma tissues. Results: We detected the ERBB2 kinase domain mutations in 9 of 180 gastric carcinomas (5.0%), in 3 of 104 colorectal carcinomas (2.9%), and in 4 of 94 breast carcinomas (4.3%). All of the detected ERBB2 mutations except for one in-frame deletion mutation were missense mutations. Of the 16 ERBB2 mutations detected, 4 affected Val777 in the exon 20 site, and 3 affected Leu755 in the exon 19 site. We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA, and BRAF genes in the 16 samples with ERBB2 mutations, and found that all of the 3 colorectal carcinoma samples with ERBB2 mutations harbored K-RAS mutations. Conclusion: This study showed that in addition to lung adenocarcinomas, ERBB2 kinase domain mutation occurs in other common human cancers such as gastric, breast, and colorectal cancers, and suggested that alterations of ERBB2-mediated signaling pathway by ERBB2 mutations alone or together with K-RAS mutations may contribute to the development of human cancers.

The JAK2 V617F mutation in de novo acute myelogenous leukemias

A missense somatic mutation in JAK2 gene (JAK2 V617F) has recently been reported in chronic myeloproliferative disorders, including polycythemia vera, essential thrombocythemia and myelofibrosis with myeloid metaplasia, strongly suggesting its role in the pathogenesis of myeloid disorders. As activation of JAK2 signaling is occurred in other malignancies as well, we have analysed 558 tissues from common human cancers, including colon, breast and lung carcinomas, and 143 acute adulthood leukemias by polymerase chain reaction – single strand conformation polymorphism analysis. We found three JAK2 mutations in the 113 acute myelogenous leukemias (AMLs) (2.7%), but none in other cancers. The mutations consisted of two V617F mutations and one K607N mutation. None of the AML patients with the JAK2 V617F mutation had a history of previous hematologic disorders. This is the first report on the JAK2 gene mutation in AML, and the data indicated that the JAK2 gene mutation may not only contribute to the development of chronic myeloid disorders, but also to some AMLs.

BRAF and KRAS mutations in stomach cancer

Ras proteins control signaling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumor development. As there have been no data on the BRAF mutation in stomach cancer, we analysed the genomic DNAs from 319 stomach carcinomas for the detection of somatic mutations of BRAF. Overall, we detected BRAF mutations in seven stomach carcinomas (2.2%). Five of the seven BRAF mutations involved Val 599, the previously identified hotspot, but the substituted amino acid (V599 M) was different from the most common BRAF mutation (V599E). The remaining two mutations involved a conserved amino acid (D593G). One tumor had both BRAF and KRAS mutations. This is the first report on BRAF mutation in stomach cancer, and the data indicate that BRAF is occasionally mutated in stomach cancer, and suggest that alterations of RAS pathway both by RAS and BRAF mutations contribute to the pathogenesis of stomach cancer.

Somatic mutations of the ERBB4 kinase domain in human cancers.

The EGFR family consists of 4 receptor tyrosine kinases, EGFR (ERBB1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4). Recent reports revealed that the kinase domains of both EGFR (ERBB1) and ERBB2 gene were somatically mutated in human cancers, raising the possibility that the other ERBB members possess somatic mutations in human cancers. Here, we performed mutational analysis of the ERBB4 kinase domain by polymerase chain reaction–single‐strand conformation polymorphism assay in 595 cancer tissues from stomach, lung, colon and breast. We detected the ERBB4 somatic mutations in 3 of 180 gastric carcinomas (1.7%), 3 of 104 colorectal carcinomas (2.9%), 5 of 217 nonsmall cell lung cancers (2.3%) and 1 of 94 breast carcinomas (1.1%). The 12 ERBB4 mutations consisted of 1 in‐frame duplication mutation and 8 missense mutations in the exons, and 3 mutations in the introns. We simultaneously analyzed the somatic mutations of EGFR, ERBB2, K‐RAS, PIK3CA and BRAF genes in the 12 samples with the ERBB4 mutations and found that 1 gastric carcinoma with ERBB4 mutation also harbored K‐RAS gene mutation. Our study demonstrated that in addition to EGFR and ERBB2, somatic mutation of the kinase domain of ERBB4 occurs in the common human cancers, and suggested that alterations of ERBB4‐mediated signaling pathway by ERBB4 mutations may contribute to the development of human cancers.

Somatic mutations of CASP3 gene in human cancers

Failure of apoptosis is one of the hallmarks of cancer. As an execution-phase caspase, caspase-3 plays a crucial role during apoptosis. To explore the possibility that the genetic alterations of CASP3, which encodes caspase-3, might be involved in the development of human tumors, we analyzed the entire coding region and all splice sites of human CASP3 gene for the detection of somatic mutations in a series of 944 human tumors, including 165 stomach carcinomas, 95 colon carcinomas, 76 breast carcinomas, 80 hepatocellular carcinomas, 181 non-small cell lung cancers, 45 acute leukemias, 28 multiple myelomas, 12 medulloblastomas, 15 Wilms’ tumors, 12 renal cell carcinomas, 40 esophagus carcinomas, 33 urinary bladder carcinomas, 33 laryngeal carcinomas, and 129 non-Hodgkin lymphomas. Overall, we detected 14 somatic mutations of the CASP3 gene, including six missense and four silent mutations, two mutations in the introns, one mutation in the 5′-untranslated region, and one mutation in the 3′-untranslated region. The mutations were observed in four of 98 colon carcinomas (4.1%), four of 181 non-small cell lung cancers (2.2%), two of 129 non-Hodgkin lymphomas (1.6%), two of 165 stomach carcinomas (1.2%), one of 80 hepatocellular carcinomas (1.3%), and one of 28 multiple myelomas (3.6%). This is the first report on CASP3 gene mutations in human tumors; these data indicate that the CASP3 gene is occasionally mutated in human tumors.

Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas

Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, χ2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.

Caspase-8 gene is frequently inactivated by the frameshift somatic mutation 1225_1226delTG in hepatocellular carcinomas.

Evidence exists that alterations of the genes encoding apoptosis-related proteins contribute to either development or progression of human cancers. Caspase-8 plays a crucial role in the initiation phase of apoptosis. To explore the possibility that the genetic alteration of caspase-8 gene is involved in the development of hepatocellular carcinomas (HCCs), we have analysed the entire coding region of human caspase-8 gene for the detection of somatic mutations by polymerase chain reaction-single-strand conformation polymorphism in 69 HCCs with low-grade dysplastic nodule (LGDN, n=2) or high-grade dysplastic nodule (HGDN, n=2) or without any dysplastic nodules (n=65). Overall, we detected a total of nine somatic mutations in 69 HCCs (13.0%). Interestingly, all of the nine mutations were an identical frameshift mutation with two base-pair deletion (1225_1226delTG), which would result in a premature termination of amino-acid synthesis in the p10 protease subunit. In a patient sample, we detected the 1225_1226delTG mutation both in HCC and LDGN lesions, suggesting that caspase-8 mutation could be involved in the early stage of HCC carcinogenesis. We expressed the tumor-derived caspase-8 mutant in the cells and found that the mutant abolished cell death activity of caspase-8. Our data indicate that caspase-8 gene is frequently mutated in HCC and the majority of the mutations may be the frameshift mutation 1225_1226delTG. Also, the data suggest that caspase-8 gene mutation might lead to the loss of its cell death function and contribute to the pathogenesis of HCC.