Kei Fujii

The Role of S100A6 in Pancreatic Cancer Development and Its Clinical Implication as a Diagnostic Marker and Therapeutic Target

Recent microarray analyses showed that the S100 family contains members that are candidate diagnostic markers or therapeutic targets. In the present study, to evaluate the involvement of S100A6 in pancreatic cancer and its clinical usefulness for diagnosis, we examined S100A6 mRNA expression in pancreatic tissues and pancreatic juice from patients with different pancreatic diseases. To investigate the role of S100A6 in carcinogenesis of pancreatic cancer and the potential of S100A6 as a diagnostic marker for early detection of pancreatic cancer, we did immunohistochemistry and microdissection-based mRNA analysis of pancreatic normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. We also used in vitro experiments and microarray analysis with RNA interference to evaluate the functional role of S100A6 and its potential as a therapeutic target for pancreatic cancer. S100A6 mRNA levels were significantly higher in carcinoma specimens than in nonneoplastic tissues. In pancreatic juice, there was a significant difference in S100A6 expression between patients with carcinoma and those with nonneoplastic disease. Receiver operating characteristic curves revealed that S100A6 might be a useful marker for diagnosis of pancreatic cancer. Immunohistochemistry and microdissection-based analysis showed differential expression of S100A6 among normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. In vitro data showed that inhibition of S100A6 decreased proliferation and invasiveness of cancer cells, and these findings were supported by microarray data. Our present results suggest that quantitation of S100A6 mRNA is a promising tool for diagnosis of pancreatic cancer, and that S100A6 may be a promising therapeutic target for pancreatic cancer.

S100P Is an Early Developmental Marker of Pancreatic Carcinogenesis

Purpose: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis. Experimental Design: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples. Results: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P < 0.017 and P = 0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P < 0.0001) and PanIN (P = 0.0031), although S100P expression did not differ between IDC and PanIN (P = 0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P < 0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve = 0.837; 95% confidence interval, 0.749-0.903). Conclusion:S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.

Quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice for preoperative diagnosis of pancreatic cancer

Pancreatic juice is a promising type of diagnostic sample for pancreatic cancer, and members of the mucin (MUC) family are diagnostic candidates. To evaluate the utility of MUC family members as diagnostic markers, we measured MUC mRNA expression in pancreatic tissues and pancreatic juice obtained from patients with different pancreatic diseases as well as in pancreatic cancer cell lines by real‐time PCR. Furthermore, to support the possibility of early diagnosis by quantification of MUC1 and MUC5AC, immunohistochemistry and microdissection‐based quantitative analysis of mRNA were carried out. There was no significant correlation between MUC1 and MUC5AC expression in cell lines. When β‐actin was used as a reference gene, median MUC1 and MUC5AC mRNA expression levels were remarkably greater in tumoral tissues than in non‐tumoral tissues, but median MUC4 and MUC6 mRNA expression levels were not. Receiver operating characteristic curve analysis showed that quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice is better diagnostic modality than that of MUC4 and MUC6 mRNA. Immunohistochemistry showed that MUC1 and MUC5AC were highly expressed in invasive ductal carcinomas (IDC) and moderately expressed in high‐grade pancreatic intraepithelial neoplasia (PanIN); no staining was observed in normal ducts. Analysis of cells isolated by microdissection showed stepwise upregulation of MUC1 and MUC5AC in the development of high‐grade PanIN to IDC. Our results suggest that MUC1 and MUC5AC are upregulated stepwise in pancreatic carcinogenesis and that quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer.

The role of the DNA damage checkpoint pathway in intraductal papillary mucinous neoplasms of the pancreas

Purpose: Intraductal papillary mucinous neoplasms (IPMN) are known to show a transition from adenoma to carcinoma accompanied by several molecular abnormalities. ATM-Chk2-p53 DNA damage checkpoint activation, which is involved in prevention of the progression of several tumors, was analyzed to evaluate the role of the DNA damage checkpoint in the progression of IPMNs. Experimental Design: One hundred and twenty-eight IPMNs were classified into four groups (intraductal papillary mucinous adenoma, borderline IPMN, noninvasive intraductal papillary mucinous carcinoma, and invasive intraductal papillary mucinous carcinoma) and stained immunohistochemically using antibody for Thr68-phosphorylated Chk2. Expression of ATM, Chk2, and p21WAF1 and accumulation of p53 were also analyzed. Results: Chk2 phosphorylation was shown in all adenomas and showed a significant decreasing trend with the progression of atypia (P < 0.0001 by the Cochran-Armitage test for trend). Expression of p21WAF1 also exhibited a decreasing tendency (P < 0.0001), reflecting DNA damage checkpoint inactivation. p53 accumulation was mostly detected in malignant IPMNs. It was suggested that the DNA damage checkpoint provides a selective pressure for p53 mutation. Conclusion: Our findings indicate that DNA damage checkpoint activation occurs in the early stage of IPMNs and prevents their progression. It is suggested that disturbance of the DNA damage checkpoint pathway due to Chk2 inactivation or p53 mutation contributes to the carcinogenesis of IPMNs.

Expression of GalNAc-T3 and its relationships with clinicopathological factors in 61 extrahepatic bile duct carcinomas analyzed using stepwise sections—Special reference to its association with lymph node metastases—

Extrahepatic bile duct carcinomas (EBDCs) still result in an unfavorable prognostic outcome, and little is known about their biological aggressiveness. Recently, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl transferase-3 (GalNAc-T3) was reported to be associated with differentiation and malignant potential of human carcinomas. Here, we investigated 61 EBDCs for their detailed clinicopathological features and GalNAc-T3 expression by immunohistochemistry, and then evaluated the relationships between the clinicopathological features and GalNAc-T3 expression patterns. Most of the EBDCs were massively invasive tumors with frequent vascular or perineural invasion and lymph node metastases. GalNAc-T3 expression was detected in all 61 EBDCs, and the expression patterns could be classified into granular and diffuse types. All four noninvasive or minimally invasive EBDCs were the granular type. Among the 58 minimally or massively invasive EBDCs, the GalNAc-T3 expression pattern at the luminal surface was the granular type in 38 cases (66%) and diffuse type in 20 cases (34%), while the expression pattern at the invasive front was the granular type in 26 cases (45%) and diffuse type in 32 cases (55%). Among the 38 cases with granular-type expression at the luminal surface, 26 cases (68%) remained the granular type and 12 cases (32%) became the diffuse type at the invasive front. All 20 cases with diffuse-type expression at the luminal surface remained the diffuse type at the invasive front. Diffuse-type GalNAc-T3 expression at the invasive front was significantly associated with lymph node metastasis (P<0.05). There were no significant correlations between the GalNAc-T3 expression patterns and other clinicopathological factors, including tumor differentiation, depth of invasion or overall survival. In conclusion, EBDCs alter their GalNAc-T3 expression pattern during tumor growth, and the difference in the GalNAc-T3 expression pattern may be associated with lymph node metastasis. Clinically, preoperative evaluation of GalNAc-T3 expression is considered to be useful for decisions regarding operative procedures.

Frequent microsatellite instability in non-Hodgkin lymphomas irresponsive to chemotherapy.

Microsatellite instability (MSI) in haematopoietic malignancies has been controversial. Particularly in non-Hodgkin lymphoma, the data published to date lack unity. Using a unique fluorescent technique, we found MSI in eight (14%) tumours in a panel of 59 carefully selected non-Hodgkin lymphoma patients. Our fluorescent technique also reveals two qualitatively distinct modes of MSI, i.e. Type A and Type B. Based on our previous studies using DNA mismatch repair (MMR) gene-knock out animals, we have concluded that Type A MSI is a direct consequence of defective MMR. MSI observed in non-Hodgkin lymphomas was uniformly Type A, which implies that MMR deficiency occurs in this malignancy. Intriguingly, in non-Hodgkin lymphoma patients treated by CHOP/VEPA-based therapies, response to chemotherapy was significantly worse in those with microsatellite-unstable tumours (p=0.027). As a consequence, the patient outcomes at 1 year after treatment were significantly less favourable in this population (p=0.046), although the survival difference was not statistically confirmed in a longer term. These findings suggest that in some non-Hodgkin lymphomas MMR deficiency may lead to drug resistance in tumour cells and, consequently, to poor patient outcomes. In non-Hodgkin lymphoma, MSI may be a potential biomarker that predicts the tumour response against chemotherapy.

Simulation-based analyses reveal stable microsatellite sequences in human pancreatic cancer

Genomic analysis using tissue samples is an essential approach in cancer genetics. However, technical and biological limits exist in this approach. Microsatellite instability (MSI) is frequently observed in human tumors. MSI assays are now prevalent and regarded as commonplace. However, several technical problems have been left unsolved in the conventional assay technique. Indeed, the reported frequencies of MSI differ widely in each malignancy. An example is pancreatic cancer. Using a unique fluorescent technique, we found that MSI is extremely infrequent in this malignancy, despite the relatively high frequencies in some reports. In a series of simulations, we have demonstrated that the extremely low frequency was derived neither from less sensitive assays nor from a scarcity of cancer cells in tissue samples. Furthermore, analyzing laser-capture microdissection (LCM)-processed cell populations of a microsatellite-unstable colorectal cancer cell line, HCT116, we have shown that MSI can be detected only when comparing two cell populations that have grown independently to a sufficiently large size. When MSI is not detected in analyses using tissue samples, LCM is not advisable. We therefore did not extend our study to LCM of tissue specimens. We conclude that microsatellite sequence alterations are not detectable in human pancreatic cancer.

Heterochronous occurrence of microsatellite instability in multiple myeloma – an implication for a role of defective DNA mismatch repair in myelomagenesis

Kaname Miyashita , Kei Fujii, Youko Suehiro, Kenichi Taguchi, Naokuni Uike, Mitsuaki A. Yoshida and Shinya Oda Department of Hematology, National Kyushu Cancer Center, Fukuoka, Japan; Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Clinical Research Institute, Cancer Genetics Laboratory, National Kyushu Cancer Center, Fukuoka, Japan; Department of Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University, Aomori, Japan

A specific mode of microsatellite instability is a crucial biomarker in adult T-cell leukaemia/lymphoma patients.

PurposeMicrosatellite instability (MSI) has been a long-standing biomarker candidate for drug resistance in tumour cells. Despite numerous clinical studies, the data in the literature are not conclusive. The complexity of the MSI phenomenon in some malignancies may, at least partly, account for the discrepancy. In addition, methodological problems are also pointed out in the assay techniques. We previously established a unique fluorescent technique in which the major methodological problems in conventional assays are overcome. Application of this technique has revealed two distinct modes of microsatellite alterations, i.e. Type A and Type B. More importantly, we demonstrated that Type A MSI is the direct consequence of defective DNA mismatch repair (MMR) that causes cellular resistance against antineoplastic agents.MethodWe first applied this technique to adult T-cell leukaemia/lymphoma (ATLL).ResultsThe MSI phenomenon was indeed observed in ATLLs (4/20, 20%). Intriguingly, the observed microsatellite alterations were invariably Type A, which implies that the tumours were MMR-defective. Indeed, clinical outcomes of patients with these MSI+ tumours were significantly worse. Furthermore, multivariate analysis revealed that Type A MSI is an independent prognostic factor.ConclusionThese observations strongly suggest the possibility of Type A MSI as a prognostic and potentially predictive biomarker in ATLL.