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DNA mismatch repair (MMR) is an evolutionally conserved and major mechanism in cells that counteracts the occurrence of point mutations on the genome. Defective MMR confers the ‘mutator’ phenotype ...

Purpose Microsatellite 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. Method We first applied this technique to adult T-cell leukaemia/lymphoma (ATLL). Results The 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. Conclusion These observations strongly suggest the possibility of Type A MSI as a prognostic and potentially predictive biomarker in ATLL.

// Asuka Murata 1, * , Yoshifumi Baba 1, * , Takatsugu Ishimoto 1 , Keisuke Miyake 1 , Keisuke Kosumi 1 , Kazuto Harada 1 , Junji Kurashige 1 , Shiro Iwagami 1 , Yasuo Sakamoto 1 , Yuji Miyamoto 1 , Naoya Yoshida 1 , Manabu Yamamoto 2 , Shinya Oda 3 , Masayuki Watanabe 4 , Mitsuyoshi Nakao 5 , Hideo Baba 1 1 Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Japan 2 Department of Surgery, National Hospital Organization Kyushu Cancer Center, Japan 3 Department of Cancer Biology, National Kyushu Cancer Center Clinical Research Institute, Japan 4 Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Japan 5 Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan * These authors have contributed equally to this work Correspondence to: Yoshifumi Baba, e-mail: y-baba@kumamoto-u.ac.jp Keywords: epigenetics, markers, demethylation, TET Received: April 17, 2015 Accepted: May 26, 2015 Published: June 08, 2015 ABSTRACT Mammalian DNA is epigenetically marked by 5′-cytosine methylation (5-methylcytosine [5-mC]). The Ten-eleven translocation (TET) enzymes (TET1, TET2, and TET3) are implicated in DNA demethylation, through dioxygenase activity that converts 5-mC to 5-hydroxymethylcytosine (5-hmC). Although decreased TET is reportedly associated with decreased 5-hmC levels in various cancers, functions of 5-hmC and TET expression in esophageal squamous cell carcinoma (ESCC) are unclear. We used ELISA and immunohistochemistry tests to analyze 5-hmC status in ESCC tissues, RT-qPCR to analyze TET family mRNA expression in normal and tumor tissues, and pyrosequencing to quantify LINE-1 (i.e., global DNA methylation) levels. ELISA and immunohistochemical testing showed 5-hmC levels were significantly lower in ESCC than in paired normal tissues ( P < 0.0001). TET2 expression was significantly lower in ESCCs than paired normal tissues ( P < 0.0001), and significantly associated with 5-hmC levels in ESCCs ( P = 0.003, r = 0.33). 5-hmC levels were also significantly associated with LINE-1 methylation level ( P = 0.0002, r = 0.39). Patients with low 5-hmC levels had shorter overall survival than those with higher levels, although not significantly so ( P = 0.084). In conclusion, 5-hmC expression was decreased in ESCC tissues, and was associated with TET2 expression level. TET2 reduction and subsequent 5-hmC loss might affect ESCC development.

The aim of this study was to evaluate the efficacy of the auditory evoked potential (AEP) index (aepEX) as an assessment tool for hypnosis during induction of various anesthetic drugs, and to compare its performance to that of the bispectral index (BIS). A total of 45 cases were divided into three groups based on the drugs used for anesthesia. Before anesthetic induction, BIS and AEP monitors were initiated. Anesthesia was induced through intravenous injection (IV) as follows: MP (n = 15) group, midazolam (0.1 mg/kg IV); TP (n = 15) group, thiopental (4 mg/kg IV); and KP (n = 15) group, ketamine (2 mg/kg IV). After loss of response (LOR), an infusion of 3 μg/ml propofol via a target-controlled infusion was initiated in all groups. AepEX and BIS were measured in the waking state (baseline) and at LOR (1 min after LOR), pre-intubation (1 min after previous intubation) and post-intubation (1 min after tracheal intubation finished). The value of aepEX significantly decreased in all groups with LOR and that of BIS also decreased except of KP group. No significant difference were observed in BIS values between baseline and LOR in the KS group. The aepEX might be more useful than BIS for hypnosis monitoring during anesthetic induction, particularly when drugs such as ketamine are used.

The herpes simplex virus 1 (HSV-1) Us8A gene overlaps the gene that encodes glycoprotein E (gE). Previous studies have investigated the roles of Us8A in HSV-1 infection using null mutations in Us8A and gE; therefore, the role of Us8A remains to be elucidated. In this study, we investigated the function of Us8A and its phosphorylation at serine 61 (Ser-61), which we recently identified as a phosphorylation site by mass spectrometry-based phosphoproteomic analysis of HSV-1-infected cells, in HSV-1 pathogenesis. We observed that (i) the phosphorylation of Us8A Ser-61 in infected cells was dependent on the activity of the virus-encoded Us3 protein kinase; (ii) the Us8A null mutant virus exhibited a 10-fold increase in the 50% lethal dose for virulence in the central nervous system (CNS) of mice following intracranial infection compared with a repaired virus; (iii) replacement of Ser-61 with alanine (S61A) in Us8A had little effect on virulence in the CNS of mice following intracranial infection, whereas it significantly reduced the mortality of mice following ocular infection to levels similar to the Us8A null mutant virus; (iv) the Us8A S61A mutation also significantly reduced viral yields in mice following ocular infection, mainly in the trigeminal ganglia and brains; and (v) a phosphomimetic mutation at Us8A Ser-61 restored wild-type viral yields and virulence. Collectively, these results indicate that Us8A is a novel HSV-1 virulence factor and suggest that the Us3-mediated phosphorylation of Us8A Ser-61 regulates Us8A function for viral invasion into the CNS from peripheral sites. IMPORTANCE The DNA genomes of viruses within the subfamily Alphaherpesvirinae are divided into unique long (UL) and unique short (Us) regions. Us regions contain alphaherpesvirus-specific genes. Recently, high-throughput sequencing of ocular isolates of HSV-1 showed that Us8A was the most highly conserved of 13 herpes simplex virus 1 (HSV-1) genes mapped to the Us region, suggesting Us8A may have an important role in the HSV-1 life cycle. However, the specific role of Us8A in HSV-1 infection remains to be elucidated. Here, we show that Us8A is a virulence factor for HSV-1 infection in mice, and the function of Us8A for viral invasion into the central nervous system from peripheral sites is regulated by Us3-mediated phosphorylation of the protein at Ser-61. This is the first study to report the significance of Us8A and its regulation in HSV-1 infection.

DNA polymerase lambda, pol lambda, is a eukaryotic member of the X-family DNA polymerases that is involved in two modes of DNA repair, i.e. base excision repair (BER) or non-homologous end joining (NHEJ). Using immunohistochemical approaches, we have observed pol lambda expression in human tissues, particularly in the respiratory system of lung cancer patients. pol lambda proteins were distributed in the nuclei of the epithelial cells in the bronchi, bronchioles and alveoli. Intriguingly, the level of pol lambda expression in the bronchiolar epithelia significantly correlated with the amount of habitual smoking in the individuals. Conversely, pol lambda expression in cancer tissues did not correlate with the smoking status of the patients. Pol lambda expression was sometimes discrepant between the tumor tissues and adjacent bronchioles. More importantly, tumors without pol lambda expression that occurred in heavy smokers significantly tended to be at an advanced clinical stage. Pol lambda may thus be involved in the DNA repair processes counteracting DNA damage caused by tobacco smoke in the respiratory system.

Malignant peripheral nerve sheath tumor (MPNST) is an uncommon soft tissue neoplasm with a poor prognosis, occurring sporadically or associated with neurofibromatosis type 1 (NF1); however, the histogenesis of MPNST remains unclear, especially in sporadic tumors. There are two major forms of genomic instability in human cancer: chromosomal instability (CIN) and microsatellite instability (MSI). An inverse relationship has recently been demonstrated between CIN and MSI in colorectal cancers. CIN and MSI are suggested to be individual pathways, which are involved in the pathogenesis and which may lead to specific clinical and pathological characteristics. To elucidate the chromosomal aberration as a consequence of CIN and MSI status of MPNST, we karyotyped 10 MPNSTs from nine patients, and examined the MSI of seven microsatellite markers using high-resolution fluorescence microsatellite analysis; 2 out of 10 cases (20%) had normal karyotypes, and 8 out of 10 cases (80%) revealed structural and numerical chromosomal aberrations. Three of the 10 cases (30%) showed near triploidy. The most frequent aberration was -22 (40%), followed by +2, +14, -13, -17, and -18 (30% each). An MSI-low status was observed in 30% of cases; the remaining cases showed microsatellite stability. These findings suggest that chromosomal aberration as a consequence of CIN has a greater role in the pathogenesis of MPNST than does that due to MSI.

Background Hypoxia-inducible factor 1α (HIF-1α) plays a key role in responses to hypoxia and expression of HIF-1α downstream genes leads to both an adapted metabolism and increased oxygen supply. We investigated the clinical significance of HIF-1α expression in gastric carcinoma. Methods We examined HIF-1α, vascular endothelial growth factor (VEGF), and insulin-like growth factor-2 (IGF-2) expression patterns immunohistochemically in 126 specimens of gastric carcinoma. CD34 antigen levels were also examined by immunohistochemistry to determine microvessel density (MVD) within tumors and correlations between HIF-1α expression, clinicopathological features, and survival were examined. Results HIF-1α expression correlated with tumor size (P

Summary Background : In non-small cell lung cancer, a loss of heterozygosity (LOH) is frequently observed; however, few studies have investigated the differences in the LOH status between adenocarcinoma and squamous cell carcinoma. Patients and methods : In a consecutive series of 49 patients with adenocarcinomas and 22 patients with squamous cell carcinomas, the LOH in tumors was analyzed using polymerase chain reaction employing 5 fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, D13S175) and an autosequencer. Results : LOH was more frequently observed in squamous cell carcinoma (20 of 22, 90%) than in adenocarcinomas (33 of 49, 67%) ( P = 0.0348 ), and the number of LOH per patient was also higher in the patients with squamous cell carcinoma (2.2±1.4) than in those with adenocarcinoma (1.5±1.2, P = 0.037 ). In adenocarcinomas, the number of LOH per patients correlated significantly with the pack-year index, whereas the pathological stage significantly affected the number of LOH in squamous cell carcinomas. Conclusion : The presence of LOH is relatively uncommon in adenocarcinoma of the lung; however, the incidence of LOH tends to be associated with the smoking status.

Synchronous tumors of the ovary and endometrium are a well-known phenomenon. There are histological criteria for defining double primary tumors or metastasis from one organ to another, but in some cases a precise diagnosis is difficult. In this study we reviewed 17 cases of synchronous ovarian and endometrial adenocarcinoma by previously reported histological criteria and performed a microsatellite analysis, combined with X-linked clonality analysis. We also analyzed 8 cases of endometrial adenocarcinoma with pelvic lymph node metastasis as a control. Five dinucleotide microsatellite markers were selected, and microsatellite analysis was performed by a high-resolution method using fluorescence-labeled polymerase chain reaction and laser scanning. In synchronous tumors, 11 ovarian carcinomas (65%) and 10 endometrial carcinomas (59%)demonstrated microsatellite instability (MSI). In total, 13 of the 17 patients demonstrated MSI in the ovarian tumor, the endometrial tumor, or both. Four cases of endometrial carcinoma with pelvic lymph nodes metastases displayed MSI, and MSI findings of the endometrial tumor and lymph node metastasis were same in all cases. Based on these findings, we considered that similar MSI findings indicate metastatic tumors. According to the MSI findings, 13 of the 17 patients (76%) had single or double clonal tumors, 11 (67%) with double primary tumors and 2 (13%) with metastatic tumors. Using X-linked clonality analysis, 3 patients were diagnosed with double primary tumors. The molecular diagnosis corresponded with the histological criteria in all but 1 case. In conclusion, using both MSI and X-linked clonality analysis, most patients (82%) could be diagnosed as having single or double clonal tumors. The histological criteria are accurate and useful in most cases; however, in some cases where the relationship between the 2 tumors is difficult to determine, high-resolution MSI analysis may be helpful.

Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous tumor categorized as a tumor of intermediate malignancy, and its progression in some cases to fibrosarcoma is well known. However, molecular analysis of tumor progression has been limited. The present study investigated microsatellite instability (MSI) of 7 microsatellite markers through high-resolution microsatellite analysis in addition to a mutational analysis of the p53 gene in 44 tumors in 36 patients. The patients were divided into 2 groups: 9 patients with a fibrosarcomatous component in the primary or recurrent/metastasized tumor, designated as the DFSP+FS group, and the remaining 27 patients, designated as the DFSP group. Cases in which the percentage of markers with an additional peak among the markers successfully analyzed was more than 30% was considered MSI high (MSI-H); cases in which microsatellites were stable at all of the successfully examined markers were considered microsatellite stable (MSS); and the remaining cases were considered MSI low (MSI-L). MSI-H cases were observed more frequently in the DFSP+FS group (4 of 9 cases) than in the DFSP group (1 of 27 cases) (P = 0.028, Fischer's exact test). The MSI status of recurrent or metastatic tumors in both the DFSP+FS and the DFSP groups was the same as that in the corresponding primary neoplasms. Furthermore, there was no difference in MSI status between an ordinary DFSP area and a fibrosarcomatous area in 7 tumors that exhibited both areas. p53 mutational analysis revealed 10 point mutations, composed of 4 missense mutations and 6 silent mutations, in 6 of 36 cases (16.7%). A missense mutation was more frequently observed in the DFSP+FS group (3 of 4) than in the DFSP group (1 of 4). Among 3 cases of a missense mutation in the DFSP+FS group, 2 had a mutation only in a fibrosarcomatous area and 1 had a mutation only in a metastatic tumor progressing to fibrosarcoma. These results suggest that MSI and p53 mutations are involved in tumor progression of DFSP to fibrosarcoma as early and late events, respectively.

Background: Various studies have analyzed the relationships between new biological parameters and DNA ploidy for human cancer. However, the biological significance of DNA ploidy remains unclear, since there have so far been few studies analyzing DNA ploidy in relation to the combination of multiple such biological parameters. Methods: Samples for the analysis of DNA ploidy, estrogen receptor (ER), progesterone receptor (PgR), p53 protein and epidermal growth factor receptor (EGFR) were prepared from the same frozen specimens of 498 primary breast cancers. DNA ploidy was determined by flow cytometry, while ER and PgR were determined by an enzyme immunoassay. Both p53 protein and EGFR were determined by immunohistochemical analyses. Results: Aneuploidy was significantly correlated with the absence of ER, the absence of PgR, the positivity of p53 protein and the positivity of EGFR, and it was also significantly correlated with the combination of the absence of ER and PgR and the combination of the positivity of p53 protein and EGFR. A deviation in the four parameters was defined as the absence of ER, the absence of PgR, the positivity of p53 protein or the positivity of EGFR. As the number of deviations of these four biological parameters increased, the incidence of aneuploidy increased significantly (p < 0.0001). Multivariate analysis also indicated that deviation of two, three or all four parameters were significant factors for DNA ploidy, while the p value decreased and the odds ratio increased as the number of deviations of the four biological parameters increased. Conclusions: DNA aneuploidy based on flow cytometric analysis reflects the accumulation of deviations in the four biological parameters investigated in the present study. The two facts that DNA aneuploidy reflects the accumulation of the aggressiveness of these biological parameters and that DNA aneuploidy consisted of heterogeneous characteristics which were represented by the deviation of each biological parameter should be taken into consideration when selecting treatment strategies for breast cancer.

In one third of colorectal cancer patients, tumours occur in the rectum. Unique aetiologies may underlie the increased carcinogenesis in this region of the colorectum. Microsatellite instability (MSI) was analysed in specimens obtained from 121 colorectal carcinoma patients, using five dinucleotide markers and a new fluorescent system. The incidence of microsatellite alterations in the proximal colon, the distal colon and the rectum was 44.4% (16/36), 37.2% (16/43) and 23.8% (10/42), respectively. Patterns of microsatellite alterations could be classified into two subtypes, one showing relatively small changes within 6 bases (type A) and the other exhibiting drastic changes over 8 bases (type B). All the changes observed in tumours in the rectum were type A, and no type B mutation was noted. There was a close correlation between type B mutations and high-frequency MSI (≧2 markers), MSI-H, and between type A mutations and low-frequency MSI (1 marker), MSI-L. The type B/MSI-H phenotype significantly correlated with the proximal localisation of tumours. In the rectum, there was no tumour with the type B/MSI-H phenotype. These findings suggest that cancers occurring in the colon and the rectum have a differential molecular background for carcinogenesis.

Direct evidence of tumour seeding in distant organs at the time of surgery for gastric cancer is not available. An immunocytochemical assay for epithelial cytokeratin protein may fill this gap since it is a feature of epithelial cells that would not normally be present in bone marrow. The bone marrow of 46 patients with primary gastric cancer was examined for tumour cells, using immunocytochemical techniques and antibody reacting with cytokeratin, a component of the intracytoplasmic network of intermediate filaments. The monoclonal antibody CK2 recognises a single cytokeratin polypeptide (human cytokeratin no. 18) commonly present in epithelial cells. The expression of tumour-suppressor genes p53 and RB for the primary lesion was also determined using the monoclonal antibodies PAb 1801 and 3H9 respectively, and the proliferating activity was determined by the Ki-67 antigen labelling index for MIB-1 antibody staining. Of these 46 patients, 15 (32.6%) presented with cytokeratin-positive cells at the time of primary surgery. The positive findings were related to the undifferentiated tissue type and to the prominent depth of invasion, but not to other clinicopathological factors. In 2 of 15 (13.3%) patients, the depth of invasion was limited to the mucosa. The metastatic potential to bone marrow did not relate to expressions of p53 and RB genes, or to the proliferating activity of MIB-1 staining for the primary lesion of gastric cancer. As tumour cells in bone marrow are indicative of the general disseminative capability of an individual tumour, this technique may be useful for identifying patients at high risk of metastasis from a gastric tumour. Images Figure 1

We report a case of cranial subdural hematoma with intracranial hypotension. A 34-year-old woman had laparoscopic ovarial cysterectomy under general anesthesia combined with epidural anesthesia. Two days later, she developed a severe headache and nausea. She underwent cranial magnetic resonance imaging (MRI) scanning, and was diagnosed with cranial subdural hematoma with intracranial hypotension. The patient had had no anticoagulant therapy before the surgery. She was managed conservatively with bed rest and additional intravenous infusion. Her symptoms gradually improved except a slight headache, and she was discharged on the 38th postoperative day. Intracranial hypotension is a syndrome characterized by orthostatic headaches and hypovolemia of cerebrospinal fluid (CSF). There were typical findings on MRI, which include linear enhancement of the pachymeninges, pituitary hyperemia and subdural hemorrhage. We thought that these were due to epidural anesthesia first, but there was no evidence of dural puncture. It was also considered that it is influenced by change in CSF pressure, and intracranial venous engorgement may be due to Trendelenburg position for several hours. Because cranial subdural hematoma is a life-threatening complication, it is necessary to reconsider application of epidural anesthesia for laparoscopic surgery with Trendelenburg position.

Genomic sequences encoding the 3′ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.

Detection of occult cancer cells in peripheral blood or bone marrow has recently received a great deal of attention regarding the prediction of postoperative recurrence of the cancer, and for novel strategies of adjuvant therapy. This study addresses the detection of circulating tumor cells in peripheral blood in patients with gastric cancer using Quantitative RT-PCR.Common mRNA targets for RT-PCR for detection of small numbers of cancer cells in gastric cancer are CK18, CK19, CK20, and CEA. Ten milliliter of peripheral venous blood was taken from 14 healthy Japanese volunteers and 101 patients with gastric cancer. Samples were analyzed using real-time TaqMan technology and a Model 7700-sequence system. The group of gastric cancer patients included 69 individuals with curative disease on preoperative diagnosis and 32 individuals with a non-curative operation or recurrence of the disease.The number of CK19 and CK20 mRNA copies was significantly increased in patients with a non-curative operation or recurrence of gastric cancer (CK19; p=0.0087, CK20; p=0.0022) compared with healthy volunteers. Cut-off levels of CK19 or CK20 copy numbers were determined by the maximum value of healthy volunteers. For CK19, there were 61 (88.4%) negative cases and 8 (11.6%) positive cases in 69 individuals with curative gastric cancer. There was a significant difference in tumor stages between CK19 positive and negative patients with curative disease on preoperative diagnosis. For CK20, there were 59 (85.5%) negative cases and 10 (15.5%) positive cases. There was no statistical difference between CK20 positive and negative cases for all clinicopathological factors. On postoperative day 14, there was a significant difference between positive and negative cases regarding tumor size, tumor stage, and lymph node metastasis for CK19, and tumor stage and lymph node metastasis for CK20. Five-year survival rates of patients with CK19 positive or negative cases were 50.0% or 79.0%, respectively (p=0.0347). While, for CK20, 5-year survival rates for positive cases was 51.9%, and for negative cases 78.9% (p=0.0490).Micrometastases of gastric cancer can be detected in circulating peripheral blood using quantitative real-time RT-PCR. CK19 is a better marker than CK18, CK20 and CEA, and could be clinically useful to estimate prognosis or to make a postoperative strategy of adjuvant treatment.

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.

Microsatellite instability (MSI) is regarded as reflecting defective DNA mismatch repair (MMR). MMR defects lead to an increase in point mutations, as well as repeat instability, on the genome. However, despite the highly unstable microsatellites, base substitutions in representative oncogenes or tumor suppressors are extremely infrequent in MSI-positive tumors. Recently, the heterogeneity in MSI-positive colorectal tumors is pointed out, and the 'hereditary' and 'sporadic settings' are proposed. Particularly in the former, base substitution mutations in KRAS are regarded as relatively frequent. We sequenced the KRAS gene in a panel of 76 human colorectal carcinomas in which the MSI status has been determined. KRAS mutations were detected in 22 tumors (28.9%). Intriguingly, all of the KRAS-mutant MSI-H (high) tumors harbored sequence alterations in an essential MMR gene, MLH1, which implies that KRAS mutation more frequently and almost exclusively occurs in MMR gene-mutant MSI-H tumors. Furthermore, in contrast with the prevailing viewpoint, some of these tumors are derived from sporadic colorectal cancer patients. The tight connection between MMR gene mutation and KRAS mutation may suggest previously unrecognized complexities in the relationship between MSI and the mutator phenotype derived from defective MMR.

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.

A loss of heterozygosity (LOH) is a major cause of lung carcinogenesis, and it is considered to be related to tobacco smoking in central type lung cancer. We investigated the relationship between LOH in lung adenocarcinoma and tobacco smoking. In a consecutive series of 50 patients with lung adenocarcinoma who underwent a surgical resection, cancer tissue specimens and corresponding normal peripheral lung and central bronchial tissue specimens were analyzed for LOH at the regions of D3S1234 (FHIT), D3S1300 (FHIT), D9S171 (CDKN2), and D17S796 (p53) by polymerase chain reaction using four fluorescence-labeled dinucleotide markers. To examine how cells are influenced by smoking, the A549 cell line was exposed to benzo[a]pyrene (B[a]P) for 24 weeks and then was subjected to the above analysis. The LOH in cancer tissue was thus detected in four (17%) patients at D3S1234, six (14%) at D3S1300, and seven (18%) at D17S796, but no LOH was detected in any normal tissue specimens. The incidence of LOHs in cancer tissue specimens from active smokers was 21% at D3S1234, 11% at D3S1300, and 19% at D17S796, whereas that of LOHs from nonactive smokers was 0% at D3S1234, 19% at D3S1300, and 14% at D17S796. Analyzing the relationship between the pack-year index and the presence of LOH, a significant difference was found among the active smokers. Besides, in the A549 cell line exposed to B[a]P, LOH was de novo detected in one (D2S123) of the nine regions examined. The incidence of LOH could be influenced by tobacco smoking in lung adenocarcinoma, thus suggesting the presence of an important event in the carcinogenesis of this disease.

Sinonasal carcinomas arise from the respiratory epithelium that lines the nasal and paranasal cavities, and are histologically composed of either squamous or cylindrical cell carcinoma. However, molecular analysis with the purpose of distinguishing sinonasal carcinomas from other head and neck squamous cell carcinomas (HNSCCs), which arise from squamous epithelium, has been limited. Moreover, a wide range of frequency of microsatellite instability (MSI) in HNSCC has been reported. Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied microsatellite alterations in 34 patients with sinonasal carcinoma. As a control, 24 oral squamous cell carcinomas were used. MSI was detected in 14 patients with sinonasal carcinoma (41%), but not in any with oral squamous cell carcinoma (p=0.002). Furthermore, in sinonasal carcinoma, 11 out of 17 (65%) T1-T3 sinonasal carcinomas demonstrated MSI, whereas only 3 out of 15 (20%) T4 tumors demonstrated MSI. Immunohistochemically, sinonasal carcinoma showed a higher MIB-1-labeling index and more frequently showed cytokeratin 18 expression when compared with oral squamous cell carcinoma. These findings suggest that sinonasal carcinoma and HNSCC have quite different molecular backgrounds regarding carcinogenesis, and the role of MSI is relatively minor in cases of advanced sinonasal carcinoma.

Alterations of the size of microsatellite DNA sequences, namely microsatellite instability (MSI), have been demonstrated in some types of malignancies. We analyzed the MSI of five microsatellite markers in 40 cases of soft tissue sarcoma (STS) using high resolution fluorescent microsatellite analysis. In addition, we examined the expression of hMLH1 and hMSH2 proteins of DNA mismatch repair (MMR) genes by immunohistochemistry, and promoter methylation of the hMLH1 gene by methylation-specific PCR (MSP). MSI was recognized in 10 of 40 STS cases (25%), which consisted of 2 MSH-high (MSI-H) tumors and 8 MSI-low (MSI-L) tumors. A loss of hMLH1 expression was recognized in 7 of 40 STS cases (18%), and loss of hMSH2 expression was recognized in 3 of 40 STS cases (8%). One case showed a loss of both hMLH1 and hMSH2 expression. Promoter hypermethylation of the hMLH1 gene was detected in only 3 of 40 STS cases (8%). Of 10 cases with MSI, 5 (50%) showed a loss of hMLH1 and/or hMSH2 expression. There was a statistically significant correlation between MSI-positive tumors and the loss of hMLH1 and/or hMSH2 expression (p=0.0286). Although the frequency of MSI (25%) or a loss of hMLH1 and/or hMSH2 expression (23%) was relatively low in STS cases, a loss of hMLH1 and/or hMSH2 was recognized in 5 out of 10 MSI-positive cases (50%). These findings suggest that the inactivation of MMR gene expression might be the cause of MSI in STS cases.

Oxidative damage to diverse physiological molecules, including proteins, lipids, and nucleic acids, is an inevitable outcome of various cellular activities in living organisms. In particular, some oxidised forms of nucleotides cause miscoding of genetic information, and have therefore been present as a major threat for cells. Multigene systems to counteract such oxidative damage have evolved in diverse organisms. In E coli , several mutants designated ‘mutator’ have been isolated, and in these cells the mutation rate is significantly elevated, due to disruption of genes regulating the spontaneous mutation rate on the genome. Previous studies using these mutators have identified three genes that function in the system to counteract mutagenic oxidative damage. The mutT− strain is one of the mutators that exhibit the highest spontaneous mutation rate. Maki H et al have shown that the product of the mutT gene hydrolyses an oxidised form of guanine nucleotides, 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP).1 8-oxo-dGTP incorporated into the genome stably pairs with adenine as well as cytosine in the template strand, accumulation of this oxidised form of guanine nucleotides leads to an increase in base substitution mutations—that is, A:T to G:C and G:C to T:A transversions. In the mutT− strain, the rate for A:T to C:G transversion is indeed elevated 1000 fold over the wild type level.2 The other two genes that function in cooperation with mutT are mutM (fpg) and mutY , both of which encode a DNA glycosylase to excise deleterious bases on the genome.3 The former excises 8-oxo-guanine in the opposite site of cytosine on the genome, the latter removing adenine that pairs with 8-oxo-guanine. Thus, even in cells lacking MUT , G:C to T:A transversions are suppressed low.4 Multiplicity of cellular anti-mutagenic systems guarantees the spontaneous mutation rate on the genome at an extremely low …

Reported frequencies for microsatellite instability (MSI) in oesophageal cancer differ widely in the literature, perhaps due to the high incidence of loss of heterozygosity (LOH) in this cancer. Using high-resolution fluorescent microsatellite analysis (HRFMA), we analysed microsatellite alterations in detail in 50 Japanese and 50 Chinese patients with squamous cell carcinoma in the oesophagus. In HRFMA, several devices have been developed to improve the detection characteristics, reproducibility of polymerase chain reaction and the migration accuracy of electrophoresis. All the alterations observed were separable into MSI, LOH and alterations ambiguous for both. MSI was rare in these panels of oesophageal carcinomas. The frequencies of MSI in the Japanese and Chinese subjects were 8 and 4%, respectively. All the alterations were mild (within 2 base pairs) and were observed in a limited number of markers. More drastic types of MSI, such as those typical in colorectal cancer, were not observed. On the other hand, the incidence of LOH was high, reaching 50% for the Japanese and 70% for the Chinese subjects. In many of these cases, LOH was observed in multiple microsatellite markers. The frequency of LOH in each marker was not apparently biased. Although in many cases MSI and LOH were clearly distinguished with use of the sensitive and quantitative fluorescent assay, theoretically indistinguishable patterns were noted in some cases. In conclusion, MSI is rare and LOH predominates in squamous cell carcinoma in the oesophagus.

We recently developed a novel system for detecting microsatellite alteration, which is an important process in carcinogenesis. In patients with non-small cell lung cancer (NSCLC), loss of heterozygosity (LOH) is frequently observed and causes functional disorders of tumor suppressor genes.In a consecutive series of 51 patients with NSCLC who had undergone a surgical resection, microsatellite instability (MSI) and LOH in tumors were analyzed by polymerase chain reaction using five fluorescence-labeled dinucleotide markers (D2S123, D5S107, D10S197, D11SS904, and D13S175) and an autosequencer.MSI was detected in only one patient (2.0%) with only one marker. LOH was detected in at least one chromosomal region that was tested in 39 patients (76%). The mean (+/- SD) number of LOHs detected by each marker was 1.74 +/- 1.40, with 1 LOH detected in 10 patients, 2 LOHs detected in 15 patients, 10 LOHs detected in 3 patients, 1 LOH detected in 4 patients, and 3 LOHs detected in 5 patients. The number of LOHs detected in each patient was significantly associated with the pack-year index (rho = 0.501; p = 0.0004), although there was no relationship with having a history of multiple cancers and familial cancer. Patients with stage IA disease showed a significantly lower number of LOHs than did patients with other stages of disease (1.15 vs 2.38, respectively; p = 0.0013).LOH is very common in patients with NSCLC, and the number of LOHs increases with increases in smoking, suggesting the presence of an important event in lung carcinogenesis.

Background and Objectives An overexpression of c-erbB2 has been reported to be associated with a poor clinical outcome in breast cancer, however, its prognostic value remains controversial especially in patients with node negative breast cancer, and regarding the estrogen receptor (ER) status. Methods Immunohistochemical staining for c-erbB2 was performed on the primary breast cancer from 698 Japanese patients with a mean follow-up duration of 54 months. Results The c-erbB2 expression was positive in 120 (17.2%) of 698 cases, which inversely correlated with the ER status. Both univariate and multivariate analyses indicated the c-erbB2 expression to be a significant prognostic factor for the disease-free survival (DFS) and overall survival (OS), while the same efffect was also seen in the patient groups with node negative as well as node positive breast cancer. A univariate anlysis also indicated a subgroup with the positive c-erbB2 and negative ER to have both a worse DFS and OS than the other subgroups. The patients with positive c-erbB2 had a significantly worse DFS and OS than the patients with negative c-erbB2 in all patient groups stratified according to the adjuvant therapies, while the prognostic significance of c-erbB2 on DFS was also found in the patients with the node negative breast cancer who received adjuvant tamoxifen alone. Conclusions The c-erbB2 expression is an independent significant factor for breast cancer and the prognostic significance remains in the node negative as well as node positive breast cancer, while the same effect was also found in all subgroups stratified according to the adjuvant therapies. In addition, the combination of c-erbB2 and ER made it possible to identify the subgroup with the worst clinical outcome. J. Surg. Oncol. 2002;79:216–223. © 2002 Wiley-Liss, Inc.

An epidermal growth factor receptor (EGFR) has been reported to be associated with a poor clinical outcome in breast cancer, while its prognostic value remains controversial. Immunohistochemical staining for EGFR was performed on frozen sections of primary breast cancer from 1029 patients with a mean follow-up duration of 46 months. EGFR was positive in 277 (26.9%) of 1029 cases which inversely correlated with the estrogen receptor (ER) status. A univariated analysis indicated that EGFR had a significant prognostic value in both the disease free survival (DFS) and the overall survival (OS), while the same effect was also found in node negative as well as node positive breast cancer. A multivariate analysis indicated that EGFR was an independently significant prognostic factor for DFS (p = 0.0174) and OS (p = 0.0105) in all patients, but that EGFR demonstrated a prognostic significance only for DFS (p = 0.0241) in node negative and only for OS (p = 0.0333) in node positive breast cancer. When all patients were stratified for EGFR and ER, a multivariate analysis indicated that the combination of EGFR(+)/ER(-) was an independently significant factor for both DFS and OS in node negative as well as node positive breast cancer. In conclusion, the prognostic value of EGFR was demonstrated by a multivariate analysis in a large series of breast cancer patients, but the value of EGFR was somewhat insufficient to achieve statistical significance for both DFS and OS in the subgroups divided by nodal status. On the other hand, the prognostic value of combination of EGFR and ER was sufficient to achieve statistical significance based on a multivariate analysis for both DFS and OS in the subgroups of node negative as well as node positive breast cancer patients.

Background. The prognostic value of DNA ploidy has been extensively studied in breast cancer; however, the results remain controversial. Flow cytometry (FCM) studies of DNA ploidy on frozen sections have not yet been performed in a large series of Japanese women with node-negative breast cancer. Methods. An FCM analysis of DNA ploidy was performed on frozen sections of node-negative breast cancer from 653 Japanese women, with a mean follow-up duration of 46 months. Results. Three hundred and twenty-four (49.6%) patients showed a diploid tumor, while 329 (50.4%) showed an aneuploid tumor. There was a significant correlation between DNA ploidy and estrogen receptor (ER) status. Patients with an aneuploid tumor had a significantly worse outcome in terms of both disease-free survival (DFS; P = 0.0116) and overall survival (OS; P = 0.0492) than those with a diploid tumor, while the same effect, in terms of DFS, was also seen in ER-positive breast cancer. Multivariate analyses indicated DNA ploidy to be an independent prognostic factor for DFS, while DNA ploidy and tumor size were found to be independent prognostic factors for OS. DNA ploidy was also shown to be an independent prognostic factor for DFS in ER-positive breast cancer. Conclusion. Our findings demonstrated DNA ploidy, based on FCM, to have an important prognostic value in Japanese women with node-negative breast cancer.

Although the prognostic value of p53 protein has been extensively studied in breast cancer, there have so far been few immunohistochemical studies of p53 protein using frozen sections in a large series of Japanese women with breast cancer.Immunohistochemical staining for p53 protein was performed on frozen sections from 514 Japanese patients with breast cancer with a mean follow-up duration of 31 months.Two hundred and eight (40.5%) of 514 cases showed nuclear accumulation of p53 protein. There was a significant inverse correlation between p53 protein and estrogen receptor (ER) status. The patients who were positive for p53 protein had a significantly worse outcome in terms of both disease free survival (DFS) (p0.0001) and overall survival (OS) (p=0.0411) than those negative for p53 protein. The same effect on DFS was seen in subgroups divided either by nodal status or ER status. Multivariate analyses indicated that nodal status, ER and p53 protein were all independent prognostic factors for DFS. The nodal status, ER and tumor size were independent prognostic factors for OS, and p53 protein status was still an independent prognostic factor for DFS in subgroups divided either by nodal status or ER status.Our findings demonstrated the prognostic value of p53 protein expression for the early recurrence of breast cancer, and the prognostic value of p53 protein expression was independent from that of both the nodal status and ER status in breast cancer.

The incidence of gastric cancer is much higher in Japan than in other countries even though diagnostics and treatments of such patients have improved. The objective of this study was to present an overview of the past, present and future of surgical treatment for our patients with gastric cancer. We analysed data on 2152 Japanese men and women with gastric cancer who underwent surgical resection from 1965 to 1995 at Kyushu University in Fukuoka, Japan, based on a univariate and the multivariate analysis. We focused on time trends of surgical treatment and the postoperative outcome. Over the years, there have been favourable changes in the numbers of patients with early gastric cancer. In all cases of gastric cancer, the rate of 18% in the first six year period (group 1) was 57% in the last 5 year period (group 6). Size of the tumour was smaller, well-differentiated tumour tissue was more common, and lymphatic involvement was less frequent. Lymph node metastasis, liver metastasis and peritoneal dissemination all decreased. Extensive lymph node dissection was more frequently done and the rate of curative resection (curability A and B) increased. With increases in identifying the early stage of cancer and better perioperative care, mortality rates 30 days after the surgery greatly decreased. Multivariate analysis revealed that the 10 factors of depth of invasion, lymph node metastasis, lymph node dissection, tumour size, liver metastasis, peritoneal dissemination, lymphatic invasion, vascular invasion, lesion in the whole stomach and lesion in the middle stomach were independent factors for determining the prognosis. Detection of the tumour in an early stage, standardized surgical treatment, including routine lymph node dissection, close follow-up schedules and better perioperative management are expected to increase survival time for patients with this malignancy. © 2000 Cancer Research Campaign

In breast cancer, the rates of positivity for microsatellite instability (MSI), vary greatly in the literature. Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied microsatellite alterations in 75 patients with sporadic breast cancer. In this system, several devices were prepared to improve reproducibility of polymerase chain reaction products, migration accuracy of electrophoresis, and characteristics of the detection system. Precise and objective analyses of microsatellite alterations are made feasible using HRFMA. Seven of the 75 cases were judged to be positive for MSI, the rate of positivity being 9.3%. This rate is relatively low compared to the data in the literature. All the microsatellite changes observed in this system can be classified into two types: type A with relatively small changes in microsatellite sequences observed in limited loci and type B with drastic and widely dispersed changes. The former was thought to be connected to abnormal activity in DNA mismatch repair (MMR). Among the 7 cases, 6 (8.0%) had type A alterations, which means that the tumors may have an abnormal MMR activity. Application of precise and objective systems for microsatellite analysis is expected to be clinically useful to detect patients at high risk for cancers.

Angiogenesis has an important role in the growth and metastasis of solid tumors. Several angiogenic factors have been identified, one being platelet-derived endothelial cell growth factor (PD-ECGF), which is identical to thymidine phosphorylase (dThdPase). We investigated the activity of dThdPase in 84 samples of 42 human gastric cancers, by liquid chromatography. The dThdPase activity significantly correlated to the microvessel density assessed by immunostaining to CD-31 antigen (P

Deficient repair of nucleotide mismatches in the genome is considered a major factor in tumorigenesis. Such deficiency is evidenced by alterations in dinucleotide repeats of microsatellite sequences, specifically microsatellite instability (MSI) or replication errors. We investigated the frequency of MSI in human gliomas in terms of patient outcome. Frequency of MSI was estimated by examining five loci on chromosomes 2, 5, 10, 11, and 13 in 31 gliomas using high-resolution fluorescent microsatellite analysis. MSI was found at all loci in only 2 malignant gliomas (6.5%). MSI was detected at the D10S197 locus in 3 of 11 glioblastomas (27.2%) and 4 of 8 anaplastic astrocytomas (50%), while no MSI was detected in low-grade gliomas. Among patients with anaplastic astrocytoma, the 4 with MSI at D10S197 died from local recurrence less than 18 months after surgery, while 3 of the patients without MSI survived for more than 20 months. MSI at D10S197 may be a prognostic marker for patients with anaplastic astrocytomas.

To evaluate the relation between the degree of lymph node metastasis and the growth potential of tumour cells and the local immune function in gastric cancer, we analyzed data on 444 patients with advanced serosally invasive gastric cancer who underwent curative gastrectomy. Tumour growth potential was evaluated based on the value proliferating cell nuclear antigen (PCNA) in the primary tumour, and dendritic cell infiltration into the tumour was determined as an indicator of local immune function. The values of PCNA labeling in the primary tumour increased and the infiltration of dendritic cells into the tumour decreased in relation to the extent of lymph node metastasis. High growth potential and low immune function were seen in cases with n3 lymph node metastasis. There was a reverse relation between the PCNA labeling index and dendritic cell infiltration. A variety of forms of recurrence was noted in patients with lymph node metastasis while the prognosis was less favorable, in relation to the degree of lymph node metastasis. Thus, the potential for nodal spread appears to be associated with the growth potential of tumour cells and with the local immune status of the tumour.

Background. Even after curative resection of an early gastric cancer, some patients die of a recurrence. It is our view that patients with early gastric cancer who died of their disease had occult micrometastases in perigastric lymph nodes at the time of the original diagnosis. In an attempt to identify these micrometastases, lymph nodes dissected from early gastric cancer lesions were stained after operation with a monoclonal antibody against cytokeratin, an essential constituent of the cytokeleton of epithelial cells. Methods . The 420 dissected lymph nodes from 34 patients with node-negative early gastric cancer who died of a recurrence were examined for the presence of tumor cells. We used immunocytochemical techniques and an antiserum to epithelial membrane antigen. The monoclonal antibody CAM 5.2 recognizes cytokeratin polypeptides (human cytokeratin numbers 8 and 18) commonly present in epithelial cells. Clinicopathologic characteristics and prognosis were determined for patients with cytokeratin-positive cells in the lymph nodes. Results . Of 420 lymph nodes, 15 (3.6%) nodes and 23.5% (8 of 34) of the patients presented with cytokeratin-positive cells at the time of primary operation. The presence of cytokeratin positivity was not related to various clinicopathologic factors. The histologic stage of eight cytokeratin-positive cases was upstaged by the group of cytokeratin-positive lymph nodes from stage I to three of stage II, four of stage III, and one of stage IV, hematogenous recurrences were common, and the prognosis was poorer. Conclusions . Immunohistochemical techniques aid in identifying micrometastatic disease in lymph nodes missed in routine hematoxylin-eosin staining. Cytokeratin staining of the dissected lymph nodes is recommended to precisely determine tumor stage and prognosis for patients with early gastric cancer.

In 323 of 1,620 patients with gastric cancer, the lesion was > or = 10 cm in maximum diameter. Studies were done with respect to clinicopathologic features and the prognosis. Patients with tumors > or = 10 cm were younger and women patients were more numerous compared to those with tumors < 10 cm. Tumors were more advanced and noncurative resection was usually done for these patients. A multivariate analysis showed that serosal invasion, liver metastasis, lymph node metastasis, operative curability, tissue differentiation, and extended lymph node dissection to be independent prognostic factors. This retrospective study showed that patients with a larger tumor are at an increased risk for tumor advancement and that they will benefit from curative resection and extended lymph node dissection.

The clinicopathological features and prognosis of gastric cancer in 344 patients aged 70 years or older who underwent gastrectomy between 1965 and 1990 were determined. Over the years the mean size of the tumour decreased, differentiated tumour tissue was more common, depth of penetration was less prominent, lymphatic and vascular involvement was less frequent, and the rate of lymph node metastasis and peritoneal dissemination decreased. Extensive lymph node dissection was more frequently carried out and the rate of curative resection rose. Survival rates improved with early detection of gastric cancer and there was no increase in operative morbidity and mortality rates. As age alone is not a contraindication to surgery for patients with gastric carcinoma, early detection of the lesion and surgical treatment are expected to increase the survival of elderly patients with this malignancy.