In spite of administration with combined chemotherapy, more than half of patients fall into the refractory status in hematological malignancy. Various cellular and molecular markers suggesting the independent prognostic factors were identified and utilized as subclassification of refractory cases. We demonstrated that cyclinD1 over-expression in mantle cell lymphoma and CD5 positive expression in diffuse large B-cell lymphoma, which should be recommended myeloablative therapy in early phase, show distinct, significantly poor prognostic factors. Using personal molecular and cellular profiles including drug sensitivity, order-made medicine might be offered for each individual patient as an optimal therapeutic strategy.

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We investigated the correlation between tumor sensitivity to 5-fluorouracil (5-FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) using human tumor xenografts and human gastric cancer specimens. DPD levels significantly correlated with 5-FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5-FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5-FU sensitivity. The tumor DPD mRNA level may be a useful indicator in predicting tumor sensitivity to 5-FU.

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We evaluated HER-2/neu status in both primary tumor and corresponding lymph node metastasis in 56 cases of invasive ductal carcinoma of the breast by immunohistochemistry using three different antibodies (monoclonal antibodies CB11 and TAB250 and DAKO polyclonal antibody) and FISH (31 cases of primary tumor and 20 cases of lymph node metastasis) in serial tissues sections of archival materials. There was a significant positive correlation in immunohistochemical scoring between primary and lymph node metastasis with all three antibodies employed (R2=0.51, p<0.0001 for CB11, R2=0.24, p<0.001 for TAB250, R2 =0.39, p<0.0001 for DAKO). This finding indicated that the assessment of HER-2/neu status in the primary tumor specimens in the archival materials for determining the application of trastuzumab may be justified at the time of carcinoma recurrence. HER-2/neu was interpreted as amplified in 6/31 cases (19%) in primary tumor and 4/20 cases (20%) in lymph node metastasis. In primary tumor, a significant positive correlation was detected between immunohistochemistry and FISH only in CB11 (p<0.0001) but not in TAB 250 (p<0.0625) and DAKO (p=0.1056). In lymph node metastasis, a significant positive correlation was detected in CB11 (p=0.0018) and TAB250 (p=0.0012) but not in DAKO. This relatively inappropriate high immunohistochemical positivity of HER-2/neu and a lack of correlation with results of FISH analysis in breast carcinoma using a DAKO polyclonal antibody indicated that antibodies other than this polyclonal antibody should also be prospectively validated in future standardization of HER-2/ neu immunohistochemical analysis.

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c-erbB-2 protein levels in tissue extracts and sera were determined in158 patients with breast carcinoma by a sandwich enzyme immunometric assay (EIA) using monoclonal antibodies directed to the extracellular domain of the c-erbB-2 oncogene protein (ErbB-2). In an analysis of tissue extracts, 48 samples (30.3%) showed ErbB-2 levels exceeding 18.0 ng/mg protein (group A), while in 110 samples these levels were below 18.0 ng/mg protein (group B). The level of ErbB-2 in tissue extracts was significantly associated with immunohistochemistry and ErbB-2 levels in preoperative sera. During follow-up, 48 patients (30.3%) developed recurrent disease: 17 in group A (35.4%) and 31 in group B (28.2%). The sensitivity of serum ErbB-2 in patients with relapsed breast cancer was 84.6% (11/13) in group A and 43.5% (10/23) in group B. In an analysis of serum samples taken before relapse, 90.9% (10/11)of the subjects in group A and 26.7% (4/15) of those in group B were shown to be positive for serum ErbB-2. Serum ErbB-2 in group A was a more sensitive marker than other tumor markers such as CEA, CA15-3 and NCC-ST-439. Thus, the determination of ErbB-2 in tissue extracts of breast carcinoma may be useful for assessing c-erbB-2 protein expression in the primary tissue, and indicates that serum ErbB-2 may be a sensitive marker for monitoring tumor relapse.

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Nd2 is murine monoclonal antibody derived from purified mucin of human pancreatic cancer cell line SW1990. We prepared mouse/human chimeric antibody Nd2(cNd2) and reported that cNd2 could induce antibody dependent cell-mediated cytotoxicity(ADCC) in vitro. In this study, we investigated the effect of cNd2 in nude mouse, and the enhancement of ADCC of cNd2 by IL-2 stimulated lymphocytes in vitro. In nude mouse, intraperitoneal injection of cNd2 inhibited the tumor growth in xenografted model, elongated the survival in orthotopically transplanted model, and suppressed the number of liver metastasis in spleno-injected model. In vitro study, IL-2 enhanced ADCC of cNd2.

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By analyzing expression of 9216 genes in 20 esophageal-cancer patients who were treated with the same adjuvant chemotherapy, we identified 52 genes that were likely to be correlated with prognosis and possibly with sensitivity to anti-cancer drugs. We developed a "drug response score (DRS) " on the basis of differential expression of these 52 genes and found a significant correlation between DRS and individual patients' prognoses. Our results indicated that this scoring system, based on microarray analysis of selected genes, is likely to have potential for predicting the response of individual cancer patients to chemotherapy.

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We applied the cDNA microarray technique to clarify gene expression profiles in hepatoma derived cell lines and tissue lesions of the chronic viral hepatitis, liver cirrhosis and hepatocellular carcinoma. 7% of genes analyzed were differentially expressed in tissues of chronic hepatitis and liver cirrhosis compared to normal liver tissue. 3% of genes analyzed were differentially expressed in tissues of hepatocellular carcinoma compared to surrounding non tumor lesions. The hierarchical clustering analysis revealed that expression profiles in chronic hepatitis B tissue lesions differed from those in chronic hepatitis C tissue lesions. Furthermore, AFP producing cell lines shared a distinct expression profiles of genes from those in AFP non producing cell lines. Our results demonstrated the usefulness of cDNA microarray technique to evaluate the genetic alteration of chronic hepatitis and the development of hepatocellular carcinoma.

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Urine samples and tumor tissues were obtained from 68 patients with transitional cell carcinoma (TCC) of the bladder. Allelic loss on chromosome 9 and/or chromosome 17p in urine samples showed 47 (69%) cases. Especially, 92% cases with invasive cancer showed LOH (loss of heterozygosity) on chromosome 17p in urine samples. Of 30 cases with atypia and no malignancy in cytological examination, 19 (63%) cases showed LOH in urine samples. And of 14 superficial cancers showing LOH in urine samples, 9 showed intravesical recurrence after TUR. LOH in urine samples was the useful method for diagnosis of bladder cancer and was the predictive factor of recurrence.

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In this study, we examined which tumor marker reflects the status of pTNM classification in ovarian epithelial cancers. Tumor markers assayed were CA125, CA602, CA546, CA19-9 and GAT. Out of these tumor markers, CA125, CA602 and CA546 were elevated according to the tumor progression represented by T of pTNM. Although the properties such as positive rates of CA125 and CA602 were almost identical, it was in CA602 that significant differences were recognized between N0 and N1 or between M0 and M1.

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The mean serum sICAM-1 titer of 224 patients with gastric cancer was not different from that of 44 healthy controls. However, the serum sICAM-1 titer of patients with hematogenous metastasis was significantly higher than that of patients without hematogenous metastasis. The patients with a high titer showed a significantly worse prognosis than patients with low titer.

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Decreased syndecan-1 expression of colorectal cancers was regulated transcriptionally and closely correlated with lymph node metastasis or liver metastasis. In the patients with colorectal cancer, loss of syndecan -1 expression may be an independent predictive factor for prognosis and useful marker in the decision on local resection alone or additional therapy. (Preliminary Note)

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The aim of this study was to evaluate the clinical significance of serum p53-Abs in gastrointestinal cancer patients. Four-hundred and thirty-three consecutive patients with gastrointestinal cancer (esophageal cancer:147 patients, stomach cancer:135 patients and colorectal cancer:151 patients) were studied for serum p53-Abs by enzyme-linked immunoabsorbent assay before treatment. Conventional tumor markers (CEA, CA19-9, SCC-Ag and CYFRA21-1) were assessed to compare their sensitivities with serum p53-Abs. Serum p53-Abs were positive for 41.5% of the patients with esophageal cancer, 17.8% for stomach cancer patients and 39.7% for colorectal cancer patients. Relatively few patients tested positive for the other tumor markers. Surveillance of serum p53-Abs is superior to the other tumor markers for detecting early gastrointestinal cancer.

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Multiple genetic and epigenetic alterations of cancer-related genes and molecules are involved in the course of the development and progression of gastrointestinal cancers. These include telomerase activation, genetic instability and abnormalities of oncogenes, tumor suppresor genes, cell cycle regulators, cell adhesion molecules and DNA repair genes. Alterations of tumor suppressor gene such as p53 and APC are good genetic markers for differential diagnosis. Gene amplification and overexpression of oncogenes and growth factors/receptors such as c-met, c-erbB2, TGF α, EGF receptor and cyclins are biological markers of malignancy. Microsatellite instability is an indicator for high susceptibility of cancers like herediatry non-polyposis colorectal cancer as well as high risk for developing multiple cancers. Many of the genetic changes can be analyzed on paraffin-embedded biopsy specimens taken for the purpose of histopathological diagnosis. Since 1993, a system of molecular-pathological diagnosis was established and has been performed as a routine service in collaboration with Hiroshima City Medical Association Clinical Laboratory. More than 10, 000 cases of gastrointestinal biopsy and surgery have been analyzed and the reports of molecular diagnoses have been sent routinely to the clinicians. Additional new information of differential diagnosis and biological malignancy to the pure histopathological diagnosis could be obtained in about 15% of the cases by our molecular diagnosis strategy. Our diagnostic approach may contribute to the new "personalized medicine" in the next century by characterizing the individuality of cancer according to its genetic and epigenetic alterations. DNA chip technology will greatly promote the new direction of cancer diagnosis and therapeutics.

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To improve the molecular diagnosis for pancreatic carcinoma(Pca) and biliary tract carcinoma(BTCa), we analyzed K-ras and p53 gene mutations in the supernatants of pancreatic juice(PJ) and bile. K-ras mutations were examined by mutant allele specific amplification. Enriched-SSCP and direct sequencing were used for analysis of p53 mutation. In the supernatants of PJ from Pca patients, K-ras mutations were detected in 89% (17/19) and positive rate of p53 mutation was 35%(7/20). Using a combination assay with both supernatant and sediment, the mutations were found in up to 100% and 50%, respectively. In the bile supernatants from BTCa cases, K-ras and p53 mutations were detected in 40% (2/5) and 46%(6/13), respectively. In combination with sediment, positive rate for K-ras and p53 improved to 60% and 46%, respectively. Therefore, simultaneous analyses of K-ras and p53 mutations in the supernatant and sediment of PJ and bile are suggested to enhance the genetic diagnoses of Pca and BTCa.

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Human telomerase reverse transcriptase (hTERT) is regarded as a catalytic component of telomerase and a rate-limiting determinant of the enzymatic activity of human telomerase. The incidence of hTERT mRNA in pancreatic juice (PJ) revealed 6 (33%) of 18 patients with pancreatic carcinoma, whereas hTERT mRNA was not identified in PJ of 7 patients with mucin-producing tumor of pancreas and 24 chronic pancreatitis. Compared with clinicopathological parameters, the incidence of hTERT mRNA in PJ in the TS1 (less than 2 cm in greatest dimension of the PC tumor size) was 75% (3/4), although the number examined was small. These results suggest that detection of hTERT mRNA in PJ may be a useful marker for diagnosis of pancreatic carcinoma.

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SCC antigen is squamous cell specific serine protease inhibitor. To detect squamous cell carcinoma cells in peripheral blood of uterine cervical cancer patients, we applied fluorescence-based RT-PCR system to measure SCC antigen mRNA level in peripheral blood. Positive rates of SCC antigen mRNA were 80.6% (25/31) in invasive cancer and 0.7% (2/30) In healthy volunteers. Furthermore, 33% (3/9) of early stage (CIS) of cervical cancer showed positive values. The copy number of SCC antigen mRNA in cancer patients was dramatically decreased after treatment. These data suggest that this system might be useful for diagnosis and monitoring of squamous cell carcinoma.

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We have developped a simple and sensitive diagnostic method with CEA extraction for lymph node metastasis in cancer patients. CEA extracted from 67 regional lymph node groups of 9 primary lung cancer patients were examined. The amount of CEA in 5 histological positive lymph node groups (58.5±80.9 ng/ml) was significantly higher than that in 62 histological negative lymph node groups (0.60plusmn;1.45 ng/ml). Among histological negative groups, one group was CEA positive. Micrometastasis in this lymph node which were missed with standard histological examination might be detected with this method. These results implied that this CEA extraction method was feasible for the diagnosis of lymph node metastasis in lung cancer patients.

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Murine double minute-2 (MDM2) is an oncoprotein that inhibits p53 tumor-suppressor protein. Overexpression of MDM2 gene encoding a nuclear protein was shown to enhance the tumorigenic potential of cells. We made a serological survey of anti-MDM2 antibodies in sera of patients with cancers and healthy donors using EIA with synthesized MDM2 antigen. Circulating antibodies against MDM2 protein were detected in sera from patients with breast cancer, colon cancer, gastric cancer and lung cancer. In gastric cancers, immunohistochemical expression of MDM2 and p53 were observed. From results of the serological detection, we suggested clinical usefulness of circulating autoantibody to MDM2 as a tumor marker for male patients with colon cancer, gastric cancer and lung cancer and female patient with breast cancer, especially early breast cancer.

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We studied of plasma TGF-β1 levels in colon cancer patients toward their progression and significance as a tumor marker. From the results, non-activated plasma TGF-β1 levels were significantly higher than controls. And also, these levels were markedly associated with clinico-pathological stages in colon cancer. Plasma TGF-β1 levels were decreased after curative resection of the cancer. These results suggested that non-activated plasma TGF-β1 levels might be useful as a tumor marker in colon cancer patient.

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There is no adequate serum prognostic and predictive biomarker to identify the activity of renal cell carcinoma (RCC). We investigate the correlation of serum hepatocyte growth factor (HGF) levels with clinical parameters in untreated RCC patients. We measured the serum levels of HGF in 44 consecutive patients with untreated RCC and 45 healthy controls using the enzyme-linked immunosorbent assay (ELISA). Patients with RCC had significantly higher serum HGF concentrations than healthy subjects (p<0.0001). Serum HGF levels significantly correlated with tumor stage, histological grade, and prognosis of RCC. In patients with high stage and/or grade 2 RCC, higher serum HGF group had significantly worse cause specific survival (p<0.03 and p=0.0086).

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We evaluated the potential role of expression status of retinoblastoma gene protein (pRb) and cyclin D1 by immunohistochemical staining and clinicopathological features of 64 Japanese patients with transitional cell carcinoma (TCC) of the bladder. Low protein levels of pRb and cyclin D1 are associated with higher tumor stage and/or grade and with a shortened survival rate. These results suggested that pRb and cyclin D1 may be useful prognostic markers in the diseases.

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Using anti-carbonyl reductase (CR) antibody, immunohistochemical staining was performed on 73 epithelial ovarian cancers and 25 benign ovarian tumors. The combined rate for strongly and weakly positive reactions for CR was 32.0% for benign tumors and 61.6% for ovarian cancers. The CR positive rate was 35.7% for ovarian cancers with retroperitoneal lymph node (RLN) metastasis and 67.8% for those without RLN metastasis, the former being lower than the latter (p<0.05). While the expression of vascular endothelial growth factor (VEGF) and Flt-4 observed by immunoblotting was coincidently altered in the individual cancer samples, the amounts of VEGF and Flt-4 were augmented according to the decrease of CR expression.

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In this study, we immunohistochemically demonstrated that expression of plasminogen activator inhibitor-1 (PAI-1) protein in ovarian clear cell adenocarcinoma cells was affected by the presence of concomitant pelvic endometriosis and reduced the positive rate of peritoneal cytology in patients with ovarian clear cell adenocarcinoma. The prognosis of them with highly expressed PAI-1 tended to be better than that of those with lowly expressed one. Theses data suggest that the expression of the PAI-1 have some influence on progression of the ovarian clear cell adenocarcinoma.

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Fine-needle aspiration specimens from 102 breast lesions, including 10 ductal carcinoma in situ (DCIS), 78 invasive ductal carsinoma, 7 intraductal papilloma (IDP), 6 fibroadenoma and one mastopathy, were examined for numerical aberrations on chromosomes 1, 11, 17 by using FISH technique. Numerical aberrations on these chromosomes were detected most frequently in DCIS while only one out of 7 IDP cases showed abnormality. FISH analysis seems to be useful for diagnosis of intraductal breast lesions.

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We examined the frequency of several short forms of spliced Murine double minute 2 (MDM2) transcripts and their correlation with clinicopathological features in human breast cancer. There was no significant correlation between the expression of splice variants and tumor size or hormone receptor status. However cases with spliced MDM2 transcripts tended to be of a more aggressive type with axillary lymph node involvement and extensive necrosis in the tumors. We anticipate that the variants will be confirmed as a novel prognostic marker.

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The elevation of α1, 3fucosyltransferase activity in sera from patients with various types of malignancy have been investigated. To identify the origin of such an enzyme, blood samples were obtained from both the drainage vein and the artery in colon cancer patients and their α1, 3fucosyltransferase activities were measured. The enzyme levels in the drainage vein were observed to be higher than those in the artery circulated. The elevated enzyme activities were found to reduce after curative removal of tumors. Further, most of the enzyme activities elevated in both samples were neutralized by the addition of anti-FUT6 rabbit serum raised against the human FUT6 enzyme. It was therefore suggested that the origin of serum αl, 3fucosyltransferase whose activities elevated in cancer must be tumor rather than liver which has been reported to be the origin of serumα1, 3fucosyltransferase, and that the tumor-associated enzyme was coded mainly by the FUT6 gene.

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We found a GlcNAc:-6sulfotransferase (SulT-b) in human colonic mucinous adenocarcinoma. SulT-b is biochemically different from the sulfotransferase in human normal colonic mucosa (SulT-a). SulT-b is expected to be a good marker for the detection of mucinous adenocarcinomas.

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Previously we showed that the sialyl 6-sulfo Le^x determinant, the L-selectin ligand on high endothelial venules in lymph nodes, is also expressed in human colonic tissues. The sialyl 6-sulfo Le^x determinant was preferentially expressed in non-malignant colonic epithelia than in cancer cells. We next investigated the amount of mRNA for 6-sulfotransferases (6ST) isoenzymes in human colorectal cancer tissues. The most remarkable finding was that the message of HEC-GlcNAc6ST was prominently increased in cancer tissues compared to nonmalignant mucosa. On the other hand, I-GlcNAc6ST mRNA expression was significantry higher in non-malignant mucosa rather than in cancer tissues.

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N-acetylglucosaminyltransferase V (GnT-V) catalyzes the formation of β1-6 branching in N-glycans which is linked to tumor metastasis . In the present study, expression of GnT-V was investigated in 99 cases of hepatocellular carcinoma (HCC) tissues and 103 cases of colon cancer tissues, using immunohistochemical methods. While GnT-V was not expressed in normal liver, it was enhanced in HCC tissues, especially a differentiated type. In colon cancer tissues, approximately 26% cases showed positive staining of GnT-V, which was highly associated with the distant metastasis. These results indicate that GnT-V could be useful for a tumor marker for HCC and colon cancer.

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We investigated molecular basis of transcriptional regulation of Lewis type 1 antigen synthase (β3Gal-T5) gene using luciferase assay and gel-shift assay, and examined expression level of β3Gal-T5 transcript in normal and cancerous colonic tissue with real-time RT-PCR method. The data strongly suggested that intestine-specific homeobox proteins were involved in the regulation ofβ3Gal-T5 gene.

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We have established an ELISA system for assessing the tumor-associated antigen MK-1 and determined serum levels of MK-1 in cancer patients, which had scarcely been elucidated. MK-1 was increased in sera of about 10 percent of the patients tested, while it was undetectable in sera of healthy volunteers, suggesting that MK-1, a transmembrane protein, can be released from tumor cells in certain cases. This should be taken into consideration when MK-1 is used as a target molecule for in vivo immunodetection or immunotherapy.

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We have established a cell line, KY, of human ovarian clear cell carcinoma. An in vitro study using APODIREKT KIT demonstrated apoptosis induced by paclitaxel and etoposide, although no significant effect was observed in case of cisplatinum (CDDP). On the other hand, the 3 drugs showed significant antitumor effects in nude mice bearing KY tumor. These results suggested that CDDP might activate immunological system in animals. Immunohistochemical studies were performed using antibodies recognizing the products of p53, bax and bcl 2. The results revealed that mutant p53 was expressed in KY, but others were not, and only KY tumors treated with paclitaxel expressed bax. Paclitaxel seemed to induce apoptosis without wild p53.

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Structural changes of glycoconjugates on the cell surface have been observed in association with malignancy. Le^b and Y antigens are abnormally expressed in colorectal tumors which could be specifically detected by YB-2 monoclonal antibody established recently. Mouse colon26 cells in which these difucosylated antigens are never expressed were transfected with human FUT genes and alteration of their cellular characteristics was investigated. Increased resistance to apotosis caused by UV irradiation, serum withdrawal from medium and 5-FU treatment was demonstrated in FUT1 and FUT3 transfected colon26 cells. These transfected cells were then induced to react with YB-2 antibody and suffer a high complement-dependent cytotoxicity.

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A series of chemically synthesized oligosaccharides have been examined for developing drags which show anti-peritoneal dissemination activities using mouse greater omentum and gastric and colorectal cancer cells. A new ex vivo assay method has also been developed and it could be conveniently used for evaluation of each oligosaccahride with a relatively longer incubation period. Galβ1, 3[3OMeGalβ1, 4GlcNAcβ1, 6] αBn was found to possess the strongest anti-adhesion activity bewteen MKN 45, colon26 cells and greater omentum even though the compound did not show any anti-proliferation activities against those cells at all. Further, the formation of tumors in the greater omentum of Balb/c mice which were intraperitoneally inoculated with colon26 and Galβ [3OMeGalβ 1, 4GlcNAcβ1, 6] GalNAcαBn was observed to be significantly reduced in comparison with that in mice inoculated only with colon26. It must therefore be possible to develop anti-adhesion therapy with this novel oligosaccahride.

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Interleukin 12 (IL-12) plays an important role in anti-tumor immune response by the induction of interferon- γ (IFN γ) production by Tcells and NK cells, and by activation of cytotoxic T cells and NK cells. IL-12-induced IFN- γ production was evaluated in vitro in peripheral blood mononuclear cells from hepatocellular carcinoma patients(HCC), chronic hepatitis patients(CH), liver cirrhosis patients (LC) and healthy individuals (HI). There were not significant differences in clinical background between HCC and LC. HCC exhibited the reduced responsive IFN γ production compared to CH, LC and HI. IFN γ production correlated with neither a-fetoprotein nor PIVKA-II.

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We compare several urinary tumor marker (BTAtest, BFP, NMP22) with voided urine cytology to determine the usefulness for diagnosis of urinary tract cancer .These urinary marker are simple to perform and are more sensitive than urine cytology for detecting urothelial cancer.

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Lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein(AFP-L3)has been reported to be a useful marker in the early diagnosis of hepatocellular carcinoma (HCC).The aim of this study was to clearify the relationship between the tumorous tissue level of the AFP-L3 and the clinicopathological factors.In the present study, there was no significant correlation between the AFP-L3 level in tumorous tissue and tumor size, histological grade, capsular invasion and serum level of the AFP-L3.

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Overall survival rate in AFP-L3 positive patients after treatment was significantly lower than that in negative patients. No significant difference in overall survival rate was observed between AFP-L3% negative patients after treatment and patients showing a complete response depicted by diagnostic imaging. AFP-L3% after treatment was the most significant prognostic factor for patients with HCC after treatment.

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