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To screen colorectal cancers, fecal occult blood test(FOBT), conducted to detect occult blood present in the feces, has been shown to reduce colorectal cancer mortality in many large-scale colorectal cancer screening programs. However, the sensitivities of FOBT for early-stage colon cancer, colon cancer located at the right colon and colorectal adenoma tend to be low. Additionally, some colorectal cancers which could not detect by repeated FOBT screening are actually existed. Beyond those disadvantages, FOBT is the most promised colorectal cancer screening tool for large-scale cohort. To overcome the weak points of FOBT to screen colorectal tumors, the strategy for capturing and recovering tumor-derived DNA in feces has been developed and applied for clinical usage. In this study, we summarized the series of strategies for capturing and recovering fecal tumor-derived DNA to screen colorectal neoplasia.

To date, liquid biopsies have generated much interest as they involve minimally invasive blood tests, pro- vide an ongoing picture of a patient's cancer, and offer valuable insight into the best treatment. Liquid biop- sies detect circulating tumor cells (CTCs), fragments of tumor DNA and exosomes that are shed into the blood from the primary tumor and from metastatic sites. Liquid biopsies offer what tissue biopsies cannot due to risks to the patients and costs. Liquid biopsies allow the monitoring of genomic changes in tumors for the diagnosis of early and recurrent cancer and drug effects. In the future, instead of extensive imaging and invasive tissue biopsies, liquid biopsies could be used to guide cancer treatment decisions and even screen for tumors that are not vet visible on imaging.

Cancer is known to be a genetic disease, which is caused by abnormalities of oncogenes and/or tumor-suppressor genes. Genetic abnormalities include mutation, deletion or amplification of DNA sequences. Based on the findings of the genetic abnormalities in malignant tumors, many molecular-targeting agents, e.g. Bcr-Abl kinase inhibitors and EGFR kinase inhibitors, have been developed and approved. In addition to genetic abnormalities, epigenetic abnormalities, e.g. DNA methylation, histone methylation and histone acetylation, are also involved in carcinogenesis and tumor development. Based on the findings of the epigenetic abnormalities in malignant tumors, the novel epigenetic molecular-targeting agents, e.g. DNA(DNMT) methyltransferase inhibitors and histone deacetylase (HDAC) inhibitors, have also been developed and approved. Moreover, histone methyltransferase(HMT) inhibitors and histone demethylase(HDM) inhibitors have been also discovered and some agents are in clinical trials.

The fecal occult blood test (FOBT) is widely used for colorectal cancer (CRC) screening to reduce the mortality rate associated with this cancer. However, several problems exist, as FOBT results can contain some false-negative CRC patients and some-false positive healthy subjects. Thus, to resolve these problems, several fecal biomarkers based on fecal protein, fecal DNA, and fecal RNA have been reported. Fecal calprotectin, which indicates intestinal bleeding or inflammation of the colon mucosa, and fecal tumor M2-PK, which is produced by cancer cells, have been extensively investigated as fecal protein biomarkers. To detect small amounts of CRC-specific proteins, the chemiluminescent enzyme immunoassay (CLEIA), which is a highly sensitive protein detection method using immunomagnetic beads, will be used. DNA mutation of APC, KRAS, and TP53 genes and DNA methylation of VIM, TFPI2, BMP3, NDRG4, and SFRP2 genes were reported as fecal DNA biomarkers. Consequently, a fecal DNA test named Cologuard from Exact Sciences was approved by the FDA in August 2014. Fecal COX2, MMP7, miR-106a, miR-92a, and miR-223 were also reported as fecal RNA biomarkers. This review article summarizes fecal biomarkers using fecal samples for CRC diagnosis.

Cells maintain their homeostasis by checkpoint mechanisms that exist at each phase of the cell cycle. Impairment of the checkpoint mechanisms induces DNA mutation and asymmetric segregation of chromosomes, which result in genomic instability, leading to malignant cell transformation. DNA-damaging agents, which are still the first-line chemotherapeutic drugs for various cancers, have recently been demonstrated to induce death in cancer cells alone just because these cells have abnormalities in their checkpoint mechanisms. The mitotic checkpoint, in particular, has been found to play a key role in the drug-induced cell death. An understanding of the molecular mechanisms of chemotherapy will enable us to use conventional drugs more appropriately for cancer treatment.

The author developed a sensitive yeast-based color assay which expresses APC-ADE2 (reporter) fusion protein in yeast and can screen almost the entire coding region of the APC gene. In this assay, the wild-type APC coding sequence of 8.5 kb is divided into 5 overlapping regions which are respectively ligated in-frame with an ADE2 open reading frame. The resulting five constructs containing a part of wild-type APC gene preserve the ADE2(+) phenotype (white yeast colony) when introduced into the yeast, whereas the yeast transfected with plasmids containing frameshift mutations of the APC gene shows an ADE2(-) phenotype (red yeast colony). Six human colon cancer cell lines were analyzed by this yeast color assay. HCT116 cells with wild-type APC and normal colonic mucosa gave low percentages of red colonies (0-9.9%) in all the regions. On the other hand, more than 96% red colonies were observed in one of the five regions in SW480, Colo201 and Colo320DM cells. Sequence analysis demonstrated the clonal APC mutations at codon 1,338 in SW480, 1,554 in Colo201 and 811 in Colo320DM. Moreover, the assay detected a germline mutation of the APC gene in polyps of a familial adenomatous polyposis (FAP) patient which gave about 50% red colonies. For testing the assay for clinical utilization, 18 colon cancer tissues were subjected to the assay. Eleven cancers (61%) gave more than 10% red colonies (17-57%) and clonal mutations were detected in all these samples. The same mutations were demonstrated in both DNA and RNA samples derived from idendical tissues. These results suggest that this APC yeast color assay is powerful means for detection of APC mutations in clinical samples.

To compare clinical and histopathological findings to tumor DNA copy number changes, comparative genomic hybridization (CGH) was performed on 18 primary oral squamous cell carcinomas. Copy number increases were most frequently observed on 8q, 3q, 13q, 11p, and 11q, while copy number decreases most frequently on 10q, 1p, 18q, 9p, and 19q. Copy number changes in relationship to WHO grading were examined with the result that DNA copy number increase on chromosome 6p23-25 was characteristically observed in the groups of Grade II and Grade III, and DNA copy number decreases on chromosomes 9p21 and 11p11-13 were observed in the same groups. Furthermore, comparison of DNA copy number changes to TNM classification indicated that the decreases on chromosomes 1p36 and 10q25-26 might be related to tumor progression. Moreover, the relationship of DNA amplification or deletion to metastasis was investigated. It was found that the majority of the metastasis-positive tumors showed increases on 3q26 and 17q12-21 and showed decreases on chromosome 18q21. The data suggested that these DNA copy number changes on each chromosome in the three categories might be associated with tumor cell differentiation, tumor size, and lymph node metastasis.

The cellular DNA content of certain malignancies is regarded as a prognostic parameter. The mutant p53 is thought to destabilize centrosome replication, which leads to aberrant mitosis and chromosome instability. We investigated the relationship among DNA ploidy pattern type, numerical aberrations of chromosome 7 and p53 overexpression in 20 transitional cell carcinomas of the urinary bladder. The DNA ploidy pattern type was determined by laser scanning cytometry, while p53 overexpression was investigated immunohistochemically. Using fluorescence in situ hybridization with chromosome-specific probes, the copy number of chromosome 7 was counted by touch preparations of interphase nuclei. The cytometric analysis revealed that the DNA patterns were highly correlated with both the numerical aberrations of chromosome 7 (p = 0.0002) and the overexpression of p53. The incidences of p53 overexpression in DNA aneuploid tumors and DNA diploid ones were 78% and 10%, respectively (p = 0.0017). Our results suggest that the overexpression of abnormal p53 protein induces DNA aneuploidy in bladder cancer.

The deletion of region 36 in the short arm of chromosome 1 (1p36) was investigated in 18 patients with oral squamous cell carcinoma by means of the double-target fluorescence in situ hybridization (FISH) method. 1p36 has been suggested to harbor a putative tumor suppressor gene, and thought to carry several genes such as p58, FRAP and TNF-R II. The deletion of 1p36 may play a part in cell regulation, apotosis, promotion and progression of carcinoma cells. The double-target FISH was completed by the use of a biotin-labeled chromosome 1 centromeric repetitive probe and a digoxigenin-labeled 1p36 probe in biopsied specimens obtained from these patients before the treatment. The ploidy pattern of the nuclear DNA of these specimens was also examined by flow cytometry. The deletion of 1p36 was observed in 3 patients with poor prognosis showing aneuploidy in their nuclear DNA. These results may indicate that the deletion of 1p36 can be a reliable factor in the poor prognosis of patients with oral squamous cell carcinoma.

The prognosis for pancreatic cancer is extremely poor, because there are usually invasion of surrounding tissues and metastases to lymph nodes, liver or peritoneum at the time of diagnosis. Many studies from the molecular-biological stand point have demonstrated this poor prognosis. However, the mechanisms underlying these invasive and metastatic capabilities have not yet been clarified. We reviewed several reports for prognostic factors of pancreatic cancer in molecular-biological examinations, and introduced our recent study on the adhesion molecule CD44. The CD44 variant 6 (v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors. We examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 v6 and v2 was observed only in tumor cells, if at all. The expression of CD44 v6 and v2 was correlated with decreased overall survival. A significant correlation was obtained between CD44 v2 and vessel invasion. These results suggest that CD44 v2 and CD44 v6 may be useful markers of a poor prognosis.

Fifty-nine primary breast cancers were analyzed by flow cytometric cellular DNa-RNA contents stained by acridine orange simultaneous DNA-RNA double staining. Forty-three cases (72.9%) out of 59 had abnormal stemlines (DNA aneuploidy), and 16 normal ones (DNA diploidy). RNA indices widely ranged from 1.41 to 9.02 (2.99). There was no correlation between DNA indices and RNA indices. The patients with DNA diploidy had a better prognosis than those with DNA aneuploidy, but the differences were not significant. The patients with a high RNA index of more than 4.0 had a significantly poorer prognosis than those with an index of less than 4.0. Those results suggest that RNA contents, and DNA contents are independent prognostic factors, and especially RNA contents may be a good prognostic factor in long-term survival in breast cancer.

Purpose: The present study was undertaken to investigate the possibility of determining a prognosis for gastrointestinal tract leiomyosarcoma with the use of DNA analysis and MIB-1 staining., Subjects and Methods: Malignant tumors originating in smooth muscle of the gastrointestinal tract, surgically excised from 23 lesions in 17 patients (stomach; 8 cases, 12 lesions; small intestine: 6 cases, 8 lesions; colon: 3 cases, 3 lesions) and embedded in paraffin, were examined. DNA was analyzed using flow cytometry to produce a DNA histogram, and aneuploidy and diploidy were found. MIB-1 staining was done in conformity with the ABC method., Results: 1. An investigation of prognoses using the Kaplan-Meier method revealed a tendency for more favorable prognoses in patients determined to be aneuploid through DNA analysis. However, this was not significantly better than those exhibiting diploidy. 2. All patients who died had a MIB-1 staining positivity rate of over 10%, while all patients who had no recurrence within one year or survived had a MIB-1 staining positivity of less than 10%. 3. No consistent trends were observed between MIB-1 positivity rate and DNA analysis, MIB-1 positivity rate and size of tumor, or DNA analysis and size of tumor. 4. The MIB-1 positivity rate of patients with remote metastases was significantly greater than that of patients with no remote metastases., Conclusion: From the fact that patients with MIB-1 positivity rates of greater than 10% had a poor prognosis, while those with rates of less than 10% had a favorable prognosis, we conclude that a MIB-1 positivity rate of 10% is an important value in determining the prognosis of patients with gastrointestinal tract leiomyosarcomas.

It is not easy to make a differential diagnosis among adenoma, hyperplasia and carcinoma with hyperparathyroidism (HPT). We investigated the flowcytometric nuclear DNA content and analysis of cell cycle for 29 patients (39 parathyroid glands) with HPT and 16 normal parathyroid glands at the Department of Surgery II, Fukushima Medical College. Flowcytometry was performed by EPICS 751 flowcytometer (Coulter Co.) in paraffin-embedded tissue. Each fraction of cell cycle was analyzed by the PARAI software program. All controls and secondary hyperplasia showed diploid. Aneuploid pattern was found in 27.6% of all HPTs (50% of carcinomas, 30% of adenomas and 6.7% of primary hyperplasia). The S phase fraction (SPF) and proliferative index (PI) were increased in carcinomas. PIs, especially the fraction of G2M, in primary and secondary hyperplasias were more decreased than those in normal controls and adenomas. Preoperative intact-PTH, c-PTH, ionized calcium, ALP and weight of parathyroid gland had no significant correlation with DNA ploidy, DNA index and SPF. PI had tended to correlate only with the weight of parathyroid gland. When DNA content shows aneuploid, high SPF or PI in parathyroid adenoma and hyperplasia, strict follow-up is required because of the malignant potential. If G2M or PI is lower, we should pay attention not to leave any remaining glands in parathyroidectomy in the light of hyperplasia.

The DNA ploidy pattern from fresh frozen specimens and survival rate was investigated in 91 primary breast cancers. Diploid patterns were found in 32 (35.2%) and aneuploid patterns in 59 (64.8%). The 5-year overall survival rate was significantly lower in aneuploid cases (76.3%) than diploid cases (93.8%) (p = 0.042), while there was no significant difference in disease-free survival between the two groups. there were negative nodes, no significant differences in 5-year overall or disease-free survival between patients with diploidy and aneuploidy. In contrast, when there were positive nodes, the 5-year overall and disease-free survival rates in patients with aneuploidy were 60.6% and 48.5%, which were significantly lower (p = 0.048 and p = 0.030) than the corresponding percentages of 92.3% and 84.6%, in those with diploidy. When the ploidy pattern was compared with other factors, a very close correlation was found between the ploidy pattern and histological grading (p < 0.0001). The ploidy pattern determined by flow cytometric DNA analysis may reflect the grade of malignancy of the breast cancer.

We examined the relationship between apoptosis induced by 5-fluorouracil (5-FU) that was given preoperatively to colorectal cancer patients and DNA ploidy pattern, and investigated the cell cycle changes, and the expression of Ki-67. Twenty-nine patients with advanced colorectal cancer were divided into four groups, 3 days, 5 days, 7 days, and 10 days. Groups received continuous intravenous 5-FU at 500 mg/body/day preoperatively. Then, patients were divided into two groups by DNA ploidy pattern, diploid(D) and aneuploid(A). Apoptotic cells were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The expression of Ki-67 was examined by immunohistochemical staining. We used flow cytometry (FCM) for analysis of cell cycle distribution. Apoptosis of cancer cells was mostly increased in 7 days 5-FU administration in both D and A groups. The expression of Ki-67 was reduced according to the prolongation of the term of 5-FU administration in both D and A groups. We assessed S-phase fraction (SPF) to evaluate the cell cycle changes by 5-FU. Tumor samples of all patients after injection of 5-FU showed S-phase accumulation. The ratio of SPF (after 5-FU/before 5-FU) was the highest in the 5-day 5-FU administration group in both D and A groups. We concluded that apoptosis and S-phase accumulation were increased, and proliferative activity was decreased by preoperative 5-FU administration in colorectal cancer patients. However, there was no clear correlation between DNA ploidy pattern and these changes.

Orbital or ocular metastatic tumors may originate from breast cancer. Few studies have been made regarding their histopathological classification. A 71-year-old female noted a tumor in the right orbital region. She had had bilateral breast cancer 2 years before and gastric cancer 5 months before. Histopathology had shown stage II invasive ductal cancer (scirrhus) in the right breast and stage III invasive lobular cancer in the left. Signet-ring cells were present in the breast and gastric cancers. Biopsy of the right lower eyelid showed poorly differentiated adenocarcinoma with signet-ring cells. Indian file pattern, which is specific for invasive lobular cancer, was also present, suggesting that the orbital tumor had metastatized from the left breast cancer. Genetic analysis of the gastric cancer using polymerase chain reaction showed a mutation at exon 8 of the p53 tumor suppressor gene, indicating the cancer to be metastatic. These results led to the conclusion that invasive lobular cancer of the left breast was the primary lesion for the gastric and orbital metastases. This case also illustrates that signet-ring cells, which are usually seen in gastric cancer, may be present in invasive lobular breast cancer and in orbital metastasis.

After resection of a non-invasive thymoma, two metastatic lung tumors were found in the left upper lobe and the left lower lobe. There were differences in tumor doubling time (TDT) and invasion between the metastatic tumor in the lower lobe and the one in the upper lobe. The TDTs of the tumors were 834.1 days and 328.3 days, and the tumor of the left lower lobe invaded the left lateral basal segmental bronchus (B9). The differences in the two tumors were determined by pathological findings and nuclear DNA pattern. Pathologically, the resected thymoma and both of the metastatic tumors were mixed type, which consisted of epithelial cells and lymphocytes. But by epithelium form, the resected thymoma and the tumor in the lower lobe were classified as cortex type, and only the slow growing tumor in the upper lobe contained a spindle-cell component. The DNA pattern of the resected thymoma was aneuploid and the two metastatic tumors were diploid. As this case is very interesting clinically and pathologically, we reported it.

Seventy one patients with renal cell carcinomas were examined for a variety of markers associated with tumor malignancy: nuclear DNA ploidy, AgNORs, PCNA and c-erbB-2. Usefulness of the markers in predicting the prognosis was studied by analyzing the relationship between each of these markers and the prognosis of the patients with renal cell carcinomas. DNA ploidy was analyzed by flow cytometry. AgNORs were stained by the silver colloid method. PCNA and c-erbB-2 were detected by immunohistochemistry. In all the patients examined, DNA ploidy, AgNORs, PCNA and c-erbB-2 were significant predictors of the prognosis. Of the patients with grade 2 carcinomas, the survival rate was significantly higher in the patients with the PCNA-positive cells of lower than 35.0% than in those with the positive cells of more than 35.0%. The patients without the expression of c-erbB-2 exhibited a significantly higher survival rate than those with the expression. In the patients with grade 2 carcinomas, however, neither DNA ploidy nor AgNoRs was a significant predictor of the prognosis. These findings suggest that PCNA and c-erbB-2 provide more accurate information than the others to understand the biological characteristics of the grade 2 carcinomas and are useful in predicting the prognosis of the patients with grade 2 renal cell carcinomas.

Fluorescence in situ hybridization with biotinylated repetitive DNA probe specific for the centromeric region of chromosome 17 (p17H8: Oncor) was applied to suspended nuclei which were isolated by Shutte's method from formalin-fixed paraffin-embedded tissue. The tissues were obtained from surgically resected specimens from nine patients with non-small cell lung carcinoma. The isolated nuclei were prepared with 0.05% pepsin/0.1NHCl for 15 minutes at 37 degrees C. Subsequently, these were immersed in 70% acetic acid for 10 seconds at room temperature. After heat denature with hybridization mixture which contained 3 mu 1 DNA probe for 10 minutesat 70 degrees C, 1 x 10(6) nuclei were incubated overnight at 37 degrees C. After washing with 60% formamide/2 x SSC, the hybridized probes were labeled by FITC conjugated avidin. A number of centromeric signals of chromosome 17 wasevaluated by fluorescence microscopy (BH-2, Olympus). Furthermore, a probe-related FITC intensity was quantified using flow cytometry (FACScan, Becton Dickinson). As the results, there was good correlation between a relative fluorescence intensity determined by flow cytometry and a relative fluorescence signal by fluorescence microscopy (p < 0.05).

The DNA ploidy patterns of fresh specimens from 145 patients with squamous cell carcinoma of the oral cavity and the pharynx were analyzed. The specimens were embedded in paraffin and the DNA ploidy patterns were analyzed mainly by flow cytometry. Aneuploid patterns were found in 70%, and they were found more frequently in hypopharyngeal carcinomas than in oral cavity carcinomas. Correlation between DNA ploidy and tumor size was statistically significant but metastasis to lymph nodes and clinical stage were not correlated. In stage I and II oral cavity carcinomas and stage III and IV epi and oropharyngeal carcinomas, survival was much poorer in the aneuploid than in the diploid group. Analysis of 30 patients showed better response to preoperative chemotherapy in the aneuploid than in the diploid group; the mean efficacy rates were as high as 68% in the aneuploid and 48% in the diploid group. Aneuploidy with a low DNA index did not respond well to chemotherapy compared with those with a high DNA index. DNA heterogeneity was found in 22% (4/18) of the oral cavity carcinomas and in 50% (6/ 12) of the hypopharyngeal carcinomas.

Background: The object of this study is to evaluate the efficacy of selective renal tumor biopsy in patients with renal cell carcinoma. The accuracy of histological diagnosis and DNA content of biopsies in predicting those of surgical specimens was assessed., Methods: From 1985 to 1994, 41 patients with renal cell carcinoma underwent selective renal tumor biopsy before partial or radical nephrectomy. DNA content of renal cell carcinoma in 41 patients in biopsy specimens and surgical specimens were examined in paraffin blocks with static cytofluorometry (SCM) and flow cytometry (FCM)., Results: In 39 out of 41 cases, by SCM, as well as in 37 out of 41 cases by FCM, DNA content of biopsy could be analyzed. Findings of selective renal tumor biopsy for RCC coincided with those of surgical specimen in 27/39 (accuracy 69%, kappa 0.398) in cell types, 29/39 (accuracy 74%, kappa 0.432) in structural types, 26/39 (accuracy 67%, kappa 0.341) in gradings and 27/39 (accuracy 69%, kappa 0.532) in ploidy patterns., Conclusion: It was assumed that DNA histograms might help the histological diagnosis by biopsy.

We studied the relationship between DNA index, numerical abberation of chromosome 17 and alterations of the p53 gene in 23 non-small cell lung cancers. Diploid and aneuploid cells from 23 non-small cell lung cancer with DNA aneuploidy were flow sorted into each cell population using FACStar(plus). They were examined by PCR-SSCP analysis for p53 mutation and by microsatellite analysis for loss of heterozygosity at TP53 locus (17p13.1). They were also analyzed by FISH for copy number of chromosome 17. p53 mutations were found in aneuploid cells from the 11 cases (48%). Among them, 8 cases were informative at TP53 locus, and all showed loss of heterozygosity. Aneuploid cells from 16 cases exhibited gain of chromosome 17 copy number in FISH analysis. DNA index was significantly associated with th mean copy number of chromosome 17, suggesting that the number of chromosome 17 changed with DNA index. There were no associations between p53 mutation and DNA index or mean copy number of chromosome 17. In some cases, we also analyzed structural abberation of short arm of chromosome 17 by FISH using p53-cosmid (17p13.1) and Distal 17p (17p13.3-ptel) probes. The p53 gene located on the increased chromosome 17 was lost due to point mutation or deletion of 17p.

We report a protocol which can analyze DNA by the dideoxy method. First, we prepared DNA from paraffin specimen of colon cancer and normal tissue by the method using proteinase and phenol. Polymerase chain reaction (PCR) was performed as follows. The primers used were oligonucleotides corresponding to the sequence of exon 5 on p53. An initial denaturing step was carried out at 94 degrees C for 2 min. Products were amplified for 40 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Specific PCR products derived from p53 gene were purified. Protocol for the PCR-sequencing reaction: The reaction mixture was divided into four 4 microliters fraction. Each fraction was mixed with 2 microliters of NTP solution including non-RI dideoxynucleotides (TOYOBO). PCR was carried out as follows: an initial denaturing step at 94 degrees C for 1 min, then 30 cycles at 94 degrees C for 1 min, 60 degrees C for 1 min, and 72 degrees C for 1 min. Prior to loading in a denaturing 8% polyacrylamide-6M Urea gel, the samples were heated to 94 degrees C for 2 min then quickly chilled in ice-water. Electrophoresis was carried out at 1000V for 3hr and transcribed to a nylon membrane. The ladders of DNA were obtained by Non-RI Detection Kit (TOYOBO). We determined the sequence of 167 nucleotides. Results indicated that the point mutations in DNA could be easily detected.

Early Recurrence of Hepatoma: PCNA Labeling Index and DNA Ploidy Pattern Sixty-four cases of recurrent hepatocellular carcinoma (HCC) after hepatectomy were divided into two groups; E-group with recurrence within one year, and L-group with recurrence after 1 year. Clinicopathological features and surgical curability were the same in both groups. E-group had significantly higher positive rates of portal invasion, intrahepatic metastasis and rate of patients with more than 40% on PCNA labeling index. While the similar recurrence mode and the same treatment modalities were done, cumulative survival rates after recurrence in E-group had a poorer prognosis than L-group. These results suggest the possibility of lower response for the treatment on the recurrent lesion would be manifest in the E-group. New modalities for prevention of early recurrence of HCC after resection should be developed.

Twenty-one cases of surgically resected early gastric carcinoma with lymph nodal involvement (4 mucosal and 17 submucosal carcinomas) and 37 cases of that with no lymph nodal involvement (14 mucosal and 23 submucosal) were investigated by means of flow cytometry and immunohistochemical staining in order to clarify the correlation between lymph node metastasis and DNA ploidy pattern as well as overexpression of p53 protein. DNA aneuploidy was found to show a significantly higher frequency in submucosal carcinomas (60.0%) than in mucosal ones (22.2%), and also a significantly higher frequency in node positive cases (76.2%) than in node negative ones (32.4%). Meanwhile, the overexpression of p53 protein showed higher frequency in submucosal carcinomas (52.5%) than in mucosal ones (22.2%), and also higher frequency in node positive cases (47.6%) compared with that in node negative ones (40.5%). However there was no significant difference either in relation to the depth of tumor invasion or the lymph node metastasis. Thus, DNA aneuploidy showed a significant correlation to the depth of cancer invasion as well as lymph node metastasis, which was regarded as a useful indicator for the preoperative estimation of the depth of tumor invasion as well as lymph node metastasis.

We examined microsatellite instability (MSI) and loss of heterozygosity (LOH) in regions of several important genes in 25 signet-ring cell carcinomas of the stomach. The relationship between microsatellite analysis and DNA ploidy pattern was also investigated. MSI was observed in 15% (2/13) of early carcinomas and in 17% (2/12) of advanced carcinomas. Although LOH in the region of APC gene was found in 16% (4/25) and LOH of p53 was found in 12% (3/25), 15% (2/13) of early carcinomas and 33% (4/12) of advanced carcinomas showed LOH in regions of E-cadherin gene. Cyto-fluorometrical study revealed that 85% (11/13) of early carcinomas were diploid pattern, and aneuploid components were demonstrated in 50% (6/12) of advanced carcinomas. However, no MSI-positive cases contained aneuploid components, and in contrast, all p53-LOH cases contained aneuploid components. Our results suggest that gene abnormalities which have been frequently reported in differentiated adenocarcinomas are rare events in signet-ring cell carcinomas other than those associated with cell adhesion, and that MSI is not related to the occurrence of aneuploid cells.

A 58-year-old male who had a left hemicolectomy for descending colon cancer on July 1, 1993 was admitted to our hospital. A CT scan revealed a homogeneous low-density mass in the inferior portion of the spleen. Under the diagnosis of solitary splenic metastasis, a splenectomy was performed on August 10, 1995. Histologically, splenic tumor revealed moderately differentiated adenocarcinoma consistent with the primary tumor. Flow cytometric analysis revealed DNA diploidy in the primary tumor, and DNA aneuploidy (DNA index = 1.76) in the metastatic tumor. Using fluorescence in situ hybridization with p17 H8, numerical aerrations of chromosome 17 were observed in the primary tumor.

Background: Chromosomal aberration and DNA ploidy pattern are regarded as prognostic markers of the bladder cancer. In this paper, numerical chromosomal aberrations were analysed by interphase fluorescence in situ hybridization (FISH) for the cases with bladder cancer., Methods: FISH was carried out on touch smear of 35 cases of transitional cell carcinoma of the urinary bladder. DNA probes were digoxigenin labeled alpha/beta-satellite probes for chromosome 1, 7, 9, 11, 17, Y. FISH signals were counted using confocal laser microscope (LSM GB 200, OLYMPUS. Touch smear preparations were also examined with image cytometer (CAS 200, Becton Dickinson) and DNA index was obtained., Results: Numerical chromosomal aberrations were observed in 9 of 21 (42.9%) diploid cases and in all 14 non-diploid cases. Among 6 kinds of chromosome examined imbalance was observed and most common imbalance was loss of chromosome 9 (11 cases, 32.4%)., Conclusion: Even in the diploid tumors, chromosomal aberration was observed. Combined analysis of chromosomal aberration and DNA ploidy pattern was easily and quickly performed in our series. Clinical application could be possible and possess diagnostic and prognostic value.

The prognostic factors for esophageal cancer from the viewpoint of molecular biology are reviewed. Among several oncogenes and suppressor genes erbB, int2/hst1/Cyclin D1 and MDM2 gene amplifications are significant prognostic factors for esophageal cancer. The value of p53 mutation, and expression of matrix metalloproteinase (MMPs) in the prediction of patients' survival are controversial, so further research is needed. High expression of tumor proliferation-related factors (Ki67, PCNA, and AgNOR), abnormalities of adhesion molecule (E-Cadherin, alpha-Catenin), activation of autocrine mechanism of growth factor (EGFR-TGF alpha, EGF), and DNA ploidy pattern, which is thought to be the result of an accumulation of genomic abnormalities are also prognostic factors for esophageal cancer.

Curatively resected specimens of 124 cases of colorectal carcinoma, and 6 cases of colorectal carcinoma showing hematogenous metastasis were used in this study. Each carcinoma was cut out in stepwise section through the entire tumor. DNA ploidy for each section was determined by flow cytometry, and the intratumor DNA ploidy distribution pattern was decided, and divided into 5 types (Type A-E). These 5 types were broadly divided into 2 types; predominantly diploidy type (Type A, C) and predominantly aneuploidy type (Type B, D, E). A statistically significant difference was seen in the 5-year survival between 25 cases of the predominantly diploidy type (100%) and 99 cases of the predominantly aneuploidy type (76.7%). However, there was no statistically significant difference in survival between these 2 types in any Dukes stage. The intratumor DNA ploidy distribution pattern in 6 cases of colorectal carcinoma showing hematogenous metastasis comprised 3 cases of Type E, 2 Type D and 1 Type B. And that of 8 cases of curatively resected colorectal carcinoma showing hematogenous metastasis postoperatively comprised 5 cases of Type D, 2 Type E and 1 Type B. Thus, a close correlation between the aneuploidy predominant type, especially Type D as well as Type E, and hematogenous metastasis, was suggested.

To investigate whether genetic changes of the p53 gene and genetic defects in DNA-mismatch repair systems are involved in progression of colorectal carcinomas (CRCs), we examined loss of heterozygosity (LOH) on 17p and mutations in exon 5 through 8 of the p53 gene as well as replication errors (RER) at four microsatellite loci in DNAs from 108 CRCs at all clinical stages (20 Dukes A, 40 Dukes b, 48 Dukes C). We observed that LOH on 17p and/or p53 mutation were detected in 93% of Dukes A carcinomas and in 84% of Dukes C carcinomas, suggesting that the p53 gene is mutated and/or deleted before carcinoma has been produced. RER-positive phenotype was observed in approximately 30% of CRCs, irrespective of clinical stage. These results suggest that genetic instability is likely to play an important role in development of a subpopulation of sporadic CRCs, but not in progression of CRCs. In addition, we found no significant association between genetic alterations and progression of CRCs. We consider that genetic defects in DNA-mismatch repair pathway do not necessarily promote genomic instability at the p53 sequences in CRCs.

Purpose: Clinical significance of DNA ploidy pattern and its DNA heterogeneity is examined in prostate cancer., Methods: Fresh needle biopsy specimens were analyzed with flow cytometry and were compared with histopathological findings in 42 patients., Results: Seven patients had stage B (1 case of B1, 6 cases of B2), 14 had stage C and 21 had stage D disease respectively. Histopathologically, 18 of the cases were well, 12 were moderately and 12 were poorly differentiated adenocarcinoma. Diploid was observed in 20, aneuploid in 22. Though 22 cases showed no DNA heterogeneity, 14 had type A heterogeneity with aneuploid in association with diploid pattern, and 6 cases had type B heterogeneity in which multiple aneuploid patterns with different D.I. values were observed. The heterogeneity was observed in 28% of well, 58% of moderately and 67% of poorly differentiated adenocarcinoma, respectively, and also in 14% of stage B, 50% of stage C and 57% of stage D patients, respectively., Conclusion: We conclude that DNA heterogeneity in prostate cancer is more frequently seen in poorer differentiated ones on histologic grade and in more advanced ones on clinical stage.

Surgically resected specimens of 139 cases of large-bowel carcinoma were analysed in this study. Each carcinoma was cut out in stepwise section through the whole tumor, and flow cytometric DNA measurement was performed for each section. The intratumor DNA ploidy distribution pattern decided in this way was classified into 5 types (Type A-E). The 139 cases of carcinoma comprised 19 cases of Type A, 27 Type B, 11 Type C, 37 Type D and 45 Type E. The intratumor DNA ploidy distribution pattern showed a statistically significant correlation to tumor size, gross type, depth of invasion, growth pattern at the tumor margin, venous permeation of visceral wall, DNA Index and Dukes stage. Among these 5 types of carcinoma, carcinoma showing Type E was seen most frequently, even in the earlier stage, and found most frequently among the cases showing invasive growth pattern at the tumor margin, positive venous permeation in the visceral wall and DNA Index of more than 1.7. Therefore, the intratumor DNA ploidy distribution pattern seemed to reflect the degree of tumor malignancy as well as that of tumor advancement. Moreover, Type E pattern of carcinoma appeared to reveal the highest grade of malignancy, and early detection of this type seemed to be necessary in order to improve survival after surgery.

RDA is an efficient approach to the identification of the differences between complex genomes and could be valuable as the first step for the positional cloning of genes of interest. RDA is a method of DNA subtraction, which compares representation of the whole genome produced by whole genomic PCR. It can be used for the detection of genetic lesions in cancer, discovery of unknown pathogens, isolation of polymorphic markers linked to a trait and so on. We successfully applied RDA to identify the chromosomal deletion in cancer DNA. Homozygous deletions have been found in the cervical, renal and gastric carcinoma, demonstrating that RDA is an efficient method to identify the possible location of a tumor suppresser gene.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a major cancer susceptibility syndrome known to be caused by the inheritance of mutations in DNA mismatch repair genes, such as hMSH2, hMLH1, hPMS1 and hPMS2. Germline mutations in the hMSH2 and hMLH1 genes were detected in 9 and 11 Japanese or Korean HNPCC kindreds, respectively. These data establish a basis for the presymptomatic diagnosis of HNPCC patients. To determine the relation between the mutation of the TGF-beta type II receptor gene and genomic instability in the tumorigenesis of HNPCC, we screened genomic DNA of tumors from HNPCC patients. Seventeen of the 24 (71%) genomic instability-positive HNPCC tumors carried one or two A deletions in the (A)10 repeat, while none of the 14 genomic instability-negative tumors did. These deletions inactivate the receptor through a frameshift mutation and the resultant protein truncation. These data suggest that the TGF-beta type II receptor gene is a major target of genomic instability in HNPCC tumorigenesis.

The clinical and genetic studies have been very few on a familial predisposition to gastric cancers. We defined the criteria as familial gastric cancer (FGC) in which at least three relatives in two generations have gastric cancers, with one of the relatives having been diagnosed at less than 50 years of age. Other hereditary tumors, such as cancer family syndrome of hereditary nonpolyposis colorectal cancer(HNPCC), should be excluded. To clarify the carcinogenesis in FGC, we examined genetic alterations in six cancers from four FGC kindreds. Four (67%) cancers showed replication error, indicating that microsatellite instability is highly associated with not only HNPCC but also FGC. However, no germline mutation was found in the whole coding sequences of hMSH2 and hMTH1, or in the conservative regions of hMLH1 in any patients. Only few alterations were found at the small repeated sequences in the transforming growth factor-beta type II receptor gene in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.

Background: Renal cell carcinomas (RCCs) develop in 8-63% of von Hippel-Lindau disease (VHL) patients, and loss of 3p segments, chromosome aberrations found in 90% of sporadic RCCs, has also been observed in RCCs associated with VHL. In fact, comparative analysis showed that the chromosome aberrations in RCCs associated with VHL are similar to those found in sporadic RCCs. VHL patients have the whole spectrum of tumors from small early lesions to large ones in the same kidney, providing a unique opportunity to analyze tumors in different stages of development. Subsequently deoxyribonucleic acid (DNA) content in RCCs of VHL patients was examined and correlated to their tumor size to gain some insight in the progression of sporadic RCCs., Methods: From 1988 to 1991, we have experienced 6 cases of RCCs associated with VHL who underwent partial or radical nephrectomy. A total number of 52 paraffin-embedded samples from 33 RCCs from 6 patients with VHL was analyzed by flow cytometry., Results: The sizes of tumors ranged from 0.2 to 8.2 cm. DNA aneuploid patterns demonstrated in none of 9 tumors less than 1.6 cm, 4 of 14 tumors (29%) as large as 1.6 to 2.5 cm, and 5 of 10 tumors (50%) larger than 2.5 cm (p < 0.05). Twelve tumors less than 1.8 cm showed DNA diploid, so the smallest size of aneuploid tumors was 1.8 cm., Conclusion: These data suggest that DNA ploidy change (diploid to aneuploid) in RCCs probably takes place as tumors grow approximately 1.8 cm in size.

A novel in situ histochemical method of screening genetic alterations of human malignant cells was reported. The method is based on subtraction in situ hybridization of whole genomic DNA from a healthy volunteer or DNA extracted from non-neoplastic tissues. The method can widely examine the possible DNA defects in situ without employing specific probes employing colorimetric reaction in routinely processed surgical pathology materials, i.e. 10% formalin-fixed and paraffin embedded tissue sections. Results can be correlated with histopathological findings. In this report, we also describe the results on genetic alterations of human esophageal squamous cell carcinoma, using this DNA subtraction in situ hybridization.

The recent remarkable progress in molecular biology has revealed that various kinds of genetic alteration occur in cancers. Recently, many genes that cause hereditary cancer have been identified. For example, hMSH2 and hMLH1, which are known as DNA mismatch repair genes have been found to cause HNPCC (hereditary non-poliposis colorectal cancer). Mutation of RET oncogene has been recognized in the families of MEN (multiple endocrine neoplasia) type II. Mutations of the tumor suppressor genes are the most common changes in the genes of familial cancer. BRCA1 and BRCA2 are tumor suppressor genes that have recently been identified as familial breast and ovarian cancer, familial breast cancer genes. This paper reviewed the hereditary cancer families in which genetic alterations have been revealed and the recent progress in mapping and cloning of familial breast cancer candidate genes which have not been identified.

Expression of major histocompatibility complex of lung cancer was examined to study the malignant potential of the tumor using immunochemical staining (anti-HLA class I monoclonal antibody; w6/32). Nuclear DNA contents and the labeling index of proliferating cell nuclear antigen (PCNA) were measured as well. Forty three advanced (p-stage IIIa) lung cancers resected by lobectomy (33 cases), pneumonectomy (9 cases), and segmentectomy (1 case) entered the study. Mediastinal lymph node dissection was performed in every case during the operation. Of the 43 cases, expression of HLA class I was positive in 17 and negative in 26. The patients with positive HLA expression showed significantly better prognosis than those with negative HLA expression in terms of 5 year survival (p < 0.01). The patients with aneuploid pattern and positive HLA class I expression had the best prognosis on the survival curve (p < 0.01). When the PCNA positive rate was high, the prognosis was poor. And the patients with negative PCNA and positive HLA class I expression had significantly better prognosis than the others (p < 0.01). These findings suggest that the expression of HLA class I on the tumor cells is an important prognostic factor in the patients with NSCLC.

We experienced a case of renal leiomyoma. A 51-year-old woman was referred to the Department of Urology, Tochigi Cancer Center with a complaint of right renal incidentaloma pointed out on CT scan. The renal mass was removed by enucleation. Histological examination revealed the tumor composed of monotonous proliferation of spindle shaped cells without atypia. Thus, the diagnosis of leiomyoma was confirmed. DNA flow cytometric analysis of this tumor was performed and diploid pattern was shown.

This study was designed to evaluate the clinical relevance between the DNA ploidy and histopathology, and the incidence of the DNA heterogeneity in patients with bladder cancers. Flow cytometry(FCM) was used to study the DNA ploidy in 63 patients who underwent total cystectomy. The DNA ploidy and DNA index were analyzed by FCM in total 478 paraffin embedded samples (7.6 samples per case on the average). The DNA ploidy of 52 bladder cancers, that had coexisted after total cystectomy, showed that 24 cases, 46% were DNA aneuploid and 18 cases, 35% had DNA heterogeneity. The other 11 cases which were found to be pT0 after cystectomy were all DNA diploid. There were significantly good correlation among DNA ploidy pattern and intravesical involvement (lymph-duct involvement and venous involvement), but were not among the DNA ploidy pattern and tumor grade and stage. With regard to the evaluation of two vertically devided samples of tumors, DNA aneuploid had been not always recognized in the deeper samples, therefore, the author did not determine that there was good correlation between the DNA ploidy and the tumor invasion. In lymph node metastases, the 39 diploid tumors had given rise to lymph node metastases in only 5 cases(13%), whereas 11 cases(46%) of the 24 aneuploid tumors had metastasized(p < 0.01). Eleven of 16 lymph node metastases were DNA diploid and the others were DNA aneuploid. These data suggest that although the incidence of DNA heterogeneity in bladder cancers (35%) is thought to be relatively small, the DNA ploidy will be able to be the important prognosticating factor in bladder cancers.