We report a case of polyglandular autoimmune syndrome (PGA) complicated by Duane's syndrome. The patient was 44-year-old female with marked limitation of abduction in the left eye, lethargy, nonhomogeneous facial pigmentation, goiter, and oligomenorrhea. A diagnosis of chronic thyroiditis was first made to explain the patient's symptoms. Laboratory examinations were performed. Plasma ACTH level was high and plasma cortisol was low, and there was no response to the ACTH stimulation test. The presence of primary adrenocortical deficiency was confirmed. Moreover, primary gonadal failure was also present, and the diagnosis of PGA type II was made. The patient's elder sister had myasthenia gravis which is a condition known to occur with PGA type II. Therefore, the sister was also suspected to have PGA type II, as the syndrome can occur in family members. However, since she had been receiving large doses of steroids for her myasthenia gravis, laboratory findings were inconclusive. Duane's syndrome, which is characterized by congenital oculomotor disturbance, was also seen in the sister. It is still unknown whether the familial occurrence of Duane's syndrome has a genetic basis. There have been reports of congenital disorders occurring in combination with autoimmune diseases. Further investigation into the relationship between congenital anomalies and autoimmune diseases is necessary.

The authors have performed fluorescence in situ hybridization (FISH) in tissue sections of archival paraffin-embedded blocks of seven cases in adenoma and ten cases in carcinoma in order to clarify which chromosomal aberration occurred in association with multi-step carcinogenesis in colorectal carcinomas, using alpha-satellite DNA probes to chromosome 11 and 17, D11Z1 and D17Z1, respectively. Monosomy of chromosome 11 was most frequented (5/7, 71.4%) in adenoma, and trisomy of chromosome 17 was predominant (9/10, 90.0%) in carcinoma. The numerical chromosomal aberrations can be already detected at the stage of adenoma, and monosomy of chromosome 11 was mainly observed in adenoma. Furthermore, malignant transformation arising from adenoma accounted for most of the trisomic change in chromosome 17. Consequently, applying the FISH technique to tissue sections from archival paraffin embedded specimen, it would be possible to distinguish between the cancerous and non-cancerous regions in view of chromosomal numerical aberrations. The authors emphasized that intratumoral heterogeneity could be elucidated at the chromosomal level.

We evaluated the DNA ploidy in 23 lesions of colorectal carcinoma in adenoma (CIA) and 90 adenomas without carcinomas by flow cytometry using fresh samples. DNA ploidy of carcinoma and adenoma components were assessed, respectively, with 17 paraffin-embedded samples of CIAs. The incidence of DNA aneuploidy (AP) was significantly higher in CIAs than in adenomas (47.8% vs. 12.2%, p < 0.01). Even in adenoma components of CIAs, AP tended to be found more frequent than in adenomas (41.2% vs. 12.2%). The incidence of AP in adenoma components was similar to that in carcinoma components (35.3%) in CIAs. In conclusion, DNA aneuploidy in adenomas may be a marker of malignant potential.

To investigate a possible relation between the nuclear DNA content and lymph node metastasis of submucosal early gastric cancer, DNA content was analyzed for 46 patients with lymph node metastasis and 67 patients without nodal metastasis. DNA aneuploidy was found in 20 (43.5%) of 46 patients with lymph node metastasis and in 31 (46.3%) of 67 patients without it. There was no statistical difference in the incidence of aneuploidy between the 2 groups. Among the cases with DNA diploidy, the mean value of S phase fraction was 6.82% in patients with lymph node metastasis and 5.65% in those without metastasis. The mean value of S phase fraction was significantly higher in patients with nodal involvement (p < 0.05). Furthermore, among the cases with DNA aneuploidy, the mean value of G2/M phase fraction was 11.03% in patients with lymph node metastasis and 7.54% in patients without metastasis. The mean value of G2/M phase fraction was significantly higher in patients with nodal involvement (p < 0.05). These findings suggest the significant value of the S and G2/M phase fraction for the prediction of lymph node metastasis in patients with submucosal early gastric cancer.

In order to investigate whether or not DNA ploidy was altered in intramucosal gastric carcinomas, nuclear DNA content of biopsy specimen was measured using flow cytometry in 38 intramucosal carcinomas. DNA aneuploidy was detected in 27 of 38 lesions (71.1%), and noted more frequently in differentiated carcinomas than in undifferentiated ones (83.0% vs. 20.0%, p < 0.01). There was no significant relationship between the frequency of DNA aneuploidy and macroscopical type or tumor size. DNA aneuploidy was even found in two of three minute carcinomas (5 mm or less in diameter). DNA indices showed 1.2 or lower values in 40% of the lesions with DNA aneuploidy. The average value of DNA index was significantly larger in depressed type than in elevated type (p < 0.01). In conclusion, DNA ploidy is altered in most differentiated intramucosal carcinomas. A high resolution method is essential for accurate determination of DNA ploidy in intramucosal carcinomas, especially elevated ones.

The study was carried out on 18 cases of oral cavity carcinoma and 12 cases of hypopharyngeal carcinoma to assess the degree of intratumoral heterogeneity and the relation to tumor size, lymph metastasis and histological type. In oral cavity carcinoma, 6 cases (33%) were aneuploid, while in hypopharyngeal carcinoma all were aneuploid. DNA heterogeneity was identified in 4 cases (22%) in oral cavity carcinoma, end in 6 cases (50%) of hypopharyngeal carcinoma. No correlation was found between DNA heterogeneity and tumor size, lymph metastasis and histological type. However, even early carcinomas have intratumoral DNA heterogeneity. It was supposed that the result which hypopharyngeal carcinoma had higher degree of aneuploid and DNA heterogeneity comparing with oral cavity carcinoma supported that hypopharyngeal carcinoma had higher clinical malignancy from the aspect of proliferative movement. The data indicate that it is impossible to analyze correct DNA content by only one specimen and it is important to investigate intratumoral DNA heterogeneity to research the mechanism of multiplicity and clinical malignancy of carcinoma.

The mutagenicity of quercetin, a flavonoid, was examined by means of DNA fingerprint analysis using the Pc-1 and Pc-2 minisatellite probes that efficiently detect mutations due to recombination. Treatment of FM3A and BMT-11 tumor cells with 55 microM quercetin resulted in gain and loss of bands in the fingerprints in both cell lines. The frequencies of the clones having undergone mutation were 9/26 and 2/11, using Pc-1 probe, respectively, in the two lines. These results seem to provide a molecular basis for the phenotypic variations of BMT-11 tumor cells induced by quercetin, giving direct evidence of genetic instability of the tumor cells. Moreover, we examined for a possible correlation between frequencies of DNA recombinational mutations and cancer malignancy in human colon cancers. DNA of four human colon cancer tissues and corresponding peripheral blood cells were prepared, respectively, and examined by DNA fingerprint analysis using hPc-1 polymorphic minisatellite probe. These four specimens exhibited no extra-bands resulting from recombination and/or DNA slippage at present. We would explain how the prognosis of cancer patients is related to frequencies of DNA recombinational mutation.

We studied the relationship between intratumoral DNA heterogeneity and clinicopathological findings in 85 colorectal cancers. DNA ploidy was analyzed in 5 specimens from different sites of each tumor. When the difference in the DNA index (D.I) between several peaks in the same tumor was more than 0.1, the tumor was considered to consist of heterogeneous subpopulations with different DNA clones. DNA heterogeneity was found in 34 cases (40.0%). There was no relationship between DNA heterogeneity and depth of tumor invasion, lymph node metastasis or stage. The incidence of heterogeneity was significantly higher in the tumor with lower differentiation, liver metastasis or vessel invasion.

Significance of flow cytometric DNA analysis in metastatic lymph nodes for assessing malignant potential of colorectal cancer was investigated using paraffin-embedded materials of primary lesions and metastatic lymph nodes from 65 patients who had been treated between 1975 and 1990. The DNA ploidy patterns of metastatic nodes were identical in 61.5% with those of primary lesions. Diploid cancers were significantly more frequent in metastatic nodes than in primary lesions. There were significantly more aneuploid cancers in proximal nodes than in distant nodes. There was no relation between ploidy patterns in primary lesions and survival. However, a significant relation was found between ploidy patterns in metastatic nodes and survival. Diploid cancer in metastatic nodes had a significantly better survival than aneuploid cancer, in all patients as well as those with curative resection. In patients with stage III and in those with the same depth of invasion, the survival rate of diploid cancer in metastatic nodes was significantly higher. There was no correlation between ploidy patterns in metastatic nodes and clinicopathological variables in primary lesions, such as histological type, depth of invasion, nodal involvement, peritoneal or hepatic involvement and stage. These results suggest that nuclear DNA content in metastatic lymph nodes may be a prognostic indicator in colorectal cancer with nodal involvement.

We performed DNA flow cytometry analysis of colon carcinomas from the viewpoint of DNA heterogeneity. The materials were 25 colon carcinomas and 17 metastatic lymph nodes. Four different regions were examined in each primary tumor. The DNA index (DI) was classified into four groups. We classified the primary tumors into homogeneous and heterogeneous group according to the DI of the four regions of each case. Ten of 25 (40%) were classified as homogeneous and 15 (60%) as heterogeneous. The heterogeneous group tended to show more aggressive clinicopathologic characteristics. The corresponding rate between the maximum DI in the primary tumor and the DI in the metastatic lymph node was 65% (11/17). The maximum DI of the primary tumors was mostly seen in the metastatic lymph nodes, suggesting high metastatic potentiality of the higher DI clones. The examination of DNA heterogeneity may be useful for detecting the more precise character of the colon carcinomas. In heterogeneous cases, the higher corresponding rate of DI between primary and metastatic lesions suggested that metastasis occurred frequently in primary lesions containing gathering larger DI cells.

To evaluate the clinical significance of DNA ploidy heterogeneity (DH), four or more fresh tissue specimens were obtained from a tumor in 68 resected early gastric cancers. DNA content was measured by flow cytometry and the presence of DH was prospectively investigated. The incidence of DH correlated to invasion depth (m < sm), lymph vessel invasion (negative < positive) and tumor size (10 mm or less in diameter < more than 10). When the criteria of indication for minimum surgery were determined as the intramucosal cancer without n, ly and v factor, 85% of contraindication cases demonstrated DH. These results indicate that DH is a useful marker of tumor progression in early gastric cancer and will be an aid for determining indications for minimum resection.

Flow cytometric determination of DNA ploidy was performed on paraffin-embedded specimens of primary tumor (Group A) and its metastatic lymph nodes (Group B) in 30 surgically resected cases, and the DNA index (DI) was compared between these two groups. In 25 of 30 cases (83.3%), all DI observed in Group B were also found in Group A; and in the remaining 5 cases, a series of DI not found in Group A were observed in group B. They were all poorly differentiated adenocarcinomas, 4 of which exhibited serosal invasion and prominent lymphatic permeation in their wall. In 28 cases (93.3%), the modal DI value in the primary tumor was also found in their corresponding metastatic lymph node, the frequency of which revealed a statistically significant difference from 18 cases (60%) in terms of median DI value, and from 10 cases (33.3%) in terms of maximum DI value (p < 0.01). It was suggested that the modal DI value in the primary tumor could signify an important clone relating to lymph node metastasis. No significant DI difference was observed in comparison of a series of DI seen in n1(+) and n2(+) group of metastatic regional lymph nodes.

In order to assess clinical significance, the DNA content in gastric cancer with serosal invasive exposure from 50 patients was determined by flow cytometry. The DNA histograms could be measured in two different vertical portions, on the mucosal and serosal sides. DNA heterogeneity was found in 15 patients (30.0%). These cases were divided into 4 groups according to the combination of DNA ploidy patterns. There were no significant differences in clinicopathological characteristics or survival rate between these 4 subgroups. Furthermore, the serosal invasion index (SII) defined as the ratio of serosal extent to mucosal extent was examined to determine the malignancy potential. All cases were divided into 2 groups, high SII and low SII, according to the value of SII. The survival rate was significantly lower in the high SII-aneuploid group compared with the low SII-diploid group. These results suggested that DNA analysis, subclassified by the serosal invasion index, is useful for assessment of the patient's prognosis.

DNA ploidy was microspectrophotometrically investigated in 46 patients with gastric carcinoma. Measurements of DNA content and mitotic index (MI) were examined in the mucosal, submucosal, muscularis propria, and serosal layers of tumors, respectively. The frequency of cells with values exceeding hexaploid chromosome (6c) and mitotic counting analysis revealed a significantly higher value in the serosa than in the mucosa. This tendency was not evident in differentiated type adenocarcinoma but was noted in those with the undifferentiated type. There were 37 tumors (80.0%) with the same DNA distribution pattern in every layer of the stomach (homogeneous DNA ploidy). Heterogeneity of DNA ploidy was observed in nine tumors (20.0%). Carcinoma with heterogeneous DNA ploidy manifested a significantly higher incidence of metastasis to lymph nodes than did those with the homogeneous type. Characteristically, there was venous permeation preponderance in the differentiated type and peritoneal dissemination preponderance in the undifferentiated type. This evidence of DNA heterogeneity in gastric carcinoma tissue suggests a possible correlation with metastatic behavior.

We quantitatively analyzed the c-myc and p53 products using flow cytometry in 28 cases of resected lung cancer and one case each of chorio-carcinoma, plasmacytoma, malignant mesothelioma and sclerosing hemangioma. In the lung cancer cases, c-myc and p53 products were detected in 10 cases (35%) and 7 cases (21%), respectively. These rates are higher than the DNA abnormal expression rates of the c-myc and p53 genes (15% and 12%, respectively) in our own data. In the adenocarcinoma of lung cancer cases, c-myc and p53 products were detected in 9 cases (53%) and 5 cases (29%), respectively. Among the squamous cell carcinoma cases, there were one case (11%) of c-myc expression and one case (11%) of p53 expression. DNA content analysis of the lung cancer patients revealed 7 cases of DNA diploidy and 21 cases of DNA aneuploidy. All 10 c-myc-positive cases showed DNA aneuploidy; thus the positive rate for c-myc products in the DNA aneuploidy cases was significantly different compared with the DNA diploidy cases (p < 0.05). In the sclerosing hemangioma case, we detected both c-myc and p53 products. Sclerosing hemangioma has been thought to be a benign tumor, but it may be a malignant tumor.

Flow cytometric nuclear DNA analysis was performed on 36 preoperative endoscopic biopsy specimens and 89 surgically resected specimens of esophageal carcinomas without preoperative radiotherapy. Carcinomas with aneuploid or DNA stem-line heterogeneity had a higher frequency of lymph node metastasis (p < 0.01). The correspondence rate of nuclear DNA ploidy patterns was 97% between biopsy and resected specimens, and that of heterogeneity was 72%. Though only six cases showed heterogeneity in biopsy specimens out of 12 cases which showed heterogeneity in resected specimens, preoperative detection of heterogeneity was supposed to be more accurate by an increase of biopsy specimens. DNA analysis of biopsy specimens may be a possible indicator of the malignant potential of esophageal carcinoma.

Overexpression of p53 protein was examined by immunohistochemical assay. In 143 primary breast cancer patients, consisting of 18 patients with family history of breast cancer or past history of breast cancer and 125 patients with no such histories. In the later 125 patients, overexpression of p53 was shown in 42 patients (33.6%), however in the former 18 patients, p53 overexpression was found in 15 (83.3%). It was suggested that p53 protein is frequently overexpressed in patients with high risks like history of breast cancer and family history of breast. On the other hand, regarding the value of p53 overexpression as a prognostic indicator, an univariate analysis demonstrated that the relapse free survival of patients with over 50% p53 positive all rate was significantly worse compared to that of patients with less than 50% p53 positive all rate (p < 0.05). However multivariate analysis did not demonstrate the significant value (p < 0.1), suggesting that overexpression of p53 is marginal as a prognostic indicator.

In view of the elevated risk of leukemia among A-bomb survivors, genetic alterations associated with Leukemia can be considered to have been induced by ionizing radiation. Therefore, to clarify this possibility, an examination was made to see whether genetic changes such as BCR-ABL translocation closely associated with chronic myelogenous leukemia (CML) are actually induced by radiation. BCR-ABL translocation is easily detected by means of reverse transcription polymerase chain reaction. One hundred million cells of the promyelocytic leukemia-derived cell line HL60, which do not have such a gene rearrangement, were irradiated with 100 Gy of X-ray, after which RNA was extracted and examined for any rearrangements of BCR and ABL genes. Five kinds of bands were observed in the HL60 cells irradiated with 100 Gy of X-ray, and it became clear that these positive bands contain both BCR gene and ABL gene by the direct sequencing method. Furthermore, these gene rearrangements included not only the rearrangements specifically identified with CML but also atypical rearrangements which are not generally observed clinically. The induction by X-irradiation of such gene changes characteristic of malignant tumors, which are closely associated with radiation carcinogenesis, suggests that they are the initial gene changes in radiation carcinogenesis.

The pX gene of human T-cell leukemia virus type I (HTLV-I) is known to be a potent transactivator of the viral gene and the host genes which are important for cell proliferation in vitro. It has been reported that various diseases occur in transgenic mice harboring either tax, pX, or env-pX gene, such as mesenchymal tumor, neurofibroma, thymic atrophy, muscle degeneration, exocrinopathy and arthropathy. We previously demonstrated that rat but not mouse CD4 positive T cells could be easily infected and immortalized by HTLV-I and infectious transmission of HTLV-I induced HAM/TSP-like myelopathy in WKAH rats after long incubation periods of 16 months. These observations prompted us to produce a series of transgenic rats that expressed the pX gene products under the control of mouse H-2Kd promotor in order to evaluate further the biological and pathological function of the pX gene in vivo. In various tissues of pX transgenic rats (pX rats), pX mRNA was constitutively expressed irrespective of age. PX rats developed mammary tumors with massive infiltration by neutrophils as early as 9 months of age. Pathological and immunohistochemical examination revealed that the tumors were undifferentiated carcinomas of the mammary gland origin. They were transplantable into pX rats, but not into normal syngenic rats. High levels of mRNA expression of not only the pX transgene but also the host genes such as Gro (melanoma growth-stimulatory activity/KC), MIP-2 (macrophage inflammatory protein-2) and IL-1 alpha were demonstrated in the tumor tissues. Gro and MIP-2 which were known as IL-8 families were likely to be produced by tumor cells and appeared to be responsible for neutrophil infiltration in the tumor tissues. Lastly, pX rats described here appear to be suitable animal models for elucidating mechanisms involved in the tumorigenesis and the transactivation of the cellular genes by HTLV-I, especially by the pX gene products in vivo.