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To investigate the potential role of the BCL-2 gene family (BAX, BCL-2, MCL-1, and BCL-XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi-quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL-1 in ovarian tumors was also examined. The expression levels of BAX and MCL-1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P < 0.05). In contrast, the BCL-2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P < 0.05). Expression of BCL-XL mRNA was no different between normal ovaries and ovarian tumors. Log-rank testing showed that low BAX mRNA expression and high MCL-1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P = 0.05; MCL-1, P = 0.02). Immunohistochemical analysis showed that diffuse-positive expression of MCL-1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P = 0.03). In ovarian cancer cases, diffuse-positive expression of MCL-1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P < 0.01; grade, P = 0.01; survival, P = 0.01). These results suggest that increased MCL-1 expression may play an important role in replacing the functions of increased BAX and decreased BCL-2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.

Adenosquamous carcinoma of the lung is a subset of pulmonary carcinomas, and comprises less than 4% of lung carcinomas. Its histogenesis remains unclear. The clonality of adenosquamous carcinoma from four female patients was analyzed to determine whether the clonality between the squamous cell and adenocarcinomatous components coincides. Each lesion was precisely microdissected from methanol-fixed sections. Adjacent normal lung tissue was collected as a normal control. DNA was extracted for clonal analysis based on an X-chromosome-linked polymorphic marker, the human androgen receptor gene (HUMARA). HUMARA was found to be amplified with or without previous digestion by the methylation-sensitive restriction endonuclease HpaII. All four cases were informative. Squamous cell and adenocarcinomatous components showed identical monoclonal patterns in all four patients. In one case, only the squamous cell carcinomatous component showed loss of heterozygosity of the HUMARA locus. The results suggest that the squamous cell and adenocarcinomatous components originate from the same cell.

Mxi1 is thought to negatively regulate Myc function and may therefore be a potential tumor suppressor gene. Little effort has yet been made to find alterations involving this gene in human solid tumors. We screened 31 human gastric cancers, 7 esophageal cancers, 85 bone and soft tissue tumors of various types, including 4 neurofibrosarcomas. We also examined 29 human tumor cell lines consisting of 12 esophageal cancers, 7 glioma/glioblastomas and 10 others for Mxi1 mutations in exons 1, 2, 4 (HLH domain), 5 and 6. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and subsequent sequencing revealed three distinct polymorphisms in the intron-exon boundary upstream from exon 6. We discovered a missense mutation, GCA to GTA (Ala 54 Val), in exon 2 in a neurofibrosarcoma patient (case 1), two missense mutations, AAA to CAA (Lys 118 Gln) and GAA to GGA (Glu 154 Gly) in exon 5 of another neurofibrosarcoma patient (case 2), and 3 amino acid substitutions, GTG to GCG (Val 179 Ala), GTT to GCT (Val 181 Ala) and TTC to CTC (Phe 186 Leu), in a third neurofibrosarcoma patient (case 3). In case 3, loss of heterozygosity was also demonstrated by informative (TTC)3/(TTC)2 polymorphism. Our data demonstrate that mutations occur in the Mxi1 gene in neurofibrosarcoma. Missense mutations in the functional domain of Mxi1 in these cases may be involved in the pathogenesis of neurofibrosarcoma.

Bone is one of the most common sites of recurrence of breast cancer. Therefore, it would be clinically very useful if breast cancers with a high probability of bone metastasis (BM) could be identified by histopathological examination of the primary lesions. To elucidate histological characteristics associated with predisposition to initial BM, we examined nine histopathological parameters in the primary lesions of 110 invasive ductal carcinomas (IDCs) of the breast with 0 to 3 regional node metastases. These cases had recurrence between 4 months and 10.1 years after the initial radical surgery. The first metastatic site was bone in 24 cases, whereas other sites were involved in 86 cases. IDCs growing in a strand growth pattern or with fibrotic focus (FF) had a significantly higher frequency of initial BM than those growing in a non-strand growth pattern or without FF, respectively. Strand growth pattern was a significant predictor of the initial BM in multivariate analysis. In all 54 IDCs that developed BM during the follow-up period, osteolytic metastasis was significantly more frequent in the group with FF than in that without FF. This study demonstrated that strand growth pattern and the presence of FF are significant histopathological factors associated with initial BM. The combination of those predictive factors along with prognostic factors may provide a useful approach to identify patients at high risk for initial BM, enabling early treatment for the recurrent cancer.

Tumor recurrence rate (TRR) and mortality rate (MR) of invasive ductal carcinoma (IDC) of the breast in short-term follow-up are relatively low. Nevertheless, it is extremely important to identify patients at risk of early recurrence or death after surgery. The aim of this study was to establish a new histological prognostic classification scheme for IDC in order accurately to predict the short-term outcome. The following histological parameters were analyzed in 201 IDCs: 1) tumor size, 2) structural atypia, 3) nuclear atypia, 4) number of mitotic figures, 5) fibrotic focus (FF), 6) vascular invasion, 7) tumor necrosis, 8) skin invasion, 9) muscle invasion, 10) nodal status and 11) extramammary fat invasion. Multivariate analysis showed that nuclear atypia, presence of FF, and the invasive length of fat invasion (ILFI) were the most important histological parameters correlated with TRR or MR of IDCs. Accordingly, a new histological classification based on nuclear atypia, FF and ILFI (Nucleus-Fibrotic focus-Fat invasion, NFF) was devised. Comparative studies were performed with the following existing prognostic classifications: 1) histological grade, 2) modified Scarff-Bloom-Richardson histological grade, 3) prognostic index and 4) pathological TNM (pTNM) stage classifications. Patient grouping defined by NFF classification significantly correlated with tumor recurrence or death of IDCs in all cases, cases at stages I and II, those without lymph node metastasis and those who were estrogen receptor (ER)-positive after adjustment for the other four classifications, using multivariate analysis. NFF classification appeared superior to existing prognostic classifications for the accurate prediction of the short-term outcome for patients with IDCs in low risk groups.

The expression of cytochrome P450 3A4 (CYP3A4) in the foveolar epithelium of the human stomach with intestinal metaplasia was studied using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction (RT-PCR). CYP3A4 was immunohistochemically detected in the foveolar epithelium with intestinal metaplasia, but was not detected in foveolar epithelium without intestinal metaplasia, in the pyloric gland or in the fundic gland of the stomach. Western blotting and RT-PCR demonstrated that CYP3A4 protein and mRNA were expressed in the liver and pyloric gland mucosa with intestinal metaplasia, but not in the fundic gland mucosa without intestinal metaplasia. Possible roles of CYP expression in the gastric mucosa with intestinal metaplasia in human stomach carcinogenesis are briefly discussed.

A fibrotic focus (FF) is a scar-like area within invasive ductal carcinoma (IDC) of the breast, and has been shown to be a marker of high aggressiveness of IDC. In order to investigate the mechanism of FF formation in IDC, expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor (FGFR) was studied. One hundred and forty-nine IDCs were divided into solid tumors and scirrhous tumors. Immunohistochemistry was used to determine the expression of bFGF and FGFR proteins in both tumor cells and fibroblasts forming FF. Scirrhous tumors with FF showed a significantly higher frequency of bFGF protein expression than those without (P = 0.017), whereas, in solid tumors, the presence of FF was not significantly associated with the frequency of bFGF protein expression (P = 0.143). In addition, scirrhous tumors showed a significantly higher frequency of FGFR protein expression than solid tumors (P = 0.001). Among IDCs having FF and expressing bFGF protein, a significantly larger number of fibroblasts expressing FGFR protein within FF was observed in scirrhous tumors than in solid tumors (P = 0.016). The results of this study suggest that in scirrhous tumors the interaction between tumor cells and stromal fibroblasts plays an important role in the formation of FF, and that there is a paracrine mechanism between bFGF protein from tumor cells and FGFR protein in fibroblasts.

The number of primary breast cancers occurring in elderly women is increasing in Japan. Optimization of treatment regimens in this age group requires precise evaluation of the biological aggressiveness of these tumors as well as the performance status and extent of tumor spread. In 39 breast cancer patients who were at least 80 years old, we examined several parameters; the form of surgical therapy, the lymph node status, presence or absence of distant metastases, the histological type and grade of atypia, and overexpression of the c-erbB-2 oncoprotein in the cancer cells. They were correlated with the clinical outcome of the patient. Of the 33 patients who underwent a mastectomy and axillary lymph node dissection, five died from cancer recurrence. Only one out of 22 patients without lymph node metastases died from cancer, while four out of the eight patients with metastases to three or more lymph nodes died from cancer recurrence within 2.7 years of surgery. The overall survival curves also differed between patients with low-risk histological tumors or grade 1 or 2 invasive ductal carcinoma and those with grade 3 invasive ductal/lobular carcinoma. Overexpression of c-erbB-2 also affected survival. Regional recurrence occurred in three out of the six patients for whom only lumpectomy or simple mastectomy was performed. These results indicate that, although primary breast cancer occurring in patients over 80 years old was largely of low-grade malignancy, patients with three or more lymph node metastases, invasive ductal/lobular carcinomas of grade 3, or c-erbB-2 overexpression frequently exhibited an aggressive clinical course.

Lung cancer in the young, which has the characteristics of a higher incidence of adenocarcinoma, lower male-to-female ratio of the patients, and less frequent smoking history in the patients, may possibly be associated with genetic predisposition to cancers. We studied six microsatellite loci (D2S123, D3S659, D3S966, D5S346, WT1, and TP53) in 18 surgically treated lung cancer patients aged 25-40 years and nine control patients aged 62-74 to determine the presence of microsatellite instability (MSI) and to correlate its occurrence with clinicopathological characteristics. Of the 18 patients, 11 were female and seven were non-smokers. There were 15 adenocarcinomas and three squamous cell carcinomas, 15 (83%) of which had vascular invasion. MSI was positive in seven (39%) of 18 young patients and one (11%) of nine control patients. Moreover, MSIs in a half or more of six loci examined were demonstrated in five (28%) young patients, whereas no control patients showed such a high frequency of MSI. We observed no significant differences in clinical or pathologic parameters between cases with and without MSI. This result suggests that genetic factors play an important role in the development of lung cancer in young adults.

We investigated whether the presence of a fibrotic focus (FF) in the primary lesion and in lymph node metastasis is a good predictor of early tumor recurrence or death in patients with invasive ductal carcinoma (IDC). Multivariate relative risk (RR) of tumor recurrence and death according to the presence of FF in the primary tumor was estimated using the Cox proportional hazards regression model with adjustment for other prognostic factors (histologic grade, T classification, nodal status, tumor necrosis, DNA ploidy, c-erbB-2 protein expression, p53 protein expression, and labeling index of proliferating cell nuclear antigen). For the evaluation of the metastatic status in the axillary lymph nodes, RR of multivariate analysis was adjusted for the presence of FF in the metastatic tumor and the number of lymph nodes involved (1-3 and > 3). The presence of FF increased the RR of tumor recurrence significantly for the cases in all stages, and especially for those in stages I and II (RR = 6.9, P < 0.05 and RR = 25.0, P < 0.005, respectively). All cases that died of disease had FF. Among IDCs with FF, 24 cases had FF in lymph node metastasis. Significantly higher RRs of tumor recurrence and death were observed in cases with FF in lymph node metastasis than in those without it (RR = 2.0, P < 0.001 and RR = 5.9, P < 0.05, respectively). It was suggested that the presence of FF is an important predictor of early tumor recurrence or death in patients with IDCs. The presence of FF in lymph node metastatic lesions is also a significant prognostic parameter.

We studied whether a computer-assisted system using a combination of data collection by image analysis and analysis by neural networks can differentiate benign and malignant breast lesions. Forty-six intraductal lesions of the breast were studied by pathologists and by the computer-assisted system. Histological evaluation was performed independently by three pathologists, and the lesions were classified into pathologically malignant (n = 12), undetermined (n = 13), and benign (n = 21). Computerized nuclear image analysis was performed using the CAS200 (Cell Analysis Systems, Elmhurst, IL) system to obtain data on nuclear morphometric and textural features. A neural network was constructed using the morphometric and texture data obtained from teaching cases of malignant and benign lesions. Then data for unknown cases were classified by the constructed neural network into neural network-malignant (n = 11), -undetermined (n = 5), and -benign (n = 30). The agreement rate between the diagnosis by pathologists and judgment by the computer-assisted system was 75%, excluding pathologically undetermined lesions. There were four false-negative but no false-positive results. False-negative cases had nuclei that were quite different from those of the teaching cases. The agreement rate obtained using either morphometric data or texture data only was lower than that using a combination of both. Selection of appropriate teaching data and incorporation of both morphometric and textural parameters seemed important for obtaining more accurate results. The present data suggest that development of a computer-assisted histopathological diagnosis system for practical use may be possible.

Histological examination of invasive ductal carcinoma of the breast often demonstrates the presence of an extensive central fibrotic focus (FF). The clinicopathological significance of the FF, or scar, in primary invasive ductal carcinoma is still ambiguous. One hundred and fifty-three cases of invasive ductal carcinoma (IDC) were classified into two groups, those with and those without FF. The differences in frequency of immunohistochemically detected overexpression of c-erbB-2 protein and nuclear accumulation of p53 protein, and the labeling index of proliferating cell nuclear antigen (PCNA), as well as histopathological parameters were compared between these two groups. IDCs smaller than 50 mm with FF showed a higher frequency of high-grade tumors, a higher frequency of lymph node metastasis, and a significantly higher frequency of c-erbB-2 protein overexpression than those without FF. In tumors of 20 mm or less, the incidence of nuclear accumulation of p53 protein was significantly higher in tumors with than those without FF. Tumors with FF showed a significantly higher PCNA labeling index than those without FF, regardless of tumor size. The present results indicate that the presence of FF is an important clinicopathological parameter associated with a higher degree of malignancy in IDCs, especially those smaller than 50 mm. Therefore, dividing IDCs into those with and those without FF appears to be meaningful clinicopathologically.

Epstein-Barr virus DNA was detected in a case of lymphoepithelioma-like thymic carcinoma. A homogeneous terminal structure of the viral DNA was demonstrated in this case, indicating the presence of the viral genome in clonally expanded tumor cells. Since all of 26 other thymic epithelial tumors (eight non-invasive, 13 invasive thymomas and four non-lymphoepithelioma-like thymic carcinomas) in Japanese were negative by polymerase chain reaction, it is suggested that lymphoepithelioma-like thymic carcinoma may represent a unique pathological entity distinct from Epstein-Barr virus-negative thymic epithelial tumors, which are in the majority in Japan.

Abnormalities of p53 mRNA in adult T-cell leukemia (ATL) were analyzed using reverse transcription-polymerase chain reaction-single strand conformation polymorphism analysis. Mutations were present in two of 12 ATL patients studied, but not in 3 cell lines immortalized by human T cell leukemia virus type 1 (HTLV-1) infection in vitro. Direct sequencing analysis of the p53 gene from these two patients revealed missense point mutations at codon 153 (arginine to histidine) or codon 220 (cysteine to tyrosine), respectively. Immunohistochemical analysis revealed the elevated expression of p53 proteins in ATL cells from a patient carrying the mutated p53 gene at codon 158. Neither gross rearrangement of p53 gene nor abnormal size of mRNA for the gene was demonstrated by Southern or Northern blot analyses. Thus, there is a mutated p53 in some patients with ATL. The involvement of abnormalities in some suppressor oncogenes may play a role in the development of ATL.

p53 protein has been frequently detected at high levels in the nuclei of human breast cancer cells. We analyzed immunohistochemically the association between nuclear localization of p53 protein and clinical and histological parameters of breast cancer patients. Surgically resected tissues of 73 primary breast cancers were processed by acetone fixation and paraffin embedding and examined using an anti-p53 monoclonal antibody, PAb1801. p53 immunoreactivity was detected in the nuclei of cancer cells in 17 cases (23%). The nuclear p53 immunoreaction was closely associated with overexpression of c-erbB-2 protein (P less than 0.05), high histologic grade (P less than 0.01), advanced clinical stage (P less than 0.05), and negative estrogen receptor status (P less than 0.01). When 31 cases which had been followed up for more than 50 months were examined, a positive nuclear p53 immunoreaction was found to be significantly associated with shorter overall survival of patients (P less than 0.01). These results suggest that immunohistochemical examination of nuclear p53 protein is clinically useful as an indicator of breast cancer aggressiveness.

Relationships among cytological features, doubling time, S-phase percentage, expression of myc-family oncogenes, DNA ploidy and biochemical properties were studied in thirteen small cell lung cancer cell lines. Six cell lines that grew slowly (average doubling time 99 h) and had lower S-phase percentages (average 32%) showed inconspicuous nucleoli (average area of 1.5 microns 2), and the remaining seven cell lines that grew quickly (average doubling time 45 h) and had higher S-phase percentages (average 44%) showed large and prominent nucleoli (average area of 6.1 microns 2). DNA index value obtained from flow cytometric DNA histograms showed that all cell lines except for H-69 cell line displayed aneuploidy. Ribbon-like cell arrangements were observed in the 7 cell lines that grew quickly, and in 1 cell line that grew slowly. Biochemically, six slow-growing cell lines and four fast-growing cell lines showed high levels of aromatic L-amino acid decarboxylase activity, while in the remaining three fast-growing cell lines its level was low. A high level of c-myc or N-myc oncogene expression was observed in all 7 cell lines that grew quickly, but not in any of the 6 cell lines that grew slowly. It appears that small cell lung cancer cell lines that grow quickly can be expected to have large nucleoli and ribbon-like cell arrangements and to express high levels of myc-family oncogenes, and that nucleolar size is a good indicator for growth characteristics.

Surgically resected small hepatocellular carcinomas showing "nodule-in-nodule" formation were analyzed in terms of cell proliferative activity. The analysis was achieved by successful immunohistochemical demonstration of proliferating cell nuclear antigen in formalin-fixed paraffin-embedded tissue sections. Eight nodules (up to 3 cm in diameter) examined were either atypical adenomatous hyperplasia or hepatocellular carcinoma of low histologic grade, containing a discrete inner nodular area composed of obvious hepatocellular carcinoma of higher histologic grade. In all cases, the proliferating cell nuclear antigen labeling index of the latter area was much higher than that of the former, which in turn was slightly higher than that of the non-cancerous liver of the patient in 6 cases. The data presented here provide supporting evidence that the successive emergence and expansion of a more rapidly proliferating subclone within a nodule result in the stepwise progression of malignancy of human hepatocellular carcinoma.

Thirty-one cases of Hodgkin's disease were examined for the occurrence of Epstein-Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin-fixed paraffin-embedded tissues and in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the BamHI-W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed-Sternberg (RS) cells and their mononuclear variants. Furthermore, double-labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B-marker (L26) and pan T-marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA-positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.