Immunohistochemical study of Ki-67 (MIB-1), p53 protein, p21WAF1, and p27KIP1 expression in benign, atypical, and anaplastic meningiomas.

Histologic grading of meningiomas has prognostic and clinical therapeutic implications. Meningiomas were histologically classified into 3 different World Health Organization grades. Grade II, an atypical meningioma, was defined by major and various minor histologic criteria. However, these histologic criteria sometimes are not fulfilled, and other criteria are necessary. We studied and analyzed the immunohistochemical expression of MIB-1, p53, p21WAF1, p27KIP1 proteins in 146 cases of meningiomas, including 109 benign, 27 atypical, and 10 anaplastic meningiomas. Most of the benign meningiomas expressed low MIB-1 labeling index (mean, 1.5%), and fewer cases had p53 protein expression. In contrast, the anaplastic meningiomas had a high labeling index of MIB-1 (mean, 19.5%) and always expressed p53 protein, with a mean labeling index of 6.3%. The atypical meningiomas had MIB-1 and p53 labeling indexes in the range between benign and anaplastic meningiomas, with mean labeling indexes of 8.1% and 3.5%, respectively. These expressions were statistically significant among benign, atypical, and anaplastic meningiomas (P <.001). We conclude that the immunohistochemistry of MIB-1 and p53 protein will be valuable in discriminating atypical meningiomas from benign or anaplastic meningiomas, at least in histologically borderline cases. In addition, we also found direct correlation of p21 and inverse correlation of p27 expressions in meningiomas with increasing histologic grade and proliferative index.

MR imaging of synovial sarcoma arising from the floor of the mouth: a case report.

A previously unreported synovial sarcoma arising from the floor of the mouth occurred in a 46-year-old man. Preoperative MR imaging findings of the tumor reflected the macroscopic findings of the resected mass. MR imaging findings were helpful in the diagnosis of this malignancy.

Evaluation of p53 gene mutation and loss of heterozygosity of 3p, 9p and 17p in precancerous lesions of 29 lung cancer patients.

To assess the multiple steps involved in carcinogenesis we evaluated p53 gene mutation frequencies, and loss of heterozygosity (LOH) of chromosomes 3p, 9p and 17p in hyperplasia, squamous metaplasia, dysplasia, squamous cell carcinoma in-situ and early squamous cell carcinoma of the bronchial epithelium, using immunohistochemical, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and microsatellite analysis. Overexpression of p53 oncoprotein was observed in 31% of mild dysplasia, 50% of moderate and 67% of severe dysplasia. Mutation of the p53 gene, however, was confirmed only in 2 severe dysplasias. LOH of 3p, 9p and 17p were detected in 8% of mild and 6% of moderate dysplasias. Among other types of bronchial lesions, two columnar cell hyperplasias showed LOH of 3p and 17p, respectively. These results suggest that LOH of 3p or 9p may be a rare but early event in the development of squamous cell carcinoma of the lung. In particular, deletion of 3p21.3 is a relatively common event in premalignancy. The results also suggest that p53 gene abnormalities occur during the following stage of carcinogenesis.

Gallyas-positive argyrophilic and ubiquitinated filamentous inclusions in rapidly progressive motor neuron disease: immunohistochemical and electron microscopic studies.

In an autopsy case of sporadic rapidly progressive lower motor neuron disease (MND), Gallyas-positive argyrophilic and ubiquitinated filamentous intracytoplasmic inclusions were found in the neurons. Clinically, 7 months prior to death, a 68-year-old woman experienced a history of rapidly progressive muscle weakness of all four extremities and bulbar sign, without sensory and autonomic disturbance. Two months later, she became unable to stand or walk. Four months after onset, she needed respiratory support, and subsequently died due to cardiorespiratory arrest. Neuropathological examinations revealed neuronal loss and associated gliosis in the lower motor neurons, except for ocular motor nuclei, Clark's column, and accessory cuneate nucleus, and tract degeneration was observed in the middle root zone of the posterior column and spinocerebellar tract. No Bunina bodies or Lewy body-like hyaline inclusions were found in the anterior horns. Gallyaspositive argyrophilic filamentous inclusions were found in the lower motor neurons and in nerve cells of the Clark's column, intermediate zone, posterior horn and accessory cuneate nucleus. These were positive with anti-ubiquitin antibody but negative with anti-tau (tau-2 and AT8) and neurofilament antibodies. Electron microscopic examinations disclosed randomly arranged tubular-like filamentous profiles, with a diameter of 12-14 nm, sometimes with amorphous granules in the perikaryon. This is the first report on the Gallyas-positive argyrophilic and ubiquitinated filamentous inclusions in neurons in MND.

An autopsy case of diffuse panbronchiolitis associated with lung cancer.

The prognosis of diffuse panbronchiolitis (DPB) has been remarkably improved after the development of low-dose erythromycin therapy, possibly due to anti-inflammatory rather than anti-infective mechanisms. Interestingly, DPB associated with lung cancer is quite rare. Here, we report an autopsy case of DPB who developed lung cancer after a long successful therapy with low-dose erythromycin.

Preoperative treatment with tegafur suppositories enhances apoptosis and reduces the intratumoral microvessel density of human colorectal carcinoma.

BACKGROUND: This study examined the effect of tegafur, a depot of 5-fluorouracil, in human colorectal carcinomas in terms of apoptosis, cell proliferation, and expression of p53 gene and angiogenesis-related molecules. METHODS: A total of 32 patients with colorectal carcinoma were divided into 2 groups; 20 patients received tegafur suppositories (TS) at 1 g/day for 14 days before surgery, and 12 patients did not receive any chemotherapy. Surgically removed specimens were examined immunohistochemically for Ki-67, CD34, p53, p21, Bax, vascular endothelial growth factor (VEGF), and thymidine phosphorylase (dThdPase). Apoptotic tumor cells were visualized by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) procedure. RESULTS: The mean percentage of apoptotic index (AI) was 6.9 +/- 1.2 in the 20 TS-treated tumors and 4.4 +/- 1.0 in the 12 nontreated tumors (P < 0.001). In contrast, the mean percentage of Ki-67 labeling index (KI) became significantly lower in the former group (P < 0.05). The frequency of p21 expression was significantly higher in the TS-treated group than in the nontreated group (P < 0.05), whereas no difference was detected in p53 and Bax expression between the two groups. The mean intratumoral microvessel density was 47.8 +/- 19.8 in the TS-treated tumors and 66.8 +/- 16.5 in the nontreated tumors (P < 0.01). The frequency of dThdPase expression, but not of VEGF expression, became significantly lower with the TS treatment. p53 expression did not correlate with AI, KI, IMV density, or the expression of VEGF, p21, or Bax, except for dThdPase, which was significantly higher in the 18 p53 positive tumors (P < 0.05). CONCLUSIONS: Preoperative TS treatment enhances apoptosis and suppresses angiogenesis of colorectal carcinomas in a p53-independent manner.

Numerous conglomerate inclusions in slowly progressive familial amyotrophic lateral sclerosis with posterior column involvement.

A 59-year-old woman with slow progression of the loss of motor function and predominant lower motor manifestation during a 14-year period showed familial amyotrophic lateral sclerosis (fALS) with posterior column involvement, neuropathologically. Conglomerate inclusions (CIs) were observed in the remaining neurons in various areas, including the spinal anterior horn, posterior horn, Clark's column, accessory cuneate nucleus, tegmental reticular formation, motor nucleus of the trigeminal nerve, nucleus of the facial nerve, hypoglossal nucleus, medial nucleus of the thalamus, dentate nucleus, and motor cortex (Betz cells). Immunohistochemically, it was newly identified that the CIs showed marked immunoreactions with antibodies to phosphorylated and non-phosphorylated neurofilaments and to 64, 120, and 200 kD neurofilaments. The CIs were partially immunoreactive with the anti-ubiquitin antibody, although they reacted only weakly (or not at all) with anti-Cu/Zn superoxide dismutase (SOD1) antibody. Ultrastructurally, the CIs were comprised of neurofilaments. These data suggest that this case might have been different from an example of fALS with Ile 113 Thr mutation in the SOD1 gene.

A case of group II non-specific interstitial pneumonia developed during corticosteroid therapy after acute respiratory distress syndrome.

Non-specific interstitial pneumonia/fibrosis (NSIP) is a relatively new entity among idiopathic interstitial pneumonias. This disease is pathologically characterized by varying proportions of interstitial inflammation and fibrosis that appear to occur over a single, short period of time, and is clinically characterized by a generally good prognosis. Although corticosteroid therapy is widely considered to be effective for treatment of this disease, we report a case of Group II NSIP which developed during treatment with corticosteroid 4 months after acute respiratory distress syndrome (ARDS). This case suggests that any group of NSIP may be developed after ARDS, and that low-dose corticosteroid treatment is not sufficient to prevent the development of NSIP.

Symptomatic Rathke's cleft cyst coexisting with central diabetes insipidus and hypophysitis: case report.

We describe a 48-year-old female with acute onset of central diabetes insipidus followed by mild anterior pituitary dysfunction. Magnetic resonance imaging (MRI) revealed enlargement of the hypophysis-infundibulum accompanied by a cystic component. She underwent a transsphenoidal exploration of the sella turcica. Histological examination showed foreign body type xanthogranulomatous inflammation in the neurohypophysis which might have been caused by rupture of a Rathke's cleft cyst. The MRI abnormalities and anterior pituitary dysfunction improved after a short course of corticosteroid administration, but the diabetes insipidus persisted. The histological findings in this case indicated the site of RCC rupture and the direction of the progression of RCC induced neurohypophysitis and adenohypophysitis.

Assessment of usefulness of endobronchial ultrasonography in determination of depth of tracheobronchial tumor invasion.

STUDY OBJECTIVE: We assessed the usefulness of endobronchial ultrasonography in the determination of the depth of tumor invasion of the tracheobronchial wall. METHODS: We performed a needle-puncture experiment on normal tissue of 45 specimens to determine the laminar structure of the tracheobronchial wall. In addition, we compared the ultrasonographic determinations of tumor invasion from 24 lung cancer cases with the histopathologic findings. RESULTS: The cartilaginous portions of the extrapulmonary bronchi and the intrapulmonary bronchi exhibited a five-layer structure. Starting on the luminal side, the first layer (hyperechoic) was a marginal echo, the second layer (hypoechoic) was the submucosal tissue, the third layer (hyperechoic) was the marginal echo on the inner side of the bronchial cartilage, the fourth layer (hypoechoic) was bronchial cartilage, and the fifth layer (hyperechoic) was the marginal echo on the outer side of the cartilage. In the membranous portions, the first layer (hyperechoic) was a marginal echo, the second layer (hypoechoic) was smooth muscle, and the third layer (hyperechoic) corresponded to the adventitia. Comparisons between the ultrasonograms and the histopathologic findings in 24 lung cancer cases revealed that depth diagnosis was the same in 23 lesions (95.8%) and was different in 1 lesion (4.2%). In the single case in which the findings were different, lymphocytic infiltration that protruded between the cartilage rings was mistakenly interpreted as tumor infiltration. CONCLUSIONS: This method allows visualization of the laminar structure of the tracheobronchial wall, which is impossible with other diagnostic imaging methods.

A case of bronchial squamous cell carcinoma in situ detected by sputum cytology.

A 64-year-old man underwent a medical checkup in May 1996 and was evaluated as class V using sputum cytology. Chest X-ray examination, bronchoscopy and chest computed tomography (CT) demonstrated no abnormalities. Thereafter, the patient was followed up with chest X-ray, bronchoscopy and chest CT at 3-month intervals. In December 1996, chest CT showed an increased density at the mediastinal side of the left upper bronchus, B1+2. There were no findings on bronchoscopy, but subsequent exfoliative cytology demonstrated keratinized malignant cells in samples obtained from left upper bronchus, B1+2. Although, it was difficult to identify localization of the tumor, left upper lobectomy was performed and the diagnosis of squamous cell carcinoma in situ was finally made. Here, we report on the course of this patient and discuss the diagnostic usefulness of sputum cytology as well as the pathogenesis of lung squamous cell carcinoma.

[Endobronchial ultrasonography for lung cancer].

We assessed the usefulness of endobronchial ultrasonography in the diagnosis of lung cancer. We performed a needle-puncture experiment on 45 normal tissue specimens to determine the luminar structure of the tracheobronchial wall. In addition, we compared the ultrasonographic determination of tumor invasion in 24 lung cancer patients with the histopathological findings. The cartilaginous portions of the extrapulmonary bronchi and the intrapulmonary bronchi exhibited a five-layered structure. Starting on the lumen side, the first layer (hyperechoic) was a marginal echo, the second (hypoechoic) was the submucosal tissue, the third (hyperechoic) was the marginal echo on the inner side of the bronchial cartilage, the fourth (hypoechoic) was bronchial cartilage, and the fifth (hyperechoic) was the marginal echo on the outer side of the cartilage. In the membranous portions, the first layer (hyperechoic) was a marginal echo, the second (hypoechoic) was smooth muscle, and the third (hyperechoic) corresponded to the adventitia. Comparisons between the ultrasonograms and the histopathological findings in 24 lung cancer patients revealed that depth diagnosis was the same in 23 lesions (95.8%) and different in 1 lesion (4.2%). We describe the usefulness of endobronchial ultrasonography in the diagnosis of peribronchial lymph nodes and peripheral pulmonary lesions.

Comparison of phototoxicity mechanism between pulsed and continuous wave irradiation in photodynamic therapy.

A study has been conducted in which HeLa cells are incubated with hematoporphyrin derivative (HpD) for 1 h (1 microgram/ml of HpD in PBS) to compare the use of continuous wave (CW) and pulsed laser (10 Hz repetition rate and 7-9 ns pulse width) light for photodynamic therapy. Cytotoxic effects on the cells are evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay and the fluorescein diacetate (FDA)/propidium iodide (PI) stain method using a flow cytometer. The type of cell death is estimated by analysis of the DNA content and observation of the nuclear morphology. The cytotoxicity ratio of cells irradiated by pulsed laser light is estimated to be lower than that for CW laser light. The viability of cells that received pulsed laser light gradually decreases, whereas no significant changes are found in the cells irradiated with CW laser light with the elapse of post-irradiation time. The type of cell death differs between the pulsed and CW laser light irradiations. These findings suggest that the cytotoxic efficacy of the excitation light source is displayed by the difference in the type of cell death, namely apoptosis or necrosis.

p21WAF1/CIP1 expression in primary lung adenocarcinomas: heterogeneous expression in tumor tissues and correlation with p53 expression and proliferative activities.

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, discovered to be a downstream effector of p53-dependent cell cycle regulation. In order to elucidate the significance of p21 expression in lung adenocarcinomas, we performed immunohistochemical analysis of p21, p53 and Ki-67 expression in surgically resected lung adenocarcinomas. In non-neoplastic tissue, a few bronchial and bronchiolar suprabasal and ciliated cells, and a few type II alveolar cells and alveolar macrophages in the peripheral lung, were p21 positive, but the positive rate in normal lung tissue was very low (<1%). All 91 lung adenocarcinomas examined showed p21 immunoreactivity: 39 cases (42.9%) and 52 cases (57.1%) showed high and low p21 expression levels, respectively. There was no significant correlation between p21 expression and p53 expression, the loss of heterozygosity status of the p53 gene, histological grade determined by the predominant histology, lymph node metastasis, pathological stage, tumor size, smoking history or gender. A positive, not inverse, correlation between p21 and Ki-67 expression was observed. We also observed heterogeneous expression of p21 in lung adenocarcinomas, i.e. in about two-thirds of the tumors, the tumor cells in the peripheral regions were p21 positive more frequently than were those in the central regions. More intense p21 expression tended to occur in the more highly differentiated areas. These results suggest that p21 is involved in tumor cell differentiation and the physiological mechanism that protects against tumor extension.

Proliferative activity, p53 expression and loss of heterozygosity on 3p, 9p and 17p in atypical adenomatous hyperplasia of the lung.

We examined the preneoplastic nature of atypical adenomatous hyperplasia (AAH) of the lung by comparing the proliferative activity, p53 oncosuppressor gene product and loss of heterozygosity (LOH) on 3p, 9p and 17p of 20 AAH lesions (8 cases) with corresponding normal peripheral lung tissue and adenocarcinoma from the same cases. Analysis of proliferative activity with the Ki-67 labelling index and argyrophilic nucleolar organizer regions (AgNORs) score indicated that AAH had a proliferative activity intermediate between that of normal cell and adenocarcinoma. Although low level expression of p53 was detected in 7 AAH lesions, the intensity of p53 expression in AAH was weaker than that in carcinomas. Microsatellite analysis of chromosome 3p, 9p and 17p showed LOH of 18%, 13% and 6% respectively in the AAH lesions, while the corresponding carcinomatous lesions showed LOH of 67%, 50% and 17% respectively. All AAH lesions that showed LOH had moderate or severe histological atypia. One AAH lesion with moderate atypia showed LOH both on 3p and 17p. In conclusion, these results indicated that AAH lesions with moderate or severe atypia may show the preneoplastic stage of lung adenocarcinoma.

p16/CDKN2 gene and p53 gene alterations in Japanese non-smoking female lung adenocarcinoma.

Primary lung adenocarcinomas in non-smoking females are increasing in the USA and Japan. Environmental factors such as passive smoking, asbestos, domestic radon, and hormonal effects have been implicated, but the etiology is still uncertain. We therefore analyzed point mutations of p16 gene, a newly characterized tumor suppressor gene, and compared the results with alterations of p53 gene in 28 primary lung adenocarcinomas in non-smoking Japanese females. There were no cases with somatic point mutation of p16 gene, except for one case with two germline mutations (silent mutations). In contrast, six out of 16 informative cases showed loss of heterozygosity of p53 gene using a TP53 microsatellite marker and 19 out of 28 cases showed expression of oncoprotein using DO-7 immunohistochemistry. These findings suggest that p16 gene alteration is a rare event in primary lung adenocarcinomas in Japanese non-smoking females, compared with alterations of the p53 gene.

Role of point mutation of the K-ras gene in tumorigenesis of B6C3F1 mouse lung lesions induced by urethane.

In order to elucidate the role of point mutation of the K-ras gene in the tumorigenetic process of lung tumors, an experimental model of lung lesions in mice induced by the administration of urethane was used. A total of 135 B6C3F1 male mice, 6 weeks old, were given urethane in the drinking water at 0, 6, 60, 600 or 1200 ppm, and were then killed after varying periods of time. The lung lesions were histologically characterized as hyperplasia, adenoma and adenocarcinoma. Point mutations in codons 12 and 61 of the K-ras gene were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and confirmed by using dideoxy sequencing analysis. K-ras gene mutation was identified in 9 (23.7%) of 38 lesions classified as hyperplasia, 31 (46.3%) of 67 adenomas, and 3 (50%) of 6 adenocarcinomas. The most frequent mutation was an AT-to-TA transversion at the second base of codon 61 and this pattern accounted for 65% of the three mutant forms observed. These results suggest that the point mutation of K-ras gene is involved in all stages of mouse lung tumorigenesis, i.e., activation of this gene can also influence the later stages of lung lesions.

Quantitative risk assessment of carcinogenicity of urethane (ethyl carbamate) on the basis of long-term oral administration to B6C3F1 mice.

A carcinogenicity study of urethane was performed for quantitative assessment of its risk in humans. Three hundred 6-week-old male B6C3F1 mice were divided into 6 groups, each consisting of 50 mice, and urethane was given ad libitum in drinking water at levels of 0 (control), 0.6, 3, 6, 60 and 600 ppm for 70 weeks. The tumors with a clear dose-response relationship were lung tumor (alveolar/bronchiolar adenoma or carcinoma) and liver tumor (hemangioma or angiosarcoma). The incidences of these two types of tumor were applied to estimation of the virtually safe dose (VSD) at the level of 10(-6) by using four mathematical models (Logit, Probit, Weibull and Multistage models). The VSD based on the incidences of lung tumor by using the Logit model was estimated to be 1.8 x 10(-4) mg/kg body weight/day. On the other hand the VSD based on those of liver tumor by using the Weibull model was 7.2 x 10(-5) mg/kg body weight/day. Thus, the VSDs based on the incidences of the two different types of tumor using the most compatible mathematical model in each case, as judged from the P-values, were similar.

Severed nerve stumps around a laser-irradiated locus in the deep muscular plexus of the guinea-pig small intestine.

Small foci of photocoagulation necrosis (diameter about 0.5 mm) were produced in the wall of the guinea-pig small intestine by argon laser irradiation applied to the serosal surface. Features of the nerve elements around the necrotic masses were examined by immunocytochemistry, scanning electron microscopy, and transmission electron microscopy. The severed ends of nerve strands were examined at various intervals from 10 to 60 h after the laser irradiation, and the acute cytological responses of the nerve terminals, glial cells and connective tissue cells were studied. The laser irradiated foci in the deep muscular plexus (plexus muscularis profundus: CAJAL, 1911) had concentric configuration: 1) central necrotic area, 2) inner transitional zone, 3) outer transitional zone, and 4) normal area. In the necrotic area, there were inflammatory cells such as neutrophile leukocytes and macrophages. Contours of the smooth muscle cells and nerve strands were well preserved in spite of the damage to their subcellular structures. In the inner transitional area, individual nerve fibers gathered to form bundles. Some of these nerve bundles were devoid of a glial cell framework. In the outer transitional zone, there were swollen nerve terminals with a strong immunoreactivity for methionine-enkephalin-Arg6-Gly7-Leu6. In transmission electron microscopy, some of these swollen nerve fibers contained many large cored vesicles. Glial cells in the outer transitional zone exhibited an S-100b protein-like immunoreactivity. Scanning electron microscopy revealed gatherings of fibroblast-like (FBL) cells in the outer transitional zone. Two subtypes of these were distinguished: the first subtype (FBL I) was characterized by short, blunt cytoplasmic processes; the second subtype (FBL II), possessing long, slender dendritic processes, assembled to form a dense network which intermeshed with that of the nerve strands. The enteric nerve plexuses severed by laser-irradiation affords a unique experimental model in the cytological investigation of the plasticity of the peripheral autonomic nervous system.