Morules and morule-like features associated with carcinomas in various organs: report with immunohistochemical and molecular studies.

BACKGROUND: Morules have been reported in pulmonary blastoma (PB), well differentiated fetal adenocarcinoma of the lung (WDFA), and uterine endometrioid carcinoma (EC), and rarely in other carcinomas. beta Catenin gene mutation has been associated with morule formation. AIMS: To compare and clarify the cellular characteristics of morules in carcinomas in various organs and show that morules are distinct from epithelial cellular nodules. METHODS: Twenty tumours were studied: two PBs, three WDFAs, three papillary lung adenocarcinomas, 11 ECs, and one papillary thyroid carcinoma. Numerous epithelial cell, oncofetal, and neuropeptide antibodies were used for immunohistochemistry. beta Catenin gene mutation was investigated. RESULTS: Morules in PBs and ECs were uniform cell clusters distinct from squamous differentiation. All were immunonegative for epithelial cell and oncofetal antigens, but those in ECs were positive for neurone specific enolase gamma (NSEgamma). Synaptophysin, encephalin, and somatostatin were sporadically immunopositive in PB morules. Morules were not seen in the other carcinomas and WDFAs, although morule-like features closely resembling morules histopathologically were seen. These were positive for epithelial cell and oncofetal antigens, and showed squamous differentiation. Their nuclei were more atypical and slightly larger than those in morules. Morule-like features were seen in WDFAs. beta Catenin gene mutation was demonstrated in one EC and PB, and in two WDFAs. CONCLUSION: Morules were non-epithelial cell clusters showing neuronal differentiation. There were two types: endometrioid type, expressing NSEgamma, and blastoma type, expressing neuropeptides. In contrast, similar morule-like features were epithelial nodules. Although the number of cases was small, the presence of morules showed no clear prognostic correlations.

Recent striking changes in histological differentiation and rate of human papillomavirus infection in squamous cell carcinoma of the lung in Okinawa, a subtropical island in southern Japan.

AIMS: The incidence of lung cancer in Okinawa has been the highest in Japan since 1975, and squamous cell carcinoma (SCC), especially the well differentiated form, is the most prevalent form in Okinawa, although well differentiated SCC is relatively rare in mainland Japan. Furthermore, a high proportion of SCC of the lung in Okinawa was positive for human papillomavirus (HPV). In this study, we report recent striking changes in histological features and in the incidence of HPV infection. METHODS: In Okinawa between 1986 and 1998, 1109 surgically resected lung tumours were examined histopathologically. In addition, human papillomavirus infection was detected by the polymerase chain reaction and Southern blot analysis in SCC cases reported in 1993 and 1995-8. Non-isotopic in situ hybridisation of HPV DNA was also carried out. RESULTS: Up until 1994 SCC, especially the well differentiated form, was the most prevalent type of tumour. However, since 1995 the number of such cases has diminished steadily, accompanied by a slight rise in the incidence of adenocarcinoma. Although most present and past patients are heavy smokers, the incidence of SCC, especially the well differentiated form, continues to decrease steadily. Furthermore, in 1993, HPV was detected in 79% of all cases, and was particularly prevalent in the well differentiated form, but the rate fell to 68% in 1995, 35% in 1996, 23% in 1997, and 24% in 1998. The age distribution of patients, the male to female ratio, and the number of tumours overexpressing p53 protein did not change significantly over the study period, and thus did not correlate with changes in the differentiation of SCC. CONCLUSIONS: The decreasing incidence of viral infection correlates strongly with the falling numbers of SCC cases, especially well differentiated cases. These findings suggest that HPV might be involved in the development of SCC of the lung, affecting the histological differentiation of SCC in particular, at least in Okinawa, a subtropical island in southern Japan.

Human papillomavirus DNA in adenosquamous carcinoma of the lung.

AIM: To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features. METHODS: Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out. RESULTS: 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas. CONCLUSIONS: HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma.

Human papillomavirus DNA in squamous cell carcinoma of the lung.

AIM: To compare the incidence of squamous cell carcinoma (SCC) of the lung in Okinawa with that in Niigata on the mainland. METHODS: All patients presenting with SCC of the lung in Okinawa and Niigata in 1993 were included in the study. Diagnoses were confirmed by conventional histological examination of paraffin wax sections. Human papillomavirus (HPV) was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for the E6 and E7 regions of the HPV genome. PCR products were analysed by Southern and dot blotting. RESULTS: The incidence of well differentiated SCC of the lung was high in patients from Okinawa compared with moderately and poorly differentiated types, and compared with the incidence of SCC in patients from Niigata. This is despite similar patterns of age, sex (predominatly male), and smoking habit. More patients from Okinawa, however, were positive for HPV DNA by PCR (79%) and NISH (53%). Many patients haboured HPV types 6, 16, and 18. Only 30% of patients from Niigata were positive for HPV DNA by PCR and 20% by NISH. These patients all harboured one HPV type only. CONCLUSION: Surprisingly large numbers of patients from Okinawa were positive for HPV DNA. The detection of HPV DNA was strongly associated with well differentiated SCC. This was particularly true for HPV types 6 and 16. There was no correlation between either smoking and detection of HPV DNA, or smoking and histological differentiation.

Prognostic implication of human papillomavirus infection in squamous cell carcinoma of the lung.

On the subtropical island of Okinawa, squamous cell carcinoma (SCC), particularly the well-differentiated form, is the most frequent type of lung cancer, while this form is relatively rare on the Japanese mainland and in other countries. Furthermore, in Okinawa, in 1993, 80% of SCC cases of the lung were found to be infected with human papillomavirus (HPV). We studied the prognosis of SCC of the lung with HPV infection (n = 25) and compared it with non-HPV-infected SCC (n = 16). Using the Kaplan-Meier method (Wilcoxon analysis), the prognosis of HPV-infected cases was found to be better than that of the non-infected cases. In the virus-infected cases, apoptosis and infiltration of a large number of Langerhans cells were demonstrated. In addition to these findings, the virus-infected tumors were demonstrated to be histologically well-differentiated, perhaps contributing to the favorable prognosis. However, among the virus-infected cases, the type 16 virus-infected cases showed a poorer prognosis, compared to those infected with other HPV types. p53 gene mutation was also examined, and was considered to be an unfavorable prognostic factor, as reported elsewhere. However, in Okinawa, HPV-positive cases with p53 mutations showed a slightly better prognosis than did non-viral infected cases with p53 mutations. The TNM staging system was also useful for categorizing the virus-infected cases. The prognosis of stage III (A and B) cases was poor. All of our present cases received surgical treatment. Chemotherapy and radiation therapy were not performed. Such treatment, however, might be effective, because virus-infected uterine cervical carcinomas have been routinely treated with chemotherapy and radiation. Furthermore, if the immunological basis of increased Langerhans cell infiltration in HPV-infected cases is elucidated, a clinical trial with immunotherapy may be favorable for the clinical outcome.

[Pompe's disease--acid alpha-glucosidase deficiency--a review].

The importance of the role of acid alpha-glucosidase in the lysosomal degradation of glycogen has been emphasized because the deficiency of this enzyme in glycogenosis type II causes glycogen accumulation in lysosomes. Three clinical variants are distinguished. The infantile type has its onset shortly after birth and is known as generalized glycogen storage disease. The adult variant manifests itself mostly after the second decade of life and is characterized by progressive skeletal muscle weakness. The other is childhood type which is usually fatal by the second decade of life. Many biochemical reports of acid alpha-glucosidase have been published. Martiniuk et al reported the cDNA and amino acid sequence of human acid alpha-glucosidase. In prior studies, they reported that the lysosomal acid alpha-glucosidase was polymorphic with three alleles. The rarer allele GAA2 allozyme had a lower affinity for glycogen and starch. We also reported the enzyme heterogeneity in its affinity to Sephacryl S-200 gel. Whereby the enzyme separated into two fractions, S1 and S2. Each fraction contained 76 kDa and 67 kDa components on SDS/PAGE. The spleen enzyme consisted mainly of S1 fraction, containing only a 76 kDa component. In previous extensive studies, different mutations of Pompe's disease have been inferred from alterations in biochemical parameters. More recently Martiniuk et al, Hoefsloot et al and Van der Ploeg et al reported the analysis of cDNA and mRNA. These studies have revealed an absence or abnormal size of mRNA in large numbers of patients and altered restriction endonuclease fragments in a few patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Glycogenosis type II; acid alpha-glucosidase deficiency].

Knowledge of the general enzymology of lysosomal acid alpha-glucosidase (acid maltase) including that of the intracellular processing mechanism and of the cDNA sequence has advanced recently. Genetic heterogeneity of the mRNA of acid alpha-glucosidase deficient patients has also been reported. Referring to these recent advances, and based on our previously reported results, the pathologic aspects of the disease are reviewed. Heterogeneity of the enzyme molecules themselves and their characteristics in various tissues are also demonstrated.

Extremely high Langerhans cell infiltration contributes to the favourable prognosis of HPV-infected squamous cell carcinoma and adenocarcinoma of the lung.

AIMS: The infiltration of Langerhans cells in adenocarcinomas and squamous cell carcinomas of the lung was examined in relation to prognostic implications and human papillomavirus (HPV) infection. METHODS AND RESULTS: Samples from 62 adenocarcinoma and 59 squamous cell carcinoma patients in 1995-97, the prognosis of which had been followed up, were used. The Langerhans cells were demonstrated immunohistochemically using anti S100a and CD1 antibodies. Human papillomavirus (HPV) infection was examined by polymerase chain reaction (PCR) and nonisotopic in-situ hybridization (NISH) methods. Statistical analysis was carried out using the Kaplan-Meier method (Wilcoxon analysis) and multiple regression analysis. HPV infection was demonstrated in 12 cases (19.4%) of adenocarcinoma. The HPV-infected adenocarcinomas had abundant faintly eosinophilic cytoplasm, and were immunohistochemically positive for the surfactant apoprotein A. In the 59 cases of squamous cell carcinomas 19 were of the well differentiated form, and 29 and 11 were moderately and poorly differentiated cases, respectively. HPV was detected in 29 cases (49.2%) (13 well and 16 moderately differentiated cases). In all HPV-infected adenocarcinoma and squamous cell carcinoma cases, extremely large numbers of Langerhans cells (more than 100 per high-power field) were demonstrated in the tumour nests. In contrast, in the non-HPV-infected adenocarcinomas and squamous cell carcinomas, only a few (less than about 10 per high-power field) Langerhans cells were observed. The squamous cell carcinoma cases with high Langerhans cell infiltration, which were also infected with HPV, showed a significantly good prognosis (P = 0.007). The adenocarcinoma cases with high Langerhans cell infiltration tended to have a better prognosis than the cases with low Langerhans cell infiltration, but the difference was not statistically significant. The low number of highly infiltrated cases was insufficient for an adequate statistical analysis. Furthermore, there was no significant correlation between either Langerhans cell infiltration and smoking, or HPV infection and smoking, in either squamous cell carcinoma or adenocarcinoma cases. CONCLUSIONS: It was considered that the extremely high Langerhans cell infiltration in the tumours was caused by HPV infection. The extremely large number of Langerhans cells in the tumours contributes to the favourable prognosis for HPV-infected lung cancer.

Pathologic studies and comparison of the virulence of herpes simplex virus type 2 from Okinawa, Japan and Chiang Mai, Thailand.

The virulence of four herpes simplex virus type 2 (HSV 2) strains (K1-K4) isolated in Okinawa, Japan was investigated, and compared with four strains (C1-C4) from Chiang Mai, Thailand and a standard laboratory strain SAV. Virulence was tested on BALB/c and C57/black mice. After viral inoculation intraperitoneally, the distribution with the passage of time of the virus in the brain and other organs was also studied using the polymerase chain reaction (PCR) method and immunohistochemistry. Generally, Okinawan HSV 2 (K1, K3 and K4) were less virulent than Chiang Mai strains (C1-C4). Among the Okinawan strains, K2 was the most virulent, but slightly less so than C3 and C4. All four Chiang Mai strains (C1-C4) and one Okinawan strain (K2) were more virulent than SAV strain. The virulence of K3 was very weak and no animals died from the intraperitoneal inoculation. In the brain, viral DNA from each strain was demonstrated at 1-9 days after inoculation by the PCR method. However, K3 strain was detected in the brain only between one day and 3 days after virus inoculation, and not after day 5. Immunohistochemically, the virus antigen was first demonstrated around the 3rd ventricle at one day after viral inoculation, then strongly at the ventral hypothalamus and the temporal lobe at 3 days after viral inoculation, and slightly in the frontal lobe, hippocampus, pons and cerebellum (on day 5 after inoculation). Furthermore, in Kupffer cells in the liver and macrophages in the spleen, numerous viral antigens were demonstrated from one to 9 days after viral inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma of the lung in Okinawa, Japan: with special reference to squamous cell carcinoma and squamous metaplasia.

In Okinawa, a subtropical island in southern Japan, squamous cell carcinoma (SCC), especially the well-differentiated form, is prevalent, while this form is relatively rare in both the mainland and other countries (e.g. United States of America). More patients with SCC from Okinawa, moreover, were positive for human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) (79%), and harbored HPV types 6, 16 and 18, in combination. On the other hand, less than 30% of the mainland patients were positive for HPV DNA by PCR. Those patients who were positive all harbored only one HPV type. Furthermore, in Okinawa, there were a significant number of cases with adenosquamous carcinoma, and they too were positive for HPV DNA. The SCC and the adenocarcinoma cells adjacent to the SCC component in these cases were also positive for HPV DNA, and such adenocarcinoma cells were enlarged in size with relatively wide cytoplasm. The authors postulate that HPV infects adenocarcinoma cells and changes them to enlarged cells, followed by squamous metaplasia. In this report, HPV DNA was transfected to adenocarcinoma cells (cultured cell lines) and this showed that HPV causes squamous metaplasia. In addition, aberrant expression of p53 was demonstrated in a large number of the SCC cases in Okinawa. The enlarged adenocarcinoma cells adjacent to the SCC components in adenosquamous carcinomas also showed aberrant expression of p53. The recent advances in the studies of anti-oncogenes, p53, etc. and oncogenes are outlined. It is to be noted that the molecular mechanisms of carcinogenesis in the lung have been studied in general, classifying lung tumors into two groups, namely, small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). However, because human lung cancer is represented by a wide variety of histologic types, molecular genetic studies according to a more detailed histological subclassification is needed.