, were histopathologically analyzed. Hematoxylin-eosin staining, immunohistochemistry for type A influenza nucleoprotein antigen, and real-time reverse transcription-PCR assay for viral RNA were performed on formalin-fixed and paraffin-embedded specimens. In addition, the D222G amino acid substitution in influenza virus hemagglutinin, which binds to specific cell receptors, was analyzed in formalinfixed and paraffin-embedded trachea and lung sections by direct sequencing of PCR-amplified products. There were several histopathological patterns in the lung according to the most remarkable findings in each case: acute diffuse alveolar damage (DAD) with a hyaline membrane (four cases), organized DAD (one case), acute massive intra-alveolar edema with variable degrees of hemorrhage (three cases), neutrophilic bronchopneumonia (five cases) and tracheobronchitis with limited histopathological changes in alveoli (four cases). In two cases, the main findings were due to preexisting disease. Influenza virus antigen was only detected in the respiratory tract in 10 cases by immunohistochemistry. The antigen was detected in type II pneumocytes (three cases) in the epithelial cells of the trachea, bronchi and glands (six cases), and in the epithelial cells in both of the above (one case). The four cases with acute DAD presented with antigen-positive type II pneumocytes. In one case, the D222G substitution was detected in the lung as a major sequence, although 222D was prominent in the trachea, suggesting that selection of the viral clones occurred in the respiratory tract. In five

Aim-To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lungwhich 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 diVerent 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. (J Clin Pathol 1998;51:741-749)

In Okinawa, a subtropical island in Southern Japan, the incidence of oral squamous cell carcinoma is 1.5 times higher than that in mainland Japan. Sixty cases of oral squamous cell carcinoma from 1993 to 1996 in Okinawa and 42 cases over the same period in Sapporo were examined histologically. Human papillomavirus (HPV) and Epstein-Barr virus (EBV) were detected by polymerase chain reaction (PCR) amplification with primers specific for HPV and EBV. In situ hybridisations of the viruses were also carried out. In the case of Epstein-Barr virus, in situ PCR was also performed. Thirty-five (58.3%) Okinawan tumours were well-differentiated in type, but in Sapporo, 18 (42%) were of such type. In Okinawa, tumours of the mouth floor (10 cases, 16.7%) and oropharynx (12 cases, 20%) were frequently observed, whereas in Sapporo only five cases (12%) of each were found. HPV was demonstrated in 78% of Okinawan cases and 26.2% of Sapporon cases by PCR or non-isotopic in situ hybridisation (NISH). There were 76.6% (46 cases) of Okinawan and 38.1% (16 cases) of Sapporo cases positive for EBV by PCR. In only 12 Okinawan cases and 4 Sapporon cases, were positive signals demonstrated by in situ PCR on the cancer cells themselves. EBV was demonstrated in the large number of infiltrating lymphocytes, most of which were CD3+, and a few were CD19+. In Okinawa, HPV might be an important causative factor of oral squamous cell carcinoma and EBV a less important factor, whereas in Sapporo HPV and EBV might play only a small part in the aetiology of the tumour.

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.

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