Yukinori Inadome

Malignant lymphoma of bronchus‐associated lymphoid tissue (BALT) coexistent with pulmonary tuberculosis

A case in which malignant lymphoma occurred in association with a tuberculosis focus in a 70‐year‐old man is reported. Surrounding the epithelioid cell granulomas with caseous necrosis was a dense and diffuse monotonous infiltration of atypical lymphoid cells. Acid‐fast bacilli were found in the granulomas and pulmonary tuberculosis was diagnosed. The infiltrating atypical lymphoid cells occasionally invaded the respiratory epithelium producing lymphoepithelial lesions. Immunohistochemically, the lymphoid cells were positive for CD20, and clonal rearrangement of the immunoglobulin heavy chain gene was demonstrated by polymerase chain reaction (PCR). We diagnosed the lesion as a pulmonary malignant lymphoma of bronchus‐associated lymphoid tissue (BALT) occurring in the background of tuberculosis. This is the first reported case of pulmonary BALT lymphoma coexistent with pulmonary tuberculosis.

Clonal proliferation of B lymphocytes in the germinal centers of human reactive lymph nodes: possibility of overdiagnosis of B cell clonal proliferation.

Clonal expansion of the germinal center B cells of human reactive lymph nodes was analyzed. By micromanipulation, 28 germinal centers were microdissected from three nonneoplastic lymph nodes that had been fixed with formalin. Immunoglobulin heavy chain variable (V) region gene rearrangement was examined by seminested polymerase chain reaction (PCR) using two sets of primers (FR2-J and FR3A-J). An oligoclonal development (one to five clones) was found in each germinal center. Depending on the primer used, four or five (16%) of the germinal centers showed a single rearrangement band. The average number of B-cell clones in each germinal center was approximately 2.5. Next, the authors analyzed 50 endoscopic biopsy specimens from 6 patients with non–mucosa-associated lymphoid tissue (MALT) type gastric lymphoma, 25 patients with chronic gastritis, and 19 patients with nonspecific colitis. In addition to the samples from the 6 patients with malignant lymphoma, 8 of 44 biopsy samples (18.2%) from patients diagnosed as having chronic gastritis or nonspecific colitis showed one or two amplified bands. These results indicate that PCR analysis of immunoglobulin heavy chain V region gene rearrangement in small biopsy specimens could be misleading, causing overdiagnosis of reactive lymphoid tissue as B-cell clonal proliferation.

Establishment of an immortalized cell line from a precancerous lesion of lung adenocarcinoma, and genes highly expressed in the early stages of lung adenocarcinoma development

Atypical adenomatous hyperplasia (AAH) is classified as a precancerous lesion of lung adenocarcinoma. We established an immortalized AAH cell line (PL16T) and a human non‐neoplastic bronchial epithelial cell line (PL16B) from the same patient by transfection with the gene for SV40 large T antigen. The expression profile of PL16T was compared with that of PL16B by the suppression subtractive hybridization method. From 704 selectively hybridized clones, we finally selected 25 fragments of mRNA that showed transcription levels more than three times higher in PL16T than in PL16B. Thirteen (52%) and eight (32%) of them encoded tumor‐associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 (S100A2), respectively. The high transcription of TACSTD2 and S100A2 in PL16T was confirmed by in situ hybridization. In normal lung tissue, both TACSTD2 and S100A2 were expressed at very low levels, but seven and five of 14 AAH were positive for TACSTD2 and S100A2, respectively. The frequency of TACSTD2 positivity was increased in 16 of 22 bronchioloalveolar carcinomas (BAC) and adenocarcinoma with mixed subtype with BAC component (mixed BAC). Positivity for S100A2 occurred in four of 22 BAC and mixed BAC. The abnormal transcription of TACSTD2 and S100A2 are thought to be unique molecular markers of the preinvasive stage of lung adenocarcinoma.(Cancer Sci 2005; 96: 668 – 675)

DNA methylation and expression of p16INK4A gene in pulmonary adenocarcinoma and anthracosis in background lung

The p16 (CDKN2/MTS‐1/INK4A) tumor‐suppressor gene is frequently inactivated by DNA methylation in lung carcinomas. To clarify whether background anthracosis may play a role in DNA methylation and inactivation of the p16 gene, we examined DNA methylation of the p16‐promoter region by methylation‐specific polymerase chain reaction, and p16 expression immunohistochemically, and compared the results with the level of background anthracosis which was measured by an original quantitative method. At autopsy, DNA methylation of the p16 gene was observed in 6/19 tumors (32%) from patients who had died of pulmonary adenocarcinoma. The degree of background anthracosis (the effect of extrinsic carcinogenic factors) (mean absorbance value, A = 0.715) of the cases with p16‐gene methylation was significantly higher than that without methylation (mean A value = 0.298). p16 expression was inactivated in all tumors with p16‐gene methylation. The mean A value of black dust matter deposition in cases with normal expression of p16 (A = 0.151) was significantly lower than cases with abnormal expression of p16 (A = 0.531). These results indicate that the level of background anthracosis is closely associated with inactivation of p16 expression and also DNA methylation of the p16‐gene promoter region in pulmonary adenocarcinogenesis. Int. J. Cancer (Pred. Oncol.) 84:609–613, 1999.

Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material

The patterns of DNA degradation in frozen, methanol-fixed, and formalin-fixed tissues were investigated by high-performance liquid chromatography (HPLC). The chromatograms all yielded one major peak with or without several extra minor peaks representing molecular weights of preserved genomic DNA. The most characteristic differences were in the retention times of the major peaks, with the earliest major peak occurring in the formalin-fixed tissues, and followed by the methanol-fixed, and frozen tissue samples, in that order. This means that the molecular weight of the DNA from formalin-fixed tissue is much shortened than that recovered from methanol-fixed tissue and frozen tissue. The results also indicated that a small amount of higher molecular weight DNA is still preserved in formalin-fixed tissues. To improve the amplification efficiency of polymerase chain reaction (PCR) analysis of formalin-fixed material, we isolated the higher molecular weight DNA from formalin-fixed, paraffin-embedded tissue from four different organs and compared the amplification efficiencies with those of the crude DNA extract. We used eight sets of oligonucleotide primers producing 262 to 989 base pair (bp) fragments of &bgr;-globin. The results showed that the PCR amplification analyses were more efficient with the isolated higher molecular weight DNA than with the crude DNA extract. Our study demonstrated that not all the DNA in formalin-fixed, paraffin-embedded tissue samples is totally degraded but that a small amount of higher molecular weight DNA persists. The feasibility of molecular diagnosis using formalin-fixed material can be improved by isolating the preserved higher molecular weight DNA by HPLC.

Overexpression of Dickkopf 3 in hepatoblastomas and hepatocellular carcinomas

Dickkopf 3 (Dkk3) is a protein expressed at a very early stage of hepatogenesis. In this study, we examined whether Dkk3 was related to a premature or dedifferentiated nature in hepatoblastomas (HBLs) and hepatocellular carcinomas (HCCs). It was demonstrated that Dkk3 was overexpressed in HBLs and HCCs and that its expression was more frequent in the former than in the latter, being consistent with the fact that most HBLs show an embryonal or fetal hepatic histology, whereas there was no distinct relationship between Dkk3 expression and clinical data or histology. All of the HBLs expressed Dkk3, alpha-fetoprotein (AFP), or both proteins, suggesting that, similar to AFP, Dkk3 is another potentially useful biomarker detecting a wide range of HBLs. Furthermore, Dkk3 and AFP were expressed reciprocally in the tumors. These results suggest that Dkk3 may be related to the premature or dedifferentiated nature of HBLs and HCCs, whereas AFP may be related to a more differentiated nature. Thus, assessment of Dkk3 and AFP may be useful in the diagnosis of hepatic tumors.

Effectiveness of Steroid Treatment for Hoarseness Caused by Idiopathic Fibrosing Mediastinitis: Report of a Case

A 65-year-old woman was referred to our department for investigation and treatment of hoarseness. A chest computed tomography (CT) scan showed a mediastinal mass spreading into the aortopulmonary window. This finding and that of flexible laryngoscopy suggested that the hoarseness was being caused by left recurrent nerve involvement resulting in left vocal cord paralysis. Thus, we performed a mediastinoscopic biopsy via a parasternal incision. Pathological examination revealed dense fibrous tissue infiltrated with varied inflammatory cells. Because no etiological pathogen or neoplastic lesion was identified, we diagnosed idiopathic fibrosing mediastinitis and began treating the patient with prednisolone. After a course of treatment, the mediastinal lesion showed a remarkable response. The hoarseness resolved as the lesion became smaller. Laryngoscopy confirmed recuperation of vocal cord function. This report shows that steroid therapy is a treatment option for hoarseness caused by recurrent laryngeal nerve involvement of fibrosing mediastinitis, if administered under close observation.

MMP-2 activation and stepwise progression of pulmonary adenocarcinoma: Analysis of MMP-2 and MMP-9 with gelatin zymography

Small pulmonary adenocarcinomas can be classified on the basis of their histological characteristics and prognosis, and when classified as such, the prognosis of replacing‐type adenocarcinoma with active fibroblast proliferation is significantly worse than adenocarcinoma without fibroblast proliferation. In order to clarify the biological mechanisms of the key to the morphological changes associated with active fibroblast proliferation, we examined the activities of matrix metalloproteinase (MMP)‐2 and MMP‐9, which are important enzymes in the stromal invasion by cancers. The active MMP‐2 and MMP‐9 content of 40 pulmonary adenocarcinomas that were less than 20 mm in diameter was measured by the gelatin zymography method. The quantity of active MMP‐2 in the pulmonary adenocarcinomas with active fibroblast proliferation was higher than in the pulmonary adenocarcinomas without proliferation (P < 0.001), but there were no correlations between the histological features and the activation of MMP‐9. The presence of active fibroblast proliferation in small pulmonary adenocarcinomas suggests that the cancer cells have acquired the ability to invade through the action of active MMP‐2, and this is thought to be one of the reasons for the worse prognosis of pulmonary adenocarcinoma with active fibroblast proliferation.

Neuronatin Expression and Its Clinicopathological Significance in Pulmonary Non-small Cell Carcinoma

Introduction: Neuronatin is a protein that is specifically expressed in the nervous system in the course of embryonal brain development, and its expression is limited to the pituitary gland in normal human adults. Neuronatin expression has been reported in some types of tumor. The purpose of this study was to clarify the significance of neuronatin expression in pulmonary non-small cell carcinoma. Methods: We determined the frequency of neuronatin expression in surgically resected samples from non-small cell lung carcinoma (51 adenocarcinoma and 41 squamous cell carcinoma) by immunohistochemical staining, and investigated the correlations between expression level and various clinicopathological features. Results: Expression of neuronatin was observed more frequently in squamous cell carcinoma (63%) than in adenocarcinoma (25%). In most cases, nontumorous lung tissue did not react with the antibody against neuronatin. In both adenocarcinoma and squamous cell carcinoma, less differentiated tumors expressed neuronatin more frequently than did differentiated tumors. In adenocarcinoma, but not squamous cell carcinoma, the prognosis of neuronatin-positive cases was significantly worse than that of neuronatin-negative cases. Conclusion: Neuronatin expression is specific for tumor tissue and was detected in both pulmonary adenocarcinoma and squamous cell carcinoma at high frequency, particularly in less differentiated tumors. Neuronatin expression is associated with poor prognosis in patients with adenocarcinoma, and may be useful as a prognostic marker for lung adenocarcinoma.

Retroperitoneal lymphangioma with a duodenal lesion in an adult.

A multilocular-cystic and cavernous, retroperitoneal tumor was found in a 40-year-old man whose past medical history was unremarkable. On admission, he complained of a large and still growing intra-abdominal mass associated with dull pain and a low-grade fever. Laboratory findings revealed leukocytosis and C-reactive protein elevation, compatible with inflammation of the tumor. Percutaneous aspiration of the tumor was performed under transabdominal ultrasonographic guidance, and continuous drainage of fluid from within the tumor ameliorated his symptoms. From preoperative examinations, including radiological imaging, fluid aspiration, and endoscopy with biopsy, a diagnosis of retroperitoneal lymphangioma was made. Laparotomy revealed extensive adhesions between the tumor and both the duodenum and the pancreatic head. A pancreaticoduodenectomy was therefore performed. At 3-year follow-up, there was no sign of recurrence. Retroperitoneal lymphangioma is an uncommon disorder, and the cavernous type is extremely rare. The duodenal lesion was an important feature of the present case, and endoscopic biopsy of this lesion facilitated precise preoperative diagnosis of retroperitoneal lymphangioma.