Hirohisa Hirabayashi

Inversely correlated expression of p16 and Rb protein in non‐small cell lung cancers: An immunohistochemical study

Cdk4‐mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non‐small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16‐positive specimens did not stain for Rb. Only 1 of the p16‐negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein.

Overexpression of ADAM9 in Non-Small Cell Lung Cancer Correlates with Brain Metastasis

The “a disintegrin and metalloprotease” (ADAM) family contributes to regulation of the cell–cell and cell–matrix interactions that are critical determinants of malignancy. To determine the relationship between metastasis and ADAM proteins, we compared the mRNA levels of ADAM9, -10, -12, -15, and -17 in sublines of an EBC-1 lung cancer cell line that were highly metastatic to either brain or bone. ADAM9 mRNA levels were significantly higher in highly brain-metastatic sublines than in the parent or highly bone-metastatic sublines. To elucidate the role of ADAM9 in brain metastasis, we stably transfected A549 and EBC-1 cells with a full-length ADAM9 expression vector. Compared with mock-transfectants, ADAM9 overexpression resulted in increased invasive capacity in response to nerve growth factor, increased adhesion to brain tissue, and increased expression of integrin α3 and β1 subunits. Administration of the anti-β1 monoclonal antibody attenuated this increase in invasive and adhesive activity. Intravenous administration of ADAM9-overexpressing A549 cells to mice resulted in micrometastatic foci in the brain and multiple metastatic colonies in the lungs. In contrast, administration of parent and mock-transfected A549 cells to mice resulted in lung tumors without brain metastasis. These results suggest that ADAM9 overexpression enhances cell adhesion and invasion of non-small cell lung cancer cells via modulation of other adhesion molecules and changes in sensitivity to growth factors, thereby promoting metastatic capacity to the brain.

New prognostic indicator for non‐small‐cell lung cancer, quantitation of thymidylate synthase by real‐time reverse transcription polymerase chain reaction

Thymidylate synthase (TS) is an enzyme that catalyzes an important DNA biosynthesis process. The gene expression of TS has not been reported in non‐small‐cell lung cancer (NSCLC) patients. To clarify the correlation between TS mRNA levels and clinicopathological features of NSCLC, we examined 70 Stage I and II NSCLC patients for intra‐tumoral expression of TS using TaqMan reverse transcription polymerase chain reaction (RT‐PCR) assay and immunohistochemistry methods. We also investigated the TS promoter 28 bp polymorphism in 48 cancer tissues using PCR amplification of genomic DNA. Lung cancer tissue showed higher TS mRNA levels than normal lung tissue (Mann‐Whitney U‐tests; p = 0.0020). Further, TS mRNA expression was correlated with immunohistochemical TS expression (p = 0.029). We obtained 2 different DNA fragments, which indicated triple‐repeat (3R) and double‐repeat (2R) type alleles. Cancer tissues with the 3R/3R genotype showed significantly higher TS mRNA levels as compared to those with other genotypes (p = 0.0019). The TS genotype was also correlated with immunohistochemical TS expression (χ2 test; p = 0.0079). The disease‐free survival of the low TS mRNA level group was significantly better than those with high TS mRNA levels (log‐rank test; p = 0.010), however, there were no significant differences found by immunohistochemical evaluation (p = 0.34) or TS genotype analysis (p = 0.11). A multivariate analysis revealed that high TS mRNA levels independently contributed to disease‐free survival. The quantitation of TS mRNA levels is clinically more sensitive and useful for determining the prognosis of Stage I and II NSCLC patients than an immunohistochemical evaluation.

p16INK4, pRB, p53 and cyclin D1 expression and hypermethylation of CDKN2 gene in thymoma and thymic carcinoma

There have been few reports on genetic alterations in thymomas. To investigate the expression of p16INK4A, RB, p53 and cyclin D1 in thymomas, we first examined 36 thymomas (non‐invasive type, 16 cases; invasive type, 20 cases) and 3 thymic carcinomas, using immunohistochemistry. Abnormal expression of p16INK4A, RB, p53 and cyclin D1 was observed in 18, 8, 10 and 7 cases, respectively. Only a subgroup of invasive thymomas and thymic carcinomas showed an inverse correlation between p16INK4A and RB expression. Subsequently, we examined the 36 thymomas and 4 thymic carcinomas for mutations in p53 and CDKN2 genes, using PCR‐SSCP and direct‐sequencing analyses. No mutation of these genes was detected in the thymomas and thymic carcinomas examined. A polymorphism in the 3′ untranslated region of exon 3 of CDKN2 was detected in 5 cases of thymoma. We searched for hypermethylation in the promoter region of CDKN2, observing it in 4 thymomas and 1 thymic carcinoma. Our data suggest that, unlike other more common cancers, alteration of the p53 gene may not play a significant role in the tumorigenesis of thymoma. However, inactivation of p16INK4A and RB may play a role in the progression of thymoma and thymic carcinoma. Int. J. Cancer 73:639–644, 1997.

Disruption of the RB pathway and cell-proliferative activity in non-small-cell lung cancers.

The pathway consisting of retinoblastoma protein (pRB), cyclin D1 and p16 (RB pathway) which is involved in the phosphorylation of pRB plays an important role in G1/S progression. The disruption of this RB pathway has been reported in several types of human neoplasm. An immunohistochemical study of 101 non‐small‐cell lung cancers (NSCLCs) showed loss of p16 is in 47 tumors (46.5%) and loss of pRB in 42 tumors (41.6%). In 79 of 101 NSCLCs (78.2%), the expression of p16 and pRB was complementary (p < 0.0001). Methylation of the cdkn2 gene was detected in 50% of p16‐negative tumors and in 11% of p16‐positive tumors. Aberrant expression of cyclin D1 was found in 45 tumors (44.5%). The cyclin‐D1‐positive tumors had significantly higher Ki‐67 indices than the cyclin‐D1‐negative tumors irrespective of the tumor p16 or pRB expression. Thus, 91 (90%) of 101 NSCLCs showed disturbed expression of at least 1 of the 3 components of the RB pathway. Our results suggest that the disruption of the RB pathway plays an important role in tumorigenesis in NSCLCs and that increased cyclin‐D1 expression leads to strong proliferative activity which may over‐ride the suppressive effect of p16 and pRB. Int. J. Cancer (Pred. Oncol.) 79:111–115, 1998.© 1998 Wiley‐Liss, Inc.

Bilateral Cervicomediastinal Neurofibroma Originating from the Vagal Nerve in a Patient with von Recklinghausen's Disease : Report of a Case

Abstract.A 19-year-old woman with von Recklinghausens disease was admitted with symptoms of hoarseness. A computed tomography scan showed a bilateral cervicomediastinal tumor. An extirpation of the left cervicomediastinal tumor was performed for the purpose of diagnosis and treatment. On thoracotomy, the tumor, which measured 9 × 8 × 4 cm in size, arose from the intrathoracic vagal nerve and the left tumor was resected with a segment of the vagal nerve and recurrent nerve. The pathological diagnosis of the tumor was a neurofibroma. The tumor on the right side was left untreated due to concerns about possibly causing palsy of the bilateral recurrent nerve and also because of the asymptomatic state of the right tumor. Mediastinal neuofibroma in a patient with von Recklinghausens disease often arises from the intrathoracic vagal nerve. To our knowledge, this is the first report of bilateral cervicomediastinal neurofibroma originating from the vagal nerves.

Appearance of thymoma 15 years after extended thymectomy for myasthenia gravis without thymoma

Several cases of thymoma recurrence after resection have been reported. However, thymoma appearance following an extended thymectomy for non-thymomatous myasthenia gravis is very rare. We report a case of thymoma in a 48-year-old woman, 15 years after an extended thymectomy for non-thymomatous myasthenia gravis. The importance of a complete dissection of mediastinal adipose tissue during the extended thymectomy as well as careful follow-up for such patients is also noted.

Benefits of Home Parenteral Nutrition Before Lung Transplantation: Report of a Case

A 54-year-old woman, who was severely malnourished and awaiting lung transplantation for chronic respiratory failure, was given nutritional support by home parenteral nutrition (HPN). The patient’s emaciated state was further compromised by the fact that she had undergone a distal gastrectomy 4 years earlier. We decided that HPN was indicated because her oral intake was so poor and drinking enteral formula reduced her appetite. After 1 year of HPN, her body weight increased and her nutritional, anthropometric, and biochemical values were well maintained by the time of transplantation. Moreover, her progress after lung transplantation was remarkably good, indicating the effectiveness of nutritional support by HPN during the protracted waiting period.