Mitsugu Hironaka

Alterations of the c-kit gene in testicular germ cell tumors

Expression and gain‐of‐function mutation of the c‐kit gene, that encodes a receptor tyrosine kinase (KIT), have been reported in mast cell tumors and gastrointestinal stromal tumors (GISTs). Among human testicular germ cell tumors (GCTs), seminomas and seminoma components of mixed GCTs have also been shown to express KIT, but only one study has found the c‐kit gene mutation at exon 17 in seminoma. To elucidate the frequency and location of the c‐kit gene mutation of testicular GCTs, we analyzed the whole coding region of the c‐kit complementary DNA along with 4 mutational hot spots (exons 9, 11, 13 and 17) of the c‐kit genomic DNA by polymerase chain reaction and direct sequencing. Somatic mutations were found in 4 pure seminomas of 34 testicular GCTs (11.8%). One mutation was found in exon 11 (W557R) and the others were observed in exon 17 (D816H and D816V). These types of mutations were reported in GISTs (W557R), seminoma (D816H) and mastocytosis (D816V) and were considered to be gain‐of‐function mutations, although there were no differences of any clinicopathological factors or outcome between patients with and without mutations. Additionally, we also demonstrated coexpression of Gly‐Asn‐Asn‐Lys510–513 (GNNK)+ and GNNK‐ isoforms of the c‐kit gene with dominance of the GNNK‐ transcript in all testicular GCTs. The mutations and/or preferential expression of GNNK‐ isoform of the c‐kit gene might play an important role in the development of testicular GCTs, and these tumors may also be targets for STI571, which is a promising drug for advanced and metastatic GISTs.

Mutations in c-kit gene exons 9 and 13 in gastrointestinal stromal tumors among Japanese

Gain‐of‐function mutation in c‐kit proto‐oncogene exon 11 has been described in about 20‐50% of gastrointestinal stroma tumor (GIST). Recently, additional mutational hot‐spots in exon 9 and exon 13 of the c‐kit gene have been reported in GISTs without mutations of exon 11, but a subsequent report in a Western population indicated that only a small portion of GISTs (eight of 200 GISTs, 4%) showed mutations in these regions. In this study, we evaluated mutations in exon 9 and exon 13 of the c‐kit gene by both polymerase chain reaction‐single strand conformation polymorphism analysis and direct sequencing in 48 GISTs in a Japanese population, for which the clinicopatho‐logical and immunohistochemical features and mutations in exon 11 had previously been reported. C‐kit gene mutation in exon 9, representing insertion of GCC TAT, was identified in only 4 of 48 GISTs (8%), and none of the GISTs had mutations in exon 13. All four GISTs with mutation in exon 9 were high‐risk, and the patients died of multiple tumor metastasis. Mutations in exon 9 and exon 13 of the c‐kit gene were also rare events in Japanese GISTs and were related to a poor prognosis. These results in Japanese are consistent with those in Western populations, although a preferential occurrence of GISTs with exon 9 mutation in the small intestine, which was suggested in a previous report, was not observed.

Clonality and K-ras mutation analyses of epithelia in intraductal papillary mucinous tumor and mucinous cystic tumor of the pancreas

Abstract. Histological criteria for subclassification of intraductal papillary mucinous tumor (IPMT) and mucinous cystic tumor (MCT) of the pancreas remain ambiguous in the absence of apparent invasion or metastasis. To elucidate this issue, we evaluated clonality and K-ras mutations in 11 cystic tumors of the pancreas from female patients, including 7 IPMTs and 4 MCTs. The analyses were performed on DNA from laser microdissected epithelia showing different degrees of atypia as well as normal-appearing epithelia (NAE) in the individual tumors. The grades of atypia were classified into three groups on conventional hematoxylin-eosin staining. Clonality was assessed using the methylation-induced polymorphic inactivation of the X-linked phosphoglycerate kinase gene. The incidence of monoclonality increased with the grades of atypia: 27% for NAE, 43% for grade 1, and 100% for grades 2 and 3. In three of four MCTs, foci of NAE were polyclonal, while monoclonality was seen in each one of grades 1 and 2. The frequency of K-ras mutation depended on the grades of atypia: 0% for NAE, 29% for grade 1, 50% for grade 2, and 75% for grade 3. Polyclonal epithelia were devoid of K-ras mutation in 92% of sites, while monoclonality was associated with both wild and mutational types in an approximately equal ratio. Both IPMT and MCT seem to arise from polyclonal epithelia and to be replaced by monoclonal neoplastic cells as they undergo dysplastic changes and K-ras mutation. These data suggest that the monoclonal expansion precedes K-ras mutation.

c‐kit gene mutations in intracranial germinomas

Gain‐of‐function mutations of the c‐kit gene and the expression of phosphorylated KIT are found in most gastrointestinal stromal tumors and mastocytosis. Further, almost all gonadal seminomas/ dysgerminomas exhibit KIT membranous staining, and several reports have clarified that some (10–25%) have a c‐kit gene mutation. But, whether intracranial germinomas also have a c‐kit gene mutation remains unsolved. To elucidate the presence, frequency, and location of c‐kit gene mutations in intracranial germinomas, we analyzed five mutational hot spots (exons 9, 10, 11, 13, and 17) in the c‐kit genomic DNA of 16 germinomas using polymerase chain reaction and direct sequencing. We found c‐kit gene mutations at exon 11 (W557C) or 17 (D816V, D820V, and N822Y) in four germinomas (25.0%), although no statistically significant difference in any clinicopathological factor was found between patients with or without mutations. These results are similar to those seen in gonadal seminoma/dysgerminoma patients, and confirm that intracranial germinomas are exact counterparts of gonadal seminomas/dysgerminomas, as would be expected on histological and immunohistochemical grounds. Moreover, molecular targeting drugs such as imatinib mesylate (STI571), which is a selective inhibitor of KIT, might be promising agents for the treatment of intracranial germinomas with c‐kit gene mutations.

Pulmonary fibrosis and lung carcinoma : A comparative study of metaplastic epithelia in honeycombed areas of usual interstitial pneumonia with or without lung carcinoma

Usual interstitial pneumonia (UIP), or idiopathic pulmonary fibrosis, has been considered to be associated with a high risk for lung carcinoma. To investigate this well‐known but still equivocal relationship, we reviewed the clinical features of UIP autopsy cases with or without lung carcinoma (n = 32 and 38, respectively), and compared the morphology and cell kinetics of metaplastic epithelia in the honeycombed areas (n = 11, each group). Thirty‐two of 70 UIP autopsy cases showed lung carcinomas. Clinically, UIP with lung carcinoma showed a male predominance (P = 0.001), a higher rate of smoking history (P = 0.001) and a later onset of UIP (P = 0.02), compared with UIP without lung carcinoma. Most of the carcinomas were peripheral in origin (90%), and 65% were topographically associated with honeycombed areas or the border between honeycombing and non‐fibrotic areas. Quantitative assessment of the metaplastic epithelia in the honeycombed areas revealed that squamous metaplasia, but not cuboidal cell metaplasia or bronchial cell metaplasia, occurred more frequently in UIP with lung carcinoma than in UIP without lung carcinoma (P = 0.02). There were no significant differences between the two groups with regard to the labeling indexes of Ki‐67 and p53 in the metaplastic epithelia, including squamous metaplasia. The degree of atypical squamous metaplasia was not different between the two groups. The quantitative predominance of squamous metaplasia in the honeycombed areas may not be a precursor for lung carcinoma, but might reflect a constitutional susceptibility of UIP patients to develop a lung carcinoma.

Pulmonary gangliocytic paraganglioma: case report and comparative immunohistochemical study of related neuroendocrine neoplasms.

The authors report a case of gangliocytic paraganglioma of the lung, which has not yet been described in a pulmonary neoplasm. A 75-year-old man underwent right middle and lower lobe lobectomy. A slightly yellowish mass was located at the bifurcation between the lower and middle lobe bronchus, protruding into the truncus intermedius. The neoplastic cells were composed of three cellular elements: uniform endocrine cells in a Zellballen arrangement, large ganglion-like cells within the nests of endocrine cells, and spindle-shaped cells arranged in streams to surround the nests. Each component exhibited the characteristic immunohistochemical properties, which were similar to those of the corresponding neuroendocrine neoplasms: Endocrine cells were positive for CAM 5.2, chromogranin A, and synaptophysin, like carcinoid tumor; ganglion-like cells were positive only for neurofilament, like ganglioneuroma; and spindle-shaped cells were positive for neurofilament and S-100 protein, like paraganglioma. These results agreed with those in gangliocytic paraganglioma of the duodenum. Pulmonary gangliocytic paraganglioma is similar to that in the duodenum, and is a hamartomatous proliferation of epithelial endocrine and neuronal cells of the bronchus.

p16 inactivation in small-sized lung adenocarcinoma: Its association with poor prognosis

p16, an inhibitor of cell cycle machinery, is frequently inactivated in non‐small cell carcinoma of the lung (NSCCL). To clarify the significance of p16 inactivation in the progression of lung adenocarcinoma, we immunohistochemically evaluated p16 protein status and Rb, p53 and cyclin D1 expression in 51 surgically resected adenocarcinomas that were less than 3 cm in diameter (median follow‐up period: 52.5 months). Twenty‐one of 51 adenocarcinomas showed negative immunostaining for p16. Twenty adenocarcinomas were also negative for Rb, while 31 and 13 were positive for p53 and cyclin D1, respectively. Loss of p16 expression was significantly correlated with scar grade, lymphatic permeation, lymph node metastasis and clinical stage. Rb protein expression was also inversely correlated with scar grade, pleural involvement and vascular invasion. When the cases were stratified according to the expression of both proteins, the Rb−/p16− subset (7/51) consisted of poorly differentiated adenocarcinoma with a higher grade of invasion. While Rb, p53 and cyclin D1 protein status showed no significant correlations with prognosis, p16 inactivation was significantly correlated with poor prognosis, and the prognosis of Rb−/p16− was the worst among the 4 subsets. Inactivation of p16 may play a role in accelerating scar formation and lymph node metastasis, and may contribute through these mechanisms to poor prognosis in patients with small‐sized lung adenocarcinoma. Int. J. Cancer (Pred. Oncol.) 84:49–53, 1999.

High‐grade neuroendocrine carcinoma of the lung: Comparative clinicopathological study of large cell neuroendocrine carcinoma and small cell lung carcinoma

Large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC) are high‐grade neuroendocrine carcinomas. In order to clarify the similarities and differences between these cancers, 22 cases each of LCNEC and SCLC were collected and a comparative pathological study was carried out. First, their clinicopathological characteristics were confirmed, which were very similar to those previously reported. The 5 year survival rate of LCNEC and SCLC patients was 38.3% and 29.7%, respectively. The morphological characteristics of LCNEC and SCLC were then reviewed with regard to the morphology previously used to differentiate these cancers. As a result, many morphological indicators, such as tumor cell size, nuclear/cytoplasmic ratio, nuclear molding, rosette formation, prominent nucleoli and karyolysis were confirmed to be significant indicators for distinguishing LCNEC from SCLC. On comparative immunohistochemistry, LCNEC had significantly high staining scores for the expression of keratin 7 and 18, E‐ and P‐cadherins, β‐catenin, villin 1, retinoblastoma protein (pRB), c‐met and α‐enolase. These results might reflect the differentiation or deviation of LCNEC toward an epithelial nature irrespective of neuroendocrine tumor lineage. In conclusion, the present comparative study of LCNEC and SCLC defined the similarities and differences between these cancers, and showed the biologically and clinicopathologically overlapping spectrum of the tumor lineage.

Cancer risk to the gastric corpus in Japanese, its correlation with interleukin-1β gene polymorphism (+3953*T) and Epstein-Barr virus infection

Polymorphisms of interleukin‐1 (IL‐1) genes have been reported to modify the risk of gastric carcinoma (GC) in Caucasians. The significance of IL‐1β gene polymorphisms was evaluated in Japanese GC patients with or without infection of Helicobacter pylori and Epstein Barr virus (EBV) with special reference to the topographic features of GC. IL‐1β gene polymorphisms at positions ‐511 and +3953 were evaluated by PCR‐RFLP and a penta‐allelic polymorphism of IL‐1RA by PCR in healthy controls (n = 103) and GC (n =140; corpus 95, antrum 45). EBV‐infection was determined in the neoplastic tissues by EBER1 in situ hybridization, and H. pylori infection in nonneoplastic gastric mucosa by PCR targeting of the H. pylori urease A gene. GC consisted of EBV‐associated (n = 24) and EBV‐negative (n = 116) patients, whereas H. pylori infection was positive in 130 cases. Among IL‐1β gene polymorphisms, genotype IL‐1β+3953 C/T was more frequent in the EBV‐negative (21%) and corpus GC (23%) patients, compared to the controls (10%), respectively, although there was no genotype IL‐1β+3953 T/T in either group. Thus, the effect of IL‐1β+3953 T was statistically significant in logistic regression models adjusted for age in EBV negativity (odds ratio [OR] 2.27, 95% confidence interval [CI] 1.02–5.05) and in the corpus GC (2.70, 1.19–6.12) with highest OR 3.55 (1.54–8.23) in EBV‐negative corpus GC. There was no significant influence of IL‐1 gene polymorphism in EBV‐associated GC, but it occurred predominantly in the corpus (24/24) compared to EBV‐negative GC (71/116) (p = 0.00002). There was no correlation between H. pylori infection and IL‐1 gene polymorphism in GC. The cancer risk of the gastric corpus in Japanese is influenced by IL‐1β+3953 polymorphisms. On the other hand, the risk of EBV‐associated GC, which occurs predominantly in the corpus, is not influenced by this pro‐inflammatory polymorphism.

MEN1 gene mutations in sporadic neuroendocrine tumors of foregut derivation

Foregut‐derived neuroendocrine (NE) tumors occur sporadically or in association with multiple endocrine neoplasia type 1 (MEN1) syndrome. Thirty‐nine sporadic NE tumors of foregut derivation (six thymic, 21 bronchial, three gastric, and nine pancreatic tumors) as well as two hindgut‐derived rectal carcinoids for somatic MEN1 gene mutation were analyzed by direct sequencing analysis. Five tumors showed mutations: nonsense mutations (Q393X and R98X) in thymic and pancreatic NE tumors, respectively, a 4 b.p. deletion (357del4) in a gastric NE carcinoma, and missense mutations (D172Y and S178Y) in pancreatic NE tumors. No mutation was identified in pulmonary or rectal NE tumors. In a patient with a pancreatic NE tumor (D172Y), the corresponding germline DNA showed the same mutation, suggesting that sporadic MEN1 syndrome was masked in this case. Somatic MEN1 gene mutations and deletions may play a crucial role in the tumorigenesis of a subset of foregut‐derived NE tumors. Sporadic MEN1 syndrome may occur as a sporadic NE tumor of the pancreas.