Yasunori Enomoto

9p21 deletion in the diagnosis of malignant mesothelioma, using fluorescence in situ hybridization analysis.

Homozygous deletion of 9p21, the locus harboring the p16 gene, has been reported as one of the most common genetic alterations in malignant mesotheliomas (MMs). Previous studies showed that this alteration might be useful for differentiating benign from malignant mesothelial tumors in cytology and surgical specimens. Although the diagnostic utility of 9p21 homozygous deletion by fluorescence in situ hybridization (FISH) analysis has been reported only recently, it has not been well demonstrated. The purpose of this study is to evaluate the diagnostic utility of 9p21 homozygous deletion assessed by FISH in mesothelial neoplasm and hyperplasia of Japanese patients using paraffin‐embedded tissue. Simultaneously, p16 protein immunoexpression was explored as a potential diagnostic aid. FISH analysis demonstrated 9p21 deletion in 35 of 40 cases with MM (88%) (P < 0.001). In contrast, no cases of adenomatoid tumor, benign mesothelial multicystic tumor, reactive mesothelial hyperplasia or pleuritis showed 9p21 deletion (P < 0.005). 9p21 homozygous deletion correlated well with p16 protein expression in the tumor cells. Our study suggests that 9p21 homozygous deletion assessed by FISH on paraffin‐embedded tissue may be very useful for differentiating MM from reactive mesothelial proliferation.

Invasive growth of hepatic angiomyolipoma; a hitherto unreported ominous histological feature

Aims : Although histological features of hepatic angiomyolipoma (AML) are highly variable, true malignant change is extremely rare. The aim was to review the histological features of invasive growth and clinical outcomes in 39 cases of hepatic AML.

Genomic gains and losses in malignant mesothelioma demonstrated by FISH analysis of paraffin-embedded tissues

Aims Malignant mesothelioma (MM) results from the accumulation of a number of acquired genetic events at the onset. In MM, the most frequent changes were losses in 9p21, 1p36, 14q32 and 22q12, and gains in 5p, 7p and 8q24 by comparative genomic hybridisation analysis. Although the diagnostic utility of 9p21 homozygous deletion by fluorescence in situ hybridisation (FISH) analysis in MM has been reported recently, alterations of other genes have not been examined to any great extent. This study analysed the frequency of various genomic gains and losses in MM using FISH analysis. Materials and methods The authors performed a FISH analysis using paraffin-embedded tissues from 42 cases of MM. Results Chromosomal losses in MM were found at 9p21 (83%), 1p36 (43%), 14q32 (43%) and 22q12 (38%), whereas gains were found at 5p15 (48%), 7p12 (38%) and 8q24 (45%). There were no cases of adenomatoid tumour, benign mesothelial multicystic tumour, reactive mesothelial hyperplasia or pleuritis showing any gains or losses. At least one genomic abnormality was identified in all cases of MM. Among various histological subtypes, the chromosomal abnormality tended to be more common in cases showing sarcomatous elements (biphasic or pure sarcomatoid) than in cases showing an epithelioid histology. Conclusions The authors found various genomic gains and losses in MM by FISH analysis. The frequency of each genomic gain or loss examined in MM by FISH analysis almost agreed with the comparative genomic hybridisation technique in previous studies. This study suggests that genomic evaluation by FISH analysis might be helpful in distinguishing MM from benign mesothelial proliferation.

Angiomyolipoma of the liver: a reappraisal of morphological features and delineation of new characteristic histological features from the clinicopathological findings of 55 tumours in 47 patients.

Nonomura A, Enomoto Y, Takeda M, Takano M, Morita K & Kasai T 
(2012) Histopathology 61, 863–880

Epidermal growth factor receptor mutations in malignant pleural and peritoneal mesothelioma

Background Epidermal growth factor receptor (EGFR) gene mutation at the kinase domain and EGFR gene amplification are reported to be predictors of the response to EGFR tyrosine kinase inhibitors in lung cancer cases. In malignant mesothelioma (MM), the role of EGFR is less clear. Methods Thirty-eight MM specimens were submitted to EGFR mutation evaluation, and compared with the results of immunohistochemical staining and fluorescence in situ hybridization (FISH) analysis. DNA was extracted from paraffin blocks and PCR was performed to amplify exon regions 18–21 of the EGFR gene. Direct sequencing of the purified PCR products was performed. Results Five EGFR missense mutations were detected in six of the 38 patients (16%); two of these mutations were novel, two were originally detected in non-small cell lung carcinoma, and one resembled a location previously noted for malignant peritoneal mesothelioma. Conclusion As far as the authors are aware there has been no report of the EGFR mutation of MM in Japanese cases, but in this study EGFR missense mutations were detected in some cases. EGFR mutation results were not related to immunohistochemical and FISH analysis.

A comparison of epidermal growth factor receptor expression in malignant peritoneal and pleural mesothelioma.

An evaluation of epidermal growth factor receptor (EGFR) phenotypic expression in malignant pleural and peritoneal mesothelioma was undertaken, using immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) analysis. Thirty‐eight malignant mesothelioma (MM) specimens were subjected to IHC staining and FISH to evaluate the expression of EGFR protein and gene status. Overall positive IHC reaction was detected in 20/38 (53%) cases, in 11/22 (50%) pleural MM, and in 9/16 (56%) peritoneal MM. Our study confirmed that EGFR membranous expression is a common feature in MM, but not in benign mesothelial lesion. Thirty‐seven cases did not show a gene copy number gain. Only one case showed a copy number gain. The protein overexpression of EGFR was not related to a gene copy number gain.

Serous borderline tumor of the paratestis.

Reported herein is a case of serous borderline tumor (SBT, ovarian epithelial type tumor) of the paratestis, involving the tunica vaginalis, in a 64‐year‐old man. The patient complained of right hydrocele; puncture cytology of the turbid fluid pointed to an adenocarcinoma. Right orchiectomy was performed and multiple micronodules were grossly observed in the paratestis. On microscopy small papillary epithelial lesions were found with psammoma bodies and intraglandular papillary lesions were irregularly recognized in the stroma of the paratestis, similar to SBT of the ovary. The tumor cells had often short microvilli. Mucin production was evident on PAS and colloid iron staining. Both papillary and glandular epithelial cells were positive on immunohistochemistry for Ber‐EP4/epithelial antigen, low‐molecular‐weight cytokeratin (CAM5.2), cytokeratin 7 and estrogen and progesterone hormone receptors, but negative for CEA, cytokeratin 20 and calretinin. The average proliferative index was approximately 10.5% as assessed on Ki‐67 (MIB‐1) staining. Ultrastructurally, the cells did not demonstrate any well‐developed microvilli or secretory granules and immunohistochemical findings supported SBT of Müllerian type (ovarian epithelial type tumor), while excluding a papillary type of malignant mesothelioma. The lesion in the present case was concluded to be a testicular serous tumor of Müllerian type, similar to SBT of the ovary.

Autopsy case of primary myelofibrosis in which myeloid sarcoma was the initial manifestation of tumor progression

Myeloid sarcoma (MyS) is defined as an extramedullary tumor‐forming neoplasm consisting of immature myeloid cells with/without maturation. We experienced a case involving a 68‐year‐old Japanese male patient who had been followed‐up for four years with a diagnosis of chronic idiopathic myelofibrosis/primary myelofibrosis (PMF) and noticed a painful mass in his left axilla. A wedge biopsy characterized the lesion as MyS that displayed megakaryoblastic/megakaryocytic differentiation. As his complete blood count included a few myeloid blasts (1% of WBC) and a bone marrow biopsy detected fibrosis without evidence of acute myelogenous leukemia (AML), a diagnosis of extramedullary blastic transformation of PMF was made, which was confirmed later by V617F mutation in Janus kinase‐2 in both initial bone marrow biopsy and axillary tumor biopsy specimens. The patient died of pneumonia eight months after developing the axillary tumor. At autopsy, multiple MyS masses were detected in his soft tissue, but his bone marrow only contained fibrosis. Although MyS rarely develops before the leukemic transformation of PMF, no evidence of AML could be found in the patients bone marrow at any point during the course of his disease. Thus, it is possible that the blasts in his peripheral blood were derived from the remaining MyS. Furthermore, the present case indicates that extramedullary blastic transformation, which is occasionally seen in CML, can also occur in PMF. Therefore, it is important to recognize that there is a wide variation in the pathogeneses of MyS and PMF.

Expression of c-Jun N-terminal kinases after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus

Abstract. c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) are a subgroup of mitogen-activated protein kinases (MAPKs), and important mediators of signal transduction from the cell surface to the nucleus. JNK phosphorylates the transcription factor c-Jun. c-Jun is one of the earliest and most consistent markers for neurons that respond to nerve-fiber transection. To elucidate the c-Jun metabolism after axotomy, we investigated the expression of JNKs mRNA and of JNKs and c-Jun proteins following vagus and hypoglossal nerve transection. We found that JNK 1, JNK 2 and JNK 3 mRNA were positive in the cytoplasm of neuronal and glial cells. JNK 1 and JNK 2 protein were distributed mainly in the cytoplasm of neurons and glial cells, while only JNK 3 immunoreactivity was observed intensely in the nuclei of neuronal cells. Activated JNK was also observed intensely in the nuclei of neuronal cells. In sham-operated animals, the cytoplasm of a few glial cells showed moderate immunoreactivity for activated JNK, while after axotomy the cytoplasm of all perineuronal microglial cells were stained intensely. Up-regulated c-Jun and phosphorylated c-Jun immunoreactivities were found in the nuclei of neuronal cells in the severed side of both the dorsal motor and hypoglossal nuclei. These results indicate that the principal activity of JNKs in neurons is contributed largely by JNK 3 under both normal and axotomized conditions, and that JNKs play an important role in signal transduction of perineuronal microglial cells after axotomy.

A case of gastric cancer with non-islet cell tumor hypoglycemia detected by insulin-like growth factor II

To the Editor: We describe herein a case of gastric cancer with severe non-islet cell tumor hypoglycemia (NICTH) in association with liver recurrence, being detected by insulin-like growth factor II (IGF-II) immunohistochemically and with immunoblotting technique. A 61-year-old Japanese man, complaining of epigastric discomfort, was endoscopically diagnosed as Borrmann’s type 3 gastric cancer at the middle body to cardiac portion of the stomach. The gastric biopsy was histologically adenocarcinoma (poorly differentiated or moderately differentiated tubular type). He underwent total gastrectomy with lymphadenectomy. On preand post-surgical laboratory data, blood glucose and potassium were within the normal range (127–129 mg/dL) and 4.2 mmol/L (3.6–5.0), respectively. Two months after surgery, he was emergently admitted after losing consciousness, and blood glucose demonstrated hypoglycemia of 21–49 mg/dL, followed by slight decrease of blood potassium, 3.5 mmol/L. On the abdominal computed tomography (CT) scan, multiple huge tumor shadows were detected in the liver, and was diagnosed as liver metastases from gastric cancer. The hypoglycemia was refractory to high concentrate glucose and steroid therapy. The patient died of liver insufficiency with hypoglycemia, but an autopsy was not permitted. Laboratory data: IGFBP-3:1.41 ng/mL (1.99–3.89), IGF-I (somatomedin C): 26 ng/mL (75–218), IGF-II: 599 ng/mL (459–873), IGF-II/IGF-I = 599/26 = 23.0 (<10, a criteria by Hizuka et al.). After total gastrectomy, the gastric tumor grossly measured approximately 9.0 x 9.0 cm, and the resected specimens histologically demonstrated the solid type of poorly differentiated adenocarcinoma (por1), with focal areas showing moderately tubular differentiation (tub2) (Fig. 1a). There were histologically no lymph node metastases, resulting in pT3N0M0, ly1, v1, infb. Immunohistochemically, the resected gastric cancer tissues were positive for CEA, CAM5.2, chromogranin A (Fig. 1b), synaptophysin, and IGF-II (Amano Pharmaceutical Co., Nagoya, Japan, Fig. 1c,d), but negative for N-CAM (CD56), insulin or somatostatin. The distribution of CEA and IGF-II were mainly positive for tubular type (tub2) but almost negative for solid type (por1). Two neuroendocrine markers of chromogranin A and synaptophysin were focally positive in adenocarcinoma and judged to express much different distribution in tumor cells, compared with that of IGF-II-positive cells. It is suggested that IGF-II-positive tumor cells might have developed separately from some neuroendocrine cells in gastric adenocarcinoma. Western immunoblot analysis of IGF-II in serum from the present patient detected IGF-II levels approximately 2-fold higher per molecular weight (11 to 18 kDa) compared with that in normal serum, or recombinant human IGF-II (7.5 kDa). This suggests the production of big IGF molecule (Fig. 2). NICTH is a syndrome defined by the presence of solid tumor and fasting hypoglycemia caused by an insulinindependent pathway. In recent studies, over two hundreds of cases of extra pancreatic tumors have been described in the English literature, half of these cases were mesenchymal tumors such as mesothelioma, leiomyosarcoma and fibrosarcoma, followed by hepatocellular carcinoma, adrenocortical carcinoma and carcinoma of the digestive organs including gastric carcinoma, rarely. NICTH was first noted in 1929 when Nadler and Wolfer reported a patient with hepatic cell carcinoma and hypoglycemia. In 1963, the initial report of NICTH associated with gastric cancer was described by Gonzales, et al. Since then, nine reports (17 cases after excluding duplication) within our referential search of gastric cancer cases with hypoglycemia, have been found in the English literature and are still relatively rare. In 1988, Daughaday et al.confirmed elevated concentration of IGF-II in a patient of leiomyosarcoma with tumor-associated hypoglycemia, suggesting the primary hormonal mediator of NICTH. Shiraishi et al. reported that in 41 of 57 gastric cancer cases, the expression of IGF-II mRNA was greater in tumor tissue than in normal tissue, and that immunohistochemical staining demonstrated positive IGF-II in the cancer cells themselves. They suggested that IGF-II might play an important role in tumor invasion, especially in lymph vessel permeation, in expanding-type gastric cancers. Recently, it has been reported by Kato et al. that a case of gastric carcinoma showed severe hypoglycemia (NICTH) when the tumor recurred with multiple liver metastases. Our case confirmed a similar case of NICTH in gastric carcinoma with liver recurrence, but it is still rare. In our case, the serum IGF-II value remained within the normal range of 599 ng/mL (459–873), followed by low value of IGF-I. Therefore, the ratio of IGF-II/IGF-I was significantly high, 23.0 (599/26), compared with the normal value of 10 (by Hizuka’s criteria). It has been described by Hizuka et al. that the mean ratio of IGF-II/IGF-I was 35.0 1 2.2 in patients with big IGF-II, which was significantly higher than NICTH without big IGF-II (11.5 1 2.4) or normal patients (3.3 1 0.2). On Western immunoblot analysis, big IGF-II, demonstrating molecular weight of 11 to 18 kDa, was detected in serum from the present patient. Pathology International 2010; 60: 595–597 doi:10.1111/j.1440-1827.2010.02563.x