Tsuyoshi Tange

Quantitative detection of micrometastases in the lymph nodes of gastric cancer patients with real-time RT-PCR: A comparative study with immunohistochemistry

Histologic examination lacks the sensitivity to detect micrometastases in gastric cancer lymph nodes. In the present study, we applied a real‐time RT‐PCR approach to the quantitative detection of micrometastases in gastric cancer lymph nodes and compared diagnostic power with routine histology and immunohistochemistry. We studied 392 lymph nodes from 21 gastric cancer patients who underwent curative surgery. Real‐time quantitative RT‐PCR was performed on a LightCycler instrument using a hybridization probe for carcinoembryonic antigen (CEA) and cytokeratin‐20 (CK20) as marker genes. Immunohistochemistry with antibodies to wide‐keratin was also performed in the lymph nodes to compare the sensitivity and specificity. Median (average) values of CEA mRNA in lymph nodes in patients with histology+, immunohistochemistry+/histology−, immunohistochemistry−/histology− and negative control results were 4,600 (16,000), 200 (400), 0 (9.8) and 0 (0.6), respectively. There were some false‐negative results with simple CEA and CK20 real‐time RT‐PCR due to the presence of low gene‐expressing gastric cancers as revealed by CEA and CK20 immunohistochemistry. CEA in combination with CK20 (duplex) real‐time RT‐PCR partially covered this weakness. Consequently, all 71 histology+ lymph nodes were positive for duplex real‐time RT‐PCR as well as wide‐keratin immunohistochemistry. Positivity rates by histology, wide‐keratin immunohistochemistry and duplex real‐time RT‐PCR were 18.0% (71/392), 20.9% (82/392) and 25.8% (101/392), respectively. In 2 of 8 patients with pT1N0, positive lymph nodes were observed by real‐time RT‐PCR but not by immunohistochemistry. These results indicate that duplex quantitative real‐time RT‐PCR is the most sensitive method for detecting micrometastases and useful for evaluating the prognostic significance of lymph node micrometastasis in gastric cancer patients.

Chondrosarcoma of the hand secondary to multiple enchondromatosis; report of two cases

Abstract. Although malignant transformation to chondrosarcoma may occur in some patients with multiple enchondromatosis, this event rarely occurs in the hand. We encountered two patients with chondrosarcoma of the hand secondary to multiple enchondromatosis. One patient was a 27-year-old man and the other, a 76-year-old man. Both patients manifested multiple osteolytic lesions in the hand on the plain radiographs. Severe bone destruction associated with a large soft-tissue swelling of the proximal and middle phalanges of the little finger was seen in case 1. In case 2, tremendous expansion and bone destruction of the middle phalanx of the ring finger was seen. Magnetic resonance images of the tumour in both patients showed low signal intensity on T1-weighted and high signal intensity on T2-weighted images. Amputation was performed in each patient. Histological examination revealed that the tumour was a grade 2 chondrosarcoma in case 1 and a grade 1 chondrosarcoma in case 2 accompanied by enchondromata. From these findings, the diagnosis of chondrosarcoma secondary to multiple enchondromatosis was made. Because quite a few patients with multiple enchondromatosis develop secondary chondrosarcoma, although rarely in the hand, the enchondromata should be curetted, unless impractical, before malignant transformation occurs.

Studies of 13C-urea breath test for diagnosis of Helicobacter pylori infection in patients after partial gastrectomy.

Background/Aims: Many of the reports on the diagnostic efficacy of the 13C-urea breath test (13C-UBT) for the detection of Helicobacter pylori in the residual stomach have shown negative results. We conducted an evaluation to establish a standardized protocol and an appropriate cutoff value for 13C-UBT in partial gastrectomy patients. Methods: Forty-two patients undergoing partial gastrectomy were included. Three gastric biopsies from the anastomotic site and mid-to-high body were taken at panendoscopy for histology, culture and rapid urease test (RUT). The 13C-UBT protocol included ingestion of 100 mg 13C-urea, use of mouthwash, and the body in a horizontal position on the left side. Six breath samples were taken after ingestion. Results: The Δ13CO2 values were significantly elevated in infected patients at all time points, and values were higher at 20 min and thereafter than at an earlier time point. The sensitivity of 13C-UBT was 96.3% with the cutoff of 2.0‰ at 40 min. The accuracy rates were highest with 13C-UBT, culture, RUT and histological tests, in that order. Conclusion: Forty minutes and a cutoff of 2.0‰ were found to be optimal for the test, with the body position horizontal on the left side. In the present protocol 13C-UBT appears to be a reliable tool with the same accuracy rate as other routine tests in patients with a remnant stomach.

Frequent expression of the variant CD30 in human malignant myeloid and lymphoid neoplasms.

We earlier identified a variant of CD30 (CD30v) that retains only the cytoplasmic region of the authentic CD30. This variant is expressed in alveolar macrophages. CD30v can activate the nuclear factor-kappaB (NF-kappaB) as CD30, and its overexpression in HL-60 induced a differentiated phenotype. To better understand the physiological and pathological functions of CD30v, expression of this variant was examined using a multiple approach to examine 238 samples of human malignant myeloid and lymphoid neoplasms. Screening by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of CD30v transcripts in 52 of 72, 7 of 11, 63 of 90, and 7 of 30 samples of acute myeloid leukemia (AML), myeloid blast crisis of myeloproliferative disorders (MBC), and lymphoproliferative disorders (LPDs) of B- and T-cell origin, respectively. CD30v expression was high in monocyte-oriented AMLs (FAB M4 and M5), B-cell chronic lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Using the specific antibody HCD30C2, prepared using a peptide corresponding to the nine amino acids of the amino-terminal CD30v, expression of CD30v protein was detected in 10 of 25 and 2 of 10 AML and ALL samples, respectively. In AMLs, immunocytochemical detection of CD30v revealed the presence of loose clusters of CD30v-expressing cells dispersed amid a population of CD30v-negative blasts. Finally, the parallel expression of CD30v mRNA and protein, as evidenced by Northern and Western blotting, was confirmed in selected cases of AMLs and LPDs. A significant correlation was found between expressions of CD30v and CD30 ligand transcripts in AML and LPD (P = 0.02, odds ratio = 3.2). The association of CD30v with signal-transducing proteins, tumor necrosis factor receptor-associated factor (TRAF) 2, and TRAF5 was demonstrated by coimmunoprecipitation analysis, as was demonstrated for authentic CD30 protein. Expression of transcripts for TRAF1, TRAF2, TRAF3, and TRAF5, as demonstrated by RT-PCR, was noted in leukemic blasts that express CD30v. Collectively, frequent expression of CD30v along with TRAF proteins in human neoplastic cells of myeloid and lymphoid origin provide supportive evidence for biological and possible pathological functions of this protein in the growth and differentiation of a variety of myeloid and lymphoid cells.

Utility of [13C] Urea Breath Test for Helicobacter pylori Detection in Partial Gastrectomy Patients

Many reports on the diagnostic efficacy of the [13C] urea breath test ([13C] UBT) for the detection of Helicobacter pylori in the residual stomach have shown negative results. We previously reported on the utility of [13C] UBT and conducted an evaluation to establish a standardized protocol with a shorter sampling time for [13C] UBT in partial gastrectomy patients. Sixty-two patients who had undergone partial gastrectomy were included. The [13C] UBT protocol included ingestion of 100 mg [13C] urea, use of mouthwash, and the body in a horizontal position on the left side. The sensitivity of [13C] UBT was 95.7%. Thirty minutes and a cutoff of 2.0‰ were found to be optimal for the test, with the body position horizontal on the left side. In the present protocol [13C] UBT appears to be a reliable and convenient tool with the same accuracy rate as other routine tests in patients with a remnant stomach.

Replacement of m-calpain by μ-calpain during maturation of megakaryocytes and possible involvement in platelet formation

Localization of calpains in human bone marrow cells was studied immunohistochemically employing monoclonal antibodies against the high-Ca(2+)-requiring form (m-calpain) and the low-Ca(2+)-requiring form (mu-calpain). Most cells were stained with anti-m-calpain more strongly than with anti-mu-calpain, and staining with anti-mu-calpain was prominent only in megakaryocytes. To confirm the result, megakaryoblastic cell line (T-33) cells were subjected to immunoblot analysis. However no immunoreactivity to mu-calpain was seen in T-33 cells. Bone marrow from a patient with idiopathic thrombocytopenic purpura showed immature megakaryocytes (stage II) strongly stained by anti-m-calpain antibody while mature cells (stage III) were strongly stained by anti-mu-calpain antibody. These results suggest that mu-calpain plays a crucial role in mature megakaryocytes, possibly in platelet production.

Establishment and characterization of a new human mesothelioma cell line (T‐85) from malignant peritoneal mesothelioma with remarkable thrombocytosis

A mesothelioma cell line, termed T‐85, was established from a patient with malignant peritoneal mesothelioma and remarkable thrombocytosis (1.4 × 106/mm3). Electron microscopically, two types of mesothelioma cells have been characterized; the major type of cells with dense‐cored granules in the cytoplasm and the minor one with evenly dense granules. Immunologically, the cells showed staining for inter‐leukin‐6 (IL‐6), cytokeratin, collagen type IV, vimentin, laminin, fibronectin and Factor VIII‐related antigen. Quanti‐tation by ELISA revealed a high concentration of IL‐6 in T‐85 cell culture supernatants. RT‐polymerase chain reaction of T‐85 cells showed two positive bands of cDNA at 628 and 251 base pairs indicating the constitutive expression of IL‐6 and IL‐6 receptor mRNA. Moreover, prominent pro‐platelet process formation activity in T‐85 cell culture supernatants indicated the presence of a thrombopoietic activity due mainly to IL‐6 but not the c‐MpI ligand or erythropoietin. However, the fact that 15% of PPF activity remained in the supernatants treated with anti‐IL‐6 antibody indicated the presence of another thrombopoietic substance. T‐85 is so far the first mesothelioma cell line derived from a case with remarkable thrombocytosis.

Synergistic effects of erythropoietin and interleukin-6 on the in vitro proplatelet process formation of rat megakaryocytes

The racombinant hemopolettc factors of megakaryocyte potentlator (MEG‐POT) were studied to compare their activity In stimulating proplatelet process formation (PPF) with thrombopoletln (TPO, c‐MpI Itgand). For the assay, a highly enriched (>95%) population of more than 90% viable megakaryocytes was Isolated from rat bone marrow using the Immunomagnetic beads method and cultured with fetal calf serum (FCS) or In a serum‐free condition. Megakaryocytes developing slender beaded cytoplasmic processes (proplatelet processes) were observed on both inverted phase contract microscopy and scanning electron microscopy. A large number of proplatelet process clusters were dose‐dependently formed with the addition of varying doses of recombinant erythropoietin (rEpo) and interleukin‐6 (rlL‐6) as well as TPO. Epo and IL‐6 were demonstrated to act synergistically solely at low doses in the development of PPF (P<0.05). Other recombinant factors such as IL‐11, leukemia inhibitory factor (LIF) and erythroid differentiation factor (EOF) appeared weak or ineffective. From these In vitro observations, It was suggested that a synergism of Epo and IL‐6 might play a significant role in the terminal stage of megakaryocyte maturation leading to platelet release.

GLOMERULAR LESIONS IN MULTIPLE MYELOMA

An autopsy case of multiple myeloma (IgG, lambda type), clinically characterized by decreased glomerular filtration rate, is reported with particular emphasis on changes in the glomeruli of kidneys. Histologically, the glomeruli revealed slight increase in mesangial matrix and focal thickening of tuft capillary wall. Electron‐microscopically, deposits were observed in a subendothelial location in the glomerular capillary walls, and inclusions were noted in the cytoplasm of the visceral epithelial cells. Histoimmunofluo‐rescent study of the kidney demonstrated intense focal and slight diffuse positivity against labelled antisera of anti‐IgG and anti‐lambda type of light chain on the capillary wall of the glomerular tufts. Other immunoglobulins were not demonstrable in capillary walls. These findings represent the intraglomerular deposition of paraprotein of multiple myeloma without amyloidosis.

Des-gamma carboxy prothrombin (PIVKA-II)- and alpha-fetoprotein (AFP)-producing gastric cancer

To the Editor: Since Liebman et al.1 reported, in 1984, that protein induced by vitamin K absence or antagonist-II (PIVKA-II) was increased in the plasma of hepatocellular carcinoma (HCC) patients, PIVKA-II has been regarded as a new tumor marker of HCC. Although case reports of α-fetoprotein (AFP)-producing gastric cancer have been gradually increasing, PIVKA-IIproducing gastric cancer is very rare. In this report, we describe a case of PIVKA-IIand AFP-producing gastric cancer. A 61-year-old man was hospitalized with the chief complaint of appetite loss. Barium study and upper digestive tract endoscopy revealed a 10-cm-sized Borrmann type-3 gastric cancer. Hepatic masses up to 8 8 6-cm in size were also detected by abdominal computed tomography and ultrasonography. AFP and PIVKA-II values in the serum were abnormally high (AFP, 495.2 ng/ml [L2 subfraction, 21.4%; L3 subfraction, 75.6%]; normal value, 20 ng/ml; PIVKA-II, 635 mAU/ml; normal value, 40 mAU/ml). Thus, total gastrectomy and liver biopsy were performed. In the resected specimen, an 8.0 7.0/9.5 8.5-cm-sized Borrmann type-3 gastric cancer occupied mainly the posterior wall from the upper gastric body to the pyloric antrum (Fig. 1). Histologically, tumor cells proliferated to form tubular, papillary, trabecular, and solid structures. Immunohistochemically, AFPpositive tumor cells and PIVKA-II-positive tumor cells were found; the AFP-positive tumor cells were distributed mainly in the trabecular or solid structure and the PIVKA-II-positive tumor cells were distributed mainly in the tubular structure (Fig. 2). The tumor in the liver showed a structure similar to that of the gastric tumor; however, immunohistochemical staining for AFP and PIVKA-II was negative. Adjuvant chemotherapy was performed; however, the residual hepatic tumor enlarged and the patient died 6 months after the diagnosis. We made a pathological diagnosis of PIVKA-IIand AFPproducing gastric carcinoma with liver metastasis for this patient. Although AFP and PIVKA-II production in the liver tumor was not confirmed immunohistochemically, the following findings suggested that it was a metastatic tumor: (1) the histological structures of the gastric and hepatic tumors were similar, (2) hepatitis virus markers were all negative, (3) there was no remarkable change in the nontumorous liver tissue pathologically. The cause of the negative staining for AFP and PIVKA-II in the liver tumor was probably sampling limitations. In recent years, the mechanism of AFP production in most cases of AFP-producing gastric carcinoma has been assumed to be retrodifferentiation to yolk sac tumor or fetal gastrointestinal tract, as determined by the investigation of AFP-lectin combination. However, the AFP subfraction pattern in the present patient resembled that of HCC; so, hepatoid metaplasia may have been the cause of the AFP production in this particular patient. The mechanism by which gastric cancer acquires the ability to produce PIVKA-II is unknown. Some researchers have suggested that the mechanism of hepatocellular metaplasia is involved. However, in Fig. 1. Macroscopic view of the gastric cancer. An 8.0 7.0/9.5 8.5-cmsized Borrmann type-3 cancer occupies mainly the posterior wall from the upper gastric body to the pyloric antrum