Biased VH family usage in primary gastric B cell lymphoma of mucosa-associated lymphoid tissue-type and the selected Vβ expression of T cells infiltrating into the lymphoma cells

Seven cases of primary gastric low-grade B cell lymphoma of mucosa-assoclated lymphoid tissue (MALT) type, two cases of high-grade B cell lymphoma with a low-grade component and three cases of pure high-grade lymphoma were selected for the current study. The Ig VH gene use of lymphoma cells and the Vβ repertoires of infiltrating T cells were Investigated. The VH gene analysis showed multiple VH family usage In 12 cases, but the MALT-type lymphoma cell usage was found to be biased for the families that have a low number of VH genes (VHIV and V). Another analysis of lymphoma-lnfiltrating T cells showed restricted expressions of the Vβ repertoire in all seven low-grade cases and three high-grade cases. In those 10 cases, a considerable number of CD4-postttve T cells Infiltrated Into lymphoma cells and RAG-1 was also prominently expressed. Based on these findings, It was thus assumed that the normal counterpart of gastric B cell lymphoma of MALT type is different from the conventional B cell lymphoma, and the restricted expression of Vβ repertoires Is therefore considered to be a characteristic finding in low-grade B cell lymphomas of MALT type as well as in a proportion of high-grade lymphomas (the so called 'high-grade lymphoma of MALT type').     

FAS (CD95) mutations are rare in gastric MALT lymphoma but occur more frequently in primary gastric diffuse large B-cell lymphoma

A loss of FAS (CD95) function has been proposed to constitute an important step in early mucosa-associated lymphoid tissue (MALT) lymphoma development and FAS mutations have been recognized in malignant lymphomas, in particular at extranodal sites. Since primary gastric lymphomas frequently exhibit resistance to FAS-mediated apoptosis, we investigated whether FAS is mutated in 18 gastric MALT lymphomas and 28 diffuse large B-cell lymphomas (DLBCL). We detected seven mutations in five lymphomas, one MALT lymphoma and four DLBCL; two DLBCL had two mutations. The MALT lymphoma exhibited a point mutation in the splice donor region of intron 3. Three DLBCL had missense mutations in exon 2, which encodes a signal peptide and a portion of the extracellular FAS ligand-binding domain. One DLBCL carried a point mutation in the splice donor region of intron 8, which would result in exon skipping. Two DLBCL harbored a missense mutation in exon 9, which encodes the intracellular death domain. The two death domain mutations inhibited FAS ligand-induced apoptosis in a dominant-negative mode, when transiently expressed in human T47D breast carcinoma and Jurkat T cells. A signal peptide and an extracellular domain mutation, however, failed to inhibit apoptosis in these transfection assays. They are likely to reduce apoptosis in lymphoma cells solely by a loss of function. In summary, our data show that FAS mutations are rare in primary gastric MALT lymphomas (5.6%) but occur in a subset of primary gastric DLBCL (14.3%) and suggest that these mutations contribute to the pathogenesis of gastric lymphomas by rendering lymphocytes resistant to apoptosis.     

Mutation of the myelin Po gene in Charcot -- Marie -- Tooth neuropathy type 1B

We had previously reported that the mutations of the myellnPo gene were completely linked with Charcot—Marie—Toothneuropathy type 1B (CMT1B) in two families. In this study wefound a different mutation In another family with CMT1B. Themutation, a methlonine substitution for Isoleucine at aminoacid position 30, Is located in the extracellular domain, whichconstitutes an Immunoglobulin domain responsible for the functionof Po as an adhesion molecule. The results confirmed that PoIs a gene responsible for CMT1B.     

Comparative Ultrastructural Analysis and KIT/PDGFRA Genotype in 125 Gastrointestinal Stromal Tumors

GISTs are the most common mesenchymal neoplasms of the digestive tract and are thought to originate from or differentiate toward the interstitial cell of Cajal lineage. Almost all GISTs express KIT protein and the majority show activating mutations in either KIT or PDGFRA proto-oncogenes. Ultrastructurally, these tumors have been shown to have either a smooth muscle, neuronal, dual, or null phenotype. The objective of this study was to investigate the relationship between ultrastructural features and genotype in a large series of 125 histologically confirmed and CD117 positive GISTs. PCR analysis for the presence of KIT exon 9, 11, 13, and 17 and PDGFRA exon 12 and 18 mutations was performed. There were 62 (50%) tumors located in the stomach and 45 (36%) in the small bowel. Overall, KIT mutations were detected in 93 (75%) patients: 86 (69%) in exon 11, and 7 (6%) in exon 9. A PDGFRA mutation was detected in 7 (6%) cases and 25 (19%) cases had no mutation. Ultrastructurally, skeinoid fibers were seen in 55 (44%) cases and were more common in small bowel than stomach GISTs, and occurred in only in 1 of 16 patients with an ITD (KIT) exon 11 or PDGFRA mutation. Focal actin microfilaments were identified in 82 (65%) cases and did not correlate with location or mutation type. Rare neurosecretory-type granules (NS-G) were seen in 34 (27%) of cases, but were seen in most of the cells in only 5 (4%) cases. GISTs showing both NS-G and microtubules were associated with KIT exon 11 genotype and spindle cell morphology. PDGFRA mutated cases were associated with gastric location, predominantly epithelioid morphology and lacked NS-G.     

Molecular characterization of the porcine TIM-3 gene

TIM-3 is a member of the TIM (T-cell immunoglobulin domain and mucin domain) family, which plays an important role in TH1 responses and autoimmune diseases. In this study, we cloned and characterized the porcine TIM-3 gene. Real-time PCR showed little expression of porcine TIM-3 in muscle and stomach, low expression in kidney, brain, stomach and muscle, moderate expression in liver, small intestine and lymph, and high expression in spleen and lung. Transient transfection indicated that porcine TIM-3 fusion protein was found to localize on the cell membranes or cytoplasm. Association analysis indicated that the SNP AccI in exon2 was significantly associated (P < 0.05) with red blood cell count mean corpuscular haemoglobin, packed cell volume, Lymphocyte percentage and Lymphocyte modulus. In conclusion, our results provide some information for conducting further studies on the functions of porcine TIM-3 gene in type I diabetes and suggest that SNP AccI in exon2 may be utilized as a marker for molecular-assisted selection in animal breeding.     

Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.     

Comparative ultrastructural analysis and KIT/PDGFRA genotype in 125 gastrointestinal stromal tumors

GISTs are the most common mesenchymal neoplasms of the digestive tract and are thought to originate from or differentiate toward the interstitial cell of Cajal lineage. Almost all GISTs express KIT protein and the majority show activating mutations in either KIT or PDGFRA proto-oncogenes. Ultrastructurally, these tumors have been shown to have either a smooth muscle, neuronal, dual, or null phenotype. The objective of this study was to investigate the relationship between ultrastructural features and genotype in a large series of 125 histologically confirmed and CD117 positive GISTs. PCR analysis for the presence of KIT exon 9, 11, 13, and 17 and PDGFRA exon 12 and 18 mutations was performed. There were 62 (50%) tumors located in the stomach and 45 (36%) in the small bowel. Overall, KIT mutations were detected in 93 (75%) patients: 86 (69%) in exon 11, and 7 (6%) in exon 9. A PDGFRA mutation was detected in 7 (6%) cases and 25 (19%) cases had no mutation. Ultrastructurally, skeinoid fibers were seen in 55 (44%) cases and were more common in small bowel than stomach GISTs, and occurred in only in 1 of 16 patients with an ITD (KIT) exon 11 or PDGFRA mutation. Focal actin microfilaments were identified in 82 (65%) cases and did not correlate with location or mutation type. Rare neurosecretory-type granules (NS-G) were seen in 34 (27%) of cases, but were seen in most of the cells in only 5 (4%) cases. GISTs showing both NS-G and microtubules were associated with KIT exon 11 genotype and spindle cell morphology. PDGFRA mutated cases were associated with gastric location, predominantly epithelioid morphology and lacked NS-G.     

Mucosa-associated lymphoid tissue lymphoma coexisting with epithelioid granulomas in the stomach of a patient with systemic sarcoidosis

Malignant lymphoma arising in the stomach of a 23-year-old Japanem man with systemic sarcoldosis is presented. The patient was followed because of systemic sarcoidosis involving the lungs, eyes, and lymph nodes. Biopsy specimens from the stomach were repeated because of recurrent eplgastraigia and multiple ulcerations. Some of the specimens revealed epithelloid granuiomas with no caseous necrosis, which confirmed gastric involvement of sarcoidosis. Three years after the initial diagnosis, biopsy specimens taken from the stomach were diagnosed as malignant lymphoma of the large cell type. The resected stomach revealed muiticentric mucosa-associated type malignant lymphoma of low-grade B cell type, with foci of high-grade transformation coexisting with numerous epithelioid granulomas with no caseous necrosis. Epithelloid granulomas were observed in all the respected lymph nodes, liver, and appendix, which had been obtained at operation, whereas malignant lymphoma was limited to the stomach. Hellcobacter pylori (H. pylori ) infection was also observed in the stomach. Consequently, the present report is a rare case of coexistence of malignant lymphoma and involvement of sarcoidosis in the stomach. Both H. pylori infection and active sarcoid noduies may play a role in the development of malignant lymphoma, although the exact mechanism remains undear.     

Simultaneous Detection of Gaucher's Disease and Renal Involvement of non-Hodgkin's Lymphoma: the First Asian Case Report and a Review of Literature

Gaucher's disease (GD) is a rare autosomal recessive (AR) disorder characterized by a deficiency of glucocerebrosidase (glucosylceramidase, acid β-glucosidase). This enzyme deficiency results in an accumulation of sphingolipids in the cells of GD patients, which may contribute to the dysregulation of the immune system, B-cell dysfunction and expression of specific cytokines such as interleukin (IL) -1, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF). Accumulated substrate may directly affect the patient's immunity and pose a higher risk for cancer, especially hematologic malignancies. However, recent large-scale studies suggest that the relative risks of GD and hematologic malignancies are not statistically significant and, therefore, their association with each other remains controversial. In this report, we present the first Asian GD case where the patient was simultaneously diagnosed with a non-Hodgkin's lymphoma. A renal biopsy confirmed that the patient had diffuse large B-cell lymphoma (DLBCL). A bone marrow study during lymphoma staging revealed Gaucher cells with abundant fibrillary, blue-gray cytoplasm and a wrinkled, tissue paper-like appearance. Subsequently, an acid β-glucosidase (GbA) gene mutation study demonstrating two heterozygote mutations, G202R (c.721G>A; p.G241R), a known pathogenic mutation, and a novel mutation R277C (c.946C>T; p.R316C) prompted the diagnosis of GD. Previous case reports have demonstrated concurrent GD and lymphoma in type 1 GD patients, with 40% of patients diagnosed with GD when a lymphoma is detected during disease evaluation. In Korea, GD cases with the G202R gene mutation have been reported in neuropathic patients with a very low frequency. To our knowledge, this case represents the first observation of the G202R mutation in a type 1 GD patient associated with lymphoma. Furthermore, this report is the first patient with DLBCL with kidney involvement along with GD.     

Mutation of RET codon 768 is associated with the FMTC phenotype

Multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are inherited cancer syndromes resulting from mutations in the RET proto-oncogene. Missense mutations of five codons in exons 10 and 11 are found in both MEN 2A and FMTC families, while mutations at codon 768 in exon 13 have been identified in three FMTC families. We report here the results of mutation analysis on a large multi-generation family with multiple cases of medullary thyroid carcinoma (MTC) or C-cell hyperplasia and two individuals with isolated adrenal medullary hyperplasia. A mutation in exon 13, which alters codon 768 from a GAG (Glu) to a GAC (Asp), was found to segregate with the FMTC phenotype in this family but not with the adrenal medullary hyperplasia. These findings suggest that the codon 768 mutation does not predispose to adrenal medullary hyperplasia, but is an accurate predictor of the MTC phenotype in this family.     

Myelin protein zero (MPZ) gene mutations in nonduplication type 1 Charcot-Marie-Tooth disease

The myelin protein zero gene (MPZ) maps to chromosome 1q22-q23 and encodes the most abundant peripheral nerve myelin protein. The P_o protein functions as a homophilic adhesion molecule in myelin compaction. Mutations in the MPZ gene are associated with the demyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), and the more severe Dejerine-Sottas syndrome (DSS). We have surveyed a cohort of 70 unrelated patients with demyelinating polyneuropathy for additional mutations in the MPZ gene. The 1.5-Mb DNA duplication on chromosome 17p11.2-p12 associated with CMT type 1A (CMT1A) was not present. By DNA heteroduplex analysis, four base mismatches were detected in three exons of MPZ. Nucleotide sequence analysis identified a de novo mutation in MPZ exon 3 that predicts an Ile(135)Thr substitution in a family with clinically severe early-onset CMT1, and an exon 3 mutation encoding a Gly(137)Ser substitution was identified in a second CMT1 family. Each predicted amino acid substitution resides in the extracellular domain of the P_o protein. Heteroduplex analysis did not detect either base change in 104 unrelated controls, indicating that these substitutions are disease-associated mutations rather than common polymorphisms. In addition, two polymorphic mutations were identified in MPZ exon 5 and exon 6, which do not alter the codons for Gly(200) and Ser(228), respectively. These observations provide further confirmation of the role of MPZ in CMT1B and suggest that MPZ coding region mutations may account for a limited percentage of disease-causing mutations in nonduplication CMT1 patients. © 1996 Wiley-Liss, Inc.     

霍奇金淋巴瘤IκBα基因第5外显子突变初步分析

目的分析单个霍奇金淋巴瘤细胞IκBα基因第5外显子的碱基序列,探讨IκBα基因突变与霍奇金淋巴瘤的关系。方法用免疫组织化学EnVision法检测7例霍奇金淋巴瘤CD30抗原,采用激光显微切割技术捕获CD30阳性表达的单个霍奇金淋巴瘤细胞和其周围反应性增生的淋巴细胞,以这些标本的基因组DNA为模板,用半巢式聚合酶链反应(PCR)方法扩增IκBα基因的第5外显子,PCR产物经纯化后直接测序。结果分析测序图发现3例霍奇金淋巴瘤细胞IκBα第5外显子出现点突变,均形成TGA终止密码子。结论霍奇金淋巴瘤中IκBα基因第5外显子出现点突变,可能是霍奇金淋巴瘤恶性转化过程中除EB病毒感染外另一重要分子机制。     

Gain-of-function mutation at the extracellular domain of KIT in gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the human gastrointestinal tract. Previous studies of GISTs found gain-of-function mutations of the c-kit gene, which encodes a receptor tyrosine kinase (KIT). All the mutations were confined to exon 11, which encodes the juxtamembrane domain. By further examination of the whole coding region of c-kit complementary DNA in 35 GISTs, two were found to show the identical mutation at exon 9, which encodes the extracellular domain. The aims of the present study were to examine the frequency of the extracellular domain mutation and to determine whether the mutation is a gain-of-function type or not. Genomic DNA was extracted from paraffin-embedded tissues of 133 GISTs and exon 9 of the c-kit gene was amplified by polymerase chain reaction. Screening of the mutation was carried out by single-strand conformation polymorphism analysis and direct sequencing was done. Mutant c-kit cDNA was transfected into 293T human embryonic kidney cells and the magnitude of autophosphorylation of the mutant KIT was examined with or without the ligand of KIT, stem cell factor (SCF). In total, seven GIST cases (approximately 5%) were found with the identical mutation at exon 9. The mutant KIT exhibited constitutive autophosphorylation without SCF stimulation. The prognosis of the patients with the extracellular domain mutation was comparable to that of the patients with the juxtamembrane domain mutation.     

Somatic and MEN 2A de novo mutations identified in the RET proto-oncogene by screening of sporadic MTC: s

Since germllne mutations in the RET proto-oncogene (RET) predisposingto tumor development In Familial Medullary Thyroid Carcinoma(FMTC), Multiple endocrine neoplasia type 2A (MEN 2A), and Multipleendocrine neoplasia type 2B (MEN 2B) were reported, It has becomepossible to identify gene carriers with a very high degree ofaccuracy. Mutations in FMTC and MEN 2A exclusively affect cystelneresidues in exon 10 and 11 of RET, whereas in MEN 2B codon 918in exon 16 is involved. This latter mutation has also been describedin a subset of apparently sporadic medullary thyroid carcinomas(MTC). Mutations in MEN 2B often occur as de novo germline mutations,whereas de novo mutations have not yet been described In FMTCor MEN 2A. We analyzed ten MTC: s and ten pheochromocytomas,all clinically judged to be sporadically occurring, by directDNA sequencing of exons 10, 11, and 16 of RET. This analysisrevealed a de novo germline mutation of codon 634 in exon 11In a patient with MTC. In addition, somatic mutations of codon918 in exon 16 in six of the remaining MTC: s were found, Interestingly,the presence of this somatic mutation was associated with asignificantly less favorable clinical outcome.     

Expression of Bruton's tyrosine kinase in B lymphoblastoid cell lines from X-linked agammaglobulinaemia patients

X-linked agammaglobulinaemia (XLA) is an immunodeficiency caused by mutations in Bruton's tyrosine kinase (Btk) and is characterized by an almost complete arrest of B cell development. We analysed expression of Btk in B lymphoblastoid cell lines (BLCL) derived from four unrelated XLA patients. In one patient, with a 3.5 kb genomic deletion encompassing the first (untranslated) exon, mRNA levels and in vitro kinase activities were very low. The patient manifested a mild phenotype with a delayed onset of the disease. Another mutation, in which the intron 3 donor splice site is lost, was also associated with very low mRNA levels and an absence of detectable Btk protein. Patients with this mutation showed extensive heterogeneity of the immunological phenotype. In the BLCL of a third patient, with an Arg₂₈₈ substitution in the SH2 domain, the mutation did not appear to affect the expression level, nor to abrogate in vitro phosphorylation activity. In the BLCL of the fourth patient, with an Arg₂₈ mutation in the PH domain, tyrosine kinase activity in BTK precipitates appeared to be decreased compared with control BLCL.