ABSTRACT
Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.
Subject(s)
Flow Cytometry , Immunotherapy, Adoptive , Receptors, Chimeric Antigen/analysis , T-Lymphocytes/cytology , Humans , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunologyABSTRACT
The 2019 13th Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of Bioanalysis, issues 22 and 23 (2019), respectively.
Subject(s)
Biological Assay/methods , Biomarkers/metabolism , Flow Cytometry/methods , Genetic Therapy/methods , United States Food and Drug Administration/standards , History, 21st Century , Humans , United StatesABSTRACT
Erythropoiesis is an important response to certain types of stress, including hypoxia, hemorrhage, bone marrow suppression, and anemia, that result in inadequate tissue oxygenation. This stress-induced erythropoiesis is distinct from basal red blood cell generation; however, neither the cellular nor the molecular factors that regulate this process are fully understood. Here, we report that type 1 conventional dendritic cells (cDC1s), which are defined by expression of CD8α in the mouse and XCR1 and CLEC9 in humans, are critical for induction of erythropoiesis in response to stress. Specifically, using murine models, we determined that engagement of a stress sensor, CD24, on cDC1s upregulates expression of the Kit ligand stem cell factor on these cells. The increased expression of stem cell factor resulted in Kit-mediated proliferative expansion of early erythroid progenitors and, ultimately, transient reticulocytosis in the circulation. Moreover, this stress response was triggered in part by alarmin recognition and was blunted in CD24 sensor- and CD8α+ DC-deficient animals. The contribution of the cDC1 subset to the initiation of stress erythropoiesis was distinct from the well-recognized role of macrophages in supporting late erythroid maturation. Together, these findings offer insight into the mechanism of stress erythropoiesis and into disorders of erythrocyte generation associated with stress.
Subject(s)
Dendritic Cells/physiology , Erythropoiesis/physiology , Stress, Physiological/physiology , Alarmins/physiology , Animals , CD24 Antigen/physiology , CD8 Antigens/analysis , Cisplatin/toxicity , Colony-Forming Units Assay , Dendritic Cells/classification , Erythroid Precursor Cells/physiology , Female , Gene Expression Profiling , HMGB1 Protein/toxicity , Hematopoietic Stem Cell Transplantation , Heterografts , Humans , Hypoxia/physiopathology , Imatinib Mesylate/toxicity , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Phlebotomy/adverse effects , Radiation Chimera , Recombinant Proteins/toxicity , Splenectomy/adverse effects , Stem Cell Factor/biosynthesis , Stem Cell Factor/geneticsABSTRACT
Signaling via the pre-T-cell receptor (pre-TCR), along with associated signals from Notch and chemokine receptors, regulates the ß-selection checkpoint that operates on CD4(-) CD8(-) doubly negative (DN) thymocytes. Since many hematopoietic malignancies arise at the immature developmental stages of lymphocytes, understanding the signal integration and how specific signaling molecules and distal transcription factors regulate cellular outcomes is of importance. Here, a series of molecular and genetic approaches revealed that the ShcA adapter protein critically influences proliferation and differentiation during ß-selection. We found that ShcA functions downstream of the pre-TCR and p56(Lck) and show that ShcA is important for extracellular signal-regulated kinase (ERK)-dependent upregulation of transcription factors early growth factor 1 (Egr1) and Egr3 in immature thymocytes and, in turn, of the expression and function of the Id3 and E2A helix-loop-helix (HLH) proteins. ShcA also contributes to pre-TCR-mediated induction of c-Myc and additional cell cycle regulators. Moreover, using an unbiased Saccharomyces cerevisiae (yeast) screen, we identified c-Abl as a binding partner of phosphorylated ShcA and demonstrated the relevance of the ShcA-c-Abl interaction in immature thymocytes. Collectively, these data identify multiple modes by which ShcA can fine-tune the development of early thymocytes, including a previously unappreciated ShcA-c-Abl axis that regulates thymocyte proliferation.
Subject(s)
Cell Proliferation , Proto-Oncogene Proteins c-abl/metabolism , Shc Signaling Adaptor Proteins/metabolism , Signal Transduction , Thymocytes/cytology , Transcription Factors/metabolism , Animals , Cell Differentiation , Cells, Cultured , Early Growth Response Protein 1/genetics , Early Growth Response Protein 1/metabolism , Early Growth Response Protein 3/genetics , Early Growth Response Protein 3/metabolism , Gene Expression Regulation, Developmental , Inhibitor of Differentiation Proteins/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , MAP Kinase Signaling System , Mice, Inbred C57BL , Mutation , Proto-Oncogene Proteins c-myc/genetics , Receptors, Antigen, T-Cell/metabolism , Shc Signaling Adaptor Proteins/genetics , Src Homology 2 Domain-Containing, Transforming Protein 1 , Thymocytes/metabolism , Transcription Factors/genetics , Up-RegulationABSTRACT
T cell development in the thymus is a highly regulated process that critically depends upon productive signaling via the preTCR at the ß-selection stage, as well as via the TCR for selection from the CD4(+)CD8(+) double-positive stage to the CD4 or CD8 single-positive stage. ShcA is an adapter protein expressed in thymocytes, and it is required for productive signaling through the preTCR, with impaired signaling via ShcA leading to a developmental block at the ß-selection checkpoint. However, the role of ShcA in subsequent stages of T cell development has not been addressed. In this study, we generated transgenic mice (CD4-Cre/ShcFFF mice) that specifically express a phosphorylation-defective dominant-negative ShcA mutant (ShcFFF) in late T cell development. Thymocytes in CD4-Cre/ShcFFF mice progressed normally through the ß-selection checkpoint, but displayed a significant reduction in the numbers of single-positive CD4(+) and CD8(+) thymocytes. Furthermore, CD4-Cre/ShcFFF mice, when bred with transgenic TCR mouse strains, had impaired signaling through the transgenic TCRs. Consistent with defective progression to the single-positive stage, CD4-Cre/ShcFFF mice also had significant peripheral lymphopenia. Moreover, these CD4-Cre/ShcFFF mice develop attenuated disease in CD4(+) T cell-dependent experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Collectively, these data identify an important role for the adapter protein ShcA in later stages of thymic T cell development and in peripheral T cell-dependent events.
Subject(s)
CD4-Positive T-Lymphocytes/cytology , Cell Differentiation/immunology , Precursor Cells, T-Lymphoid/cytology , Shc Signaling Adaptor Proteins/immunology , T-Lymphocytes/cytology , Animals , CD4-Positive T-Lymphocytes/immunology , Cell Proliferation , Encephalomyelitis, Autoimmune, Experimental , Flow Cytometry , Fluorescent Antibody Technique , Immunohistochemistry , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Precursor Cells, T-Lymphoid/immunology , Reverse Transcriptase Polymerase Chain Reaction , Src Homology 2 Domain-Containing, Transforming Protein 1 , T-Lymphocytes/immunologyABSTRACT
Cancer develops after the acquisition of a collection of mutations that together create the cancer phenotype. How collections of mutations work together within a cell and whether there is selection for certain combinations of mutations are not well understood. We investigated this problem with a mathematical model of the Ras signaling network, including a computational random mutagenesis. Modeling and subsequent experiments revealed that mutations of the tumor suppressor gene NF1 can amplify the effects of other Ras pathway mutations, including weakly activating, noncanonical Ras mutants. Furthermore, analyzing recently available, large, cancer genomic data sets uncovered increased co-occurrence of NF1 mutations with mutations in other Ras network genes. Overall, these data suggest that combinations of Ras pathway mutations could serve the role of cancer "driver." More generally, this work suggests that mutations that result in network instability may promote cancer in a manner analogous to genomic instability.
ABSTRACT
T cell development and activation are highly regulated processes, and their proper execution is important for a competent immune system. Shc SH2-domain binding protein-1 (Shcbp1) is an evolutionarily conserved protein that binds to the adaptor protein ShcA. Studies in Drosophila and in cell lines have strongly linked Shcbp1 to cell proliferation, embryonic development, growth factor signaling, and tumorigenesis. Here we show that Shcbp1 expression is strikingly upregulated during the ß-selection checkpoint in thymocytes, and that its expression tightly correlates with proliferative stages of T cell development. To evaluate the role for Shcbp1 during thymic selection and T cell function in vivo, we generated mice with global and conditional deletion of Shcbp1. Surprisingly, the loss of Shcbp1 expression did not have an obvious effect during T cell development. However, in a mouse model of experimental autoimmune encephalomyelitis (EAE), which depends on CD4(+) T cell function and mimics multiple features of the human disease multiple sclerosis, Shcbp1 deficient mice had reduced disease severity and improved survival, and this effect was T cell intrinsic. These data suggest that despite the striking upregulation of Shcbp1 during T cell proliferation, loss of Shcbp1 does not directly affect T cell development, but regulates CD4(+) T cell effector function in vivo.
Subject(s)
Cell Proliferation/genetics , Shc Signaling Adaptor Proteins/genetics , T-Lymphocytes/metabolism , Animals , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Mice , Mice, Knockout , Phenotype , Shc Signaling Adaptor Proteins/metabolism , Signal Transduction/genetics , T-Lymphocytes/cytology , Thymus Gland/cytology , Thymus Gland/metabolismABSTRACT
Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.
Subject(s)
Apoptosis , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , Phagocytes/cytology , Phagocytes/metabolism , Phagocytosis/physiology , Animals , Cell Line , Cell Size/drug effects , Cells, Cultured , Ion Channels/deficiency , Ion Channels/genetics , Membrane Potential, Mitochondrial/drug effects , Membrane Potential, Mitochondrial/physiology , Mice , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Phagocytes/drug effects , Phagocytosis/drug effects , Thymus Gland/cytology , Uncoupling Protein 2ABSTRACT
Passage through the beta-selection developmental checkpoint requires productive rearrangement of segments of the T cell antigen receptor-beta gene (Tcrb) and formation of a pre-TCR on the surface of CD4(-)CD8(-) thymocytes. How other receptors influence betabeta-selection is less well understood. Here we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associated with the pre-TCR and influenced beta-selection by regulating the steady-state localization of immature thymocytes in thymic subregions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. Our data designate CXCR4 as a costimulator of the pre-TCR during beta-selection.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/immunology , Lymphoid Progenitor Cells/cytology , Receptors, CXCR4/immunology , Thymus Gland/cytology , Animals , B-Lymphocytes/immunology , Blotting, Western , Cell Proliferation , Chemokine CXCL12/biosynthesis , Chemokine CXCL12/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescent Antibody Technique , Genes, T-Cell Receptor beta/immunology , Immunoprecipitation , Lymphoid Progenitor Cells/immunology , Mice , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/immunologyABSTRACT
Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable. This is thought to be due to the release of 'find-me' signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages and dendritic cells, leading to the prompt clearance of the dying cells. However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to that of apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or the expression of ectopic CD39) abrogated the ability of apoptotic cell supernatants to recruit monocytes in vitro and in vivo. We then identified the ATP/UTP receptor P2Y(2) as a critical sensor of nucleotides released by apoptotic cells using RNA interference-mediated depletion studies in monocytes, and macrophages from P2Y(2)-null mice. The relevance of nucleotides in apoptotic cell clearance in vivo was revealed by two approaches. First, in a murine air-pouch model, apoptotic cell supernatants induced a threefold greater recruitment of monocytes and macrophages than supernatants from healthy cells did; this recruitment was abolished by depletion of nucleotides and was significantly decreased in P2Y(2)(-/-) (also known as P2ry2(-/-)) mice. Second, clearance of apoptotic thymocytes was significantly impaired by either depletion of nucleotides or interference with P2Y receptor function (by pharmacological inhibition or in P2Y(2)(-/-) mice). These results identify nucleotides as a critical find-me cue released by apoptotic cells to promote P2Y(2)-dependent recruitment of phagocytes, and provide evidence for a clear relationship between a find-me signal and efficient corpse clearance in vivo.
Subject(s)
Adenosine Triphosphate/metabolism , Apoptosis/physiology , Phagocytes/cytology , Phagocytosis/physiology , Signal Transduction , Thymus Gland/cytology , Uridine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Line , Cells, Cultured , Chemotactic Factors/metabolism , Chemotactic Factors/pharmacology , Chemotaxis/drug effects , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/metabolism , Culture Media, Conditioned/pharmacology , Humans , Jurkat Cells , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Monocytes/cytology , Monocytes/drug effects , Monocytes/metabolism , Phagocytes/drug effects , Phagocytes/metabolism , Phagocytosis/drug effects , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2/deficiency , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y2 , Signal Transduction/drug effects , Uridine Triphosphate/pharmacologyABSTRACT
To investigate the unregulated Ras activation associated with cancer, we developed and validated a mathematical model of Ras signaling. The model-based predictions and associated experiments help explain why only one of two classes of activating Ras point mutations with in vitro transformation potential is commonly found in cancers. Model-based analysis of these mutants uncovered a systems-level process that contributes to total Ras activation in cells. This predicted behavior was supported by experimental observations. We also used the model to identify a strategy in which a drug could cause stronger inhibition on the cancerous Ras network than on the wild-type network. This system-level analysis of the oncogenic Ras network provides new insights and potential therapeutic strategies.
Subject(s)
Computer Simulation , Metabolic Networks and Pathways , Models, Biological , Neoplasms/metabolism , Signal Transduction , ras Proteins/metabolism , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line , Cell Line, Tumor , Cell Transformation, Neoplastic , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Genes, ras , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Mathematics , Phosphorylation , Point Mutation , ras Proteins/antagonists & inhibitors , ras Proteins/geneticsABSTRACT
During thymic development, the beta selection checkpoint is regulated by pre-T-cell receptor-initiated signals. Progression through this checkpoint is influenced by phosphorylation and activation of the serine/threonine kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2, but the in vivo relevance of specific upstream players leading to ERK activation is not known. Here, using mice with a conditional loss of the shc1 gene or expressing mutants of ShcA, we demonstrate that the adapter protein ShcA is responsible for up to 70% of ERK activation in double-negative (DN) thymocytes in vivo and ex vivo. We also identify two specific tyrosines on ShcA that promote ERK phosphorylation in vivo, and mice expressing ShcA with mutations of these tyrosines show impaired DN thymocyte development. This work provides the first in vivo demonstration of the relative requirement of upstream adapters in controlling ERK activation during beta selection and suggests a dominant role for ShcA.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Thymus Gland/cytology , Thymus Gland/embryology , Adaptor Proteins, Signal Transducing/genetics , Animals , Enzyme Activation , Fluorescent Dyes , Indoles , Mice , Mice, Transgenic , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Thymus Gland/enzymologyABSTRACT
The placental-umbilical unit in sickle cell disease (SCD) pregnancy was used to explore hypoxia in vivo, an important factor in the pathophysiology of this disease. Gross examination and microscopic analysis of the placentas, taken immediately after delivery, indicate good concordance between maturity and term as controls, but higher frequency of vascular injuries such as excess syncytial knots, excess fibrin deposits, congestion and villous necroses. Unexpectedly, neither leukocyte recruitment nor alteration in extraplacental membrane was observed, suggesting the absence of inflammation. Additionally, interleukin (IL)-6 and IL-8 concentrations, measured by enzyme-linked immunosorbent assay (ELISA), were similar in the placental maternal blood from controls and SCD. There were also no significant differences found in IL-6 vein blood concentrations between controls and SCD, IL-8 being not detected. Immunostaining of umbilical vein endothelium in SCD pregnancies showed redistribution of PECAM-1 (CD31), von Willebrand factor (vWF), and P-selectin to the cell surface, controls exhibiting the classical pattern. Staining quantification indicated increases in vWF (+36.2%; P=.006) and vascular endothelial growth factor (VEGF) expression (+96.0%; P=.006) over control, but a reduction in endothelial nitric oxide synthase (eNOS) (-45.5%; P=.029). These results document, for the first time, direct functional adjustments in response to hypoxia in human in vivo. The mechanism for these changes has not been clearly established, but it may reflect increased tolerance to SCD hypoxic conditions and hypoxia in general.
Subject(s)
Adaptation, Physiological , Anemia, Sickle Cell/metabolism , Hypoxia/metabolism , Placenta/metabolism , Pregnancy Complications, Hematologic/metabolism , Umbilical Veins/metabolism , Adolescent , Adult , Anemia, Sickle Cell/pathology , Biomarkers/analysis , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Fetal Blood/metabolism , Homeostasis , Humans , Hypoxia/physiopathology , Models, Biological , Placenta/pathology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Pregnancy , Pregnancy Complications, Hematologic/pathology , Umbilical Cord/blood supply , Umbilical Veins/pathology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The expression of metallothionein (MT)-3 is often markedly reduced in gastric carcinoma (GC). The molecular mechanism of this MT-3 downregulation is unknown. Transcriptional silencing of MT-3 by methylation of CpG island was investigated by nucleotide sequencing and denaturing high performance liquid chromatography (DHPLC) analyses. We found that normal brain tissue and a xenografted GC that expressed MT-3 mRNA had unmethylated regions of the CpG island in intron1 of this gene. On the other hand, gastric cancer cell lines AGS and MKN445, a xenografted GC, and a representative primary gastric cancer that had no expression of MT-3 mRNA demonstrated hypermethylation of the MT-3 intron1 CpG island. Treatment of the gastric cancer cell lines with 5-azacytidine resulted in new expression of MT-3 mRNA in these cells. A quantifying DHPLC assay was developed to determine the methylation status of this specific region of the MT-3 gene. Fifty-eight primary GC and their corresponding normal gastric epithelial tissues, and 34 normal gastric mucosa were analyzed for MT-3 methylation by DHPLC in the region of methylation abnormalities initially identified. Our DHPLC analyses of the methylated MT-3 product demonstrated that the primary gastric cancers have an average methylation percentage of 6.3% per tumor compared with 2.4% in normal gastric tissues (P < 0.05). The MT-3 was not methylated in all of eight P53-positive GCs and hypermethylated in eight of 13 P53-negative cases by immunohistochemistry staining (P = 0.007). In conclusion, the CpG island in the MT-3 intron1 are abnormally hypermethylated in many gastric carcinomas and may account for the downregulation of MT-3 in gastric carcinogenesis.