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Alternative splicing is an important mechanism that links to transcription and contributes to protein diversity. Disturbed alternative splicing is frequently observed in cancers, but its precise mechanism remains largely unknown. FUSE-binding protein (FBP) -interacting repressor (FIR) is a transcriptional repressor of the c-myc gene. Previous studies indicated that a splice variant of FIR, FIRΔexon2, that lacks exon2 in the transcriptional repressor domain, was increased in colorectal cancers, hepatocellular carcinomas, and leukemia cells. Furthermore, FIRΔexon2 activated c-myc transcription by disabling wild-type FIR as a dominant-negative form of FIR. Recently, somatic mutations of the SF3B1 (SAP155) gene, a subunit of the SF3B spliceosome complex, were found in myelodysplastic leukemia. In this study, FIR heterozygous knockout (FIR(+/-)) was established as a dominant-negative model of FIR in the C57BL/6 mouse. FIR(+/-) mice showed an increased c-myc mRNA expression level, particularly in peripheral blood, although FIR(+/-) mice had no apparent pathogenic phenotype. Therefore, an increased c-myc mRNA expression level alone is not enough for leukemogenesis. Nevertheless, FIR(+/-)TP53(-/-) mice generated acute T-cell lymphoblastic leukemia (T-ALL) with increased organ and/or bone marrow invasion. In conclusion, alternative splicing of FIR, generating FIRΔexon2, contributes to not only colorectal carcinogenesis but also leukemogenesis independent of the c-Myc activation pathway. Finally, we will discuss our hypothesis that FIRΔexon2 interferes with FBW7, that FIRΔexon2 inhibits PP1 in the EGFR pathway, and that FIR haploinsufficiency is potentially associated with protein expression through transcriptional and post-transcriptional mechanisms.

Vitamin D is pro-hormone that has important roles in calcium metabolism in the intestine, kidneys, and bone. Many studies have indicated that vitamin D deficiency causes not only rickets and osteomalacia, but also increases the risk of various diseases such as cancer and autoimmune disease. Of the many vitamin D metabolites, 25-hydroxyvitamin D[25OH-D], 1α,25-dihydroxyvitamin D[1,25(OH)2-D], and 24,25-dihydroxyvitamin D [24,25 (OH) 2-D] are important for assessing vitamin D metabolism. They are circulating, biologically active, and major inactive forms of vitamin D metabolite, respectively. Immunoassays are widely used for the measurement of serum/plasma vitamin D metabolites. With such an assay, however, the accurate quantification of these metabolites is difficult because of cross-reactivity. Liquid chromatography-tandem mass spectrometry [LC-MS/MS] is the gold standard method for analyzing these metabolites due to its high sensitivity and selectivity. Derivatization with a Cookson-type reagent is a key technique for robust and sensitive analysis by LC-MS/MS. A Cookson-type reagent rapidly and quantitatively reacts with the s-cis-diene structure of vitamin D metabolites, and markedly enhances the ionization efficiency. Using a recently developed Cookson-type reagent (DAPTAD), we successfully established an LC-MS/MS-based method for analyzing serum vitamin D metabolites including 250H-D, 1,25 (OH) 2-D, and 24,25 (OH) 2-D. Thus, LC-MS/MS can be a powerful tool for the accurate determination of vitamin D metabolism. In this review, we describe the advantages of LC-MS/MS for the determination of vitamin D metabolites.

During the past 15 years, various approaches have been adopted for medical personnel training in the Division of Laboratory Medicine, Chiba University Hospital. Medical personnel have been encouraged to enter the Graduate School of Medicine, Chiba University. At present, 14 of them have successfully completed the Ph.D. program and 16 have been awarded a master's degree. In our unit, clinical proteomics is a principal research subject, and we have identified a number of biomarker candidates in collaboration with clinical units. In Chiba University Hospital, all clinical laboratory physicians are certified as medical geneticists and are in charge of the Division of Clinical Genetics as well. We have treated a total of 1,009 patients, including those with hereditary neuromuscular diseases, familial cancers, and prenatal diagnoses. We have also encouraged medical technologists to become certified as genetic counselors, which may be a promising subspecialty for medical technologists. Mass spectrometry (MS) is a powerful analytical tool used in an increasing number of clinical laboratories around the world. Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has been used for newborn screening, toxicology, therapeutic drug monitoring endocrinology, and, more recently, for the measurement of targeted proteins and peptides. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) has proven to be a rapid and reliable tool for identifying microorganisms. The Japanese Society for Medical Mass Spectrometry has started to certify medical mass spectrometrists, which could be another promising subspecialty for medical technologists.

Alternative splicing is a fundamental process of gene regulation that contributes to protein diversity, a common phenomenon in the mammalian genome. Alternative splicing events not only happen in the normal gene regulation process, but are also closely related to certain diseases, including cancer. In this review, we briefly demonstrate the proof of concept (POC) of the relationship between alternative splicing and DNA damage, and describe the associations among alternative splicing and cancer pathogenesis, DNA damage, and gastrointestinal cancers. We discuss whether alternative splicing leads to genetic instability, which is considered to be a driving force for tumorigenesis. FUSE-binding protein (FBP) -interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2), upregulates c-myc transcription by inactivating wild-type FIR. FIR+/- mice exhibited marked c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Because the single knockout of TP53 generates thymic lymphoma, FIR+/-TP53-/- mice developed T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion and showed a poor prognosis. After describing the POC of alternative splicing of FIR in DNA damage and carcinogenesis, clinical application for cancer diagnosis and treatment by FIR/FIRΔexon2 was briefly summarized. Chiba University has prepared a biobank to support studies to develop biomarker detection, molecular diagnosis, and "Omics" research. In conclusion, alternative splicing of FIR, generating FIRΔexon2, potentially contributes to not only colorectal carcinogenesis, but also leukemogenesis, and a better understanding of the role and mechanism of alternative splicing in tumorigenesis may reveal new directions for cancer biomarker detection.

Personalized medicine has been expected to be utilized in daily practice since the international human genome project completed sequencing of the entire genome in 2003. The aim of personalized medicine is to treat patients effectively and safely based on the genome characteristics of each patient, or to choose the right drug for the right patient at the right time and at the right dose. Regulatory agencies such as the United States Food and Drug Administration(FDA) and Pharmaceuticals and Medical Devices Agency (PMDA), Japan implemented guidance to facilitate the co-development of drugs and companion diagnostics. For the practice of personalized medicine, companion diagnostic tests are essential, although their availability is limited.

Mass spectrometry has been recently used in clinical diagnostics, especially drug screening, newborn screening, and bacterial identification; therefore, the current developments in mass spectrometry platforms (i. e. GC-MS, LC-MS/MS) and applications for clinical diagnosis will be discussed. Technological advances in drug analyses and bacterial identification using mass spectrometry during recent years have provided an effective tool for the clinical laboratory. This symposium provides a comprehensive future overview of the latest technologies to quantify metabolites, drugs, and identify bacterial proteins using mass spectrometry, proposed for potential new biomarker development.

Serum cystatin C (sCysC) is valuable to evaluate the renal functions in various clinically studies. Since the reagents to detect sCysC are different among institutes or hospitals, the accidental error due to the difference of reagents is not negligible. That is why sCysC is sometimes difficult to use or interpret for patient as estimated Glomerular filtration rate (eGFR) that is one of the most popular factors to evaluate renal function. Recently, the international reference standard is gradually accepted as standardization of chemical measurement of sCysC in Japan. Japan Society of Clinical Chemistry has reported the utilities for sCysC measurement standardization in 2010. In this study, we examined the usefulness of the standardization of sCysC reagent in Chiba University Hospital. Our study indicated that the clinical usefulness of eGFR calculated by sCysC. As results, eGFR from serum creatinine (sCr) was relatively high in patients with reduced muscle mass, such as old-age patients with wheelchair and prolonged hospitalization. On the other hand, eGFR from sCysC was high in patients with hypothyroidism. Together, eGFR calculated by sCysC is clinically available for the patients with reduced muscle mass could be underestimated. Further studies are required to evaluate the validity of standardization of sCysC measurement and find the dissociation among eGFR, sCysC and sCr depending in the renal disease or pathogenesis.

In a clinical diagnostic microbiology laboratory, the current method of identifying bacterial isolates is based mainly on phenotypic characteristics, such as the growth pattern on different media, colony morphology, Gram stain, and various biochemical reactions. These techniques collectively allow high-level accuracy in identifying most bacterial isolates, but they are costly and time-consuming. In our clinical microbiology laboratory, we prospectively assessed the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify bacterial strains that were routinely isolated from clinical samples. Bacterial colonies obtained from a total of 468 strains of 92 bacterial species isolated at the Department of Clinical Laboratory at Chiba University were directly placed on target MALDI plates, followed by the addition of CHCA matrix solution. The plates were then subjected to MALDI-TOF MS measurement, and the microorganisms were identified by pattern matching by the libraries in the BioTyper 2.0 software. The identification rates at species and genus levels were 91.7% (429/468) and 97.0% (454/468), respectively. MALDI-TOF MS is a rapid, simple, and high-throughput proteomic technique for the identification of a variety of bacterial species. Since colony to colony differences and the effects of culture duration on the results are minimal, it can be implemented in a conventional laboratory setting. Although for some pathogens, the preanalytic processes should be refined and current database should be improved to obtain more accurate results, the MALDI-TOF MS-based method generally performs as well as the conventional methods and is a promising technology in clinical laboratories.

In this study, clinical utility of various renal function markers (Cystatin C, Creatinine, beta2-microglobulin, Urea nitrogen) including serum Cystatin C was evaluated by the latex turbidimetric immunoassay based on the Chronic Kidney Disease (CKD) stage classification. Serum creatinine was most correlated with estimated Glomerular Filtration Rate (eGFR), followed by serum Cystatin C. However, the level of serum Cystatin C increased significantly earlier than other renal function markers in the group of mild renal dysfunction based on the CKD stage classification. Cystatin C also had the most distinctive ability of the renal dysfunction by Receiver Operating Characteristic (ROC) and distinction characteristic analysis. Thus, it is useful to measure serum Cystatin C and serum creatinine at the same time to improve diagnostic sensitivity and specificity, eGFR was 6% higher than Ccr on stage III-V, though Ccr was 30% higher than eGFR on stage I-II by CKD stage classifications. Since eGFR and Ccr showed different results in early and severe CKD groups, it was suggested that Ccr and eGFR need to be evaluated as different renal function markers.

Development of useful biomarkers is pivotal for prediction of micro-metastasis, recurrence probability and/or prognosis of the patients. Recent studies have revealed that cancer-specific alternative splicing can be valuable for cancer cell detection. Among them, FUSE-binding protein-interacting repressor, FIR, has been reported to repress c-myc transcription and its exon2-spliced variant, FIRDelta(exon)2, is unable to repress c-myc by competing with authentic FIR in vivo and in vitro. Moreover FIRDelta(exon)2 was frequently discovered in human primary colorectal cancers, but not in the adjacent normal tissues, indicating its cancer-related expression. Thus, the expression level of FIRDelta(exon)2 mRNA in the colorectal cancer tissues as tumor marker candidates is examined. Further, to determine the interacting proteins, FIR-flag or FIRDelta(exon)2-flag stably expressing HeLa cells have been established by G418 selection and nuclear proteins were co immunoprecipitated with flag-conjugated magnetic beads. Those co-immunoprecipitated proteins with FIR or FIRDelta(exon)2 are candidates of tumor makers. In addition, substances that interfers FIR mRNA splicing should be anti-cancer drugs. Together, FIR splicing variant, FIRDelta(exon)2 mRNA or proteins and its interacting proteins are applicable for novel screening tumor markers in colorectal cancer detection.

Since the completion of the human genome project, there is growing interest in the clinical application of genome sciences. For this purpose, particular attention toward identifying at-risk individuals and understanding the complexities of the testing process are essential. In this article, I describe the importance of clinical genetics and genetic counseling, and explain how and why the division of laboratory medicine is involved in these tasks in Chiba University Hospital. Our genetic counseling team consists of a clinical laboratory physician qualified as a clinical geneticist, medical technologist qualified as a genetic counselor, clinical psychologists, and a medical social worker. We treat more than 100 cases including late-onset, incurable neurological diseases, hereditary tumors, prenatal diagnosis, and chromosomal abnormalities. The sequencing of the human genome has paved the way for comprehensive transcriptome and proteome analyses. Since the detailed understanding of biological processes, both in healthy and pathological states, requires the direct study of relevant proteins, proteomics bridges the gap between the information coded in the genome sequence and cellular behavior. Therefore, proteomics is among the most promising technologies for the development of novel diagnostic tools. Recent advances in sophisticated technologies in proteomics should identify promising ways to discover novel markers in various fields of clinical medicine. In this presentation, I will give a definition of the proteome, and outline the basic methodologies for proteome analyses. I will also present our experiences in identifying novel biomarker candidates in hepatobiliary diseases, and discuss future perspectives of clinical proteomics in laboratory medicine.

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The severity of disease varies widely from mild illness to cirrhosis and hepatocellular carcinoma. The routine diagnostic test for HCV infection is performed using anti-HCV antibodies and an enzyme immunoassay (EIA) for serologic identification and HCV-RNA assay employing the polymerase chain reaction(PCR) for genetic identification. This study assessed the sensitivity and specificity of HCV-RNA diagnostic tests on analysis of the literature. A literature search was performed using the criteria from The standards for Reporting of Diagnostic Accuracy (STARD) steering committee for the assessment of diagnostic tests. The selected studies were searched for identifying publications on the appropriate conducting and reporting of diagnostic studies, and we extracted potential items to generate an extensive list. Out of 409 studies, 38 were selected. Further studies are necessary to accurately access HCV-RNA in various populations compared with reference tests.

Serum tartrate resistant acid phosphatase 5b (TRACP 5b) is an isozyme of osteoclast origin. Indeed, measurement of TRACP 5b activity is used as an index of osteoclast activity. However, the precise mechanism of TRACP 5b maturation is unclear. This study aimed to clarify the mechanism of generation of TRACP 5b. We used a highly sensitive fiber-type DNA chip to investigate the mechanism of generation of TRACP 5b at the genetic level. Genes derived from three related cell types (monocytes, macrophages and osteoclasts) were compared. In addition, at the protein level, posttranscriptional modification was tested by Western blotting using an antiserum specific for the flexible loop region of TRACP 5. Our DNA chip study shows that genes implicated in oligosaccharide construction do not show significant differences in expression levels between the cell types under investigation. Strongly expressed Cathepsin K was observed in osteoclasts. Western blotting demonstrated that TRACP undergoes unique partial degradation during bone resorption, such that serum TRACP 5b lacks the flexible loop found in TRACP 5a. In conclusion, TRACP 5b generated by a specific posttranscriptional modification pathway undergoes partial digestion in resorption lacunae or inside osteoclasts. Serum TRACP 5b lacking the flexible loop differs from TRACP 5a in terms of optimum pH, isoelectric point, sugar chain and antigenicity. The measurement of TRACP 5b could therefore be of great use for monitoring of osteoporosis, rheumatoid arthritis and bone metastasis.

Cancer has been the disease with the highest cause of death for a decade. This is partly due to the lack of ideal tumor markers for early diagnosis, which causes cancer found at the advanced stage where no curative treatment is available. Therefore, development of tumor markers with higher sensitivity and specificity is waiting to emerge. The ideal source for the tumor markers are serum, plasma or urine samples that are routinely used as clinical laboratory test. They contain a number of proteins that could be useful for cancer diagnosis. Recent advances in proteomic technology made it possible to identify the low abundant proteins in the clinical samples and thus extensive efforts are now attempted to search for the tumor markers across the country. There are two prominent types of proteomic approaches, 2D gel-based (2DE) and MS/MS based approach. 2DE is favorable to analyze relatively high moleculer weight proteins (MW > 20kD) and MS is superior to detect low molecular proteins or peptides. In this article, we introduce our recent studies on tumor marker discovery using various proteomic approaches and will discuss future application for cancer

During the past few years, the emphasis on genomics has shifted via transcriptomics to proteomics, the science of understanding how the whole set of proteins are expressed and function at the cellular level. Recent progress in clinical proteomics is mostly thanks to sophisticated new methodologies for proteome analyses. In search for novel biomarkers for cancers and other disorders, we have taken advantage of the surface-enhanced laser/desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) and the MALDI-TOF/TOF. Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence and found that a 5.9kD peptide detected and identified by this technology could be a novel marker of heavy alcohol consumption. Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We could identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. A number of proteins with altered expression between cancer and adjacent non-cancer tissues were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting and immunohistochemistry. Clinical proteomics consists of the discovery proteomics and the measurement proteomics. Although we still stand at the discovery proteomics, undoubtedly, the clinical proteomics will be a major contributor in laboratory medicine in the near future.

Compulsory postgraduate clinical training in laboratory medicine will start on a national basis in April 2004. It remains to be discussed how departments of laboratory medicine and/or divisions of clinical laboratory should contribute to the training program. I here summarize what we are planning to do in Chiba University Hospital together with my personal views on this topic. Two points are of note in our program. First, one-week training in the division of clinical laboratory is essential as a part of the 6-month training of internal medicine. The items in the program include Gram staining, white blood cell differential counting, urinary sediment study, and representative point-of-care testing. A program to follow the entire flow of laboratory tests(from withdrawal of blood to reporting of the test results) is also included to highlight a variety of pre-analytical errors. Furthermore, trainees are encouraged to learn how to work and co-operate with co-medical personnel. Second, our program aims at training postgraduate medical students to be clinical geneticists competent at genetic counseling. Genetic counseling is the process by which patients or relatives at risk of a disorder that may be hereditary are advised of the consequences of the disorder, the probability of developing or transmitting it and of the ways to test them. Thus, our final goal is to foster genetically oriented experts in laboratory medicine and clinical geneticists with a wide knowledge of laboratory medicine.

Detection of virus genomes and their mutants by molecular technology is essential in clinical practice for patients with hepatitis B virus(HBV) and hepatitis C virus(HCV) related liver diseases. We here briefly summarize genome structures of HBV-DNA and HCV-RNA. Also, we describe representative methods to detect and quantify HBV-DNA and HCV-RNA.

Three assays for measuring hepatitis C virus(HCV) were compared with regard to sensitivity and correlations. The methods included were the Roche Amplicor HCV Monitor test(PCR), the branched DNA signal amplification assay(b-probe), and the serum concentration of HCV core protein measured by the sandwich FEIA (Core-Ag). Also, HCV serotypes were determined by the enzyme-linked immunosorbent assay. Testing 105 consecutive HCV-RNA positive samples showed that the PCR was the most sensitive assay(100% quantifiable), followed by the Core-Ag(82.4%) and the b-probe(75.3%). The values obtained by each method correlated well in serotype 1, whereas some discordance was noted in serotype 2 cases. Also, serotype 2 samples were less quantifiable particularly by b-probe assay than serotype 1 samples. These points have to be considered in quantification of serum HCV levels by currently available these three methods.

Current EIA for PIVKA-II is not sensitive enough to detect small Hepatocellular Carcinoma (HCC). In an attempt to increase the diagnostic threshold, the current EIA was modified in two different ways: 1) immunoreaction of PIVKA-II in the sample with its monoclonal antibody was carried out overnight at 5 degrees C instead of for two hours at room temperature (the overnight method), 2) Avidin-Biotin technique was used for the second reaction(the ABC method);and their diagnostic values were determined as compared with the current EIA(2hr method) in a total of 138 patients including 36 patients with HCC. In 27 patients with HCC(< 3 cm in diameter), the rates of abnormal values obtained by the 2hr-, the overnight- and the ABC method were 14.8, 25.9 and 29.6% respectively. False positive rates of these three methods in 69 patients with liver cirrhosis were 1.4, 8.6 and 22.9% respectively. Thus, these two modifications improved the sensitivity of the current EIA and the overnight method appears to be superior to the ABC method in terms of specificity and simplicity. This conclusion was confirmed by ROC analysis.

Pheochromocytomas synthesize and release catecholamines, which subsequently are related to various clinical manifestations of the disease. However, pheochromocytomas are not innervated and the catecholamine release and synthesis are not initiated by neural impulses. It is still unknown how catecholamine synthesis is regulated in pheochromocytomas. As a first step toward understanding the molecular mechanisms by which catecholamine synthesis is controlled in the tumor, we measured the levels of mRNA coding for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH) and catecholamines in 6 pheochromocytomas and 2 normal adrenal glands. The TH mRNA level was overexpressed and the catecholamine contents were high in 4 out of 6 pheochromocytomas. There was a close correlation between the TH mRNA level and the catecholamines content in the tumors. We also examined the gene expression of the messengers of other catecholamine synthesizing enzymes, dopamine beta-hydroxylase (DBH) and aromatic 1-amino acid decarboxylase (AADC) in pheochromocytomas. The expression of these genes was in parallel with that of TH mRNA in the tumors. These findings indicate that catecholamine overproduction in pheochromocytomas is mediated by the overexpression of genes coding for catecholamines synthesizing enzymes, TH, DBH, and AADC.

Pharmacokinetics of theophylline were determined in patients with liver cirrhosis and idiopathic portal hypertension with reference to estimated hepatic blood flow assessed by indocyanine green (ICG). Decreased plasma clearance of theophylline was noted in patients with liver cirrhosis and the clearance was significantly lower in Child C group than in Child A, B groups (17.5 +/- 3.4 ml/Kg/hr vs 27.6 +/- 8.7, p less than 0.05). Theophylline has been classified as a drug with a low hepatic extraction ratio and it has been believed that changes in hepatic blood flow have little effect on its clearance. The results of present study indicate that theophylline clearance is basically not related to ICG clearance but to theophylline extraction ratio, supporting the common belief. However, it is noteworthy that the clearance was related to decreased hepatic blood flow rather than extraction ratio in a cirrhotic patient with huge extrahepatic shunt, suggesting that hepatic clearance of this drug could be affected by hepatic blood flow under some circumstances.