Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma.

Three types of chromosomal translocations, t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32), are associated with prognosis and the decision making of therapeutic strategy for multiple myeloma (MM). In this study, we developed a new diagnostic modality of the multiplex FISH in immunophenotyped cells in suspension (Immunophenotyped-Suspension-Multiplex (ISM)-FISH). For the ISM-FISH, we first subject cells in suspension to the immunostaining by anti-CD138 antibody and, then, to the hybridization with four different FISH probes for genes of IGH, FGFR3, MAF, and CCND1 tagged by different fluorescence in suspension. Then, cells are analyzed by the imaging flow cytometry MI-1000 combined with the FISH spot counting tool. By this system of the ISM-FISH, we can simultaneously examine the three chromosomal translocations, i.e, t(4;14), t(14;16), and t(11;14), in CD138-positive tumor cells in more than 2.5 × 104 nucleated cells with the sensitivity at least up to 1%, possibly up to 0.1%. The experiments on bone marrow nucleated cells (BMNCs) from 70 patients with MM or monoclonal gammopathy of undetermined significance demonstrated the promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive compared with standard double-color (DC) FISH examining 200 interphase cells with its best sensitivity up to 1.0%. Moreover, the ISM-FISH showed a positive concordance of 96.6% and negative concordance of 98.8% with standard DC-FISH examining 1000 interphase cells. In conclusion, the ISM-FISH is a rapid and reliable diagnostic tool for the simultaneous examination of three critically important IGH translocations, which may promote risk-adapted individualized therapy in MM.

Tumor-Informed Approach Improved ctDNA Detection Rate in Resected Pancreatic Cancer.

Pancreatic cancer is one of the cancers with very poor prognosis; there is an urgent need to identify novel biomarkers to improve its clinical outcomes. Circulating tumor DNA (ctDNA) from liquid biopsy has arisen as a promising biomarker for cancer detection and surveillance. However, it is known that the ctDNA detection rate in resected pancreatic cancer is low compared with other types of cancer. In this study, we collected paired tumor and plasma samples from 145 pancreatic cancer patients. Plasma samples were collected from 71 patients of treatment-naïve status and from 74 patients after neoadjuvant therapy (NAT). Genomic profiling of tumor DNA and plasma samples was conducted using targeted next-generation sequencing (NGS). Somatic mutations were detected in 85% (123/145) of tumors. ctDNA was detected in 39% (28/71) and 31% (23/74) of treatment-naïve and after-NAT groups, respectively, without referring to the information of tumor profiles. With a tumor-informed approach (TIA), ctDNA detection rate improved to 56% (40/71) and 36% (27/74) in treatment-naïve and after-NAT groups, respectively, with the detection rate significantly improved (p = 0.0165) among the treatment-naïve group compared to the after-NAT group. Cases who had detectable plasma ctDNA concordant to the corresponding tumor showed significantly shorter recurrence-free survival (RFS) (p = 0.0010). We demonstrated that TIA improves ctDNA detection rate in pancreatic cancer, and that ctDNA could be a potential prognostic biomarker for recurrence risk prediction.

The relationship between re-endothelialization and endothelial function after DES implantation: comparison between paclitaxcel eluting stent and zotarolims eluting stent.

BACKGROUND: Several studies have reported re-endothelialization and endothelial function after drug-eluting stent (DES) implantation; however, the relationship between re-endothelialization and endothelial function after DES implantation has not been investigated yet. METHODS: A total of 14 patients underwent evaluation of re-endothelialization by optical coherence tomography (OCT) and endothelial function by incremental Ach infusion at 9 months after DES implantation (ZES: N = 7, PES: N = 7). The neointimal thickness (NIT) inside each strut, strut coverage, and malapposition at every 1 mm cross-section were evaluated by OCT and the endothelial function was estimated by measuring the coronary vaso-reactivity in response to acetylcholine (Ach) infusion into coronary arteries. RESULTS: Zotarolims eluting stent (ZES), compared with paclitaxcel eluting stent (PES), showed more homogeneous neointimal coverage of stent struts and low rate of malapposition. Vasoconstriction in response to Ach in the peri-stent region was also less pronounced in ZES than PES. In particular, vasoconstriction was more often observed in cases with inhomogeneous neointimal coverage of stent struts in the PES group. CONCLUSIONS: Our findings suggest that endothelial function seems to be better preserved with ZES than PES, and homogeneous neointimal coverage of stent struts seem to be associated with the preserved endothelial function.

Replacement of chemical rocket launchers by beamed energy propulsion.

Microwave Rocket is a beamed energy propulsion system that is expected to reach space at drastically lower cost. This cost reduction is estimated by replacing the first-stage engine and solid rocket boosters of the Japanese H-IIB rocket with Microwave Rocket, using a recently developed thrust model in which thrust is generated through repetitively pulsed microwave detonation with a reed-valve air-breathing system. Results show that Microwave Rocket trajectory, in terms of velocity versus altitude, can be designed similarly to the current H-IIB first stage trajectory. Moreover, the payload ratio can be increased by 450%, resulting in launch-cost reduction of 74%.

Harmonization of molecular monitoring of chronic myeloid leukemia therapy in Japan.

BACKGROUND: Real-time quantitative polymerase chain reaction (RQ-PCR) has been widely used for molecular monitoring for patients with chronic myeloid leukemia (CML). Currently, RQ-PCR is not based on the concept of international scale (IS) in Japan; mainly because none of the domestic laboratories have obtained their own conversion factor (CF) which makes it possible to convert locally scaled BCR-ABL (BCR-ABL (L)) value to the IS (BCR-ABL (IS)). To join the global trend of molecular assessment of BCR-ABL in CML patients, we have tried to obtain a CF in Japan. METHODS: Samples from 55 patients were exchanged between the Japanese laboratory and the reference laboratory in Adelaide, and BCR-ABL and internal control gene transcripts of the samples were measured using RQ-PCR. The patient bias conversion method was used to determine the CF for the IS using the Bland and Altman method. RESULTS: The local CF in the Japanese laboratory was determined to be 0.87. Based on this CF, 0.1% BCR-ABL (IS), defined as major molecular response, becomes equivalent to 731 copy/µg RNA BCR-ABL (L). CONCLUSION: This study is the first to introduce a laboratory-specific CF for harmonizing RQ-PCR methodology for detecting BCR-ABL transcripts to Japan, which may open new windows for molecular assessment of CML patients in Japan.

Long-term follow-up of chronic pancreatitis patients with K-ras mutation in the pancreatic juice.

BACKGROUND/AIMS: Pancreatic cancer is known to occur during the course of chronic pancreatitis in some patients. This study aimed to identify a high risk group for developing pancreatic cancer associated with chronic pancreatitis, particularly the presence of K-ras mutations in the pancreatic juice. METHODOLOGY: K-ras mutation was analyzed by enriched polymerase chain reaction-enzyme linked mini-sequence assay in endoscopically-collected pancreatic juice of 21 patients with chronic pancreatitis between 1995 and 2000. All of them were followed-up for 6.0 +/- 3.8 (mean +/- SD) years (range, 2.1-14.2 years). RESULTS: K-ras point mutation was observed in the pancreatic juice of 11 patients with chronic pancreatitis (2+, n=2; 1+, n=6; +/-, n=3). Of these, 2 chronic pancreatitis patients with 2+K-ras point mutation developed pancreatic cancer 4.5 and 10.8 years, respectively, after the examination. CONCLUSIONS: Two chronic pancreatitis patients with K-ras mutation developed pancreatic cancer 4.5 and 10.8 years later. Semiquantitative analysis of K-ras mutation in endoscopically-collected pancreatic juice appears to be a useful tool for identifying chronic pancreatitis patients at high risk for developing pancreatic cancer.

Efficacy of EGFR tyrosine kinase inhibitors in patients having EGFR-activating mutations with or without BIM polymorphisms.

PURPOSE: Patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer with BIM deletion polymorphism may have a limited response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, some results of previous reports are discordant. It is necessary to evaluate the relationship between BIM polymorphism and the efficacy of EGFR-TKIs. METHODS: We retrospectively analyzed patients treated with EGFR-TKIs. We collected serum samples from patients before EGFR-TKI administration. We analyzed BIM deletion polymorphism and BIM single nucleotide polymorphism in exon 5 c465C > T by the Invader® assay. RESULTS: BIM deletion polymorphism was identified in 27 of 194 patients (13.9%). BIM single nucleotide polymorphism was identified in 29 of 194 patients (14.9%). The overall response ratio was 81.5% in patients with BIM deletion polymorphism, 89.7% with BIM single nucleotide polymorphism, and 83.6% with BIM wild type. Median progression-free survival was 10.3 months with BIM deletion polymorphism, 8.5 months with BIM single nucleotide polymorphism, and 10.4 months with BIM wild type. Overall survival was 38.4 months with BIM deletion polymorphism, 29.1 months with BIM single nucleotide polymorphism, and 31.6 months with BIM wild type. There were no significant differences between the groups in overall response ratio, progression-free survival, and overall survival. CONCLUSIONS: BIM polymorphism does not affect EGFR-TKI efficacy.

Quantitation of viral load by real-time PCR-monitoring Invader reaction.

With its broad effective range for fluorescence detection, real-time PCR is one of the most valuable techniques for quantitation in molecular biology. A modified real-time PCR assay is described for determining viral load. The assay uses fluorescence to measure the number of PCR amplicons by monitoring the Invader reaction in four steps in the thermal cycle. The Invader reaction with its cleavase was performed at moderate temperature after the amplicon was denatured at a high temperature. The method was as effective as real-time PCR with a TaqMan probe in determining the quantity of virus in samples of human papillomavirus type 16. Importantly, the assay allows the use of a common probe for multiple reactions. Thus, this method is a rapid inexpensive assay with a common fluorescence probe that does not depend on the conformation of the target DNAs.

Application of deafness diagnostic screening panel based on deafness mutation/gene database using invader assay.

Despite progress in identification of deafness genes, clinical application has lagged due to the genetic heterogeneity of deafness. We designed and tested a comprehensive and simple diagnostic strategy to simultaneously detect deafness gene mutations based on a mutation/gene database followed by Invader assay screening of 41 known mutations of nine known deafness genes. Three hundred thirty-eight Japanese patients with congenital or childhood-onset (up to age 10) bilateral sensorineural hearing loss participated in this study. A total of 100 (29.6%) subjects had at least one mutation in GJB2, SLC26A4, and/or the mitochondrial 12S rRNA, indicating that these are the three major causative genes in Japanese deafness patients. The present study demonstrated that the Invader assay has excellent sensitivity and accuracy, and its application to deafness mutation screening will greatly improve medical management and facilitate extensive genetic counseling for hearing impairment.

Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.

Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.

Evaluation of the state of active ingredients in pharmaceutical preparations using fourier transform-Raman difference spectra.

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.

The molecular assembly of the ionic liquid/aliphatic carboxylic acid/aliphatic amine as effective and safety transdermal permeation enhancers.

In spite of numerous advantages, transdermal drug delivery systems are unfeasible for most drugs because of the barrier effect of the stratum corneum. Ionic liquids were recently used to enhance transdermal drug delivery by improving drug solubility. In the present study, safe and effective ionic liquids for transdermal absorption were obtained as salts generated by a neutralization reaction between highly biocompatible aliphatic carboxylic acids (octanoic acid or isostearic acid) and aliphatic amines (diisopropanolamine or triisopropanolamine) (Medrx Co., Ltd., 2009). The mechanism of skin permeability enhancement by ionic liquids was investigated by hydrophilic phenol red and hydrophobic tulobuterol. Further, the skin permeation enhancing effect was remarkably superior in the acid excess state rather than the neutralization state. Infrared absorption spectrum analysis confirmed that ionic liquids/aliphatic carboxylic acid/aliphatic amine are coexisting at all mixing states. In the acid excess state, ionic liquids interact with aliphatic carboxylic acids via hydrogen bonds. Thus, the skin permeation enhancing effect is not caused by the ionic liquid alone. The "liquid salt mixture," referred to as a complex of ingredients coexisting with ionic liquids, forms a molecular assembly incorporating hydrophilic drug. This molecular assembly was considered an effective and safety enhancer of transdermal drug permeation.

Colonic Paneth cell metaplasia is pre-neoplastic condition of colonic cancer or not?

ABSTRACTS: BACKGROUND: The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer. METHODS: Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS (D2S123, D17S250 and D5S346) of PaM was examined by high resolution fluorescenced labeled PCR primers. RESULTS: K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) (D2S123: 35.4%, 17/48 regions, D17S250: 13.7%, 7/51 regions, and D5S346: 0%, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls (Colorectal mucosa with no PaM). CONCLUSIONS: Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.

Mucin phenotypic expression and p53 gene abnormality of gastric super-minute well-differentiated adenocarcinoma: re-evaluation with relationship between histogenesis of well-differentiated adenocarcinoma and intestinal metaplasia in distal stomach.

BACKGROUND: Although the gastric well-differentiated adenocarcinoma in the distal stomach has been thought to develop via a intestinal metaplasia-carcinoma sequence, there are some disproofs from new mucin examinations for minute-size lesions in same type carcinoma. The current study was performed and pointed out the new findings for the solution to the problem according to the point described above. METHODS: 12 super-minute lesions (less than 1 mm in maximum diameter) of well-differentiated adenocarcinoma in distal stomach (SMCa), which were detected from the pathological examinations of 210 surgically resected stomach specimens, and the mucosa adjacent to these carcinoma lesions, were examined by immunohistochemical mucin stainings (MUC2 and CD-10: intestinal phenotype, 45M1 and MUC6: gastric phenotype) and p53-overexpression. And the analyses of the replication error of the microsatellites in chromosome 17 related p53 gene (TP53 and D17S786) (RER-p53MS) were performed in SMCa lesions, adjacent mucosa to each lesion and other gastric mucosa with intestinal metaplasia, because all SMCa lesions showed p53-overexpression immunohistochemically, described below. RESULTS: 1. The carcinoma cells in all SMCa lesions were positive for 45M1 and p53. On the other hand, no positive carcinoma cells for MUC6 were seen although the pyloric glands and the remnant pyloric gland in the SMCa lesions in the same slides were positive for MUC6. Ten lesions (83%) had intestinal phenotypic mucin (10 lesions: MUC2 (+), 4 lesions: CD10 (+)). Two lesions (17%) were positive for only 45M1 (gastric phenotypic mucin). 2. All of the mucosa adjacent to SMCa showed intestinal metaplasia (complete type: 7 regions, incomplete type: 5 regions). 3. RER-p53MS was confirmed in 42% (5/12 regions) of SMCa, in 42% (5/12 regions) of the mucosa adjacent to SMCa and 14% (6/42 regions) of the other intestinal metaplasia mucosa. CONCLUSION: Most of the super-minute well-differentiated adenocarcinoma lesions in the distal stomach, which had both gastric and intestinal phenotypic mucin, are considered to develop from the tubular proliferative zone with the incomplete type of the intestinal metaplasia and p53 gene abnormality, while a part of them, which had only gastric phenotypic mucin, may derive from the gastric native tubules (non-metaplastic epithelium) with p53 gene abnormality.

Intercalated duct cell is starting point in development of pancreatic ductal carcinoma?

BACKGROUND: Although it is well known that the pancreatic ductal carcinoma may develop having a relationship to the mucous gland hyperplasia (MGH) with atypia (PanIN-1B by PanIN system), the starting point of this atypical MGH is unclear. To know it, we examined the pancreas tissue using many methods described below. METHODS: 1. Twenty-seven surgically resected pancreas tissue specimens, including pancreatic ductal carcinomas (PDC), chronic pancreatitis and normal pancreas, were investigated using immunohistochemical stainings for MUC1, MUC6, 45M1, Ki67 and p53. 2. DNA extraction and analysis of K-ras mutation at codon 12 using microdissection method: The paraffin blocks with 16 regions including the intercalated duct cell (IC) adjacent to the atypical MGH were prepared for DNA extraction. Mutation of K-ras codon 12 was analyzed and compared in enriched polymerase chain reaction-enzyme-linked mini sequence assay (PCR-ELMA). RESULTS: 1. In the normal pancreas, although no positive cell was seen in 45M1, p53, Ki67, the cytoplasm of IC were always positive for MUC1 and sometimes positive for MUC6. In the pancreas with fibrosis or inflammation, MGH was positive for MUC6 and 45M1. And atypical MGH was positive for MUC1, MUC6 and 45M1. Some IC adjacent to the atypical MGH was positive for Ki67 as well as atypical MGH. The carcinoma cells in all cases of PDC were diffusely positive for MUC1, 45M1, p53 and Ki67, and focally positive for MUC6. 2. In K-ras mutation, we examined the regions including IC adjacent to the atypical MGH, because the immunohistochemical apomucin stainings of these regions resembled those of PDC as described above. And K-ras mutation was confirmed in 12 of 16 regions (75%). All mutations were a single mutation, in 6 regions GTT was detected, in 4 regions GAT was detected and in 2 region AGT was detected. CONCLUSION: Some intercalated duct cell may be the starting point of the pancreatic ductal carcinoma, because the exhibitions of mucin expressions, Ki67, p53 and K-ras mutation in some intercalated duct cell resembled those of mucous gland hyperplasia or pancreatic ductal carcinoma.

Real-time PCR for quantification of Streptococcus mutans.

A real-time polymerase chain reaction (PCR) assay was developed for the quantification of Streptococcus mutans. Primers targeting gtf genes of S. mutans were designed and tested for their specificity using 28 oral streptococcal strains, three other bacterial strains, and human DNA. The primers could amplify specifically the target DNA fragment from a mixture of oral streptococcus genomic DNA containing about 10 fg to 10 ng of S. mutans genome DNA. The real-time PCR produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of a few copies of S. mutans' genomic DNA per reaction tube. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. mutans in saliva samples. A real-time PCR assay for Streptococcus sobrinus and Streptococcus downei was also established and produced results that corresponded well to those from conventional culture assays for S. sobrinus in saliva samples. These assays will be useful as a new means to assess one of the important risk factors for caries.

Temporal bone histopathological and quantitative analysis of mitochondrial DNA in MELAS.

OBJECTIVES/HYPOTHESIS: Although hearing loss is common in MELAS (syndrome of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes), the histopathology of the temporal bone has not been reported. The majority of cases of MELAS are linked to a mitochondrial DNA (mtDNA) mutation at nucleotide 3243. In MELAS, normal mtDNA and mutant mtDNA coexist in a heteroplasmic manner. The purpose of the study was to report the otopathological findings from two patients with MELAS and quantitative mtDNA analysis in the inner ear of one of these patients. STUDY DESIGN: Basic scientific histopathological examination and quantitative mtDNA analysis of the temporal bone. METHODS: Temporal bones were embedded in celloidin and sectioned for light microscopic study. Graphic reconstruction of the cochlea was performed using the method described by Schuknecht. For quantitative mtDNA analysis, total DNA from the membranous part of the inner ear was collected, amplified by polymerase chain reaction, and digested with the restriction enzyme. The percentage of mutant/total mtDNA was measured by the ratio of fluorescence intensity. RESULTS: Histopathological examination revealed severe degeneration of the stria vascularis and degenerative change of spiral ganglion cells in both patients. The quantitative DNA studies showed that the proportion of mutant to wild-type mtDNA was similar in both histologically affected and histologically unaffected tissues within the inner ear. CONCLUSION: Dysfunction of the stria vascularis and spiral ganglion cells causes sensorineural hearing loss in MELAS.

Effects of Nd:YAG and CO2 laser treatment and ultrasonic scaling on periodontal pockets of chronic periodontitis patients.

BACKGROUND: The aim of the present study was to compare the effectiveness of Nd:YAG and CO2 laser treatment to that of ultrasonic scaling used as monotherapies by examining clinical parameters, subgingival microflora, and interleukin-1 beta (IL-1beta) in gingival crevicular fluid (GCF). METHODS: Eighteen patients, each of whom had 2 or more sites with probing depth measuring > 5 mm, were included this clinical trial. The 41 sites were randomly assigned treatment with either Nd:YAG laser alone (n = 14, 100 mj, 20 pps, 2.0 W, 120 seconds), CO2 laser alone (n = 13, 2.0 W, 120 seconds), or ultrasonic scaling alone (n = 14, maximum power, 120 seconds). At baseline and at 1, 4, and 12 weeks, clinical measurements (plaque index, PI; gingival index, GI; probing depth, PD; clinical attachment level, CAL; and bleeding on probing, BOP) were performed and subgingival plaque and GCF sampled. A quantitative analysis of Porphyromonas gingivalis was carried out using real-time polymerase chain reaction (PCR) procedures. The amounts of IL-1beta were estimated by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Decreased inflammation and PD were observed in all 3 groups after treatment. A microbiological analysis indicated significant decreases in P. gingivalis in the Nd:YAG and scaling groups at 1, 4, and 12 weeks compared to baseline (P < 0.05). The amount of GCF significantly decreased in the Nd:YAG and scaling groups at 12 weeks. The amount of IL-1beta increased in the CO2 group from baseline to 1 week (P < 0.05). The Nd:YAG group tended to show a decrease in IL-1beta from 1 to 12 weeks, although these data were not statistically significant. CONCLUSIONS: Our data suggest that Nd:YAG laser and ultrasonic scaling treatments showed significant improvements regarding the clinical parameters and subgingival microflora compared to the baseline, but no significant difference was observed between the 3 groups.

Detection of lymph node micrometastasis in esophageal carcinoma.

BACKGROUND/AIMS: The presence of lymph node metastasis is the most important prognostic factor in esophageal squamous cell carcinoma. Molecular biology techniques have improved the ability to recognize micrometastasis in lymph nodes, bone marrow, and peripheral blood. Previous studies have demonstrated that cytokeratin 19 reverse transcriptasepolymerase chain reaction can detect tumor cells even when lymph nodes appear normal histologically. However, the presence of pseudogenes for cytokeratin 19 have reduced the specificity of reverse transcriptase-polymerase chain reaction, thereby reducing its clinical worth as a sensitive diagnostic technique. METHODOLOGY: We examined the expression of mRNA for cytokeratin 19 using a newly designed set of primers, and compared the results with data from histologic examinations. Samples were obtained from tumors, intact tissues, resected lymph nodes and in 10 patients who underwent esophagectomy via right thoracotomy with lymph node dissection in the neck, mediastinum and abdomen. RESULTS: All tumors, non-cancerous tissues were positive for cytokeratin 19 by reverse transcriptasepolymerase chain reaction. However, 2 of the 6 lymph nodes that appeared normal on histologic examination were positive for cytokeratin 19; sensitivity and specificity were 100% and 67%, respectively. CONCLUSIONS: Reverse transcriptase-polymerase chain reaction using new primers for cytokeratin 19 detected micrometastasis in specimens of lymph nodes from patients with squamous cell carcinoma. This method may increase the accuracy of tumor staging and provide clinicians with valuable information that will help individualize treatment options.

[External quality assessment of the genetic testings for hematopoietic tumor, CML].

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.