Short tandem repeat typing of cells transferred via micromanipulation with whole-genome amplification.

This study attempted to determine the minimum number of cells required to conduct DNA analyses effectively. Oral mucosal cells obtained from eight persons were suspended and individually collected by using micromanipulation technique. DNA was extracted and amplified by whole-genome amplification (WGA). Nuclear DNA was extracted to evaluate the feasibility of autosomal short tandem repeat (STR) polymorphism and Y-chromosomal STR polymorphism analyses. Tests were conducted with 20 and 30 cells, to determine the minimum number of cells required for each DNA analysis. Tests with 20 cells were repeated 5 times, to examine reproducibility. When five or 10 cells were used, loci could not be identified for most alleles. Furthermore, DNA polymorphism analyses of a single cell transferred directly to a polymerase chain reaction solution were unsuccessful. The present findings suggest that, in forensic identification, 20 or more cells are required in order to obtain clear results from autosomal and Y-chromosomal STR polymorphism analyses. Furthermore, the feasibility of sample preservation and reexamination was also confirmed by DNA amplification with WGA.

Social contribution of forensic odontology in Japan.

Half a century has passed since the department for education and research on forensic odontology was established at dentistry-related universities in Japan in 1964. In order to meet the demands of society, the number of universities with a department of forensic odontology increased up until around 2005. In 2007, the Japanese Society of Forensic Dental Science was established, and then a series of reforms such as establishment of the Study Council on Death Cause Investigation in both the National Police Agency and the Cabinet Office of the Japanese government, cabinet decision of enactment and enforcement of new laws on death cause investigation, publication of an article on the Model Core Curriculum of Dental Education, publication of the results of a fact-finding survey on education and research on forensic odontology conducted by the Ministry of Education, Culture, Sports, Science and Technology, inclusion of questions about forensic odontology in the National Board Dental Examination, and compilation of a database on dental findings by the Ministry of Health, Labor and Welfare, proceeded in succession. We introduced the half century of forensic odontology in Japan in chronological order.

DNA analysis of root canal-filled teeth.

Teeth are markedly useful as samples for DNA analysis; however, intact teeth are not always available. This study examined the possibility of identifying autosomal and Y-chromosome short tandem repeat (STR) types in samples from 34 teeth (15 intact and 19 root canal filled) that had been preserved for 10-33years after dental extraction. The aim was to explore the feasibility of individual identification by DNA analysis of samples obtained from highly decomposed and skeletonized corpses. Only one out of 24 autosomal STR loci was not identified in two of the 15 intact teeth, whereas all 23 loci of the Y chromosome STR were detected. One or two autosomal STR loci remained unidentified in eight of the 19 root-filled teeth, and four or five of the 23 Y STR loci were undetected in three cases. However, the types were identified in about 20 loci in all samples, and the composition of the root canal filling material did not appear to interfere with the PCR. This study demonstrates that the storage period of the teeth had no influence on our results indicating that root canal filled teeth can be used for DNA analysis.

Analysis of human mitochondrial DNA polymorphisms in the Japanese population.

The highly polymorphic nature and high amplification efficiency of mitochondrial DNA (mtDNA) is valuable for the analysis of biological evidence in forensic casework, such as the identification of individuals and assignment of race/ethnicity. To be useful, a mtDNA polymorphism database for the Japanese population requires an understanding of the range of haplotype variation and phylogenies of mtDNA sequences. To extend current knowledge on the haplotypes in the Japanese population, this study defines new lineages and provides more detail about some of those previously described. We compared the hypervariable regions (HVRs) of 270 healthy, unrelated Japanese individuals and demonstrated 192 haplotypes. Combining HVR1 and HVR2, the genetic diversity was 0.9935, thus providing a high level of identification capability. Haplogroup status was defined for 160 individuals using HVR1, HVR2, and particular coding region polymorphisms; these individuals belonged to 94 haplotypes, four of which were new lineages. The complete mtDNA sequence was also determined from seven individuals.

ABO blood group genotyping by quenching probe method.

We developed a multiplex ABO genotyping method with quenching probes (Q-probe). In this method, it is possible to discriminate the mutations, not only frequently used positions 261 and 796 but also position 703 in a single PCR. Each probe was designed to have cytosine residue at 5' or 3' end and labeled with three different fluorescence dyes, enabling the triplex detections of these polymorphisms. All polymorphisms were successfully detected by using fluorescence labeled Q-probe in a specifically amplified PCR product. Each Q-probe showed unique dissociation patterns depending on the polymorphism types. All of the results obtained with Q-probe were compared with standard serotyping and TaqMan PCR method and resulted in complete match with each other. Consequently, these results indicated that multiplex ABO genotyping method is quite accurate and convenient method for the determination of ABO genotype.

A case of personal identification due to detection of rare DNA types from seminal stain.

Following a rape incident in an apartment in Japan, we were requested to perform a DNA analysis on a body fluid stain left on a bath towel to determine whether it could be attributed to the suspect. The acid phosphatase and prostatic-specific antigen tests confirmed it to be a seminal stain. Based on the DNA analysis by autosomal and Y-chromosome short tandem repeat (STR) systems, no inconsistency was found with the profile of the suspect with African ancestry. In this case, allele 21 of DYS390 at the Y-STR locus was examined, as it is reported to have a distinctly lower frequency in the Japanese population. Furthermore, the haplotype combinations of Y-STR at the DYS389I, DYS389II and DYS390 loci are powerful for personal identification, as these have not yet been found in the Japanese population.

Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry.

A detailed procedure for the analysis of four beta-blockers, acebutolol, labetalol, metoprolol and propranolol, in human plasma by high-performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) using an MSpak GF column, which enables direct injection of crude plasma samples, is presented. Protein and/or macromolecule matrix compounds were eluted first from the column, while the drugs were retained on the polymer stationary phase of the MSpak GF column. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All drugs showed base peak ions due to [M + H]+ ions by LC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]+ ion by LC-MS-MS. Quantification was performed by selected reaction monitoring. The recoveries of the four beta-blockers spiked into plasma were 73.5-89.9%. The regression equations for all compounds showed excellent linearity in the range 10-1000 ng/mL of plasma, with the exception of propranolol (10-800 ng/mL). The limits of detection and quantification for each drug were 1-3 and 10 ng/mL, respectively, of plasma. The intra- and inter-day coefficients of variation for all drugs in plasma were not greater than 10.9%.

Gastrointestinal stromal tumor arising from anorectum: correlation of imprint cytology and radiologic imaging.

The diagnosis of gastrointestinal stromal tumor (GIST) is generally established on histopathologic examination of surgical specimens. Gastrointestinal stromal tumor comprises a heterogenous group of neoplasms of the gastrointestinal tract previously referred to as leiomyomas, leiomyosarcomas, or schwannomas. Gastrointestinal stromal tumor arising from anorectum is a rare instance. We report a case of GIST for the correlation of imaging and cytologic features with immunocytochemical staining. A computed tomography and magnetic resonance imaging confirmed a 2-cm tumor growing into the rectal lumen. The central portion of the tumor showed T1-weighted imaging of low signal and suspected central necrosis by the T2-weighted imaging of high signal. Imprint cytology from excised tumors showed isolated or loosely aggregated spindle cells with scanty and fibrillary cytoplasmic processes, nuclear pleomorphism, fine granular chromatin, and irregular nuclear margins. Epithelioid tumor cells showed grooves with abundant cytoplasm and several round nucleoli. Both c-kit and CD34 antigen were positive with strong and diffuse stainability in smears as well as paraffin sections by immunoperoxidase staining. We suggest that the combined use of imaging diagnosis and cytology with immunocytochemical staining are useful initial diagnosis of GIST.

Effects of p21cip1/waf1 overexpression on growth, apoptosis and differentiation in human colon carcinoma cells.

The cyclin-dependent kinase inhibitor p21cip1/waf1 negatively regulates the progression of cell cycle and the potential usefulness of p21cip1/waf1 gene is proposed in gene therapy. However, studies have demonstrated a protective role of p21cip1/waf1 against apoptosis and little is known about effects of ectopic expression of p21cip1/waf1 on differentiation of colon cancer cells. In the present study, we found diffuse p21cip1/waf1 expression in only a few clinical samples of colorectal cancer with wild-type p53 gene. To explore the role of p21cip1/waf1 in cell growth, apoptosis and differentiation, we constitutively overexpressed p21cip1/waf1 in HT29 colon carcinoma cells. Ectopic overexpression of p21cip1/waf1 was associated with inhibition of CDK2-associated kinase activity, indicating the functionality of the introduced p21cip1/waf1 gene. Overexpression of p21cip1/waf1 caused an appreciable growth inhibition in monolayer and soft agar cultures and it significantly reduced sodium butyrate- but not 5-fluorouracil-induced apoptosis. p21cip1/waf1 overexpressing cells exhibited marked decrease of intestinal differentiation when assayed with intestinal alkaline phosphatase. Our findings suggest that introduction of p21cip1/waf1 gene into colon cancer cells may be useful for inhibiting cell growth but caution should be taken regarding the increased resistance to certain apoptosis-inducing agents and dysregulation of endogenous p21cip1/waf1-mediated differentiation process.

[A case of recurrent gastric cancer with peritoneal dissemination successfully treated with DJ stents against bilateral hydronephrosis and chemotherapy].

A 69-year-old female patient underwent total gastrectomy with a D2 lymph node dissection. Her final findings were of pT2, pN0, sP0, sH0, sM0 and Stage IB. After thirty-five months from the operation, peritoneal recurrence with ascites, bilateral hydronephrosis and stenosis of colon was found. TS-1 (80 mg/day/body) was administered for four weeks followed by a 2-week rest after DJ stents were inserted into bilateral ureters. At the end of two courses of TS-1, ascites disappeared and the decrease of tumor marker was observed. During the seventh course, symptoms such as abdominal fullness and ascites became worse. She underwent a weekly administration of paclitaxel (90 mg/body) as a second-line chemotherapy. This regimen was continued for three weeks followed by a 1-week rest. After four courses of paclitaxel, ascites disappeared and the tumor marker was gradually reduced. However, multiple bone metastases were found during the eighth course, and she died about two years after the recurrence. The toxic events were mucositis (grade 1) in TS-1, and alopecia (grade 2) and leukopenia (grade 1) in paclitaxel. No major adverse effects were observed. Although the prognosis of recurrent gastric cancer with peritoneal dissemination was extremely poor, this case might suggest a possibility that intensive therapies are useful in maintaining the quality of life and improving survival.

[A case report--Efficacy of combination therapy to unresectable advanced gall bladder carcinoma--palliative operation, hepatic arterial infusion therapy, and radiation therapy].

A patient of advanced gall bladder carcinoma with liver metastases and direct invasions to the duodenum and liver underwent a palliative operation, 3 hepatic arterial infusion (HAI) therapies, and radiation therapy at the obstructive common biliary duct. (Palliative operation was a partial resection of duodenum and transverse colon, HAI therapy with 5-FU (1 g/day) was given as a continuous infusion for 6 days, radiation therapy was given 2 Gy/day for 20 times) After the combination therapy, the main tumor of gall bladder and hepatic metastases were decreased and tumor markers were normalized. (CEA 15.1 ng/ml, CA19-9 93 U/ml to CEA 4.4 ng/ml, CA19-9 29 U/ml) Then, an expandable metallic stent (EMS) could be inserted to the stenotic common biliary duct after radiation therapy. Although para-aotic lymph nodes were existent, systemic chemotherapy (UFT 300 mg/day p.o., MMC 2 mg/week div) has been performed as an outpatient with a good quality of life.

[A case of Vp3 hepatocellular carcinoma with a good QOL after multidisciplinary treatments].

A 74-year-old male was examined with abdominal CT scan because of general fatigue. Abdominal CT scan indicated enhanced tumors, 9x8 cm in size in subsegment 6/7 and 5 mm in size in subsegment 3. Tumor thrombus was observed in the right portal branch to the main portal vein. We diagnosed the patient with Vp3 hepatocellular carcinoma. A right hepatectomy with extraction of portal venous thrombus was performed. Unresectable tumor was treated with one shot arterial infusion (epi-ADM 40 mg) and TAE 3 times at an interval of three months. The side effect was only a fever and the QOL was good under the treatment. But a tumor in S1 had developed, and the patient died at about 12 months after the operation.

[Non-surgical treatment of hepatocellular carcinoma with tumor thrombus in the right atrium].

A 65-year-old Japanese man who had been suffering from severe and progressive dyspnea for more than 2 months underwent an extended right hepatectomy for hepatocellular carcinoma (HCC) in August 2001. Radiological examination, performed in August 2003, revealed the mass in the left lobe of the liver extended into the left hepatic vein, the inferior vena cava and the right atrium. Those clinical manifestations were supposedly attributed to HCC tumor thrombus in the right atrium. The decision to carry out the palliative operation for the tumor thrombus was not made because of poor prognosis in light of hemodynamic compromise indicating a reasonable liver function. A sequential course of treatments for the tumor thrombus was performed including transcatheter chemotherapy, transarterial chemoembolization and radiation therapy. Although a radiological response rate was 27% in diameter of the tumor thrombus, the clinical manifestations, such as dyspnea or edema, completely disappeared during the treatment. No surgical standard or interventional regimen for HCC tumor thrombus in the right atrium has been established. However, we here demonstrated the possibility for the treatment of the tumor thrombus with intensive combination therapies.

[Evaluation of hepatic arterial infusion chemotherapy with levofolinate (l-LV) and 5-fluorouracil (5-FU) for multiple liver metastases from colorectal cancer].

We evaluated the effect of hepatic arterial infusion chemotherapy with levofolinate (l-LV) and 5-fluorouracil (5-FU) for multiple liver metastases from colorectal cancer. All patients received drugs on an outpatient basis every six weeks, followed by no medication for two weeks. In this regimen levofolinate (200 mg/m2) was administered for two hours and 5-fluorouracil (500 mg/m2) was administered for thirty minutes as a bolus. A complete response was obtained in five patients and a partial response in five patients; the overall response rate was 40%. All patients could receive this therapy on an outpatient basis because no patient had side effects of Grade 3 or over. It is suggested that our protocol may be useful for improvement of outcome.

[A case of liver metastasis from anorectal malignant melanoma treated by chemoembolization].

Anorectal malignant melanoma is relatively rare and its prognosis is very poor because of distant metastasis via the blood or lymphatic vessels. This paper reports a case of liver metastasis from anorectal malignant melanoma treated by chemoembolization. A 68-year-old man was admitted to our hospital because abdominal enhanced computed tomography revealed multiple liver metastases. Angiography also revealed metastasis, so a chemoembolization with nedaplatin was performed. Two months later some lesions fell into necrosis but new ones appeared, and the same treatment was performed another three times. Accessory vessels from the inferior diaphragma artery developed and prevented these treatments. The patient died from the progress of metastases to the liver, bone and skin three years and two months after the operation, or one year and three months after the liver recurrence. The chemoembolization showed some effects on liver metastases from malignant melanoma, but they were temporary.

[A case of stage IV diffusely infiltrating colon cancer treated by surgical resection and chemoradiation therapy].

A 62-year-old male patient presented at the hospital because of left lower abdominal tumor. Based on preoperative examination and biopsy results, he was diagnosed with stage IV diffusely infiltrating colon cancer (scirrhous type) with paraaortic lymph node metastases. He underwent sigmoidectomy with D1 lymph node dissection and received systemic infusion of 5-FU 750 mg and l-LV 300 mg once a week. This chemotherapy produced no change in response in the paraaortic lymph node metastases for a long time. One year later, there were distant lymph node metastases including left inguinal and Virchow's lymph node, and systemic infusion of CPT-11 was performed. In addition, left inguinal lymph node was treated with irradiation therapy (total 50 Gy). The patient died of multiple organ failure 18 months after the operation. It is known that the prognosis in cases of diffusely infiltrating colorectal cancer is extremely poor. However, this case might suggest that intensive therapies with surgery and chemoradiation are useful in maintaining quality of life and improving survival.

Analysis of cyclin D1 and CDK expression in colonic polyps containing neoplastic foci: a study of proteins extracted from paraffin sections.

Overexpression or amplification of G1 cyclins has been demonstrated in various types of human cancers. Overexpression of cyclin D1, an accelerator of cell cycle, is thought to be an early event in colonic multistage carcinogenesis, but its expression at transformation from benign to neoplastic foci is not clear. We analyzed the expression of cyclin D1 and its catalystic partner CDK4 in colon polyps containing neoplastic foci. For direct comparison of the expression in adenomatous tissues and neoplastic foci, proteins in adequate amounts were extracted from paraffin embedded sections. Western blot and densitometry analyses showed that cyclin D1 expression was 2.3-times and 2.5-times higher in adenomatous tissues and neoplastic foci, compared with normal colonic mucosa. In contrast, the levels of CDK4 and CDK2 expression were only modestly increased in adenomatous tissues but significantly higher in neoplastic foci, relative to normal mucosa. Expression of PCNA, a cell proliferation marker, increased from normal mucosa to adenoma to focal cancer. Our findings suggest that overexpression of cyclin D1 may be associated with high proliferative activity in adenomatous tissues and that concurrent high expression of cyclin D1 and CDK4 may further perturb cell cycle progression and play a pivotal role in colonic carcinogenesis.

Extraction and analysis of carcinoembryonic antigen in lymph nodes: a new approach to the diagnosis of lymph node metastasis of colorectal cancer.

PURPOSE: To detect lymph node metastasis of colorectal cancer, we extracted protein from lymph nodes and determined the concentration of carcinoembryonic antigen. METHODS: In Experiment 1, a total of 237 lymph nodes from 23 colorectal cancer patients were examined histologically after immersion in 200 microl of saline for 2 hours. Concentrations of protein and carcinoembryonic antigen in each saline sample were determined by protein assay and immunoradiometric assay, respectively. Each value of carcinoembryonic antigen in the saline was divided by extracted protein, and the carcinoembryonic antigen levels in lymph nodes were represented as nanograms per milligram of protein. In Experiment 2, 63 lymph nodes from 8 colorectal cancer patients were cut into 2 pieces and immersed in 1 ml of saline for 15 minutes, and they were subjected to reverse transcriptase-polymerase chain reaction for carcinoembryonic antigen and histologic examination, respectively, after measurement of the carcinoembryonic antigen concentration in the extract. RESULTS: From 236 of 237 lymph nodes in Experiment 1, an average of 3,249.4 microg/ml protein was successfully extracted. Histologic examination revealed that 33 of 236 lymph nodes had colorectal cancer metastases, with a significantly higher concentration of carcinoembryonic antigen on average (655.5 ng/mg protein) than in the 203 lymph nodes without metastasis (18.2 ng/mg protein; P < 0.0001). In Experiment 2, 19 of 63 lymph nodes examined were positive for metastasis in both reverse transcriptase-polymerase chain reaction and histology and showed a significantly higher carcinoembryonic antigen concentration on average (1,003.9 ng/mg protein) than the 42 lymph nodes that demonstrated no metastasis by either method (18.0 ng/mg protein; P < 0.0001). The remaining two lymph nodes, which were positive by reverse transcriptase-polymerase chain reaction but negative by histology, showed high carcinoembryonic antigen concentrations of 514.7 and 61,970.5 ng/mg protein, respectively. CONCLUSION: This simple method of protein extraction and determination of carcinoembryonic antigen concentration in lymph nodes may provide an alternative tool for the diagnosis of colorectal cancer metastasis.