Monitoring gastric cancer progression with circulating tumour DNA.

BACKGROUND: Circulating tumour DNA (ctDNA) is an emerging candidate biomarker for malignancies and may be useful for monitoring the disease status of gastric cancer. METHODS: We performed targeted deep sequencing of plasma cell-free DNA (cfDNA) by massively parallel sequencing in patients with tumours harbouring TP53 mutations. The quantitative values of TP53-ctDNA during the clinical course were compared with the tumour status. RESULTS: Three out of ten patients with TP53 mutations in primary tumours showed detectable TP53 mutation levels in preoperative cfDNA. Although the cfDNA concentrations were not always reflective of the disease course, the ctDNA fraction correlated with the disease status. CONCLUSIONS: ctDNA may serve as a useful biomarker to monitor gastric cancer progression and residual disease.

Adult-onset Krabbe disease with homozygous T1853C mutation in the galactocerebrosidase gene. Unusual MRI findings of corticospinal tract demyelination.

A 51-year-old woman developed a slowly progressive spastic paraparesis and diminished vibration sense beginning at age 38. Intellectual capacity was normal. Krabbe disease was confirmed by markedly reduced leukocyte galactocerebrosidase (GALC) activity, typical inclusions in Schwann cell cytoplasm, and an identification of the homozygous point mutation T1835C (Leu618Ser) in the GALC gene. T2-weighted MRI of the brain showed symmetric high-signal-intensity lesions in the bilateral frontoparietal white matter, the centrum semiovale, and the posterior limb of the internal capsule with sparing of the periventricular white matter. This case is unusual because of the late onset, protracted clinical course, and MRI findings of demyelination confined to the corticospinal tracts.

Single-stranded conformational polymorphism analysis using automated capillary array electrophoresis apparatuses.

We describe a new environment of a single-stranded conformational polymorphism (SSCP) analysis using automated capillary array sequencers (e.g., ABI Prism 3100 and 3700). In this environment, electrophoretic conditions, settings for instrument management, and software for data analysis are adjusted for SSCP analysis. Highly reproducible results are obtained with this new system, and fragments with mutations and/or polymorphisms in different capillaries or different runs can be reliably detected. The relative peak heights between alleles are quantitative and reproducible between runs, and so allele frequencies of single nucleotide polymorphisms can be accurately estimated by a pooled DNA strategy. The method allows unattended, low-cost, and quantitative SSCP analysis using instruments that are widely accessible.

Distinct pattern of PCR-SSCP analysis of p53 mutations in human astrocytomas.

Clarification of somatic mutations during the progression of human astrocytomas is important in order to understand the mechanisms underlying the development of these tumors. We analyzed surgical specimens of human astrocytomas for mutations in the p53 gene using single-strand conformation polymorphism analysis of polymerase chain reaction product (PCR-SSCP analysis) at a low pH. Klenow fragment treatment after PCR amplification was an effective means to get rid of some extra bands on the SSCP gel. Five mutations in three of 24 astrocytomas were identified by this improved SSCP method. The frequency of p53 gene mutations in astrocytomas examined was 12.5%. Further examination by direct sequencing showed that all five mutants had single-base substitutions resulting in missense mutations. The present studies revealed a loss of heterozygosity and two point mutations on the remaining allele in one of the fibrillary astrocytomas. Finally, the improvement of PCR-SSCP analysis using Klenow treatment and low pH showed a distinct electrophoresis gel pattern and could be relevant for the prognosis of human astrocytomas.

Histological and radiographic assessment of well functioning porous-coated acetabular components. A human postmortem retrieval study.

Nine porous-coated acetabular components were retrieved post mortem. All components had been inserted at our institution and had been in situ for a mean of fifty months (range, seventeen to eighty-seven months). Clinical records revealed that all had been functioning well at the time of death, and clinical radiographs showed signs that all had been stable. Standard backscattered scanning electron microscopy was used to quantitate the amount of bone ingrowth into the porous coating. For each component, the histological appearance of the bone-metal interface was compared with the appearance on clinical radiographs. Light microscopy was used to study the non-ossified areas. Every component had growth of bone into the porous coating, with the ingrowth occupying a mean of 32 per cent (range, 3 to 84 per cent) of the fields that were examined. In areas where bone ingrowth had occurred, the mean area density was 48 per cent (range, 26 to 65 per cent). Use of radiographs consistently led to an underestimation of the presence of gap areas and an overestimation of the occurrence of bone apposition. When fibrous tissue was present in non-ossified areas, it was extremely dense and well organized. Within the limits of light microscopic examination, there was no evidence of granulomatous formation in the non-ossified regions. This is particularly encouraging since the fibrous tissue-bone interfaces seem to prohibit the deposit of particulate debris.

Characterization of the GALC gene in three Japanese patients with adult-onset Krabbe disease.

Krabbe disease, a neurodegenerative disorder of autosomal recessive inheritance, is caused by mutations in the galactosylceramidase (GALC) gene. However, its clinical manifestations in terms of time of onset and severity are heterogeneous. Thus, elucidation of the relationship of symptoms to the site and type of mutation is important, both for an understanding of the etiology of the disease and for diagnostic purposes. We examined the genomic structure of the GALC gene in three unrelated adult-onset Krabbe disease patients. One patient was homozygous for an Ile66Met mutation. Another patient who appeared to express only one mutated mRNA species was, in fact, a compound heterozygote for an Ile66Met mutation and a nonsense mutation, Tyr354ter. Because the allele with the nonsense mutation was not detectable by mRNA analysis, a rapid degradation of the mRNA caused by premature chain termination was suggested. The third patient who carried two inactiving mutations--Leu618Ser and a second resulting in exon 6 skipping--was also found to carry an intronic mutation, IVS6 + 5G > A. Transfection experiments using a GALC mini-gene proved that this intronic mutation was the cause for the exon 6 skipping.

SSCP analysis of long DNA fragments in low pH gel.

Sensitivity of single-strand conformation polymorphism analysis of PCR products (PCR-SSCP analysis) is known to be decreased when the DNA fragments are longer than 300 bp. We examined effects of buffer ions in an attempt to extend the length limit of the analysis. It has been noted that addition of glycerol to the gel containing Tris-borate buffer enhances the sensitivity, but the effects of glycerol have been left unexplained. We found that the effects of glycerol are caused by the reduction of pH of the buffer by the reaction of glycerol and borate ion. We further extended these observations and found that sensitivity of SSCP can be greatly improved by running the electrophoresis in low pH buffer systems. Using a new buffer system and running the electrophoresis at a fixed temperature, we detected 27 of 31 known mutations of factor IX gene in six different sequence contexts ranging in length from 300 to 800 bp.

Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA.

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential-for example, in linkage disequilibrium analysis.

Evaluation of initial surface apposition in porous-coated acetabular components.

The initial surface apposition of the acetabular bone-implant interface was investigated for acetabular components of three different designs: hemispherical with spikes, hemispherical, and threaded hemispherical. Four acetabular components of each design were implanted into fresh anatomic specimen acetabula that were underreamed by 1 mm to the size of the component. Each acetabular specimen then was embedded in methylmethacrylate, sectioned, and examined under magnification. Although acetabular implantation was carefully performed by an experienced surgical team in an idealized laboratory environment, less than complete interface contact was achieved in most cases. Surface contact was limited by five factors: (1) bony anatomy, (2) asymmetric acetabular reaming, (3) retention of the subchondral plate, (4) acetabular component design, and (5) incorrect version of the acetabular component. The bony acetabulum is an incomplete hemisphere because of the acetabular notch and a deep acetabular fossa, and therefore cannot be machined to a perfect hemisphere. Asymmetric reaming occurs because of the anisotropic quality of bone. Retention of the subchondral plate causes incomplete seating of components with spikes or threads. The acetabular design determines the amount of porous coating available for bony apposition and varies depending on the surface area used for screw holes, apical holes, and thread segments. Increased, stable initial surface contact of acetabular components will increase the chances of stable biologic fixation. Improved surface contact will require changes in component design, instrumentation, and technique.

Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients.

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products, initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535-573del). Two of the patients had only the I66M mutant mRNA, and one only the G27OD mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535-573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535-573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535-573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD.

ATM mutations in patients with ataxia telangiectasia screened by a hierarchical strategy.

ATM has been identified as a gene that is responsible for ataxia telangiectasia (AT), a pleiotropic disorder of autosomal recessive inheritance. While many mutations of this gene in AT patients of various ethnicities have been reported, data on Japanese patients are scarce. In this report, we present the results of a thorough survey of ATM mutations in 14 unrelated AT patients, with an emphasis on Japanese subjects. We used a hierarchical strategy in which we extensively analyzed the entire coding region of the cDNA. In the first stage, point mutations were sought by PCR-SSCP in short patches. In the second and third stages, the products of medium- and long-patch PCR, each covering the entire region, were examined by agarose gel electrophoresis to search for length changes. We found a total of 15 mutations (including 12 new) and 4 polymorphisms. Abnormal splicing of ATM was frequent among Japanese, and no hotspot was obvious, suggesting no strong founder effects in this ethnic group. Eleven patients carried either one homozygous or two compound heterozygous mutations, one patient carried only one detectable heterozygous mutation, and no mutation was found in two patients. Overall, mutations were found in at least 75% of the different ATM alleles examined. Possible reasons for the inability to detect mutations in some patients are discussed.