Yoji Kukita

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. Hum Mutat 10:400–407, 1997.

Diagnostic Accuracy of Noninvasive Genotyping of EGFR in Lung Cancer Patients by Deep Sequencing of Plasma Cell-Free DNA

BACKGROUND Genotyping of EGFR (epidermal growth factor receptor) mutations is indispensable for making therapeutic decisions regarding whether to use EGFR tyrosine kinase inhibitors (TKIs) for lung cancer. Because some cases might pose challenges for biopsy, noninvasive genotyping of EGFR in circulating tumor DNA (ctDNA) would be beneficial for lung cancer treatment. METHODS We developed a detection system for EGFR mutations in ctDNA by use of deep sequencing of plasma DNA. Mutations were searched in >100 000 reads obtained from each exon region. Parameters corresponding to the limit of detection and limit of quantification were used as the thresholds for mutation detection. We conducted a multi-institute prospective study to evaluate the detection system, enrolling 288 non-small cell lung cancer (NSCLC) patients. RESULTS In evaluating the performance of the detection system, we used the genotyping results from biopsy samples as a comparator: diagnostic sensitivity for exon 19 deletions, 50.9% (95% CI 37.9%-63.9%); diagnostic specificity for exon 19 deletions, 98.0% (88.5%-100%); sensitivity for the L858R mutation, 51.9% (38.7%-64.9%); and specificity for L858R, 94.1% (83.5%-98.6%). The overall sensitivities were as follows: all cases, 54.4% (44.8%-63.7%); stages IA-IIIA, 22.2% (11.5%-38.3%); and stages IIIB-IV, 72.7% (60.9%-82.1%). CONCLUSIONS Deep sequencing of plasma DNA can be used for genotyping of EGFR in lung cancer patients. In particular, the high specificity of the system may enable a direct recommendation for EGFR-TKI on the basis of positive results with plasma DNA. Because sensitivity was low in early-stage NSCLC, the detection system is preferred for stage IIIB-IV NSCLC.

Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA.

Prevalence of copy-number neutral LOH in glioblastomas revealed by genomewide analysis of laser-microdissected tissues

We have employed a laser-capture microdissection technique and single-nucleotide polymorphism arrays to characterize genomic alterations associated with the development of glioblastoma multiforme (GBM). Combined analysis of loss of heterozygosity (LOH) and copy number revealed that more than half (56.3%) of the 254 identified LOH loci showed no copy-number alteration, indicating the presence of copy-number neutral LOH (cnLOH). Furthermore, we found a GBM case that showed cnLOH in 18 of the 22 autosomes. These results were confirmed by quantitative real-time PCR, microsatellite analysis, and fluorescence in situ hybridization. The high rate of cnLOH suggests that epigenetic abnormalities of many genes are involved in the development and progression of GBMs.

Gastric juvenile polyposis associated with germline SMAD4 mutation

We treated a 39‐year‐old woman with hypoproteinemia and anemia who had profuse gastric polyposis. Radiographic and endoscopic examination showed numerous polyps restricted to the stomach. The patient had pulmonary arteriovenous malformations in the left lung. Histological examination of the resected stomach revealed the gastric polyposis to be composed of cystic dilatation of the glands with small areas of adenocarcinoma. These findings were compatible with gastric juvenile polyposis (GJP) accompanied by gastric cancer. Analysis of genomic DNA revealed that the patient had truncating mutation of SMAD4, a responsible gene for juvenile polyposis (JP). Our case suggests that SMAD4 is possibly a responsible gene for GJP.

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. Hum Mutat 12:186–195, 1998.

Evaluation of Haplotype Inference Using Definitive Haplotype Data Obtained from Complete Hydatidiform Moles, and Its Significance for the Analyses of Positively Selected Regions

The haplotype map constructed by the HapMap Project is a valuable resource in the genetic studies of disease genes, population structure, and evolution. In the Project, Caucasian and African haplotypes are fairly accurately inferred, based mainly on the rules of Mendelian inheritance using the genotypes of trios. However, the Asian haplotypes are inferred from the genotypes of unrelated individuals based on population genetics, and are less accurate. Thus, the effects of this inaccuracy on downstream analyses needs to be assessed. We determined true Japanese haplotypes by genotyping 100 complete hydatidiform moles (CHM), each carrying a genome derived from a single sperm, using Affymetrix 500 K Arrays. We then assessed how inferred haplotypes can differ from true haplotypes, by phasing pseudo-individualized true haplotypes using the programs PHASE, fastPHASE, and Beagle. We found that, at various genomic regions, especially the MHC locus, the expansion of extended haplotype homozygosity (EHH), which is a measure of positive selection, is obscured when inferred Asian haplotype data is used to detect the expansion. We then mapped the genome using a new statistic, XDiHH, which directly detects the difference between the true and inferred haplotypes, in the determination of EHH expansion. We also show that the true haplotype data presented here is useful to assess and improve the accuracy of phasing of Asian genotypes.

Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4-ALK fusion gene.

Introduction: Using cell line-based assays, the secretion of erythroblastic leukemia viral oncogene homologue (ERBB) ligands has been reported to contribute to resistance against crizotinib in lung cancer with the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase fusion gene. However, it is difficult to predict the role of the ligands in each patient. Here, we report an analysis of the mechanism of resistance behind crizotinib resistance using a primary culture of cancer cells from pleural effusion of an anaplastic lymphoma kinase-positive lung cancer patient who was clinically resistant to crizotinib. Methods: Primary cancer cells were prepared as cancer tissue-originated spheroids (CTOSs) according to previously described methods. CTOSs were maintained in StemPro medium, and a sensitivity assay was performed under growth factor-free conditions, or under stimulation with epidermal growth factor (EGF) or neuregulin-1/heregulin. The effect of treatment with crizotinib alone or a combination of crizotinib and erlotinib was examined. Results: Cancer cells (LB53) were established to be CTOSs from a patient who was clinically resistant to crizotinib. The CTOSs were sensitive to crizotinib under growth factor-free conditions in vitro, whereas resistant under stimulation with EGF or neuregulin-1. These ligands rescued the inhibition of intracellular signaling by crizotinib. Pleural effusion from the patient also activated EGF receptor signaling to the similar extent of EGF stimulation. The resistance to crizotinib by EGF was reversed by blocking EGF receptor signaling by erlotinib in vitro. Conclusion: Stimulation by ERBB ligands is suggested to be responsible for resistance to crizotinib in this patient. The CTOS method may enable analysis of resistance mechanism for targeted therapy in individual patients.

Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis

Mapping loss of heterozygosity (LOH) regions in the genomes of tumor tissues is a practical approach for identifying genes whose loss is related to tumorigenesis. Conventional LOH analyses using microsatellite or single nucleotide polymorphism (SNP) markers require the simultaneous examination of tumor‐ and matched normal‐DNA. Here, we improved the previously developed SNP‐based LOH assay using single strand conformation polymorphism (SSCP) analysis, so that LOH in tumor samples heavily contaminated with normal DNA can now be precisely estimated, even when matched normal DNA is not available. We demonstrate the reliability of the improved SSCP‐based LOH detection method, called the LOH estimation by quantitative SSCP analysis using averaged control (LOQUS‐AC), by comparing the results with those of the previous “LOH estimated by quantitative SSCP assay” (LOQUS) method. Using the LOQUS‐AC assay, LOH was detected at a high consistency (98.1%) with the previous LOQUS method. We then applied this new method to characterize LOH profiles in 130 meningiomas, using 68 SNPs (i.e., a mean inter‐SNP interval of 441 kbp) that are evenly distributed throughout chromosome 1p36. Benign, atypical and anaplastic meningiomas exhibited 1p36 LOH at frequencies of 48.39, 84.62 and 100.00%, respectively, using LOQUS‐AC. Subsequently, we detected a candidate common LOH region on 1p36.11 that might harbor tumor suppressor genes related to malignant progression of meningioma.