Publications by authors named "Yoji Kukita"

45 Publications

Malignant epithelioid soft-tissue tumour with NRID1-MAML1 fusion.

Histopathology 2021 Jul 16. Epub 2021 Jul 16.

Department of Outpatient Chemotherapy, Osaka International Cancer Institute, Osaka, Japan.

Epithelioid soft-tissue tumours are predominantly composed of rounded or polygonal tumour cells resembling epithelial cells. In the current WHO classification, epithelioid soft tissue tumors of uncertain differentiaton include myoepithelial tumour, alveolar soft part sarcoma (ASPS), and PEComa among others. There is only one published case of paediatric novel soft-tissue tumour with an NRID1-MAML1 fusion. We report the second case with the same NRID1-MAML1 fusion gene; however, this is the first case with malignancy in a young adult.
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http://dx.doi.org/10.1111/his.14455DOI Listing
July 2021

Signet-ring cell/histiocytoid carcinoma of the axilla: a clinicopathologic and genetic analysis of 11 cases, review of the literature, and comparison with potentially related tumours.

Histopathology 2021 Jun 22. Epub 2021 Jun 22.

Department of Dermatologic Oncology, Osaka International Cancer Institute, Osaka, Japan.

Aims: The purpose of this study was to reveal the clinicopathologic and genetic characteristics of axillary signet-ring cell/histiocytoid carcinoma (SRCHC) and the relationship between axillary SRCHC, eyelid SRCHC, and conventional apocrine carcinoma (AC).

Methods And Results: A total of 11 cases of axillary SRCHC, 4 cases of eyelid SRCHC, 8 cases of axillary AC, and 5 cases of invasive lobular carcinoma (ILC), were retrieved. Additionally, 14 axillary and 43 eyelid SRCHC cases from the literature were reviewed. Male predominance was prominent in axillary (24:1) and eyelid (42:5) SRCHCs. Axillary SRCHC formed a circumscribed plaque or nodule, unlike eyelid SRCHC. Lymph node metastasis was predominantly seen in axillary SRCHC (72%, 18/25), compared to eyelid SRCHC (19%, 9/47). Both axillary and eyelid SRCHCs were histopathologically similar and showed rare tubular formations. Immunoexpression of cytokeratin 7, cytokeratin 19, MUC1, MUC5AC, BerEP4, and androgen receptor was observed in all tested cases of four diseases. Oestrogen and progesterone receptors were negative in both SRCHCs and AC, but strongly positive in ILC cases. Complete loss of E-cadherin expression was observed in approximately a quarter of both SRCHCs as well as in all ILC cases. PIK3CA mutations were detected in all three sequenced cases (two axillary SRCHCs and one eyelid SRCHC).

Conclusion: The histopathological, immunohistochemical, and genetic findings suggest that both SRCHCs are phenotypic variants of AC, although there are differences in sex, macroscopic findings, and the frequency of lymph node metastasis among the three. In contrast, ILC differs from the three tumour types.
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http://dx.doi.org/10.1111/his.14436DOI Listing
June 2021

EML4-ALK fusion variant.3 and co-occurrent PIK3CA E542K mutation exhibiting primary resistance to three generations of ALK inhibitors.

Cancer Genet 2021 Aug 28;256-257:131-135. Epub 2021 May 28.

Department of Thoracic Oncology, Osaka International Cancer Institute, Osaka, Japan.

The ALK inhibitors are promising therapeutic agents against lung cancer harboring ALK fusion genes and are currently under development up to the third generation. However, its therapeutic effects are reported to be affected by differences in ALK variants and co-occurrent mutations. Materials and Methods; We experienced an autopsy case of an ALK-positive lung cancer patient who showed primary resistance to three generations of ALK inhibitors. The poor survival time of the case was 14 months. To reveal the mechanism of primary resistance to three generations of ALK inhibitors, we performed next generation sequencing for 12 specimes obtained from an autopsy with covering whole exons of 53 significantly mutated, lung cancer-associated genes and amplicon-based target RNA sequenceing for the ALK fusion gene. The NGS analysis revealed a rare variant.3 of ALK fusion, in which 30 bp of base was inserted at the end of ALK intron.19 and was associated with EML exon.6 [E6_ins30A20] and a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. Structural analysis of the fusion protein ALK [E6_ins30A20] showed no interferance with the binding of ALK inhibitors to the kinase domain. The NGS analysis of primary and metastatic lesions obtained from an autopsy revealed a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. The constitutive activation of PI3K-Akt signal by PIK3CA E542K mutation occurred downstream of ALK signaling pathway, could lead to primary resistance to ALK inhibitors in all generations.
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http://dx.doi.org/10.1016/j.cancergen.2021.05.010DOI Listing
August 2021

Atezolizumab with bevacizumab, paclitaxel and carboplatin was effective for patients with SMARCA4-deficient thoracic sarcoma.

Immunotherapy 2021 Jul 25;13(10):799-806. Epub 2021 May 25.

Department of Thoracic Oncology, Osaka International Cancer Institute, Osaka, Japan.

SMARCA4-deficient thoracic sarcoma (DTS) is a recently noted progressive thoracic malignancy. We recently experienced three cases of SMARCA4-DTS who were treated with atezolizumab in combination with bevacizumab, paclitaxel and carboplatin (ABCP) as the first-line therapy. Immunohistopathological analysis revealed absent expression of SMARCA4 in all cases. The tumor mutational burden was over 11/Mb and mutations in and were detected in all three cases. Partial response to ABCP treatment was observed in all three cases, with a progression-free survival of approximately 6 months or longer and a continuous response of 1 year or longer in one case. The first-line ABCP treatment demonstrated durable efficacy in SMARCA4-DTS regardless of the degree of PD-L1 expression.
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http://dx.doi.org/10.2217/imt-2020-0311DOI Listing
July 2021

Late recurrence of lung adenocarcinoma harboring EGFR exon 20 insertion (A763_Y764insFQEA) mutation successfully treated with osimertinib.

Cancer Genet 2021 Aug 10;256-257:57-61. Epub 2021 Apr 10.

Department of Thoracic Oncology, Osaka International Cancer Institute, 3-1-69 Otemae Chuoku, Osaka 541-8567, Osaka, Japan.

The EGFR-A763_Y764insFQEA is a unique mutation among EGFR exon 20 insertion mutations in that it is associated with sensitivity to conventional EGFR-tyrosine kinase inhibitors. This mutation, which was not initially covered by conventional reverse transcription polymerase chain reaction (RT-PCR) genotyping method, has only been detected in clinical practice when a next-generation sequencing (NGS)-based cancer panel is implemented. We present the case of a female patient with recurrent lung adenocarcinoma from a lung tumor resected 10 years earlier. Sequential single-gene investigations and the OncomineTM Comprehensive Assay (ver.3) analysis of the recurrent tumor did not reveal any targetable driver mutations. However, the second NGS analysis with the OncoGuideTM NCC oncopanel found the EGFR-A763_Y764insFQEA mutation after tumor progression with carcinomatous lymphangiomatosis and multiple brain metastases. Osimertinib treatment improved her condition immediately. The identical EGFR-A763_Y764insFQEA mutation was detected in the tumor resected 10 years earlier. Based on this common mutation the patient was diagnosed with late recurrence of lung cancer harboring the EGFR-A763_Y764insFQEA mutation. The OncoGuideTM NCC oncopanel covered whole exons of the EGFR gene and was able to detect this mutation. In the present clinical practice, the EGFR-A763_Y764insFQEA mutation is the only treatable mutation among EGFR Ex.20 insertion mutations. We need to understand the gene mutation profile identified by each panel and consider reexamining them for this mutation.
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http://dx.doi.org/10.1016/j.cancergen.2021.04.001DOI Listing
August 2021

Bcl-2-negative IGH-BCL2 translocation-negative follicular lymphoma of the thyroid differs genetically and epigenetically from Bcl-2-positive IGH-BCL2 translocation-positive follicular lymphoma.

Histopathology 2021 Apr 7. Epub 2021 Apr 7.

Department of Pathology, Sakai City Medical Center, 1-1-1 Ebaraji-cho, Nishi-ku, Sakai, Osaka, 593-8304, Japan.

Aims: Follicular lymphoma (FL), comprising a minor subset of primary thyroid lymphomas, is divided into two groups based on Bcl-2 expression and IGH-BCL2 translocation. The clinicopathological features exhibited by Bcl-2-negative IGH-BCL2 translocation-negative FL of the thyroid (Bcl-2 /IGH-BCL2 tFL) are different from those of conventional FL; however, its lymphomagenesis remains unclear. Here, we collected samples from seven patients with Bcl-2 /IGH-BCL2 tFL to investigate their epigenetic and genetic aberrations.

Methods And Results: The immunohistochemical profiles of epigenetic modifiers and the methylation status of histones were examined, including EZH2, MLL2/KMT2D, CBP/CREBBP, EP300, H3K27me3, and H3K4me3, in Bcl-2 /IGH-BCL2 tFL and Bcl-2-positive IGH-BCL2 translocation-positive FL of the thyroid (Bcl-2 /IGH-BCL2 tFL). Most Bcl-2 /IGH-BCL2 tFLs retained the positivity of epigenetic modifiers and lower expression of H3K27me3, although Bcl-2 /IGH-BCL2 tFLs exhibited aberrant immunohistochemical patterns of EZH2 and CBP/CREBBP and overexpression of H3K27me3. Samples from seven cases were further analysed using targeted sequencing, focusing on the exons of 409 key tumour suppressor genes and oncogenes. Bcl-2 /IGH-BCL2 tFLs do not have pathogenic mutations of epigenetic modifiers, such as EZH2, MLL2/KMT2D, MLL3/KMT2C, EP300, and ARID1A, which have been reported in FLs in the literature, whereas Bcl-2 /IGH-BCL2 tFLs are likely pathogenic/pathogenic missense mutations or frameshift mutations of these genes. Additionally, novel mutations in TET2 and EP400 were detected in Bcl-2 /IGH-BCL2 tFLs.

Conclusions: Different genetic and epigenetic abnormalities might be involved in the oncogenesis of Bcl-2 /IGH-BCL2 tFLs from Bcl-2 /IGH-BCL2 tFLs and other FLs.
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http://dx.doi.org/10.1111/his.14378DOI Listing
April 2021

Quantitative detection of ALK fusion breakpoints in plasma cell-free DNA from patients with non-small cell lung cancer using PCR-based target sequencing with a tiling primer set and two-step mapping/alignment.

PLoS One 2019 12;14(9):e0222233. Epub 2019 Sep 12.

Laboratory of Medical Genomics, Nara Institute of Science and Technology, Ikoma, Nara, Japan.

Background: Tyrosine kinase inhibitors targeted to anaplastic lymphoma kinase (ALK) have been demonstrated to be effective for lung cancer patients with an ALK fusion gene. Application of liquid biopsy, i.e., detection and quantitation of the fusion product in plasma cell-free DNA (cfDNA), could improve clinical practice. To detect ALK fusions, because fusion breakpoints occur somewhere in intron 19 of the ALK gene, sequencing of the entire intron is required to locate breakpoints.

Results: We constructed a target sequencing system using an adapter and a set of primers that cover the entire ALK intron 19. This system can amplify fragments, including breakpoints, regardless of fusion partners. The data analysis pipeline firstly detected fusions by alignment to selected target sequences, and then quantitated the fusion alleles aligning to the identified breakpoint sequences. Performance was validated using 20 cfDNA samples from ALK-positive non-small cell lung cancer patients and samples from 10 healthy volunteers. Sensitivity and specificity were 50 and 100%, respectively.

Conclusions: We demonstrated that PCR-based target sequencing using a tiling primer set and two-step mapping/alignment quantitatively detected ALK fusions in cfDNA from lung cancer patients. The system offers an alternative to existing approaches based on hybridization capture.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222233PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742348PMC
March 2020

High-throughput screening in colorectal cancer tissue-originated spheroids.

Cancer Sci 2019 Jan 20;110(1):345-355. Epub 2018 Nov 20.

Department of Clinical Bio-resource Research and Development, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.
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http://dx.doi.org/10.1111/cas.13843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317944PMC
January 2019

Selective identification of somatic mutations in pancreatic cancer cells through a combination of next-generation sequencing of plasma DNA using molecular barcodes and a bioinformatic variant filter.

PLoS One 2018 16;13(2):e0192611. Epub 2018 Feb 16.

Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

The accuracy of next-generation sequencing (NGS) for detecting tumor-specific mutations in plasma DNA is hindered by errors introduced during PCR/sequencing, base substitutions caused by DNA damage, and pre-existing mutations in normal cells that are present at a low frequency. Here, we performed NGS of genes related to pancreatic cancer (comprising 2.8 kb of genomic DNA) in plasma DNA (average 4.5 ng) using molecular barcodes. The average number of sequenced molecules was 900, and the sequencing depth per molecule was 100 or more. We also developed a bioinformatic variant filter, called CV78, to remove variants that were not considered to be tumor-specific, i.e., those that are either absent or occur at low frequencies in the Catalogue of Somatic Mutations in Cancer database. In a cohort comprising 57 pancreatic cancer patients and 12 healthy individuals, sequencing initially identified variants in 31 (54%) and 5 (42%), respectively, whereas after applying the CV78 filter, 19 (33%) and zero were variant-positive. In a validation cohort consisting of 86 patients with pancreatic cancer and 20 patients with intraductal papillary mucinous neoplasm (IPMN), 62 (72%) with pancreatic cancer patients and 10 (50%) IPMN patients were initially variant positive. After CV78 filtering, these values were reduced to 32 (37%) and 1 (5%), respectively. The variant allele frequency of filtered variants in plasma ranged from 0.25% to 76.1%. Therefore, combining NGS and molecular barcodes with subsequent filtering is likely to eliminate most non-tumor-specific mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192611PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815581PMC
April 2018

HLA genotyping by next-generation sequencing of complementary DNA.

BMC Genomics 2017 Nov 28;18(1):914. Epub 2017 Nov 28.

Laboratory of Medical Genomics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101, Japan.

Background: Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.

Methods: Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.

Results: The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.

Conclusion: The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.
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http://dx.doi.org/10.1186/s12864-017-4300-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704545PMC
November 2017

Rectal Cancer in a Patient with Bartter Syndrome: A Case Report.

Genes (Basel) 2017 May 12;8(5). Epub 2017 May 12.

Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

A woman with rectal cancer was scheduled for surgery. However, she also had hypokalemia, hyperreninemia, and hyperaldosteronism in the absence of any known predisposing factors or endocrine tumors. She was given intravenous potassium, and her blood abnormalities stabilized after tumor resection. Genetic analysis revealed mutations in several genes associated with Bartter syndrome (BS) and Gitelman syndrome, including , , , , and . Prostaglandin E2 (PGE2) plays an important role in BS and worsens electrolyte abnormalities. The PGE2 level is reportedly increased in colorectal cancer, and in the present case, immunohistochemical examination revealed an increased PGE2 level in the tumor. We concluded that the tumor-related PGE2 elevation had worsened the patient's BS, which became more manageable after tumor resection.
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http://dx.doi.org/10.3390/genes8050139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448013PMC
May 2017

Transient appearance of circulating tumor DNA associated with de novo treatment.

Sci Rep 2016 12 9;6:38639. Epub 2016 Dec 9.

Department of Thoracic Oncology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

The limitation of circulating tumor DNA (ctDNA) is its inability to detect cancer cell subpopulations with few or no dying cells. Lung cancer patients subjected to the EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment were prospectively collected, and ctDNA levels represented by the activating and T790M mutations were measured. The first data set (21 patients) consisting of samples collected in the period from before initiation of EGFR-TKI to at least 2 weeks after initiation: the ctDNA dynamics generally exhibited a rapid decrease and/or a transient increase. In 4 patients, we detected a transient increase of ctDNA bearing activating mutations not identified in biopsy samples. ctDNA with the same genotypical pattern was identified in 7 out of the 39 patients of the second data set intended to include samples until the onset of disease progression. In 6 of the 7 patients, this unique ctDNA appeared in the early period after treatment initiation, and did not reappear even after disease progression or chemotherapy. In another patient, similar ctDNA appeared upon radiation therapy. The identification of ctDNA with a unique genotype indicates the presence of cancer cell subpopulations that normally contain few or no dying cells, but generate dead cells because of the treatment.
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http://dx.doi.org/10.1038/srep38639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146655PMC
December 2016

Homozygous inactivation of is linked to a familial case of multiple primary lung cancer with accompanying cancers in other organs.

Cold Spring Harb Mol Case Stud 2016 11;2(6):a001032

Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.

In clinical practice, there are a number of cancer patients with clear family histories, but the patients lack mutations in known familial cancer syndrome genes. Recent advances in genomic technologies have enhanced the possibility of identifying causative genes in such cases. Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer and had a history of uterine myoma. The latter had initially developed prostate cancer at the age of 59 and had a history of colon cancer. Single-nucleotide polymorphism (SNP) genotyping revealed that ∼10% of the genomes were homozygous in both patients. Exome sequencing revealed nonsynonymous mutations in five genes in the runs of homozygosity: , , , , and . Evolutionary conservation of primary protein structures suggested the functional importance of the mutation, p.R474C. This mutation altered the tertiary structure of CHK2 by disrupting the salt bridge between p.R474 and p.E394. No such structural changes were observed with the other mutated genes. Subsequent cell-based transfection analysis revealed that CHK2 p.R474C was unstable and scarcely activated. We concluded that the homozygous variant was contributory in this case of familial cancer. Although homozygous inactivation of in mice led to cancers in multiple organs, accumulation of additional human cases is needed to establish its pathogenic role in humans.
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http://dx.doi.org/10.1101/mcs.a001032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111006PMC
November 2016

Early responses of EGFR circulating tumor DNA to EGFR tyrosine kinase inhibitors in lung cancer treatment.

Oncotarget 2016 Nov;7(44):71782-71789

Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

Objectives: Early evaluation of the effect of treatment is helpful in the management of cancer patients. Circulating biomarkers are an ideal tool for this if they are highly specific to tumors and respond rapidly to tumor volume changes. Circulating tumor DNA (ctDNA) is one such candidate. We conducted a prospective study to test the utility of EGFR ctDNA in early evaluation of EGFR-TKI effects.

Results: Twenty-one patients with EGFR-mutant lung cancer who were naïve to EGFR-TKI were enrolled. PM scores of EGFR ctDNA with activating mutations decreased rapidly in response to EGFR-TKI. Of the 14 patients with positive pretreatment PM scores, complete disappearance of major EGFR ctDNA was observed in 14.3%, 42.9%, and 57.1% on days 2 - 4, 8, and 15, respectively. These responses of EGFR ctDNA were most prominent among the measures used to evaluate responses, and correlated with early radiologic responses evaluated by chest X-rays.

Materials And Methods: EGFR ctDNA in serial plasma samples was amplified and 105 copies were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 105EGFR cell free DNA (cfDNA). When EGFR mutation in ctDNA was the same as that detected in cancer tissue, the ctDNA was defined as major EGFR ctDNA.

Conclusions: The results indicate the usefulness of ctDNA as a highly specific biomarker for prediction of early response to treatment and that it can be applied to various types of cancer.
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http://dx.doi.org/10.18632/oncotarget.12373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342122PMC
November 2016

Characterization of the T-cell receptor beta chain repertoire in tumor-infiltrating lymphocytes.

Cancer Med 2016 09 27;5(9):2513-21. Epub 2016 Jul 27.

Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

Tumor-infiltrating lymphocytes (TILs) are direct effectors of tumor immunity, and their characterization is important for further development of immunotherapy. Recent advances in high-throughput sequencing technologies have enabled a comprehensive analysis of T-cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, which may provide information of therapeutic importance. We developed a high-fidelity target sequencing method with the ability for absolute quantitation, and performed large-scale sequencing of TCR beta chain (TCRB) CDR3 regions in TILs and peripheral blood lymphocytes (PBLs). The estimated TCRB repertoire sizes of PBLs from four healthy individuals and TILs from four colorectal cancer tissue samples were 608,664-1,003,098 and 90,228-223,757, respectively. The usage of J- and V-regions was similar in PBLs and TILs. Proportions of CDR3 amino acid (aa) sequences occupying more than 0.01% of the total molecular population were 0.33-0.43% in PBLs and 1.3-3.6% in TILs. Additional low coverage sequencing of 15 samples identified five CDR3 aa sequences that were shared by nine patients, one sequence shared by 10 patients, and one sequence shared by 12 patients. The estimated size of the TCRB repertoire in TILs was significantly smaller than that in PBLs. The proportion of abundant species (>0.01%) in TILs was larger than that in PBLs. Shared CDR3 aa sequences represent a response to common antigens, and the identification of such CDR3 sequences may be beneficial in developing clinical biomarkers.
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http://dx.doi.org/10.1002/cam4.828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055180PMC
September 2016

Numerical indices based on circulating tumor DNA for the evaluation of therapeutic response and disease progression in lung cancer patients.

Sci Rep 2016 07 6;6:29093. Epub 2016 Jul 6.

Department of Thoracic Oncology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

Monitoring of disease/therapeutic conditions is an important application of circulating tumor DNA (ctDNA). We devised numerical indices, based on ctDNA dynamics, for therapeutic response and disease progression. 52 lung cancer patients subjected to the EGFR-TKI treatment were prospectively collected, and ctDNA levels represented by the activating and T790M mutations were measured using deep sequencing. Typically, ctDNA levels decreased sharply upon initiation of EGFR-TKI, however this did not occur in progressive disease (PD) cases. All 3 PD cases at initiation of EGFR-TKI were separated from other 27 cases in a two-dimensional space generated by the ratio of the ctDNA levels before and after therapy initiation (mutation allele ratio in therapy, MART) and the average ctDNA level. For responses to various agents after disease progression, PD/stable disease cases were separated from partial response cases using MART (accuracy, 94.7%; 95% CI, 73.5-100). For disease progression, the initiation of ctDNA elevation (initial positive point) was compared with the onset of objective disease progression. In 11 out of 28 eligible patients, both occurred within ±100 day range, suggesting a detection of the same change in disease condition. Our numerical indices have potential applicability in clinical practice, pending confirmation with designed prospective studies.
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http://dx.doi.org/10.1038/srep29093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933907PMC
July 2016

Monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor DNA of heterogeneous mutant EGFR genes in lung cancer.

Lung Cancer 2016 Apr 2;94:68-73. Epub 2016 Feb 2.

Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

Objectives: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatic effects on EGFR-mutant non-small-cell lung cancer (NSCLC). However, most patients experience disease recurrences, approximately half of which are T790M-mediated. Monitoring EGFR status with re-biopsy has spatiotemporal limitations.

Patients And Methods: EGFR circulating tumor DNA (ctDNA) in serial plasma samples was amplified and 10(5) of them were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 10(5)EGFR cell free DNA (cfDNA).

Results: PM scores of various EGFR mutations showed dynamic, case-specific changes during EGFR-TKI treatments in 52 patients. The effects of the treatment on EGFR ctDNA were evaluated in 38 patients with elevated pre-treatment PM scores. The ctDNA responses correlated well with radiologic responses in radiologic good responders, whereas correlation was poor in non-responders. In addition to the peaks for the most prevalent ctDNA, small peaks of ctDNA with different types of activating EGFR mutations or the T790M mutation (early T790M ctDNA) appeared transiently in 10.5% and 26.3%, respectively. Early T790M ctDNA disappeared in all patients, including 7 who eventually developed acquired resistance accompanied by elevated levels of T790M ctDNA.

Conclusions: Monitoring ctDNA is useful in evaluating treatment responses and monitoring driver oncogene status in NSCLC. ctDNA revealed clonal heterogeneity and genetic processes of cancer evolution in individual patients. The simple presence of the T790M mutation may be insufficient to confer EGFR-TKI resistance to tumor cells.
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http://dx.doi.org/10.1016/j.lungcan.2016.01.023DOI Listing
April 2016

Dynamics of circulating tumor DNA represented by the activating and resistant mutations in epidermal growth factor receptor tyrosine kinase inhibitor treatment.

Cancer Sci 2016 Mar 18;107(3):353-8. Epub 2016 Feb 18.

Department of Molecular and Medical Genetics of Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

Circulating tumor DNA (ctDNA) is an emerging field of cancer research. For lung cancer, non-invasive genotyping of EGFR is the foremost application. The activating mutations represent the ctDNA from all cancer cells, and the T790M-resistant mutation represents that from resistant cells. We examined the ctDNA dynamics of EGFR mutations by using deep sequencing with a massively parallel DNA sequencer. We obtained 190 plasma samples from 57 patients at various times during the treatment course and classified them according to treatment status. The mutation detection rate of exon 19 deletion/L858R in plasma was high at the initiation of treatment with epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI; P = 0.001), suppressed during EGFR-TKI treatment before disease progression, and elevated after the onset of disease progression (P = 0.023). The mutation detection rate of T790M was low until the onset of disease progression and elevated thereafter (P = 0.01). Samples across the development of disease progression were obtained from 10 patients and showed a correlation between increased ctDNA level and disease progression. Decreased ctDNA level in response to the initiation of EGFR-TKI was observed in 4 of 6 eligible patients. In two patients, the ctDNA dynamics suggested the presence of cancer cell populations only with the T790M mutation. In another patient, the T790M ctDNA represented cell subpopulations that respond to cytotoxic agents differently from the major population. Considering the high incidence, ctDNA could be a clinical parameter to complement information from image analyses.
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http://dx.doi.org/10.1111/cas.12860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814247PMC
March 2016

A definitive haplotype map of structural variations determined by microarray analysis of duplicated haploid genomes.

Genom Data 2014 Dec 24;2:55-9. Epub 2014 Apr 24.

Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

Complete hydatidiform moles (CHMs) are tissues carrying duplicated haploid genomes derived from single sperms, and detecting copy number variations (CNVs) in CHMs is assumed to be sensitive and straightforward methods. We genotyped 108 CHM genomes using Affymetrix SNP 6.0 (GEO#: GSE18642) and Illumina 1 M-duo (GEO#: GSE54948). After quality control, we obtained 84 definitive haplotype consisting of 1.7 million SNPs and 2339 CNV regions. The results are presented in the database of our web site (http://orca.gen.kyushu-u.ac.jp/cgi-bin/gbrowse/humanBuild37D4_1/).
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http://dx.doi.org/10.1016/j.gdata.2014.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535890PMC
December 2014

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

Clin Chem 2015 Sep 23;61(9):1191-6. Epub 2015 Jul 23.

Department of Thoracic Oncology.

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.
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http://dx.doi.org/10.1373/clinchem.2015.241414DOI Listing
September 2015

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.

DNA Res 2015 Aug 29;22(4):269-77. Epub 2015 Jun 29.

Department of Molecular and Medical Genetics, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan

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.
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http://dx.doi.org/10.1093/dnares/dsv010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535617PMC
August 2015

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

J Thorac Oncol 2015 Mar;10(3):527-30

*Departments of Thoracic Oncology, †Biochemistry, and ‡Molecular and Medical Genetics, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan.

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.
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http://dx.doi.org/10.1097/JTO.0000000000000381DOI Listing
March 2015

Prognostic prediction of glioblastoma by quantitative assessment of the methylation status of the entire MGMT promoter region.

BMC Cancer 2014 Aug 30;14:641. Epub 2014 Aug 30.

Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, Japan.

Background: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is reported to be a prognostic and predictive factor of alkylating chemotherapy for glioblastoma patients. Methylation specific PCR (MSP) has been most commonly used when the methylation status of MGMT is assessed. However, technical obstacles have hampered the implementation of MSP-based diagnostic tests. We quantitatively analyzed the methylation status of the entire MGMT promoter region and applied this information for prognostic prediction using sequencing technology.

Methods: Between 1998 and 2012, the genomic DNA of 85 tumor samples from newly diagnosed glioblastoma patients was subjected to bisulfite treatment and subdivided into a training set, consisting of fifty-three samples, and a test set, consisting of thirty-two samples. The training set was analyzed by deep Sanger sequencing with a sequencing coverage of up to 96 clones per sample. This analysis quantitatively revealed the degree of methylation of each cytidine phosphate guanosine (CpG) site. Based on these data, we constructed a prognostic prediction system for glioblastoma patients using a supervised learning method. We then validated this prediction system by deep sequencing with a next-generation sequencer using a test set of 32 samples.

Results: The methylation status of the MGMT promoter was correlated with progression-free survival (PFS) in our patient population in the training set. The degree of correlation differed among the CpG sites. Using the data from the top twenty CpG sites, we constructed a prediction system for overall survival (OS) and PFS. The system successfully classified patients into good and poor prognosis groups in both the training set (OS, p = 0.0381; PFS, p = 0.00122) and the test set (OS, p = 0.0476; PFS, p = 0.0376). Conventional MSP could not predict the prognosis in either of our sets. (training set: OS; p = 0.993 PFS; p = 0.113, test set: OS; p = 0.326 PFS; p = 0.342).

Conclusions: The prognostic ability of our prediction system using sequencing data was better than that of methylation-specific PCR (MSP). Advances in sequencing technologies will make this approach a plausible option for diagnoses based on MGMT promotor methylation.
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http://dx.doi.org/10.1186/1471-2407-14-641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161852PMC
August 2014

Quantitative identification of mutant alleles derived from lung cancer in plasma cell-free DNA via anomaly detection using deep sequencing data.

PLoS One 2013 21;8(11):e81468. Epub 2013 Nov 21.

Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka, Japan.

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).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081468PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836767PMC
January 2015

Conversion of a molecular classifier obtained by gene expression profiling into a classifier based on real-time PCR: a prognosis predictor for gliomas.

BMC Med Genomics 2010 Nov 10;3:52. Epub 2010 Nov 10.

Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511, Japan.

Background: The advent of gene expression profiling was expected to dramatically improve cancer diagnosis. However, despite intensive efforts and several successful examples, the development of profile-based diagnostic systems remains a difficult task. In the present work, we established a method to convert molecular classifiers based on adaptor-tagged competitive PCR (ATAC-PCR) (with a data format that is similar to that of microarrays) into classifiers based on real-time PCR.

Methods: Previously, we constructed a prognosis predictor for glioma using gene expression data obtained by ATAC-PCR, a high-throughput reverse-transcription PCR technique. The analysis of gene expression data obtained by ATAC-PCR is similar to the analysis of data from two-colour microarrays. The prognosis predictor was a linear classifier based on the first principal component (PC1) score, a weighted summation of the expression values of 58 genes. In the present study, we employed the delta-delta Ct method for measurement by real-time PCR. The predictor was converted to a Ct value-based predictor using linear regression.

Results: We selected UBL5 as the reference gene from the group of genes with expression patterns that were most similar to the median expression level from the previous profiling study. The number of diagnostic genes was reduced to 27 without affecting the performance of the prognosis predictor. PC1 scores calculated from the data obtained by real-time PCR showed a high linear correlation (r=0.94) with those obtained by ATAC-PCR. The correlation for individual gene expression patterns (r=0.43 to 0.91) was smaller than for PC1 scores, suggesting that errors of measurement were likely cancelled out during the weighted summation of the expression values. The classification of a test set (n=36) by the new predictor was more accurate than histopathological diagnosis (log rank p-values, 0.023 and 0.137, respectively) for predicting prognosis.

Conclusion: We successfully converted a molecular classifier obtained by ATAC-PCR into a Ct value-based predictor. Our conversion procedure should also be applicable to linear classifiers obtained from microarray data. Because errors in measurement are likely to be cancelled out during the calculation, the conversion of individual gene expression is not an appropriate procedure. The predictor for gliomas is still in the preliminary stages of development and needs analytical clinical validation and clinical utility studies.
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http://dx.doi.org/10.1186/1755-8794-3-52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988704PMC
November 2010

A definitive haplotype map as determined by genotyping duplicated haploid genomes finds a predominant haplotype preference at copy-number variation events.

Am J Hum Genet 2010 Jun 27;86(6):918-28. Epub 2010 May 27.

Division of Genome Analysis, Research Center for Genetic Information, Kyushu University, Fukuoka 812-8582, Japan.

The majority of complete hydatidiform moles (CHMs) harbor duplicated haploid genomes that originate from sperm. This makes CHMs more advantageous than conventional diploid cells for determining haplotypes of SNPs and copy-number variations (CNVs), because all of the genetic variants in a CHM genome are homozygous. Here we report SNP and CNV haplotype structures determined by analysis of 100 CHMs from Japanese subjects via high-density DNA arrays. The obtained haplotype map should be useful as a reference for the haplotype structure of Asian populations. We resolved common CNV regions (merged CNV segments across the examined samples) into CNV events (clusters of CNV segments) on the basis of mutual overlap and found that the haplotype backgrounds of different CNV events within the same CNV region were predominantly similar, perhaps because of inherent structural instability.
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http://dx.doi.org/10.1016/j.ajhg.2010.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032077PMC
June 2010

Impact of group IVA cytosolic phospholipase A2 gene polymorphisms on phenotypic features of patients with familial adenomatous polyposis.

Int J Colorectal Dis 2010 Mar 1;25(3):293-301. Epub 2009 Oct 1.

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan.

Objective: Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) plays a key role in tumorigenesis via generating arachidonic acids as the substrate of cyclooxygenase. The aim of this study was to elucidate the possible associations between cPLA ( 2 )alpha gene polymorphisms and phenotypic features of patients with familial adenomatous polyposis (FAP).

Patients And Methods: A tag single nucleotide polymorphisms (SNPs)-based genotype-phenotype association study of the cPLA ( 2 )alpha gene was conducted in 73 Japanese patients from 59 families with FAP. Based on the HapMap database, seven tag SNPs of the cPLA ( 2 )alpha gene were selected and genotyped by direct sequencing analysis. The genotype-phenotype association in relation to the adenomatous polyposis coli (APC) gene mutation was also assessed.

Results: The single SNP analysis showed that rs3820185 C allele [odds ratio (OR), 2.5; 95% confidence interval (CI), 1.2-4.9] and rs127446200 GG genotype (OR, 10.9; 95%CI, 1.6-69.8), were more frequent in patients with gastric fundic gland polyposis (FGP) than in those without. Rs12749354 C allele was more frequently found in patients with small intestinal adenoma (OR, 7.0; 95% CI, 1.5-30.4; p = 0.008). This association was also significant when adjusted for covariates (age, sex, and APC mutation) in a logistic regression analysis (adjusted OR, 7.4; 95% CI, 1.2-64.2; p = 0.027).

Conclusions: The cPLA ( 2 )alpha gene may be a possible disease modifier gene in FAP.
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http://dx.doi.org/10.1007/s00384-009-0808-xDOI Listing
March 2010

Estimation of SNP allele frequencies by SSCP analysis of pooled DNA.

Methods Mol Biol 2009 ;578:193-207

Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

The single strand conformation polymorphism (SSCP) method is a sensitive technique used to detect subtle sequence differences in PCR-amplified DNA fragments as separated peaks in electrophoretic analysis. In this chapter, we focus on SSCP analysis for quantifying polymorphic alleles rather than scanning for mutations. Short fragments carrying single nucleotide polymorphisms are amplified from individual and pooled DNA samples, then the products are labeled with fluorescent dyes and analyzed by automated capillary electrophoresis under nondenaturing conditions. Dedicated software, QSNPlite, interprets trace data of the electrophoresis to identify alleles of individuals and quantify these alleles in the pool. The software can also incorporate sequencing data to assign alleles at the nucleotide level. The procedures described here are being used in association studies that compare allele frequencies between cases and controls to identify genes responsible for common diseases.
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http://dx.doi.org/10.1007/978-1-60327-411-1_12DOI Listing
December 2009

Evaluation of haplotype inference using definitive haplotype data obtained from complete hydatidiform moles, and its significance for the analyses of positively selected regions.

PLoS Genet 2009 May 8;5(5):e1000468. Epub 2009 May 8.

Division of Genome Analysis, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

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.
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http://dx.doi.org/10.1371/journal.pgen.1000468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670534PMC
May 2009
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