Takehiro Matsubara

Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer

Cell‐free circulating tumor DNA (ctDNA) in serum has been considered to be a useful candidate for noninvasive cancer diagnosis. The current study was designed to estimate the clinical usefulness of genetic analysis for ctDNA by digital polymerase chain reaction in patients with pancreatic cancer.

Droplet digital PCR measurement of HER2 in patients with gastric cancer

Background:Although there are some new criteria for human epidermal growth factor receptor 2 (HER2) expression with immunohistochemistry/fluorescence in situ hybridisation (IHC/FISH) in gastric cancer, the method is still ambiguous and is somewhat dependent on the subjective qualities of the evaluator.Methods:We used droplet digital polymerase chain reaction (ddPCR) to evaluate HER2 amplification in formalin-fixed and paraffin-embedded (FFPE) samples and cell-free serum circulating tumour DNA (ctDNA) in 25 patients with gastric cancer.Results:The concordance rate of HER2 amplification examined in FFPE samples with ddPCR and IHC/FISH was 92% (23 out of 25). The concordance rate of FFPE with ctDNA was not high (62.5%); however, patients who were HER2-positive by ctDNA had significantly shorter survival compared with HER2-negative patients.Conclusions:Our results demonstrated that this ddPCR method was as effective as IHC/FISH and therefore might become a standard method for analysing not only FFPE but also ctDNA.

Low frequency of drug-resistant virus did not affect the therapeutic efficacy in daclatasvir plus asunaprevir therapy in patients with chronic HCV genotype-1 infection.

BACKGROUND The efficacy of a direct-acting antiviral agent (DAA) is compromised by the development of drug resistance. The associations between resistance-associated virus (RAV) and therapeutic outcomes have not been well-understood. METHODS A total of 30 patients with HCV genotype-1b were enrolled and treated for 24 weeks with asunaprevir (ASV) and daclatasvir (DCV). Viral sequences in non-structural (NS) regions 3 and 5A in serum and liver tissue before treatment were examined with direct sequencing, next-generation sequencing (NGS) and the PCR-invader method to evaluate the importance of drug-resistance in the prediction of the outcomes of ASV plus DCV therapy. RESULTS Of 30 patients (22 treatment-naive patients, 2 interferon-intolerant patients and 6 non-responders), 25 patients (83.3%) achieved sustained virological response (SVR) 24 weeks after the treatment. Viral breakthrough occurred in three treatment-naive patients and one non-responder. One treatment-naive patient experienced viral relapse. Among 25 patients without RAV, 24 obtained SVR, whereas 5 patients had RAV with a 1.3 to 88% frequency, resulting in various therapeutic outcomes. As for HCV compartments, similar RAVs were detected in serum and liver tissue for a patient obtaining SVR despite HCV NS5A Y93H and another developed viral breakthrough although no RAV was detected. Direct sequencing could not detect RAVs in low frequency (1.3 to 12%) for three of four patients. CONCLUSIONS Low frequency of RAVs might not affect the outcomes of ASV plus DCV therapy. Deep sequencing and PCR-invader methods can detect clinically significant RAVs for ASV plus DCV therapy.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods

Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

Relapsed infant MLL-rearranged acute lymphoblastic leukemia with additional genetic alterations

Acute lymphoblastic leukemia (ALL) in infants is a rare and aggressive disease, and has poor prognosis. Approximately 80% of patients with infant ALL harbor a mixed lineage leukemia (MLL, also known as KMT2A) gene rearrangement, which is a strong independent predictor of poor outcome.1,2 Although MLL rearrangement is considered a strong driver mutation, it may not be sufficient for leukemogenesis.3 We report two relapsed cases of infant ALL with MLL rearrangement. Case 1 is a 3-month-old female who was diagnosed as infant ALL with MLL-ENL, and Case 2 is a 4-month-old male who was diagnosed as infant ALLwithMLL-AF4. Both patients experienced a relapse twice. Somatic DNA was obtained from bone marrow at diagnosis and each episode of relapse, while germline DNA was obtained from buccal swab. Targeted sequencing was performed on MiSeq (Illumina, San Diego, CA) using HaloPlex custom panels (Agilent, Santa Clara, CA), with 163 known mutated genes in hematological diseases, which are customized to detect cancer-related genes (Supplementary Table S1). Read alignment and variant calling were performed using the MiSeq reporter (Illumina), and variants were annotated using SureCall software (Agilent). Synonymous or noncoding variants were excluded, and single nucleotide polymorphisms (SNPs) and germline polymorphisms were reported to dbSNP, unless they were found in the COSMIC database. Among the remaining variants with a variant allele frequency higher than 0.1, recurrentlymutated genes reported in a larger cohort4 were considered as candidate genes and validated by conventional Sanger sequencing. Sanger sequencingwasperformedafterPCR using specific primers, and mutations were detected by ABI PRISM 3100 (Thermo Fisher Scientific,Waltham,MA) using the primers listed in Supplementary Table S1. Thus, we identified additional gene alterations (Fig. 1). Only two mutations were detected in diagnostic samples, indicating that small number of mutations cooperate with MLL rearrangement. Among them, mutations in KRAS and FLT3 have also been recurrently reported in a study by Andersson et al. using wholegenome or whole-exome sequencing .4 To date, their study has been the largest describing the mutational profile of infant ALL. It is notable that many mutations other than those involved in the tyrosine kinasePI3K-RAS signaling pathways, which are found in approximately half of the patients, are found with low frequency. We could monitor our

Simultaneous detection of ABL1 mutation and IKZF1 deletion in Philadelphia chromosome‐positive acute lymphoblastic leukemia using a customized target enrichment system panel

Recent clinical outcomes of pediatric Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ALL) vastly improved owing to tyrosine kinase inhibitor (TKI). However, the genetic status would be different in each case with ABL1 gene mutation or copy number variants (CNVs) such as IKZF1 deletion. In particular, the TKI resistant clone with ABL1 kinase mutation remains problematic. The comprehensive assessment of genetic status including mutation, insertion and deletion (indel) and CNVs is necessary.

Comparative mutational evaluation of multiple lung cancers by multiplex oncogene mutation analysis

In patients presenting with synchronous or metachronous multiple lung cancer (MLC), it is important to distinguish between multiple primary lung cancer (MP) and intrapulmonary metastasis (IM). The present study was aimed at investigating the mutational profiles of synchronous/metachronous MLC and to compare the classification of paired tumors by multiplex gene mutation analysis with the histopathological evaluation. We carried out targeted sequencing of 20 lung cancer‐related oncogenes using next‐generation sequencing (NGS) in 82 tumors from 37 MLC patients who underwent surgical resection at our department. The patients were diagnosed as MP or IM cases based on the Martini and Melamed criteria, histopathological and gene mutational evaluations. Matching mutations between paired tumors was observed in 20 (54%) patients, who were diagnosed as IM cases by mutational evaluation. Patients who could not be clearly diagnosed by histopathological evaluation were classified as equivocal cases. Among the histopathological IM cases (n = 7), six (86%) were confirmed as IM cases also by mutational evaluation, and most of the paired tumors of these cases (n = 5) harbored multiple matching mutations. Among the histopathological MP cases (n = 17), mutational evaluation yielded a discordant diagnosis in eight (47%) cases. Of these, the paired tumors of four cases harbored multiple matching mutations, suggesting that the mutational diagnosis might be more suitable in these patients. Our findings suggest that multiplex mutational analysis could be a useful complementary tool for distinguishing between MP and IM in addition to histopathological evaluation.

PIK3R1Met326Ile germline mutation correlates with cysteine-rich protein 61 expression and poor prognosis in glioblastoma

Despite therapeutic advances, glioblastoma represents a lethal brain tumor. Recently, research to identify prognostic markers for glioblastoma has intensified. Our previous study demonstrated that median progression-free survival (PFS) and overall survival (OS) of patients with high cysteine-rich protein 61 (CCN1) expression was significantly shorter than that of patients with low CCN1 expression. To understand the molecular mechanisms that regulate CCN1 expression, we examined 147 tumour samples from 80 patients with glioblastoma and 67 patients with lower grade glioma. Next-generation and Sanger sequencing showed that PIK3R1Met326Ile was more frequent in the CCN1 high expression group (10/37 cases, 27.0%) than the CCN1 low expression group (3/38 cases, 7.9%) in glioblastoma. This mutation was also detected in corresponding blood samples. In multivariate analysis, high CCN1 expression and PIK3R1Met326Ile in glioblastoma patients were prognostic factors for OS [HR = 2.488 (1.298–4.769), p = 0.006] and [HR = 2.089 (1.020–4.277), p = 0.0439], respectively. Thus, the PIK3R1Met326Ile germline appears to be correlated with CCN1 expression and poor prognosis in glioblastoma.

Draft Genome Sequence of Bifidobacterium lemurum DSM 28807T Isolated from the Gastrointestinal Tracts of Ring-Tailed Lemurs (Lemur catta)

ABSTRACT Bifidobacterium lemurum DSM 28807T was isolated from the gastrointestinal tracts of ring-tailed lemurs (Lemur catta). Here, we report the first draft genome sequence of this organism.

Draft Genome Sequence of Probiotic Lactobacillus acidophilus Strain L-55 Isolated from a Healthy Human Gut

ABSTRACT Probiotic Lactobacillus acidophilus L-55 was isolated from a healthy human gut. Here, we report the draft genome sequence of this organism.