A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Genetic and phenotypic assessment of sugar beet (Beta vulgaris L. subsp. vulgaris) elite inbred lines selected in Japan during the past 50 years.

Genetic diversity of Japanese sugar beet elite inbred line diversity (JSBDIV) set consisting of 63 lines was investigated using 33 cleaved amplified polymorphic sequence and 38 simple sequence repeat analyses. JSBDIV set was significantly subdivided into six (pedigree information), seven (Neighbor-Joining method) or 12 (population structure analysis) groups. The highest value of a pairwise population differentiation estimate, Φ PT value, among groups was yielded from population structure analysis with explained variation 32%. Some of the groups defined in this study exhibited close association with ancestral open-pollinated varieties (OPVs), suggesting that inter-OPV cross was rare during the establishment of JSBDIV set. On the other hand, low Φ PT values between some groups suggest that genetic backgrounds of ancestral OPVs had historically overlapped to some extent. Phenotypic traits showed significant differences both among and within groups. A nearly identical group was identified as the highest sugar content group irrespective of the grouping methods. Groups with Aphanomyces root rot resistance are associated with an OPV 'Tmm-1', suggesting it as a source of this trait. 'Tmm-1' is also associated with Cercospora leaf spot resistance, but an exceptional resistant line with no association of 'Tmm-1' supports a notion that different genetic resources exist for this trait.

Easy MPE: Extraction of Quality Microplot Images for UAV-Based High-Throughput Field Phenotyping.

Microplot extraction (PE) is a necessary image processing step in unmanned aerial vehicle- (UAV-) based research on breeding fields. At present, it is manually using ArcGIS, QGIS, or other GIS-based software, but achieving the desired accuracy is time-consuming. We therefore developed an intuitive, easy-to-use semiautomatic program for MPE called Easy MPE to enable researchers and others to access reliable plot data UAV images of whole fields under variable field conditions. The program uses four major steps: (1) binary segmentation, (2) microplot extraction, (3) production of ∗.shp files to enable further file manipulation, and (4) projection of individual microplots generated from the orthomosaic back onto the raw aerial UAV images to preserve the image quality. Crop rows were successfully identified in all trial fields. The performance of the proposed method was evaluated by calculating the intersection-over-union (IOU) ratio between microplots determined manually and by Easy MPE: the average IOU (±SD) of all trials was 91% (±3).

Identification and characterization of a semi-dominant restorer-of-fertility 1 allele in sugar beet (Beta vulgaris).

KEY MESSAGE: The sugar beet Rf1 locus has a number of molecular variants. We found that one of the molecular variants is a weak allele of a previously identified allele. Male sterility (MS) caused by nuclear-mitochondrial interaction is called cytoplasmic male sterility (CMS) in which MS-inducing mitochondria are suppressed by a nuclear gene, restorer-of-fertility. Rf and rf are the suppressing and non-suppressing alleles, respectively. This dichotomic view, however, seems somewhat unsatisfactory to explain the recently discovered molecular diversity of Rf loci. In the present study, we first identified sugar beet line NK-305 as a new source of Rf1. Our crossing experiment revealed that NK-305 Rf1 is likely a semi-dominant allele that restores partial fertility when heterozygous but full fertility when homozygous, whereas Rf1 from another sugar beet line appeared to be a dominant allele. Proper degeneration of anther tapetum is a prerequisite for pollen development; thus, we compared tapetal degeneration in the NK-305 Rf1 heterozygote and the homozygote. Degeneration occurred in both genotypes but to a lesser extent in the heterozygote, suggesting an association between NK-305 Rf1 dose and incompleteness of tapetal degeneration leading to partial fertility. Our protein analyses revealed a quantitative correlation between NK-305 Rf1 dose and a reduction in the accumulation of a 250 kDa mitochondrial protein complex consisting of a CMS-specific mitochondrial protein encoded by MS-inducing mitochondria. The abundance of Rf1 transcripts correlated with NK-305 Rf1 dose. The molecular organization of NK-305 Rf1 suggested that this allele evolved through intergenic recombination. We propose that the sugar beet Rf1 locus has a series of multiple alleles that differ in their ability to restore fertility and are reflective of the complexity of Rf evolution.

A fertility-restoring genotype of beet (Beta vulgaris L.) is composed of a weak restorer-of-fertility gene and a modifier gene tightly linked to the Rf1 locus.

Cytoplasmic male sterility (CMS) is a plant trait that involves interactions between nuclear- and mitochondrial genomes. In CMS, the nuclear restorer-of-fertility gene (Rf), a suppressor of male-sterility inducing mitochondria, is one of the best known genetic factors. Other unidentified genetic factors may exist but have not been well characterized. In sugar beet (Beta vulgaris L.), CMS is used for hybrid seed production, but few male-sterility inducing nuclear genotypes exist. Such genotypes could be introduced from a closely related plant such as leaf beet, but first the fertility restoring genotype of the related plant must be characterized. Here, we report the discovery of a Japanese leaf beet accession 'Fukkoku-ouba' that has both male-sterility inducing and fertility restoring genotypes. We crossed the leaf beet accession with a sugar beet CMS line, developed succeeding generations, and examined the segregation of two DNA markers that are linked to two sugar beet Rfs, Rf1 and Rf2. Only the Rf2 marker co-segregated with fertility restoration in every generation, implying that the Rf1 locus in leaf beet is occupied by a non-restoring allele. Fertility restoration was incomplete without a genetic factor closely linked to Rf1, leading to the assumption that the Rf1 locus encodes a modifier that cannot restore fertility by itself but perhaps strengthens another Rf. We sequenced the apparently non-restoring 'Fukkoku-ouba' rf1 gene-coding region and found that it closely resembles a restoring allele. The protein product demonstrated its potential to suppress CMS in transgenic suspension cells. In contrast, 'Fukkoku-ouba' rf1 transcript abundance was highly reduced compared to that of the restoring Rf1. Consistently, changes in protein complexes containing CMS-associated mitochondrial protein in anthers were very minor. Accordingly, we concluded that 'Fukkoku-ouba' rf1 is a hypomorph that acts as a non-restoring allele but has the potential to support another Rf, i.e. it is a modifier candidate.

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BACKGROUND: Obtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line 'NK-219mm-O' is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis. FINDINGS: Five sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat. CONCLUSIONS: Efficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.

Identification of molecular variants of the nonrestoring restorer-of-fertility 1 allele in sugar beet (Beta vulgaris L.).

KEY MESSAGE: Only three variants of nonrestoring alleles for sugar beet Rf1 were found from the US maintainer lines which were the selections from a broad range of genetic resources. Cytoplasmic male sterility is widely used for hybrid breeding of sugar beets. Specific genotypes with a nonsterility-inducing cytoplasm and a nonrestoring allele of restorer-of-fertility gene (rf) are called maintainers. The infrequent occurrence of the maintainer genotype evokes the need to diagnose rf alleles. Molecular analysis of Rf1, one of the sugar beet Rfs, revealed a high level of nucleotide sequence diversity, but three variants were tightly associated with maintainer selection in Japan. The question was raised whether this small number of variants would be seen in cases where a wider range of genetic resources was used for maintainer selection. Fifty-seven accessions registered as maintainers in the USDA germplasm collection were characterized in this study. Mitochondrial DNA types (mitotypes) of 551 plants were diagnosed based on minisatellite polymorphism. A mitotype associated with sterility-inducing (S) cytoplasm was identified in 58 plants, indicating S-cytoplasm contamination. The organization of rf1 was investigated by two PCR markers and DNA gel blot analysis. Eight haplotypes were found among the US maintainers, but subsequently two haplotypes were judged as restoring alleles after a test cross and another haplotype was not inherited by the progeny. Nucleotide sequences of rf1 regions in the remaining five haplotypes were compared, and despite the sequence diversity of the gene-flanking regions, the gene-coding regions were identified to be three types. Therefore, there are three rf1 variants in US maintainers, the same number as in the Japanese sugar beet germplasm collection. The implications of having a small repertoire of rf1 variants are discussed.

Sugar beet (Beta vulgaris L.).

Creating transgenic plants is invaluable for the genetic analysis of sugar beet and will be increasingly important as sugar beet genomic technologies progress. A protocol for Agrobacterium-mediated transformation of sugar beet is described in this chapter. Our protocol is optimized for a sugar beet genotype that performs exceptionally well in tissue culture, including the steps of dedifferentiation, callus proliferation, and regeneration. Because of the infrequent occurrence of such a genotype in sugar beet populations, our protocol includes an in vitro propagation method for germplasm preservation. The starting materials for transgenic experiments are aseptic shoots grown from surface-sterilized seed balls. Callus is induced from leaf explants and subsequently infected with Agrobacterium. Plantlets are regenerated from transgenic callus and vernalized for flowering, if necessary. The efficiency of transformation was quite high; in our laboratory, the culture of only ten leaf explants, on average, generated one transgenic plant.

Prognostic significance of pathologic complete response and Ki67 expression after neoadjuvant chemotherapy in breast cancer.

BACKGROUND: Recent studies have indicated that response to chemotherapy and the prognostic impact of a pathologic complete response (pCR) after neoadjuvant chemotherapy differ among breast cancer subtypes. METHODS: Women with Stage I to III breast cancer treated with anthracycline and taxane-based neoadjuvant chemotherapy (four cycles of docetaxel every 3 weeks followed by four cycles of FEC every 3 weeks) between 2006 and 2011 were retrospectively analyzed. Trastuzumab was concurrently added to docetaxel for HER2-positive breast cancer. Expression of estrogen receptor (ER), progesterone receptor (PgR), HER2, and Ki67 was examined by immunohistochemistry in pre- and post-treatment specimens. Predictive factors for neoadjuvant chemotherapy and prognosis were analyzed by breast cancer subtype. RESULTS: Of 64 patients, 30 (47 %) were ER-positive (ER+) HER2-negative (HER2-), including eight as luminal A (Ki67 labeling index (LI) <14 %) and 22 as luminal B (Ki67 LI ≥ 14 %) subtypes, 11 (17 %) were ER+ HER2-positive (HER2+), 12 (19 %) were ER-negative (ER-) HER2+, and 11 (17 %) were ER- HER2-. The clinical response rates were significantly higher in luminal B, ER+ HER2+, and ER- HER2+ subtypes compared with luminal A subtype. Patients whose tumors contained high Ki67 expression effectively responded to neoadjuvant chemotherapy. Ki67 LI was a predictive marker for pCR, and all patients whose tumors achieved pCR are currently disease-free. Furthermore, high Ki67 expression in post-treatment tumors was strongly correlated with poor disease-free and overall survival regardless of subtype. CONCLUSIONS: It is necessary to establish additional strategies to improve survival for patients whose residual tumors show high Ki67 expression after neoadjuvant chemotherapy.

Molecular mapping of restorer-of-fertility 2 gene identified from a sugar beet (Beta vulgaris L. ssp. vulgaris) homozygous for the non-restoring restorer-of-fertility 1 allele.

KEY MESSAGE: By genetically eliminating the major restorer - of - fertility gene ( Rf ), a weak Rf gene was unveiled. It is an allele of Z , long known as an elusive Rf gene in sugar beet. In the hybrid breeding of sugar beet, maintainer-genotype selection is a laborious process because of the dependence on test crossing, despite the very low occurrence of this genotype. Marker-assisted selection (MAS) of the maintainer genotype is highly desired by sugar beet breeders. The major restorer-of-fertility gene (Rf) was identified as Rf1, and its non-restoring allele (rf1) was discriminated at the DNA level; however, some of the rf1rf1 selections retained an as yet unidentified Rf, another target locus for MAS. The objective of this study was to identify this Rf. An rfrf1 plant was crossed to a cytoplasmic male-sterile sugar beet and then backcrossed to obtain progeny segregating the unidentified Rf. The progeny exhibited partial male-fertility restoration that was unstable in single plants. The segregation ratio of restored vs. non-restored plants suggested the involvement of a single Rf in this male-fertility restoration, designated as Rf2. We confirmed the feasibility of molecular tagging of Rf2 by identifying four shared amplified fragment length polymorphism (AFLP) fragments specific to 17 restored plants. Bulked segregant analysis also was performed to screen the Rf2-linked AFLP markers, which were subsequently converted into 17 sequence-tagged site markers. All the markers, as well two additional chromosome-IV-assigned markers, were linked to each other to form a single linkage map, on which Rf2 was located. Our data suggested that Rf2 is likely an allele of Z, long known as an elusive Rf gene in sugar beet. We also discuss the importance of Rf2 for sugar beet breeding.

An assessment of the diversity of culturable bacteria from main root of sugar beet.

The partial sequences of the 16S rRNA genes of 531 bacteria isolated from the main root of the sugar beet (Beta vulgaris L.) were determined and subsequently grouped into 155 operational taxonomic units by clustering analysis (≥99% identity). The most abundant phylum was Proteobacteria (72.5-77.2%), followed by Actinobacteria (9.8-16.6%) and Bacteroidetes (4.3-15.4%). Alphaproteobacteria (46.7-64.8%) was the most dominant class within Proteobacteria. Four strains belonging to Verrucomicrobia were also isolated. Phylogenetic analysis revealed that the Verrucomicrobia bacterial strains were closely related to Haloferula or Verrucomicrobium.

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype.

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.

Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates.

Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K(m) for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K(m) values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K(m) values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K(m) value of maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.

Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.).

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Identification and Precise Mapping of Resistant QTLs of Cercospora Leaf Spot Resistance in Sugar Beet (Beta vulgaris L.).

The complex inheritance of resistance to Cercospora leaf spot (CLS), the most severe fungal foliar disease in sugar beet, was investigated by means of quantitative trait loci (QTL) analysis. Over a three year period, recombinant inbred lines (RILs) of sugar beet (Beta vulgaris L.), generated through a cross between lines resistant ('NK-310mm-O') and susceptible ('NK-184mm-O') to CLS, were field-tested for their resistance to the pathogen. Composite interval mapping (CIM) showed four QTL involved in CLS resistance to be consistently detected. Two resistant QTL (qcr1 on chromosome III, qcr4 on chromosome IX) bearing 'NK-310mm-O' derived alleles promoted resistance. Across 11 investigations, the qcr1 and qcr4 QTL explained approximately 10% and over 20%, respectively, of the variance in the resistance index. Two further QTL (qcr2 on chromosome IV, qcr3 on chromosome VI) bearing 'NK-184mm-O' derived alleles each explained about 10% of the variance. To identify the monogenic effect of the resistance, two QTL derived from 'NK-310mm-O' against the genetic background of 'NK-184mm-O', using molecular markers. The qcr1 and qcr4 were precisely mapped as single QTL, using progenies BC(5)F(1) and BC(2)F(1), respectively. The qcr1 that was located near e11m36-8 had CLS disease severity indices (DSI) about 15% lower than plants homozygous for the 'NK-184mm-O' genotype. As with qcr1, heterozygosis of the qcr4 that was located near e17m47-81 reduced DSI by about 45% compared to homozygosis. These two resistant QTL might be of particular value in marker-assisted selection (MAS) programs in CLS resistance progression.

[Safety and tolerance of docetaxel (especially 75 mg/m2) with cyclophosphamide (TC therapy) as adjuvant chemotherapy for Japanese patients with operable breast cancer].

The safety and tolerance of docetaxel (especially 75 mg/m/2) plus cyclophosphamide (600 mg/m2) (TC therapy) as adjuvant chemotherapy for Japanese patients (n=47) with operable breast cancer was evaluated. Among 47 patients who received TC therapy, 38 patients (80.8%) were treated as scheduled for 4 courses every three weeks, and 6 patients (12.8%) were dose-reduced or the interval between courses was extended due to neutropenia or wound infection. Three patients (6.4%) quit TC therapy due to stomatitis or skin toxicities of grade 3. Although febrile neutropenia of grade 3 or 4 was observed in 2 patients (4.3%), TC therapy could be prescribed for these patients. Non-hematological side effects(such as edema, myalgia and arthralgia), occurred in relatively many cases, but were not remarkable. From these results, TC therapy (75/600 mg/m2) is considered to be a safe and tolerable regimen in Japanese patients operated for breast cancer.

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet.

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants' phenotypic and molecular markers' segregation was carried out. At a highly infested field site, some 187 F(2) and 66 F(3) individuals, derived from a cross between lines 'NK-310mm-O' (highly resistant) and 'NK-184mm-O' (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 'NK-310mm-O'-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F(2) population (in the year 2002) and 65% in F(3) lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.

[Efficacy and toxicity of combination treatment with epirubicin (EPI) plus docetaxel (DOC) in advanced breast cancer].

Thirty women (mean age 50.4 years, range 31-63) with primary advanced breast cancer were given EPI 40-60 mg/m(2) and DOC 50-60 mg/m(2) intravenously every three weeks. The efficacy was evaluated after 4 cycle treatments. There were 5 complete responses (CR) and 14 partial responses (PR), giving an overall response rate of 63.3%. There were 2 pathological CR (8%) which showed complete disappearance of cancer cells. The high dose group showed a better response than the low-dose group. The most common grade 3/4 adverse events were neutropenia (26.7%) and general fatigue (6.7%). The simultaneous combination treatment of EPI and DOC is effective for primary chemotherapy and can be performed safely even for outpatients.