Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

Plant-Specific Domains and Fragmented Sequences Imply Non-Canonical Functions in Plant Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein translation. In addition, numerous aaRSs (mostly in vertebrates) have also been discovered to possess a range of non-canonical functions. Very few studies have been conducted to elucidate or characterize non-canonical functions of plant aaRSs. A genome-wide search for aaRS genes in Arabidopsis thaliana revealed a total of 59 aaRS genes. Among them, asparaginyl-tRNA synthetase (AsnRS) was found to possess a WHEP domain inserted into the catalytic domain in a plant-specific manner. This insertion was observed only in the cytosolic isoform. In addition, a long stretch of sequence that exhibited weak homology with histidine ammonia lyase (HAL) was found at the N-terminus of histidyl-tRNA synthetase (HisRS). This HAL-like domain has only been seen in plant HisRS, and only in cytosolic isoforms. Additionally, a number of genes lacking minor or major portions of the full-length aaRS sequence were found. These genes encode 14 aaRS fragments that lack key active site sequences and are likely catalytically null. These identified genes that encode plant-specific additional domains or aaRS fragment sequences are candidates for aaRSs possessing non-canonical functions.

Phase I study of the indoleamine 2,3-dioxygenase 1 inhibitor navoximod (GDC-0919) as monotherapy and in combination with the PD-L1 inhibitor atezolizumab in Japanese patients with advanced solid tumours.

Navoximod (GDC-0919) is a small molecule inhibitor of indoleamine-2,3-dioxygenase 1. This study investigated the safety, tolerability and pharmacokinetics of navoximod alone and in combination with atezolizumab in Japanese patients with advanced solid tumours. This was a phase I, open-label, dose-escalation study. Patients received monotherapy with navoximod 400 mg, 600 mg or 1000 mg orally twice daily (BID) in Stage 1 and navoximod 200 mg, 400 mg, 600 mg or 1000 mg orally BID plus atezolizumab 1200 mg intravenously every 21 days in Stage 2. Objectives included safety, tolerability, efficacy and pharmacokinetic outcomes.Overall, 20 patients were enrolled (Stage 1: n = 10; Stage 2: n = 10). No dose-limiting toxicities were observed. In Stage 1, treatment-related adverse events (TRAEs) of any grade that occurred in ≥20% of patients were chromaturia (50%) and maculopapular rash (20%). Grade ≥ 3 TRAEs were reported in two patients (20%; maculopapular rash and lipase increased). In Stage 2, TRAEs that occurred in ≥30% of patients were chromaturia (60%) and, decreased appetite (40%). Grade ≥ 3 TRAEs were reported in three patients (30%; hyponatraemia, aspartate aminotransferase increased, alanine aminotransferase increased, lymphopaenia and neutropaenia). Stable disease was observed in five patients (50%) in Stage 1 and eight patients (80%) in Stage 2. Navoximod showed linear pharmacokinetics. The recommended dose of navoximod monotherapy was determined as 1000 mg orally BID, and could be considered 1000 mg orally BID in combination with atezolizumab. Navoximod as monotherapy and in combination with atezolizumab was well tolerated in Japanese patients with advanced solid tumours.

Amino acids in conserved region II are crucial to substrate specificity, reaction velocity, and regioselectivity in the transglucosylation of honeybee GH-13 α-glucosidases.

Honeybees, Apis mellifera, possess three α-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-III preferred sucrose to maltose as substrate and formed only α-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the α-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) α-glucosidases and related enzymes.

Total biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α: heterologous expression of four biosynthetic genes in Aspergillus oryzae.

Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions. The resulting quadruple transformant carrying the genes of geranylgeranyl diphosphate synthase PbGGS, terpene synthase PbACS, and two monooxygenases (PbP450-1 and PbP450-2) produced aphidicolin. The double and triple transformants also respectively produced aphidicolan-16ß-ol and 3-deoxyaphidicolin. Alternative host Saccharomyces cerevisiae carrying the genes, PbGGS and PbACS, produced key intermediate aphidicolan-16ß-ol. This is the first example of a total biosynthesis of terpenoids using fungal hosts.

Sensitive and convenient yeast reporter assay for high-throughput analysis by using a secretory luciferase from Cypridina noctiluca.

The yeast reporter assay has been widely used in various applications such as detection of endocrine disruptors and analysis of protein-protein interactions by the yeast two-hybrid system. The molecular characteristics of the reporter enzyme are critical determinants for this assay. We herein report the establishment of a novel yeast reporter assay using a secretory luciferase, Cypridina noctiluca luciferase (CLuc), as an alternative to the conventional beta-galactosidase. The CLuc reporter assay in yeast is more sensitive and convenient than the conventional assay. A yeast high-throughput reporter assay was established with a laboratory automation system, and the transcriptional activity of hundreds of yeast promoter fragments was comprehensively determined. Our results indicate that the yeast CLuc reporter assay is a promising tool for large-scale and sensitive analysis in the development of new drugs and in various fields of biotechnology research.

Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins.

Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of the disruption of yeast-specific glycosyltransferases. This decreased protein productivity in engineered yeast strains is an obstacle to the development of efficient glycoprotein production in yeast. For economic and effective synthesis of such therapeutic glycoproteins in yeast, the development of appropriate strains is highly desirable. We applied a novel mutagenesis technique that utilized the proofreading-deficient DNA polymerase delta variant encoded by the pol3-01 gene of Saccharomyces cerevisiae or the cdc6-1 gene of Schizosaccharomyces pombe to the engineered S. cerevisiae TIY20 strain and S. pombe KT97 strain, respectively. TIY20, which is deficient in the outer chain of mannan due to the disruption of three genes (och1Delta, mnn1 Delta, mnn4 Delta), and KT97, which is an och1 disruptant, are impractical as hosts for the production of therapeutic glycoproteins since they show a temperature-sensitive (ts) phenotype, a growth defect phenotype, and decreased protein productivity. We successfully isolated YAB mutants that alleviated the growth defect of the TIY20 strain. Surprisingly, these mutants generally secreted foreign proteins better than the wild-type strain. Furthermore, we successfully isolated YPAB mutants that alleviated the growth defect of the KT97 strain, too. The development of these new mutants by the combination of genetic engineering of yeast and this mutagenesis technique are major breakthroughs for the production of therapeutic glycoproteins in engineered yeast cells.

Inactivation of recombinant human steroid sulfatase by KW-2581.

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC(50) of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner (k(inact), 0.439 min(-1); K(i, app), 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.

Novel reporter gene expression systems for monitoring activation of the Aspergillus nidulans HOG pathway.

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a beta-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogADelta. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogADelta mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.

MpkA-Dependent and -independent cell wall integrity signaling in Aspergillus nidulans.

Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Delta and mpk1Delta mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Delta and swi6Delta mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmADelta, Answi4Delta Answi6Delta, and mpkADelta strains) and analyzed mpkA and CWG transcripts after treatment with a beta-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The beta-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkADelta strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except alpha-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models.

We screened a series of 17beta-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives, and describe here a potent and selective steroid sulfatase (STS) inhibitor with antitumor effects in breast cancer models in vitro and in vivo. In biochemical assays using crude enzymes isolated from recombinant Chinese hamster ovary cells expressing human arylsulfatses (ARSs), one of the best compounds, KW-2581, inhibited STS activity with an IC(50) of 4.0 nM, while > 1000-fold higher concentrations were required to inhibit the other ARSs. The failure to stimulate the growth of MCF-7 human breast cancer cells as well as in uteri in ovariectomized rats indicated the lack of estrogenicity of this compound. In MCF-7 cells transfected with the STS gene, termed MCS-2 cells, KW-2581 inhibited the growth of cells stimulated by estrone sulfate (E1S) but also 5-androstene-3beta, 17beta-diol 3-sulfate (ADIOLS) and dehydroepiandrostenedione 3-sulfate. We found that oral administration of KW-2581 inhibited both E1S- and ADIOLS-stimulated growth of MCS-2 cells in a mouse hollow fiber model. In a nitrosomethylurea-induced rat mammary tumor model, KW-2581 induced regression of E1S-stimulated tumor growth as effectively as tamoxifen or another STS inhibitor, 667 Coumate. Dose-response studies in the same rat model demonstrated that more than 90% inhibition of STS activity in tumors was necessary to induce tumor shrinkage. STS activity in tumors has well correlated with that in leukocytes, suggesting that STS activity in leukocytes could be used as an easily detectable pharmacodynamic marker. These findings demonstrate that KW-2581 is a candidate for development as a therapeutic agent for the treatment of hormone receptors-positive breast cancer.

Inhibition of steroid sulfatase activity and cell proliferation in ZR-75-1 and BT-474 human breast cancer cells by KW-2581 in vitro and in vivo.

In the present study, we found that two hormone receptor-positive human breast cancer cell lines, ZR-75-1 and BT-474, naturally expressed steroid sulfatase (STS) protein and had catalytic activity to produce estrone from estrone sulfate (E1S) with a comparable level to those in human breast cancer tissues. E1S at physiological concentrations stimulated the growth of those cells. A novel steroidal STS inhibitor, KW-2581 inhibited the STS activity of ZR-75-1 cells with an IC(50) of 13 nM, a potency equal to or higher than that of the non-steroidal STS inhibitor, 667 COUMATE. The inhibitory effect of KW-2581 was enhanced by pre-incubation with STS enzyme, suggests being irreversible inhibition. KW-2581 inhibited the E1S-stimulated growth of ZR-75-1 cells with an IC(50) of 0.18 nM, but failed to inhibit the growth stimulated by 17beta-estradiol. Expression of E1S-induced progesterone receptors in ZR-75-1 cells was reduced by treatment of KW-2581 at concentrations as low as 0.1 nM. Oral administration of KW-2581 for 4 weeks caused tumor shrinkage in a mouse xenograft model. Tumor STS activity had been completely (>95%) eliminated by 24 hours after the last administration. These findings suggest that KW-2581 has considerable potential for therapeutic development as a novel anti-hormonal drug for treatment of breast cancer.

Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA.

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

Simulation of aortic peak enhancement on MDCT using a contrast material flow phantom: feasibility study.

OBJECTIVE: The objective of our study was to develop a flow phantom simulating aortic peak enhancement after the injection of contrast material on CT and to investigate the validity of the flow phantom by comparing the time-enhancement curves obtained for the flow phantom and humans. MATERIALS AND METHODS: We developed a flow phantom simulating the enhancement pattern of the aorta after the injection of contrast material. In protocols 1, 2, and 3 of the phantom study, 90, 102, and 150 mL of iohexol, respectively, was administered over 35 sec. In protocol 4, 102 mL of iohexol was administered over 25 sec. In phantom protocols 1', 2', and 3', the dose and contrast injection duration were the same as in protocols 1, 2, and 3; however, saline (10 mL) was injected during the 20 sec after contrast delivery. In the human study, 20 patients were randomized into four groups: Groups A, B, and C received 1.5, 1.7, and 2.5 mL of iohexol per kilogram of body weight, respectively, over 35 sec; and group D received 1.7 mL/kg over 25 sec. In patient groups A, B, C, and D, phantom protocols 1, 2, 3, and 4 were used, respectively. Single-level serial CT scans were obtained using a 16-MDCT scanner on the simulated and real aortas after the injection of contrast material. Time-enhancement curves of simulated and real aortas were generated, and aortic peak times and aortic peak enhancement values were calculated. RESULTS: Aortic peak enhancement and aortic peak times in protocols 1-4 and 1'-3' of the phantom study were 2-8% larger and 6-18% longer, respectively, than in the corresponding patient study. The shape of the time-enhancement curves before aortic peak time in protocols 1-3 and 1'-3' of the phantom study closely resembled that of the corresponding patient study. After the aortic peak time, the shape of time-enhancement curves in protocols 1, 2, and 3 of the phantom study was different from the corresponding patient study; however, it was similar in phantom protocols 1'-3' and the corresponding patient study. In all four phantom protocols, the difference between maximal and minimal aortic peak enhancement was less than the SD of the corresponding patient study. CONCLUSION: The level of peak aortic enhancement and the time to peak aortic enhancement were similar in the phantom and human studies when we used our different contrast injection protocols for MDCT.

Radiation dose reduction without degradation of low-contrast detectability at abdominal multisection CT with a low-tube voltage technique: phantom study.

PURPOSE: To reduce radiation dose from abdominal computed tomography (CT) without degradation of low-contrast detectability by using a technique with low tube voltage (90 kV). MATERIALS AND METHODS: The institutional review board approved the participation of the radiologists in the observer performance test, and informed consent was obtained from all participating radiologists. A phantom for measurement of the radiation dose and a phantom containing low-contrast objects were scanned with a 16-detector row CT scanner at 120 kV and 90 kV. For determination of the radiation dose at both 90 kV and 120 kV, the tube current-time product settings were 100-560 mAs, and the doses at the center and periphery of the phantom were measured. To assess low-contrast detectability, we used a 300-mAs setting at 120 kV and 250-560-mAs settings at 90 kV. Five observers participated in the receiver operating characteristic analysis. Area under the receiver operating characteristic curve (A(z)) values were calculated in each observer. A(z) values obtained with each of the scanning techniques were recorded, and differences were examined for significance by using the Dunnet method. RESULTS: The mean A(z) value was 0.951 at 120 kV and 300 mAs. A(z) values were 0.927-0.973 at 90 kV and 450-560 mAs, and the differences between those values and values obtained at 120 kV and 300 mAs were not significant (P = .937-.952). A value of 100% was assigned to the radiation dose delivered to the center of the phantom at 120 kV and 300 mAs. The relative dose delivered at 90 kV ranged from 65% at 450 mAs to 79% at 560 mAs. CONCLUSION: A reduction from 120 kV to 90 kV led to as much as a 35% reduction in the radiation dose, without sacrifice of low-contrast detectability, at CT.

Abdominal CT with low tube voltage: preliminary observations about radiation dose, contrast enhancement, image quality, and noise.

PURPOSE: To prospectively investigate the effect of low tube voltage on radiation dose, contrast enhancement, image quality, and image noise at abdominal dynamic computed tomography (CT). MATERIALS AND METHODS: The institutional review board approved this study. Prior informed consent was obtained from all patients. Forty patients (24 women, 16 men; mean age, 62 years) underwent initial abdominal CT at 120 kV with 100 mL of contrast material (protocol A). Then all patients were randomly assigned to one of two protocols (protocol B, CT at 90 kV with 100 mL contrast material; protocol C, CT at 90 kV with 80 mL contrast material). The CT numbers of their abdominal organs were assessed quantitatively and qualitatively. Statistical analysis was performed by using the two-tailed paired t test, Kruskal-Wallis test, and kappa test of interobserver agreement. The radiation dose was measured with a phantom that consisted of glass-rod dosimeters. RESULTS: Quantitative analysis revealed that protocols B and C yielded significantly better enhancement of the aorta, liver, pancreas, spleen, and kidney than did protocol A (P < .05). With qualitative analysis, the difference among the three protocols in regard to image quality was not significant. At 90 kV versus 120 kV, the radiation dose reduction in the center of the phantom was 56.8% (6.3 vs 14.6 mGy); in the periphery, it was 46.2% (13.6 vs 25.3 mGy). CONCLUSION: By decreasing the tube voltage, the amount of contrast material can be reduced without image quality degradation. In scans obtained with a low tube voltage, the radiation dose can be reduced as much as 56.8%, and higher contrast material enhancement can be achieved.

The use of contrast-enhanced computed tomography before neoadjuvant chemotherapy to identify patients likely to be treated safely with breast-conserving surgery.

OBJECTIVE: To select suitable candidates for breast-conserving treatment (BCT) after neoadjuvant chemotherapy (NAC), based on the classification of tumors into localized or diffuse types using contrast-enhanced computed tomography (CE-CT). SUMMARY BACKGROUND DATA: A relatively high rate of loco-regional failure after BCT has been reported with breast cancer downstaged by NAC. Accurate assessment of the suitability of BCT and the response to NAC, before the initiation of NAC, will allow the optimal selection of an appropriate therapeutic course. METHODS: We evaluated 110 consecutive patients with operable breast carcinomas measuring 3-cm or more in diameter by CE-CT after NAC treatment with doxorubicin and docetaxel at National Cancer Center Hospital, Tokyo, from May 1998 to November 2001. Lesions were classified as either localized or diffuse types by mammography (MMG), ultrasonography (US), and CE-CT. RESULTS: Tumors designated as localized type by MMG, US, and CE-CT were reduced to tumors less than 3.0 cm (P < 0.0001) in a concentric circle (P < 0.0001). Localized tumors by CE-CT were treated safely with BCT maintaining a negative margin status (P = 0.01). In contrast, diffuse type tumors shrunk into a mosaic pattern consisting of tumors larger than 3.1 cm. Tumors classified as localized by CE-CT responded better pathologically than diffuse tumors (P = 0.0365). Multivariate analysis demonstrated that morphologic type by CE-CT and histologic type were significant predictors of candidates for safe BCT. CONCLUSIONS: The classification of tumors into either localized or diffuse types, using CE-CT before NAC administration, accurately predicts which tumors will be suitable candidates for BCT after NAC.