[Diagnosis and Treatment of HBOC Syndrome by a Breast Surgical Oncologist].

Of all breast cancer cases, 5-10% occur because of inherited germline mutation. For hereditary breast and ovarian cancer (HBOC)syndrome, congenital knowledge and strategies for breast cancer treatment and risk reduction are necessary. Regarding the surgical procedure for the cancer site, the ipsilateral breast tumor recurrence rate following breast-conserving surgery in breast cancers with BRCA1/2 mutation is not significantly higher than that in sporadic breast cancers. In addition, prognosis did not differ according to surgical methods. Therefore, breast-conserving surgery for HBOC is not an absolute contraindication, although the risk of developing new primary tumors in the long term should be carefully considered. For the contralateral breast, 3 choices are available, surveillance, chemo-prevention, and risk-reducing mastectomy. Risk-reducing mastectomy is known to decrease the risk of breast cancer by 90%. Genetic counseling is essential for decision making in HBOC treatment. In this review, we reevaluated the current evidence for surgical decision making and systemic therapies for HBOC syndrome.

A model to predict upstaging to invasive carcinoma in patients preoperatively diagnosed with ductal carcinoma in situ of the breast.

BACKGROUND: The aims of this study were to determine clinicopathological factors associated with postoperative upstaging to invasive carcinoma in patients preoperatively diagnosed with ductal carcinoma in situ (DCIS) and to develop a model to predict the risk of upstaging. METHODS: Pre- and post-operative pathological diagnoses and radiological findings were assessed for 1,187 consecutive patients. RESULTS: Of the patients, 306 (25.8%) were upstaged on the surgical specimen. In multivariate analysis, the following four factors were significantly associated with upstaging: 1) the presence of sclerosing adenosis on the preoperative biopsy specimen (odds ratio [OR] 0.46, P = 0.013); 2) pleomorphic calcifications on the mammogram (OR 1.68, P = 0.009); 3) a mass suspicious for invasive carcinoma on ultrasonography and/or MRI (OR 2.13, P < 0.001); 4) tumor size ≥2 cm on ultrasonography (OR 1.80, P = 0.032). HER2-positive (OR 1.54, P = 0.062) and comedo necrosis (OR 1.42, P = 0.056) demonstrated a trend towards significance. A prediction model incorporating these variables demonstrated that the risk of upstaging was 5.1% with score 0-2 and was 58.1% with score 10. CONCLUSIONS: The prediction model incorporating clinicopathological features may be used to guide the selection of patients with DCIS for sentinel lymph node biopsy.

MRI and ultrasound evaluation of invasive lobular carcinoma of the breast after primary systemic therapy.

AIM: To assess magnetic resonance imaging (MRI) and ultrasound (US) evaluation of invasive lobular carcinoma after primary systemic therapy compared to the primary surgery group. METHODS: A total of 218 breasts with invasive lobular carcinoma (ILC), which were diagnosed by core needle biopsy and underwent surgery between 2004 and 2010 in St. Luke's International Hospital, were reviewed. Of these, 44 breasts received primary systemic therapy (PST) and the residual 174 breasts underwent surgery first. Before surgery, MRI and second-look US were performed. Patients whose extent of lesion from MRI and US was over 90° were recommended to receive total mastectomy. Diagnostic image evaluation was categorized from the final pathology. A negative margin of breast-conserving surgery (BCS) and mastectomy which has over 90° tumor extent in pathology were regarded as "appropriate". A positive margin of BCS was regarded as "underestimated". "Overestimation" was determined when the pathological extension was small enough for BCS although MRI and US diagnosis was over 90°. RESULTS: Of 44 breasts which received PST, 26 breasts received BCS as initial surgery. Of these, 14 (53.8 %) breasts were "underestimated". Of these 14, 5 breasts received additional total mastectomy. Of 174 breasts that received surgery first, 121 (69.5 %) breasts received BCS as the initial surgery. Of these, 46 (38.0 %) breasts were "underestimated" and 6 breast of these 46 received additional total mastectomy. "Underestimated" rate was similar in the two groups, but the additional total mastectomy rate was significantly higher in the PST group (p = 0.025). There were no "overestimations". The "appropriate" rate was similar between the PST (68.2 %) and surgery-first groups (73.6 %). CONCLUSIONS: Although 68.2 % of ILC patients were evaluated "appropriately" even after PST, higher additional total mastectomy rates should be considered when selecting the surgical procedure.

[Problems with topical use of Mohs paste for local recurrence of breast cancer].

Recently, several reports have been made concerning topical use of Mohs paste for local recurrence of breast cancer tumors. Since January 2008, after the introduction of Mohs paste for palliation in patients with local recurrences of breast cancer, we have encountered some problems such as pain, draining off of the paste and ulceration of the lesion. In this study, we have reported the advantages and disadvantage of using Mohs paste, the difference in the procedure depending on the purpose, and the management of problems. When using Mohs paste for the purpose of palliative therapy, these side effects are problems we should not ignore to prevent patient pain.

Direct ethanol production from barley beta-glucan by sake yeast displaying Aspergillus oryzae beta-glucosidase and endoglucanase.

Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucosidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GRI-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation. The conversion ratio of ethanol from beta-glucan was 69.6% of the theoretical ethanol concentration produced from 20 g/l barley beta-glucan. These results showed that sake yeast displaying A. oryzae cellulolytic enzymes can be used to produce ethanol from cellulosic materials. Our constructs have higher ethanol production potential than the laboratory constructs previously reported.

Alteration of substrate specificity of rat neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation.

The substrate specificity of rat brain neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the neurolysin S2' subsite, were individually and comprehensively substituted. The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2' amino acid position. Among mutant neurolysins, the Y610L mutant neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of neurolysin is the important residue to recognize the P2' amino acid of substrates.

Efficient and direct fermentation of starch to ethanol by sake yeast strains displaying fungal glucoamylases.

Aspergillus oryzae glucoamylases encoded by glaA and glaB, and Rhizopus oryzae glucoamylase, were displayed on the cell surface of sake yeast Saccharomyces cerevisiae GRI-117-UK and laboratory yeast S. cerevisiae MT8-1. Among constructed transformants, GRI-117-UK/pUDGAA, displaying glaA glucoamylase, produced the most ethanol from liquefied starch, although MT8-1/pUDGAR, displaying R. oryzae glucoamylase, had the highest glucoamylase activity on its cell surface.

Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain.

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.

Yeast cell-surface expression of chitosanase from Paenibacillus fukuinensis.

To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.

Development of high-throughput screening system by single-cell reaction using microchamber array chip.

To demonstrate the practical use of a novel high-throughput screening system by a single cell reaction for the rapid screening of combinatorially mutated beta-glucosidase 1 from Aspergillus oryzae, a yeast cell chip microchamber array in combination with yeast cell surface engineering was successfully developed.

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface.

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.

Enhancement of substrate recognition ability by combinatorial mutation of beta-glucosidase displayed on the yeast cell surface.

Recently, in family 3 beta-glucosidase (BGL), the catalytically important Asp nucleophile has been identified in the SDW segment of the SDWG sequence by site-directed mutagenesis. However, the details about the roles of each amino acid residue of the SDWG sequence have not been investigated. W293 of the SDW segment, which is the residue next to the nucleophile (D292) in family 3 BGL, is very important for hydrolytic reaction as a binder to a substrate. G294 of the SDWG sequence might play an important role in catalysis. In this study, to obtain a functional BGL1 mutant by the substitution of G294 using a genetic engineering method, the library of mutant BGL1 from Aspergillus oryzae was rapidly constructed by yeast cell surface engineering, and the hydrolytic activities of mutants were comprehensively detected. Consequently, G294F, G294W, and G294Y, in which G was substituted with aromatic amino acids, showed higher activities for substrate recognition than the parent strain (1.5-, 1.5-, and 1.6-fold, respectively). These results suggest the presence of some interaction between the sugar rings and aromatic ring of W293 at the entrance of the catalytic pocket, which enhances the substrate recognition of beta-glucosidase.