Eiichi Shiba

Endoscopic thyroid surgery through the axillo-bilateral-breast approach.

&NA; We developed a new endoscopic thyroid surgery by the axillo‐bilateralbreast approach (ABBA) method, which is different from the previously described breast approach (BA) in that the port sites are modified to obtain a better view and to prevent the interference of surgical instruments. This modification also improves cosmetic results by eliminating the parasternal incision, which results in hypertrophic scar in a significant number of cases treated with BA. Twelve patients with benign thyroid tumors successfully underwent endoscopic thyroid surgery by ABBA, and their clinical outcomes were compared with those of four patients treated with BA. The mean operation time was significantly shorter in the ABBA group than in the BA group (188 minutes vs. 270 minutes; P < 0.01). Furthermore, the mean blood loss in the ABBA group (53 mL) was half of that in the BA group (108 mL). Neither conversion to open surgery nor significant intraoperative complications were experienced. The operative scars by ABBA became inconspicuous in a few weeks. These results seem to indicate that ABBA is a better method than BA and can be a feasible option, particularly for young patients who opt for the better cosmetic outcome.

Involvement of up-regulation of 17β-hydroxysteroid dehydrogenase type 1 in maintenance of intratumoral high estradiol levels in postmenopausal breast cancers

Estradiol (E2) and estrone (E1) levels as well as mRNA expression levels of aromatase, sulfatase and 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) in breast cancer tissues were studied to elucidate the mechanism involved in the maintenance of the intratumoral high E2 levels in postmenopausal patients with very low serum E2 levels. Intratumoral E2 levels of postmenopausal patients (127.2 ± 17.5 pg/g) (mean ± SE) were not significantly different from those of premenopausal patients (110.1 ± 10.1 pg/g) (p = 0.36). The mRNA expression levels of aromatase and sulfatase, determined by a quantitative real‐time PCR, were not significantly different between premenopausal and postmenopausal breast cancers, but 17β‐HSD1 mRNA expression levels were significantly higher in postmenopausal than premenopausal breast cancers (p < 0.05). Intratumoral E2/E1 ratios were significantly higher in postmenopausal than premenopausal breast cancers (p < 0.01). These results demonstrate that the increased conversion from E1 to E2 catalyzed by 17β‐HSD1 may play an important role in the maintenance of the intratumoral high E2 levels in postmenopausal patients.

Preoperative evaluation of prognosis in breast cancer patients by [18F]2-Deoxy-2-fluoro-D-glucose-positron emission tomography

Purpose [18F]2-Deoxy-2-fluoro-D-glucose (FDG)-positron emission tomography (PET) was applied to breast cancer patients for the purpose of preoperative evaluation of patient prognosis with more accuracy than conventional TNM staging.Methods FDG-PET was performed preoperatively in 81 patients with breast cancer, and the maximum standardized uptake value (SUVmax) of tumors as well as the focal accumulation of FDG in the axillary region (PET-N status) were investigated in their association with patient prognosis.Results The SUVmax high group (n=40) showed a significantly (P=0.011) poorer prognosis than the SUVmax low group (n=41) (5-year disease-free survival (DFS) rates; 75.0% vs 95.1%). FDG-PET was more accurate in the diagnosis of axillary lymph node status than physical examination, i.e., diagnostic accuracy was 80% and 70% for FDG-PET and physical examination, respectively. The combination of high SUVmax and positive PET-N (+) was shown to be a highly significant risk factor being independent of the clinical T and N factors, i.e., patients with high SUVmax and positive PET-N (+) showed a significantly (P<0.001) poorer prognosis than the other patients (5-year DFS rates; 44.4% vs 96.8%).Conclusions These results suggest that FDG-PET is useful in the preoperative evaluation of prognosis in breast cancer patients with more accuracy than conventional TNM staging. It is expected that the indication of neoadjuvant chemotherapy can be decided more precisely by the preoperative evaluation of patient prognosis with FDG-PET due to a possible elimination of overtreatment for those who have good prognosis and, thus, need not to be treated with chemotherapy.

Comparison of FDG-PET with MIBI-SPECT in the detection of breast cancer and axillary lymph node metastasis.

PURPOSE The purpose of this work was to compare [18F]2-deoxy-2-fluoro-D-glucose (FDG) PET and 99mTc-methoxyisobutylisonitrile (MIBI) SPECT in the detection of breast cancer and axillary lymph node metastasis in the same patients. METHOD FDG-PET and MIBI-SPECT were performed within 3 days for 40 women (age range 25-86 years old) with suspected breast cancer, in whom biopsies and/or mastectomies were performed. Both images were visually assessed, and the count ratio between tumor and normal tissue (T/N ratio) was calculated. RESULTS Thirty-eight patients had breast cancer, and the remaining two had benign breast lesions. The sensitivities of FDG-PET and MIBI-SPECT were 78.9 and 76.3% for breast cancer and 50.0 and 37.5% for axillary lymph node metastasis, respectively. The T/N ratio of breast cancer was significantly higher in FDG-PET (6.01 +/- 3.08 mean +/- SD) than that in MIBI-SPECT (3.48 +/- 1.21) (p = 0.01). Nonmalignant diffuse uptake of FDG in the breasts and the accumulation of MIBI in heart and liver occasionally obscured tumor uptake. CONCLUSION These results indicate that MIBI-SPECT is comparable with FDG-PET in detecting breast cancer. Neither FDG-PET nor MIBI-SPECT is sufficiently sensitive to rule out axillary lymph node metastasis.

Frequency of BRCA1 and BRCA2 germline mutations in Japanese breast cancer families.

The purpose of this investigation is to study the frequency of BRCA1 and BRCA2 germline mutations in Japanese breast cancer families. Mutation analysis of BRCA1 and BRCA2 by SSCP was conducted on the 113 breast cancer patients (probands) with at least 1 breast cancer (site‐specific breast cancer families, n = 101) or 1 ovarian cancer (breast/ovarian cancer families, n = 12) patient in their first‐degree relatives. Fifteen deleterious mutations (13.3%), including 8 nonsense and 7 frameshift mutations, were identified in BRCA1, and 21 deleterious mutations (18.6%), including 8 nonsense, 12 frameshift and 1 splice‐site mutations, were identified in BRCA2. In site‐specific breast cancer families, mutation frequency of BRCA1 or BRCA2 was high in families with 3 or more breast cancer patients (36%, 9/25), early onset (40 ≤ years old) breast cancer patients (38%, 19/50) or bilateral breast cancer patients (40%, 6/15). In breast/ovarian cancer families, mutations of BRCA1 (58.3%, n = 7), but not BRCA2 (0%, n = 0), were observed. BRCA1 codon 63 (TTA to TAA) nonsense mutation and BRCA2 frameshift mutation (5802 del AATT) were observed in 4 and 7 independent families, respectively. Haplotype analysis has suggested that carriers of each of these mutations have common ancestors. These results demonstrate that family profiles are important determinants of risk for carrying a BRCA1 or BRCA2 mutation and that cumulative frequency of BRCA1 and BRCA2 mutations in Japanese breast cancer families (31.9%) is within the range observed in Caucasian breast cancer families. Presence of Japanese founder mutations has also been suggested. Int. J. Cancer 91:83–88, 2001.

Activation of coagulation and fibrinolysis during surgery, analyzed by molecular markers

Activation of hemostasis during surgery was investigated in 30 elective cases, who underwent either gastric (group G) or hepatic (group H) resection by a serial determination of various molecular markers such as fibrinopeptide A (FPA), fibrinopeptide B beta 15-42 (B beta 15-42) D-dimer, thrombin-antithrombin III complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC). In both groups, the values of FPA and TAT were significantly elevated intraoperatively, indicating an occurrence of hypercoagulable state. The degree of the elevation was more marked in group H, probably due to greater tissue damage during hepatic resection. Also in both groups, the values B beta 15-42 and PIC were significantly increased during surgery, while the amount of D-dimer was within normal range in most cases, indicating the occurrence of the primary fibrinolysis. These findings are compatible with our previous observations on the postoperative changes in hemostasis. There were statistically significant but variable correlations between the values of fibrinopeptides and the enzyme-inhibitor complexes. The absolute values of the molecular markers of fibrinolysis were always higher than those of coagulation, suggesting that a considerable amount of plasmin, rather than thrombin, is released by surgical tissue damages.

The effects of calpeptin (a calpain specific inhibitor) on agonist induced microparticle formation from the platelet plasma membrane.

Platelets activated by various agonists produce formation of vesicles shed from the plasma membrane (microparticles). However, the mechanism of microparticle (MP) formation has not been clarified yet. The aim of the present study was to determine the possibility of involvement of calpain (a Ca(2+)-dependent thiol protease) in MP formation. Washed platelets preincubated with calpeptin, a cell permeable calpain specific inhibitor, or with a vehicle were activated by thrombin plus collagen or by calcium ionophore A23187. Flow cytometry was used to detect the amount of microparticle formation by using murine monoclonal antibodies against GP IIb-IIIa or GP IIb and fluorescein 5-isothiocyanate labeled goat anti-mouse IgG. MP formation stimulated either by thrombin plus collagen or by A23187 was inhibited by calpeptin in a dose dependent manner. The microparticle formation from platelets activated by A23187 reached a plateau in approximately 5 min after activation, whereas that from platelets activated by thrombin plus collagen reached a plateau at 30 min following the stimulation. These time sequences corresponded well with those of degradation of actin-binding protein (ABP), a well known substrate of calpain, of platelets activated by these two stimulations. However, the inhibition of MP formation by calpeptin was more marked in the early stage (within 10 min) than in the late stage (after 30 min) of platelet activation. At 30 min after platelet activation by either two stimulations, a significant amount of microparticle formation was observed in the presence of 30 microM calpeptin, which inhibited hydrolysis of ABP almost completely. Our data suggest the involvement of calpain in the early stage (especially within 10 min) of microparticle formation.

A NOVEL ENDOTOXIN-SPECIFIC ASSAY BY TURBIDIMETRY WITH LIMULUS AMOEBOCYTE LYSATE CONTAINING BETA -GLUCAN

The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.

Proteasome and its novel endogeneous activator in human platelets

Proteasome, a high molecular weight multicatalytic protease, was purified from the cytosolic fraction of human platelets for the first time. The biochemical properties of the enzyme including substrate specificity, optimal pH and effects of various inhibitors were almost identical with those of other cells. During the purification with a Heparin-Sepharose chromatography, a novel endogenous activator of the protease was identified and was partially purified. The activator enhanced both chymotrypsin or trypsin like activities of the proteasome in a dose related manner and was inactivated by heating at 56 degrees C for 30 min. This newly identified activator may serve as an important regulator or cofactor of intracellular activities of the proteasome.

Bedside monitoring of warfarin therapy by a whole blood capillary coagulation monitor

A recently developed portable capillary coagulation monitor device allows rapid determination of prothrombin time (PT) in sec. and in International Normalized Ratio (INR) from a drop of whole blood. Considering a possible application of this device for monitoring of warfarin therapy at bedside or at outpatient clinic, a comparative study was performed in patients receiving warfarin between the whole blood capillary PT(WBC-PT) and conventional laboratory tests such as plasma PT (LAB-PT) and Thrombotest (TTO). There was an excellent positive correlation (r = 0.95, n = 49) between LAB-PT(sec) and WBC-PT(sec). As the correlation coefficient between WBC-PT(sec) and the reciprocal of LAB-PT(%) was excellent (r = 0.95, n = 49), it is feasible to convert WBC-PT(sec) to LAB-PT(%) by the following formula; y = 313.48x +8.29 (x:reciprocal value of LAB-PT(%), y:WBC-PT). As the correlation between WBC-PT(sec) and TTO(sec) was also excellent (r = 0.85, n = 63), TTO(%) may be likewise estimated by the following formula; y = 66.49x + 11.36 (x:reciprocal value of TTO(%), y:WBC-PT). From these observations it is concluded that the determination of WBC-PT is simple and accurate and that the monitoring of warfarin therapy is easily performed by the present method.