Transgenic mice overexpressing nesfatin/nucleobindin-2 are susceptible to high-fat diet-induced obesity.

BACKGROUND: Nesfatin/Nucleobindin-2 (Nesf/NUCB2), a precursor of nesfatin-1, an anorexigenic protein, is ubiquitously expressed in peripheral tissues in addition to the hypothalamus. However, the role of intracellular Nesf/NUCB2 has not been established in the periphery. METHODS: Nesf/NUCB2-transgenic (Tg) mice were generated, and chronological changes of body weight and daily food intake were measured in Nesf/NUCB2-Tg mice fed normal laboratory chow or 45% high-fat diet (HFD). In addition, changes of metabolic markers were evaluated in those mice. RESULTS: No differences were observed in daily food intake and body weight between Nesf/NUCB2-Tg mice (n=11) and their non-Tg littermates (n=11) fed normal chow. Nesf/NUCB2-Tg mice showed increased mRNA expression of oxytocin and corticotropin-releasing hormone and decreased mRNA expression of cocaine- and amphetamine-related transcript in the hypothalamus. Nesf/NUCB2-Tg mice fed 45% HFD (n=6) showed significantly higher increase in body weight than their non-Tg littermates fed the same diet (n=8); however, no difference was observed in daily food intake between these two groups. Further, Nesf/NUCB2-Tg mice fed 45% HFD showed a significant increase in the weight of the liver, subcutaneous fat, and brown adipose tissue and decrease in the expression of uncoupling protein-1 in the subcutaneous fat. Blood glucose levels of Nesf/NUCB2-Tg mice fed 45% HFD were not different from those of their non-Tg littermates fed the same diet. Insulin levels of these Tg mice were significantly higher than those of their non-Tg littermates. Histological analysis showed marked fat deposition in the hepatocytes surrounding the hepatic central veins in Nesf/NUCB2-Tg mice fed 45% HFD. CONCLUSIONS: Overexpression of Nesf/NUCB2 did not change food intake, but increased body weight only in Nesf/NUCB2-Tg mice fed HFD. The results of this study indicate that Nesf/NUCB2 was involved in the development of insulin resistance and fat deposition in the liver, independent of the modulation of energy intake.

MicroRNA-574-3p, identified by microRNA library-based functional screening, modulates tamoxifen response in breast cancer.

Most primary breast cancers express estrogen receptor α and can be treated via endocrine therapy using anti-estrogens such as tamoxifen; however, acquired endocrine resistance is a critical issue. To identify tamoxifen response-related microRNAs (miRNAs) in breast cancer, MCF-7 cells infected with a lentiviral miRNA library were treated with 4-hydroxytamoxifen (OHT) or vehicle for 4 weeks, and the amounts of individual miRNA precursors that had integrated into the genome were evaluated by microarray. Compared to the vehicle-treated cells, 5 'dropout' miRNAs, which were downregulated in OHT-treated cells, and 6 'retained' miRNAs, which were upregulated in OHT-treated cells, were identified. Of the dropout miRNAs, we found that miR-574-3p expression was downregulated in clinical breast cancer tissues as compared with their paired adjacent tissues. In addition, anti-miR-574-3p reversed tamoxifen-mediated suppression of MCF-7 cell growth. Clathrin heavy chain (CLTC) was identified as a miR-574-3p target gene by in silico algorithms and luciferase reporter assay using the 3' untranslated region of CLTC mRNA. Interestingly, loss and gain of miR-574-3p function in MCF-7 cells causes CLTC to be upregulated and downregulated, respectively. These results suggest that functional screening mediated by miRNA libraries can provide new insights into the genes essential for tamoxifen response in breast cancer.

Nesfatin/Nucleobindin-2 (NUCB2) and Glucose Homeostasis.

Anorexigenic protein, nesfatin/nucleobindin-2 (NUCB2) is ubiquitously expressed in peripheral tissues including white adipose tissue. Nesfatin/NUCB2 is selectively expressed in pancreatic ß-cell, but not ß-cells, ß-cells, PP-cells.Starvation significantly increased circulating nesfatin-1 concentrations, and refeeding restores the increase by starvation. There is an obvious dissociation in changes between nesfatin-1 releases from pancreatic ß-cells and circulating nesfatin-1, and an increase of nesfatin-1 from pancreatic islets may not be reflected to circulating concentrations of nesfatin-1.Nesfatin-1 may be a novel insulinotropic peptide, since endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity. Nesfatin-1 may also contribute to the improvement of insulin sensitivity in hyperglycemic state. An increase of circulating nesfatin-1may shift glucose uptake to peripheral organs from skeletal muscles to adipocytes. Nesfatin-1 may involve the feedback system from adipocytes to the hypothalamus via general circulation, and from the hypothalamus to adipocytes via sympathetic nervous system. The details of those molecular mechanisms should be clarified by future studies including the analysis of gene targeted animals.

Uracil-tegafur and tamoxifen vs cyclophosphamide, methotrexate, fluorouracil, and tamoxifen in post-operative adjuvant therapy for stage I, II, or IIIA lymph node-positive breast cancer: a comparative study.

BACKGROUND: It has been reported that treatment with uracil-tegafur (UFT) has shown significantly better survival and relapse-free survival (RFS) than surgery alone. Therefore, we compared UFT with a combination therapy of cyclophosphamide, methotrexate, and fluorouracil (CMF) in patients who had undergone curative surgery for axillary lymph node-positive breast cancer. METHODS: A total of 377 node-positive patients with stage I, II, or IIIA disease were registered from September 1996 through July 2000 and were randomly assigned to either 6 cycles of CMF or 2 years of UFT. In both arms, tamoxifen (TAM) was concurrently administered for 2 years. The primary end point in this study was the non-inferiority of UFT to CMF. RESULTS: No statistically significant difference between the two groups was observed with regard to the 5-year RFS rate (72.2% in the UFT and 76.3% in the CMF). Adverse event profiles differed between the two groups, with a significantly lower incidence of leukopenia and anaemia in the UFT group, as well as anorexia, nausea/vomiting, stomatitis, and alopecia, which have implications for quality of life. CONCLUSION: UFT administered in combination with TAM holds promise in the treatment of lymph node-positive early breast cancer. On stratified analysis, the recurrence rate in the UFT group was found to be better in oestrogen receptor (ER)-positive patients. Tegafur-based treatment should be evaluated by a prospective randomised trial conducted in ER-positive patients.

Extranuclear expression of hormone receptors in primary breast cancer.

BACKGROUND: Hormone receptor (HR)-positive breast cancer cells grow through estrogen receptor (ER)-signaling pathways that mediate both genomic and nongenomic actions, which cross-talk with growth factors associated with resistance to tamoxifen. The aim of this study was to explore the cross-talk between extranuclear expression of ER and progesterone receptor (PR) and growth factor signaling pathways in primary breast cancer. PATIENTS AND METHODS: The extranuclear expression of ER and PR was examined in 219 primary breast cancers by immunohistochemical staining. Specimens showing such expression were further examined for the expression of pAkt and aromatase. Staining reactions were scored on the basis of intensity and distribution in the tumors. RESULTS: Extranuclear expression of ER or PR was observed in 21 cases (9.5%), which included four cases for ER and 20 cases for PR. Among these patients, HER-2, pAkt, and aromatase-positivity were observed in 14 cases (66.6%), 13 cases (61.9%), and 14 cases (66.6%), respectively. On the basis of nuclear HR expression, 11 of these cases were categorized as ER-positive/PR-negative, while two were ER-negative/PR-positive. Of these 13 cases, increased pAkt staining was found in 11 cases (84.6%). In particular, among the 11 ER-positive/PR-negative cases, elevated pAkt and aromatase were found in 10 (90.9%; P<0.01) and nine cases (81.8%), respectively. CONCLUSIONS: PR is expressed extranuclearly more frequently than ER in primary breast cancer, and extranuclear HRs cross-talk with the Akt/HER-2-signaling pathways and activation of aromatase. These observations may explain the more beneficial effects of aromatase inhibitors than tamoxifen for ER-positive/PR-negative patients.

Significance of lymphoscintigraphic mapping with Tc-99m human serum albumin and tin colloid in sentinel lymph node biopsy in breast cancer.

Sentinel lymph node biopsy (SLNB) in breast cancer is considered in order to spare node-negative patients from axillary lymph node dissection. To assess the clinical significance of lymphoscintigraphic mapping in SLNB, we analyzed the lymphatic drain to the sentinel lymph nodes (SLNs) in terms of the pattern and direction of the hot spot. Twenty-three breast cancer patients were enrolled for SLNB. Before surgery, lymphoscintigraphic mapping of SLN was performed using Tc-99m human serum albumin (HSA) and tin colloids, and the hot spot was marked. The Tc-99m HSA and tin colloids were subcutaneously injected above the tumor and peritumor sites, respectively, and lymphoscintigraphic scanning was monitored every 5 to 10 min, for up to 2 h after injection. The SLN was identified using a combination of a blue dye, indigocalmine, and a gamma probe during surgery. The hot spot pattern and direction of the lymphatic drains were evaluated in 21 of 23 cases. Two cases did not have a hot spot. Single, double, and multiple hot spots were observed in 12 cases (52.1%), 8 cases (34.7%), and 1 case (4.3%), respectively. The positions of the hot spots were: axillary (n=17, 80.9%), axillary and sternal (n=3, 14.2%), and phrenic (n=1, 4.7%). The sensitivity and specificity rates in SLNB were 66.6% and 100%, respectively, and the overall predictive rate was 85.7%. Lymphoscintigraphy produced false negatives in three cases (33.3%), including one on the phrenic side. Lymphoscintigraphic mapping with Tc-99m HSA and tin colloids is useful for determining the SLN, and avoiding a false negative. The pattern and direction of the lymphatic drain to the SLN in scintigraphy need to be considered for the elimination of axillary lymph node dissection in node-negative patients with breast cancer.

A pitfall in the survival benefit of adjuvant chemotherapy for node- and hormone receptor-positive patients with breast cancer: the paradoxical role of Bcl-2 oncoprotein (review).

The survival benefit of adjuvant chemotherapy in node-positive patients with breast cancer compared with surgery alone has been established. The survival benefit differs considerably between hormone receptor-positive and -negative patients, and it is believed that the effectiveness of adjuvant chemotherapy can be increased by hormonal therapy with tamoxifen. In the present review, we discuss the rationale behind the effectiveness of combination treatment with anticancer drugs and tamoxifen in terms of the paradoxical role of Bcl-2 in apoptosis in breast cancer. The survival benefit between receptor-positive and -negative patients was assessed using previous reports of randomized controlled studies for postoperative adjuvant chemotherapy in node-positive breast cancer. Tamoxifen induces the anti-apoptotic gene, Bcl-2, by its effect on estradiol (E2), via an E2-response element in the promotor region of Bcl-2. The efficicacy of chemoendocrine therapy was assessed in terms of the influence of tamoxifen on the effect of anticancer drugs. Adjuvant chemotherapy, including anthracycline and non-anthracycline based regimens, has an overall survival benefit in node-positive breast cancer, with a 23.5% reduction in the annual odds of recurrence and a 15% reduction in mortality (P<0.00001). A comparison of the reduction of the relative risk indicates that the survival benefit in receptor-negative patients is superior to that in receptor-positive patients by approximately 3-fold. Further, in contrast to receptor-negative patients, there is no additional benefit from paclitaxel over doxorubicin and cyclophosphamide (AC) in receptor-positive patients. The possible reasons that the chemotherapy benefit in receptor-positive patients is small and marginal are the following: i) concurrent treatment or pretreatment with tamoxifen can increase plasma E2 levels in premenopausal patients, thereby inducing Bcl-2 in residual cancer cells, which might decrease drug-sensitivity in combination with chemotherapy; ii) induction of Bcl-2 might be involved predominantly in the resistance to taxanes, the cytotoxic action of which targets Bcl-2. Co-treatment or pretreatment with tamoxifen for adjuvant therapy might decrease the efficacy of anticancer drugs, an effect that is mediated by induction of Bcl-2, especially in premenopausal patients with node-positive breast cancer. Treatment with anticancer drugs should be followed by treatment with tamoxifen to produce a survival benefit from combination therapy in receptor-positive patients.

Pharmacokinetic and biochemical analysis in the treatment of weekly paclitaxel in relapsed breast cancer.

The mechanism(s) by which weekly paclitaxel exerted more therapeutic efficacy than the triweekly schedule in relapsed breast cancer is still unclear. To assess the rationale in therapeutic efficacy of weekly paclitaxel in relapsed breast cancer, pharmacokinetic and biochemical analyses were examined in terms of the mean peak plasma concentration at 0 min (Cmax), 30 min, and 24 h after finishing the infusion, and the extracellular domain of HER-2 in response to the treatment with paclitaxel. Twenty-five patients treated with weekly 1 h infusion of paclitaxel in the dose range from 40 mg/m(2) to 80 mg/m(2) were studied. Eleven patients responded to the treatment including 4 cases of complete response (CR) and 7 cases of partial response (PR), while 14 patients did not respond including 12 cases of no change (NC) and 2 cases of progressive disease (PD). The plasma concentration of paclitaxel and extracellular domain of HER-2 in the patients were measured by high-pressure liquid chromatography and enzyme immunoassay, respectively. The peak concentration (Cmax) and the other peaks at 30 min and 24 h in 10 patients including 3 cases of 40 mg/m(2), 3 cases of 60 mg/m(2) and 4 cases of 80 mg/m(2) in the weekly paclitaxel were compared in proportion to the increase of dose escalation, and compared to their tumor response. Further, the plasma levels of extracellular domain of HER-2 in 17 patients treated with the weekly paclitaxel were measured, and also compared to their tumor response. The mean Cmax treated with 40 mg/m(2), 60 mg/m(2) and 80 mg/m(2) in the weekly paclitaxel was 1.94, 2.18 and 1.54 microM, respectively. The dose escalation of paclitaxel and the dose intensity were not correlated with the increase of plasma concentration of paclitaxel nor with the tumor response. In contrast, the plasma level of extracellular domain of HER-2 in responders was higher than that of non-responders in the weekly paclitaxel regimen(p=0.0512, Mann-Whitney's U-test). These results suggest that tumor response to the weekly 1 h infusion of paclitaxel was not associated with the plasma concentration and the dose intensity, rather the plasma level of extracellular domain of HER-2 protein may be a predictor of tumor response in the treatment of weekly paclitaxel in relapsed breast cancer.

An analysis of relapsed breast cancer in patients previously treated with breast conserving surgery.

BACKGROUND: Recent advances in breast surgery have focused on breast conserving surgery in combination with radiotherapy. In the present study, we examine by retrospective analysis 105 patients with breast cancer who received breast conserving surgery for factors influencing disease free survival. METHODS: The analysis was performed on 105 patients with breast cancer who received breast conserving surgery in our department, including 38 patients without radiotherapy and 67 patients treated with radiotherapy. The disease-free survival of the patients was analyzed using the Kaplan-Meier method. The relapsed patients were assessed by examining pathological features and gene expression by immunohistochemical staining. RESULTS: There was no significant difference in the disease free survival at 5 years between patients without radiotherapy (89.6%) and with radiotherapy (94.5%). Relapse after breast conserving surgery was found in 6 patients including 4 patients without radiotherapy and 2 patients with radiotherapy. Local relapse and bone metastasis were found in 4 (3.8%) and 2 patients, respectively. Among the 4 local relapses, 1 patient had received radiotherapy and 3 patients had not. There was no significant difference between the type of relapse in terms of lymph node metastasis, hormone receptor, nuclear grade and intraductal component, but more vessel invasion was observed in the 2 cases with bone metastasis. The overexpression of apoptosis and angiogenesis genes such as p53, Bax, Bcl-XL, Bcl-2 and VEGF was not common in the relapsed patients, whereas the overexpression of drug resistance genes, either P-gp or MRP1, was found in the all patients. CONCLUSIONS: Although radiotherapy may reduce the incidence of local relapse and increase disease free survival after breast conserving surgery, the development of an effective adjuvant chemotherapy based on drug resistance markers, is also required.

A phase II trial of mitomycin C, 5'-deoxy-5-fluorouridine, etoposide and medroxyprogesterone acetate (McVD-MPA) as a salvage chemotherapy to anthracycline-resistant tumor in relapsed breast cancer and its mechanism(s) of antitumor action.

To assess the therapeutic efficacy in the combination of mitomycin C (MMC), 5'-deoxy-5-fluorouridine (5'-DFUR), etoposide (VP-16) and medroxyprogesterone acetate (MPA) (McVD-MPA) to anthracycline-resistant tumor as a salvage chemotherapy, a phase II trial was conducted in patients with relapsed breast cancer. Fifty-five patients were enrolled in this trial and 54 were assessable, who had all previously been treated with an anthracycline regimen. The treatment schedule was designed with the intravenous administration of MMC (6 mg/m2) on day 1 followed by peroral administration of VP-16 (75 mg/m2) on day 2, 4, 6 and the peroral administration of 5'-DFUR (600 mg/m2) and MPA (400 mg/m2) on day 1 through 21 in one cycle. The overall tumor response rate was 40.7% (22/54) including 16.6% (9 cases) in complete response and 24.0% (13 cases) in partial response, and the long no change (NC) was observed in 18.5% (10/54) out of 44.4% (24/54) in NC. Of the patients with primary resistance to anthracycline 30.0% responded to McVD-MPA therapy. Bone and liver metastases responded in 50.0% and 50.0%, whereas soft tissue and lung metastases responded in 36.8% and 35.2%, respectively. The mean time to response and response duration were 2.7 and 15.6 months, respectively. The overall survival of the patient treated with the McVD-MPA was superior to the non-treatment of second line therapy, and the median survival between McVD-MPA and non-treatment was 86 days and 50 days, respectively. The major adverse effect was observed in hematological toxicity (31.7%) such as leukopenia and thrombocytopenia and non-hematological toxicity of gastrointestinal events (31.7%), the toxicity was less than grade 2, and was tolerable during the treatment. In the experiment of MDA-MB-231 breast cancer cell line that was overexpressed with P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP), the mechanism(s) by which McVD-MPA induces the antitumor effect to anthracycline-resistant tumor may be explained at least in part as follows: i) The treatment of MMC suppressed the expression of P-gp and MRP in a dose-and time-dependent manner, connecting the increase of the intracellular concentration of VP-16; ii) The treatment of MMC enhanced the expression of thymidine phosphorylase to increase the production of 5-FU from 5'-DFUR in the antiangiogenic effect of MPA. These results indicate that the combination chemotherapy of the McVD-MPA may be an effective regimen to anthracycline-resistant tumor as a salvage chemotherapy to prolong the survival in the patient with relapsed breast cancer.

Coagulation activation in patients with Binswanger disease.

BACKGROUND: A hypercoagulable state is often associated with an acute stroke in cerebrovascular disease (CVD). However, in Binswanger disease (BD), no information is available on the coagulation-fibrinolysis pathway except for the presence of high plasma fibrinogen levels. OBJECTIVE: To determine the association of BD and coagulation-fibrinolysis pathway activation. PATIENTS AND METHODS: We examined the levels of fibrinogen, thrombin-antithrombin complex, prothrombin fragment(1+2), and cross-linked D-dimer in 17 patients with BD, 24 neurologic patients without CVD, and 26 patients with lacunar infarction in either the acute or chronic stage. RESULTS: As compared with the non-CVD and lacunar infarction groups, the patients with BD had significantly elevated levels of thrombin-antithrombin complex (P<.001), prothrombin fragment(1+2) (P<.05), and cross-linked D-dimer (P<.01). There was also a significant increase in fibrinogen levels compared with the non-CVD group (P<.05). In the BD group, 8 patients in stable condition (ie, those without obvious neurologic deficits in the past 3 months) showed normal levels or a mild increase in their fibrinogen, thrombin-antithrombin complex, prothrombin fragment(1+2), or cross-linked D-dimer levels. In contrast, 9 patients with BD with a subacute aggravation of their focal or subcortical cerebral functions (deteriorating group) showed a significant increase in their thrombin-antithrombin complex levels compared with the stable patients (P<.01). Similarly, the fibrinogen, prothrombin fragment(1+2), and cross-linked D-dimer levels were elevated in the deteriorating patients, but this trend did not reach statistical significance. CONCLUSIONS: These results indicate that the coagulation-fibrinolysis pathway is activated in patients with BD with a subacute aggravation. Coagulation activation may result in the formation of microthrombi and microcirculatory disturbances in the brains of these patients, and thus promote further biological and neurologic insults.

Effects of antithrombin on Binswanger's disease with antiphospholipid antibody syndrome.

The authors present a family with Binswanger's disease (BD) and antiphospholipid antibody syndrome (APAS). In one patient from this family, lupus anticoagulant and high levels of hemostatic markers were detected. The presence of BD and the clinicobiological improvements observed after antithrombin treatment in this patient are peculiar to this familial case of APAS.

The prognostic significance of p53, p21 (Waf1/Cip1), and cyclin D1 protein expression in esophageal cancer patients.

Recently, various cell cycle regulators have been studied as biological markers of malignant potential. These regulators might influence survival rates and the effects of adjuvant therapies. In this study, we analyzed the expression of p53, p21(Waf1/Cip1), and cyclin D1 in 64 esophageal cancer patients and their relation to clinicopathologic parameters and patient survival. For p53, p21, and cyclin D1 oncoprotein expression, we defined positive cases as those with over 10% of examined cells stained. Positive rates were 48.4%, 42.2%, and 43.8% for p53, p21, and cyclin D1, respectively. In a multivariate analysis, tumor depth, chemotherapy, and p21 expression were determined to be significant prognostic factors. Five-year survival rates of p53-negative/p21-positive and p53-positive/p21-negative patients were 53.0% and 28.5%, respectively, and were not significantly different. These results suggest that, of various cell cycle regulators, p21 might be a good predictor of prognosis for esophageal cancer patients.

A Combination Therapy with Mitomycin C, Etoposide, Doxifluridine and Medroxyprogesterone Acetate as Second Line Therapy for Advanced Breast Cancer Refractory to Combination Chemotherapy of Cyclo-phoshamide, Doxorubicin and 5-Fluorouracil.

Thirty-two patients with advanced breast cancer refractory to combination chemotherapy with cyclophosphamide (CPA), doxorubicin (ADR) and 5-fluorouracil (5-EU) (CAF) were treated with the combination of mitomycin C, etoposide, doxifluridine and medroxyprogesterone acetate as second line therapy. Observed responses included 6 patients (18.7%) with complete response (CR) and 7 (21.9%) with partial response (PR). Two (50%) out of 4 patients who had bone pain due to bone metastasis noted pain relief. CR or PR were obtained in 4 out of 12 patients who had not responded to the previous CAF therapy. While grade III myelosuppression was observed in 3 patients, other adverse effects were minimal. It is suggested that this combination therapy may be recommended for advanced breast cancer patients as a second therapy.

Relative effects of immunohistochemical expressions of c-erbB-2, p53, and bcl-2 oncoproteins on the survival of advanced breast cancer patients after endocrine therapy.

c-erbB-2, p53, and bcl-2 oncoproteins were detected immunohistochemically in 92 recurrent or advanced breast cancer tumors just before an endocrine therapy, adreno-oophorectomy. Estrogen receptors (ER) and partly progesterone receptors were concomitantly assayed in the same tumor tissues. Twenty-eight percent (26/92) of c-erbB-2, 16% (15/92) of p53, and 11% (10/92) of bcl-2 expressions were shown to be positive. There were no significant correlations of these oncoproteins with clinical background characteristics of the patients, except the inverse relation between p53 expression and ER status (p=0.0033). Of these covariates, ER status was shown to be the only predictor of response to endocrine therapy. In the absence of ER measurement, p53 expression was a significant predictor of the response. Kaplan-Meier curves showed that ER had a significant and p53 expression had a marginal effect on the overall survival length of the patients, c-erB-2 or bcl-2 were indifferent to survival or response. It is concluded that p53 immunohistochemical expression might be a supplementary predictor next to ER status of the overall survival as well as response of advanced breast cancer patients treated with endocrine therapy, and that p53 alteration might modify the ER dependent hormone-responsiveness of breast cancer.

A multivariate analysis of prognostic factors including tumor response after endocrine therapy in advanced breast cancer patients.

Although clinicians want to know how tumor response affects the survival of advanced cancer patients, the direct comparison of response with survival is severely discouraged because of biases essentially involved. In 159 patients with advanced breast cancer treated with adreno-oophorectomy, we analyzed the survival of the patients using the Kaplan and Meier method with the landmark method (landmark time of 3 months after treatment). A multivariate analysis with the Cox proportional hazard model for survival with explanatory variables including response of patients at the landmark time (3 months after therapy) was performed in order to find probable prognostic factors. By the Kaplan and Meier curves with the landmark method, response category showed a significant difference in the survival of the patients (log rank test; p < 0.0001). In the Cox model in the patients of landmark time of 3 months after therapy, we found that response was the most powerful factor for survival out of 10 variables (p = 0.0001), and the dominant site of metastasis significantly and independently modified the survival length (p = 0.001). ER status was indifferent in the analysis; however, when the response category was not included, ER was shown to be most influential. We propose that a combination of the Cox model with the landmark method would be a rational and useful approach to estimate probable prognostic factors including treatment outcome variables such as response for the survival analysis in advanced cancer patients.

Long term survival and the prognostic factors of advanced breast cancer patients treated with adreno-oophorectomy.

Longer survival data are necessary to elucidate the prognostic factors for survival in advanced breast cancer patients. Univariate and multivariate analyses were performed in 159 patients treated with adreno-oophorectomy alone as the first-line treatment for advanced or recurrent breast cancer, between 1972 and 1983. Nine clinical factors included age, menopausal status, estrogen (ER)- and progesterone receptors (PgR) in recurrent tumors, disease-free interval (DFI), number of metastatic organs, performance status, and adjuvant therapy performed. Response was evaluated according to the UICC criteria. A 31% (50/159) response with 16 CR, 34 PR, 48 NC, and 61 PD was obtained. The logistic regression model of the factors showed that ER was the single affecting factor for the response. According to the Cox proportional hazard model, ER and the dominant site of metastasis were indicated to be significant for survival. According to the landmark method, the response significantly correlated to survival. Using the backward elimination procedure of the Cox proportional hazard model in the patient group defined by the landmark time of 3 months after therapy, the survival of the patients with advanced breast cancer was shown to be primarily influenced by the tumor response which was solely affected by ER status, and the dominant site, particularly the presence of liver metastasis, independently modified the survival length. These results may be useful in future studies of total estrogen blockade trials for breast cancer.

Dissociation of dimer of bovine erythrocyte Cu,Zn-superoxide dismutase and activity of the monomer subunit: effects of urea, temperature, and enzyme concentration.

Cu,Zn-superoxide dismutase (SOD) of bovine erythrocytes is a dimeric enzyme of identical subunits associated through unusually strong noncovalent interactions. It is known not to be dissociated into subunits even in 8 M urea for 72 h at 25 degrees C [Malinowski, D.P. & Fridovich, I. (1979) Biochemistry 18, 5055-5060]. Effects of urea, temperature, and SOD concentration on the inactivation and dissociation into subunits were examined. The activation energy of the inactivation of SOD (at 0.05 mg/ml) was 64 kcal/mol at pH 7.8, and was decreased to 40 kcal/mol in the presence of urea (2.0-7.3 M). The apparent first-order rate constant (kapp) of the inactivation by urea was dependent on the SOD concentration [SOD] during the urea treatment, and SOD showed a higher resistance to the inactivation with increase in the concentration. Dissociation of SOD was monitored by gel filtration HPLC. When SOD solutions of various concentrations were incubated in 6 M urea at 45 degrees C, two monomer species (M1 and M2) were observed in addition to dimer (D). The dimer maintained full activity, while the monomers did not show the activity. The peak areas of these species were changed depending on the SOD concentration during urea treatment; at over 15 mg/ml, almost all SOD was eluted as D, and with a decrease in the SOD concentration, the peak area of D decreased and concomitantly the monomers appeared. M2 could be the sole form in infinitely diluted SOD solution, and D was considered to be converted to M2 through M1. The SOD concentration giving 50% D ([SOD]1/2) was 1.0 mg/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of epidermal growth factor receptor expression in primary breast cancer: results of a biochemical study and an immunocytochemical study.

We have conducted two series of studies, a biochemical study and an immunocytochemical study, to investigate the role of epidermal growth factor receptor (EGFR) expression in primary breast cancer patients. In the biochemical study, a consecutive 115 patients were included and EGFR was measured by a competitive binding assay with multipoint Scatchard analysis. In the immunocytochemical study comprising 126 patients, EGFR status was determined by immunostaining with anti-EGFR antibody EGFR1. Several agreements were found from these two studies. EGFR status was inversely correlated with estrogen receptor (ER) status. No significant correlation was found between EGFR status and tumor size, nodal metastases, or the expression of c-erbB-2 protein. Ki-67 immunoreactivity, a cellular proliferation marker, was enhanced in EGFR positive tumors over EGFR negative tumors, suggesting a linkage of EGFR expression to cellular proliferative activity. Post-operative follow up showed that relapse-free survival for EGFR positive patients was significantly worse than that for EGFR negative patients, particularly in node-positive patients. Multivariate analysis demonstrated a significance of EGFR status as an independent prognostic indicator in primary breast cancer. The group expressing EGFR and c-erbB-2 protein indicated a particularly high risk for relapse.