Identification of high-risk breast cancer patients from genetic changes of their tumors.

To identify the genetic prognostic markers for breast cancer, we analyzed loss of heterozygosity (LOH) at 11p, 16q, 17p, 17q, and 18q, as well as amplification of the ERBB2, INT2, and MYC genes, in 131 patients with breast carcinoma, 49 of whom had lymph node involvement, but none of whom had distant metastases. Among the several chromosome arms tested, LOH at 17q was correlated with lymph node metastasis. Amplification of the ERBB2, MYC, and INT2 genes was found more frequently in tumors from patients with lymph node metastases than in tumors from those without lymph node metastases. Univariate analysis demonstrated that LOH at 17q and INT2 amplification were factors influencing disease-free survival (DFS). A multivariate analysis was performed on 89 tumors that were able to be evaluated for both LOH at 17q and INT2 amplification, and the results showed that patients who had tumors with these genetic changes were more likely to have a poor prognosis. The findings of this study suggest that investigating genetic changes, in addition to conventional clinicopathologic factors, may contribute to defining groups of breast cancer patients with differences in prognosis.

Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

Effect of a high alpha-linolenate and high linoleate diet on membrane-associated enzyme activities in rat brain--modulation of Na+, K+- ATPase activity at suboptimal concentrations of ATP.

Semi-purified diets supplemented with either a high alpha-linolenate (n - 3) (perilla) oil or a high linoleate (n - 6) (safflower) oil were fed to rats through two generations. Rats fed safflower oil showed a decrease in docosahexaenoic acid (n - 3) and a compensatory increase in docosapentaenoic acid (n - 6) in all the brain regions and organelles examined, when compared with rats fed perilla oil. As reported previously, the safflower oil-fed rats exhibited inferior learning ability compared with the perilla oil-fed rats (N. Yamamoto et al., J. Lipid Res. 28, 144 (1987)). Using brains of rats in these dietary groups, the activities of several enzymes, Na+ , K+-ATPase, Ca2+-ATPase, 5'-nucleotidase, 2',3'-cyclic nucleotide phosphodiesterase, acetylcholinesterase, and choline acetyltransferase in membranes, were compared. The 5'-nucleotidase activity in cortex and hippocampus, and the Na+, K+-ATPase activity in myelin decreased slightly but significantly in the safflower oil group. None of the other membrane-associated enzyme activities in all the brain regions and organelles examined was affected significantly by the dietary fatty acids under optimal assay conditions in vitro. However, in the safflower oil group, the Na+, K+-ATPase activity of synaptosomes at a suboptimal concentration of ATP was 78% that in the perilla oil group. These results suggest that relatively large changes in the proportions of n - 3 and n - 6 polyunsaturated fatty acids in brain membranes caused by dietary manipulation do not provoke significant alterations in most membrane-bound enzyme activities. However, a small but significant change in Na+, K+-ATPase activity at a suboptimal concentration of ATP may be implicated in the altered learning behavior reported earlier.

CAF versus CAF plus Medroxyprogesterone Acetate for Treatment of Liver Metastases of Breast Cancer.

A controlled randomized trial was conducted to compare the effectiveness of a CAF therapy including cyclophosphamide (CPA), adriamycin (ADR) and 5-fluorouracil (5FU) with that of CAF plus medroxyprogesterone acetate (MPA) therapy including CPA, ADR and 5FU plus MPA for the treatment of liver metastases from breast cancer. A total of 34 patients with unresectable liver metastases from breast cancer were divided into two treatment groups(CAF and CAF + MPA)with stratification for estrogen receptor status. The response rate was 13%(2PR in 16 patients)for CAF therapy and 22% (1 CR and 3 PR in 18 patients) for CAF plus MPA therapy. There was no significant difference in response rates, median survival periods and survival rates between the two treatment groups. However, CAF therapy had significantly more toxicity than did CAF plus MPA therapy. The findings of this study suggested that CAF plus MPA therapy is a well-tolerated and effective treatment for patients with liver metastases of breast cancer.

Conventional Dose CAF Therapy versus Low Dose Adriamycin Therapy in the Treatment of Advanced Breast Cancer.

A controlled randomized trial was conducted to compare the effectiveness of a conventional dose of CAF therapy with that of a low dose of adriamycin (ADR) therapy for the treatment of advanced Breast Cancer. The doses of medication for the conventional CAF therapy were 100 mg/body of cyclophosphamide (CPA) p.o. daily fort wo weeks, 30 mg/m(2) of ADR and 500 mg/m(2) of 5-fluorouracil (5-FU) i.v. on days 1 and 8 for induction, and 200 mg/body of 5-FU and 20 mg/body of tamoxifen (TAM) p.o. daily for maintenance. Those for the low dose ADR therapy were 15mg/m(2) of ADR i.v. at two-week intervals for one year and 200 mg/body of 5-FU and 20 mg/body of TAM p.o. daily. Eighty patients were entered in this trial. All patients were randomly divided into two groups with stratification for estrogen receptor status. Of 78 patients among them, 38 undergoing the CAF therapy and 40 undergoing the low dose ADR therapy, were evaluated for efficacy assesment. The background factors analyzed were well balanced in both groups. The response rate was 47%(6CR, 12PR out of 38) in the CAF group and 43% (3CR, 14PR out of 40) in the low dose ADR group. There was no significant difference in response rates and survival rates as determined by the Kaplan Meier method between the two groups. The CAF therapy had significantly more toxicity than the low dose ADR therapy. Therefore, it was concluded that this low dose ADR therapy can be regarded as a treatment of choice for advanced breast cancer.

Application of a gas chromatography mass spectrometry computer system for clinical diagnosis.

Currently, one of the major clinical problems is diagnosis of inherited metabolic disorders in neonates which are caused by a defect of specific enzymes. In order to diagnose such diseases at an earlier stage, we have developed a new mass screening method, consisting of a gas chromatography mass spectrometry computer system with a newly developed 'Diagnosis Computer Program'. In this program, the chromatographic pattern of the urine of a patient with an inherited metabolic disorder is compared with that of a normal person. Internal standards are applied to simplify the comparison. The total ion current chromatogram is drawn with the relative retention times plotted on the horizontal axis and the relative intensities on the vertical axis. Diseases are diagnosed by characteristics and quantity of abnormal compounds in the urine. Reference to the stored mass spectra of abnormal compounds by a library search enables the diagnosis to be more precise. Using this program, twenty-six inherited metabolic disorders are routinely diagnosed. The diagnosis of tyrosinosis is used here as an example.