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Recommendations are presented for the routine clinical use of serum and tissue-based markers in the diagnosis and management of patients with breast cancer. Their low sensitivity and specificity preclude the use of serum markers such as the MUC-1 mucin glycoproteins (CA 15.3, BR 27.29) and carcinoembryonic antigen in the diagnosis of early breast cancer. However, serial measurement of these markers can result in the early detection of recurrent disease as well as indicate the efficacy of therapy. Of the tissue-based markers, measurement of estrogen and progesterone receptors is mandatory in the selection of patients for treatment with hormone therapy, while HER-2 is essential in selecting patients with advanced breast cancer for treatment with Herceptin (trastuzumab). Urokinase plasminogen activator and plasminogen activator inhibitor 1 are recently validated prognostic markers for lymph node-negative breast cancer patients and thus may be of value in selecting node-negative patients that do not require adjuvant chemotherapy.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1 .I .1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives.In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier.Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 pM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing r61e of the catalytic metal ions.The enzyme immobilised on agarose, with unchanged content of active sites Eur. J . Biochem. 41, 475-4841, was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogeneous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.Ahhreviations. Tes, 2-{ [tris(hydroxymethyl)methyl]amino}etha-nesulfonic acid; Tris: Tris(hydroxymethy1)-aminomethane; Mes, 2-(N-morpholino)ethanesulfonic acid. The differently substituted metallo-alcohol dehydrogenases are named as follows. The metal ions are differentiated as catalytic and noncatalytic by the characters (c) and (n) after the chemical symbol, i.e. Zn(c)zZn(n)~-enzyme = native enzyme; H4Zn(n)z-enzyme = enzyme depleted of the catalytic zinc ions; Zn(c)2Co(n)z-enzyme = enzyme substituted at the noncatalytic binding sites ('green hybrid' enzyme) ; CO(C)ZZn(n)2-enzyme = enzyme ...