Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

Possible consequences of applying guidelines to healthy women with a family history of breast cancer.

Possible effects of consistently applying published guidelines on healthy women with breast cancer in their family history were analysed. We investigated 1060 unrelated breast cancer patients and calculated the numbers of first-degree relatives that would be referred to a familial cancer clinic if the guidelines were consistently applied. A first-degree relative was considered a candidate for referral if she was female, without breast cancer at the moment of the interview, alive and over the age of 24. The criteria for referral were based on one Dutch and two British guidelines. According to the Dutch guideline, for one affected woman with breast cancer, 0.25 (95% CI 0.22-0.28) healthy first-degree female relatives should be offered a consultation at a familial cancer clinic (FCC). Application of the British guidelines would lead to a similar number of referrals. Of all healthy first-degree female relatives, who should be referred to an FCC, 34-37% had an index case among their family who was already known at a genetic department. If current guidelines are consistently applied, a sharp increase in referrals to FCCs may be expected. These guidelines, however, are arbitrary and only limited data are available on the efficacy of this surveillance for high-risk healthy women.

Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine analogue.

UCN-01 (7-hydroxystaurosporine) has been demonstrated to be a potent inhibitor of tumor cell growth both in cell culture and with in vivo xenograft models. The ability of UCN-01 to inhibit the kinase activity of recombinant protein kinase C (PKC) isozymes alpha, beta, gamma, delta, epsilon, and zeta was characterized using an in vitro kinase assay. Two distinct groups of isozymes could be defined on the basis of relative potency of kinase inhibition. UCN-01 was 15-20-fold more potent for inhibition of the Ca(2+)-dependent isozymes, compared with the Ca(2+)-independent isozymes. In contrast, UCN-02 (the diastereomer of UCN-01) and staurosporine exhibited less ability to discriminate between Ca(2+)-dependent and -independent isozymes. PKC-zeta was not inhibited by UCN-01, UCN-02, or staurosporine. IC50 values for UCN-01 inhibition of the Ca(2+)-dependent PKC-alpha, -beta, and -gamma were 29, 34, and 30 nM, respectively, and for the Ca(2+)-independent PKC-delta and -epsilon were 530 and 590 nM, respectively. IC50 values for staurosporine inhibition of the isozymes alpha, beta, and gamma were 58, 65, and 49 nM, respectively, and for the isozymes delta and epsilon were 325 and 160 nM, respectively. UCN-02 was significantly less potent for the inhibition of PKC-alpha, -beta, -gamma, -delta, and -epsilon (IC50 values of 530, 700, 385, 2800, and 1200 nM, respectively). An analysis of the inhibition by UCN-01 and staurosporine of the kinase activity of PKC-alpha and -delta indicated mixed inhibition kinetics. Increasing the ATP concentration resulted in decreased potency, as shown by increased IC50 values. In contrast, increasing the peptide substrate concentration resulted in increased potency, as shown by decreased IC50 values. Increasing concentrations of myelin basic protein as a PKC-alpha or -delta substrate also caused increased potency of inhibition by UCN-01. Because of the competitive nature of inhibition with respect to ATP and the uncompetitive nature with respect to substrate, the concentrations of these substrates can have dramatically different effects on the degree of inhibition observed. These data also suggest that UCN-01 may be an important tool for the dissection of PKC isozyme contributions to signal transduction pathways.

Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells.

UCN-01 is a derivative of staurosporine, initially developed as a potentially selective inhibitor of the Ca(2+)- and phospholipid-dependent protein kinase C, but with the capacity to inhibit a number of tyrosine and serine/threonine kinases. UCN-01 inhibits the growth of 5 breast carcinoma cell lines with a 50% inhibitory concentration range of 30-100 nM during 6 days of continuous exposure. In MCF-7, MDA-MB453, and SK-BR-3 cells, UCN-01 is 5-fold more potent in growth inhibition than its diastereomer UCN-02, but the 2 compounds are equipotent in the inhibition of MDA-MB468 and H85787 cell growth. A differential sensitivity to a 24-h period of exposure to UCN-01 followed by drug removal and growth for 5 subsequent days was observed. The rank order for persistent inhibition of cells by UCN-01 was MCF-7, MDA-MB453 >> SK-BR-3 > H85787 > MDA-MB468. MCF-7 and MDA-MB453 cells did not resume proliferation within the 5 days after brief exposure to UCN-01. In contrast, MDA-MB468 and H85787 cells showed no net growth inhibition after a 24-h pulse of UCN-01, followed by 5 more days of growth in drug-free medium. In MDA-MB468 cells, 150 nM UCN-01 retards but does not prevent cell cycle progression through S phase, but the cells are clearly blocked from exit of G1 and entry into S. Progression through S phase is completely inhibited by 600 nM UCN-01. The development of a G1 to S block by UCN-01 in MDA-MB468 cells occurs in conjunction with inhibition of [32P]orthophosphate labeling and decreased phosphotyrosine mass of discrete cellular phosphoproteins.