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Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)--negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor--induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

Background Tumorigenic breast cancer cells that express high levels of CD44 and low or undetectable levels of CD24 (CD[44.sup.+]/CD[24.sup.-/low]) may be resistant to chemotherapy and therefore responsible for cancer relapse. These tumorigenic cancer cells can be isolated from breast cancer biopsies and propagated as mammospheres in vitro. In this study, we aimed to test directly in human breast cancers the effect of conventional chemotherapy or lapatinib (an epidermal growth factor receptor [EGFR]/HER2 pathway inhibitor) on this tumorigenic CD[44.sup.+] and CD[24.sup.-/low] cell population. Methods Paired breast cancer core biopsies were obtained from patients with primary breast cancer before and after 12 weeks of treatment with neoadjuvant chemotherapy (n = 31) or, for patients with HER2-positive tumors, before and after 6 weeks of treatment with the EGFR/HER2 inhibitor lapatinib (n = 21). Single-cell suspensions established from these biopsies were stained with antibodies against CD24, CD44, and lineage markers and analyzed by flow cytometry. The potential of cells from biopsy samples taken before and after treatment to form mammospheres in culture was compared. All statistical tests were two-sided. Results Chemotherapy treatment increased the percentage of CD[44.sup.+]/CD[24.sup.-/low]cells (mean at baseline vs 12 weeks, 4.7%, 95% confidence interval [CI] = 3.5% to 5.9%, vs 13.6%, 95% CI = 10.9% to 16.3%; P < .001) and increased mammosphere formation efficiency (MSFE) (mean at baseline vs 12 weeks, 13.3%, 95% CI = 6.0% to 20.6%, vs 53.2%, 95% CI = 42.4% to 64.0%; P < .001). Conversely, lapatinib treatment of patients with HER2-positive tumors led to a non-statistically significant decrease in the percentage of CD[44.sup.+]/CD[24.sup.-/low] cells (mean at baseline vs 6 weeks, 10.0%, 95% CI = 7.2% to 12.8%, vs 7.5%, 95% CI = 4.1% to 10.9%) and a non--statistically significant decrease in MSFE (mean at baseline vs 6 weeks, 16.1%, 95% CI = 8.7% to 23.5%, vs 10.8%, 95% CI = 4.0% to 17.6%). Conclusion These studies provide clinical evidence for a subpopulation of chemotherapy-resistant breast cancer-initiating cells. Lapatinib did not lead to an increase in these tumorigenic cells, and, in combination with conventional therapy, specific pathway inhibitors may provide a therapeutic strategy for eliminating these cells to decrease recurrence and improve long-term survival.

Background: AIB1 (SRC-3) is an estrogen receptor (ER) co-activator that, when overexpressed in cultured cells, can reduce the antagonist activity of tamoxifen-bound ERs. Signaling through the HER-2 receptor pathway activates AIB1 by phosphorylation. To determine whether high AIB1 expression alone or together with HER-2 reduces the effectiveness of tamoxifen in breast cancer patients, we quantified expression of AIB1 and HER-2 in tumors from breast cancer patients with long-term clinical follow-up who received either no adjuvant therapy or adjuvant tamoxifen therapy after breast cancer surgery. Methods: AIB1 and HER-2 protein levels in tumors from 316 breast cancer patients were determined using western blot analysis. Molecular variables (e.g., expression of AIB1, ER, progesterone receptor, p53, Bcl-2), tumor characteristics, and patient outcome were assessed using Spearman rank correlation. Disease-free survival (DFS) curves were derived from Kaplan-Meier estimates, and the curves were compared by log-rank tests. The effect of AIB1 on DFS adjusted for other prognostic factors was assessed by multivariable analysis using the Cox proportional hazards model. All statistical tests were two-sided. Results: High AIB1 expression in patients not receiving adjuvant tamoxifen therapy was associated with better prognosis and longer DFS (P = .018, log-rank test). In contrast, for patients who did receive tamoxifen therapy, high AIB1 expression was associated with worse DFS (P = .049, log-rank test), which is indicative of tamoxifen resistance. The test for interaction between AIB1 expression and tamoxifen therapy was statistically significant (P = .004). When expression of AIB1 and HER-2 were considered together, patients whose tumors expressed high levels of both AIB1 and HER-2 had worse outcomes with tamoxifen therapy than all other patients combined (P = .002, log-rank test). Conclusions: The antitumor activity of tamoxifen in patients with breast cancer may be determined, in part, by tumor levels of AIB1 and HER-2. Thus, AIB1 may be an important diagnostic and therapeutic target. [J Natl Cancer Inst 2003;95:353-61]

PURPOSE: The effects of continuous or intermittent therapy with fluconazole on the recurrence of and the development of fluconazole resistance are not known. PATIENTS AND METHODS: We studied human immunodeficiency virus (HIV)-positive patients with CD4 cell count [is less than] 350 x [10.sup.6]/L and oropharyngeal candidiasis in a prospective, randomized study. After initial treatment, 20 patients (16 of whom completed 3 months of follow-up) received continuous fluconazole at 200 mg/day, and 48 patients (28 of whom completed follow-up) received intermittent therapy at the time of symptomatic relapses. Oral samples were obtained weekly during episodes of infection and quarterly as surveillance cultures. Development of resistance was defined as a fourfold rise in minimum inhibitory concentration (MIC) to at least 16 [micro]g/mL from the initial culture in the same species, the emergence of new, resistant (MIC [is greater than or equal to] 16 [micro]g/mL) species, or a significant increase in the proportion of resistant isolates. RESULTS: During a mean follow-up of 11 months, median annual relapse rates were lower in patients on continuous therapy (0 episodes/year) than in patients on intermittent therapy (4.1 episodes/year; P [is less than] 0.001). Sterile cultures were seen in 6 of 16 (38%) patients on continuous therapy compared with 3 of 28 (11%) on intermittent therapy (P = 0.04). Microbiological resistance developed in 9 of 16 (56%) patients on continuous treatment, compared with 13 of 28 (46%) on intermittent treatment (P = 0.75). However, despite isolates with increased MICs, 42 of 44 patients responded to fluconazole in doses up to 800 mg/day. CONCLUSIONS: In patients with frequent recurrences, continuous suppressive therapy significantly reduced relapses and colonization. Resistance occurred with both continuous and intermittent therapy; however, therapeutic responses were excellent. Am J Med. 1998;105:7-11. [C] 1998 by Excerpta Medica, Inc.

Study Objective: Increasing evidence indicates that routine preoperative diagnostic spirometry (pulmonary function tests [PFTs]) before elective abdominal surgery does not predict individual risk of postoperative pulmonary complications and is overutilized. [...]

Study objective: Intra-abdominal operations are relatively high risk for pulmonary complications. Previous research has more intensely investigated cardiac operative risk, but recent work suggests that significant pulmonary complications may be [...]

Background: The insulin-like growth factor (IGF)-binding proteins (IGFBPs) regulate the actions of the IGFs by influencing interactions between the IGFs and the IGF receptors. IGFBP-3, one of the six known species of IGFBPs, is the predominant IGFBP in serum and is expressed by breast cancer cells. Compared with estrogen receptor (PR)-positive samples, ER-negative breast cancer cell lines and tumors express higher levels of IGFBP-3. Therefore, expression of IGFBP-3 may be relevant in breast cancer biology, although it is unknown whether IGFBP-3 levels correlate with other breast cancer prognostic factors besides ER status. It is also not known how different methods used to measure IGFBP-3 in breast cancer correlate. Purpose: We measured IGFBP-3 messenger RNA (mRNA) and protein levels in breast tumors by different methods to test how these methods compare and to investigate the relationship between IGFBP-3 and breast cancer prognostic factors. Methods: We analyzed 40 human breast tumors and examined IGFBP-3 expression by ligand blot analysis, immunoblot analysis, immunoradiometric assay (IRMA), and ribonuclease protection assay. Another set of 40 breast tumors, selected according to ER and progesterone receptor (PR) status, S phase, and ploidy, was analyzed by IRMA. Results: In 26 (65%) of 40 samples in which RNA could be isolated, IGFBP-3 mRNA levels correlated with IGFBP-3 levels measured by IRMA (two-sided; P = .0001) but not with IGFBP-3 levels measured by ligand blot or immunoblot. Protein levels were highly correlated among all protein assays. Because the IRMA was more sensitive and accurate than the ligand blot and immunoblot assays, we used IRMA to examine IGFBP-3 levels in an additional 20 primary breast tumors with poor prognostic features (ER and PR negativity, high S phase, and aneuploidy) and in 20 tumors with good prognostic factors (opposite features). IGFBP-3 levels were threefold higher in tumors with poor prognostic features (mean [+ or -] standard deviation = 32.8 [+ or -] 25.2 versus 11.8 [+ or -] 9.7 ng/mg; two-sided; P = .003). Conclusions: These findings suggest that in human breast cancer, IGFBP-3 mRNA and protein levels are correlated and higher levels of IGFBP-3 are detectable in tumors with poor prognostic features. Implications: IGFBP-3 may be involved in the regulation of breast cancer cell growth. [J Natl Cancer Inst 1996;88:601-6]

Background: Cellular proliferation, as measured by S-phase fraction, is an important predictor of breast cancer prognosis. The insulin-like growth factors (IGFs) have been shown to regulate proliferation in both normal and neoplastic cells by interacting with specific cell surface receptors. In addition to these receptors, high-affinity extracellular binding proteins also modulate IGF action. These insulinlike growth factor-binding proteins (IGFBPs) could influence breast cancer growth and, like other biological parameters of proliferation, could be related to prognosis. Purpose: To test whether IGFBP expression was related to other biological parameters and disease-free survival, we measured IGFBP expression in 238 lymph node-negative primary breast cancer specimens. Methods: Proteins were extracted from breast cancer specimens and analyzed by semiquantitative IGF-I ligand blotting for IGFBP expression. IGFBP expression levels were compared to tumor size, age, S-phase fraction, DNA ploidy, and estrogen and progesterone receptor expression by Spearman correlation. results: Binding protein (BP)-2, BP-3, BP-4, and BP-5 were identified in breast cancer extracts. Estrogen receptor expression was positively correlated with BP-2 (Spearman correlation coefficient, [r.sub.s] = .262; P = .0001), BP-4 ([r.sub.s] = .313; P = .0001), and BP-5 ([r.sub.s] = .242; P = .0002). Similar correlations between progesterone receptor and BP-2, BP-4, and BP-5 were also found. BP-3 was inversely correlated with age ([r.sub.s] = -.251, P = .0001). BP-4 was weakly inversely correlated with tumor size ([r.sub.s] = -.141; P = .0295) and S-phase fraction ([r.sub.s] = -.216; P = .0025). Since tumor size and S-phase fraction are powerful predictors of prognosis in node-negative breast cancer, we examined the value of BP-4 as a predictor of diseasefree survival. When stratified by tumor size, patients with large (>2 cm) tumors that expressed low levels of BP-4 had improved survival when compared with patients with large tumors and high BP-4 levels (P = .001). Conclusions: IGFBPs can be detected in breast cancer specimens, and their level of expression correlates with other known biological parameters of breast cancer. Large tumors with low levels of BP-4 have relatively favorable prognoses. Implications: These data suggest that the IGFBPs may play a role in breast cancer biology and that BP-4 levels, analyzed in conjunction with tumor size, may have prognostic significance. [J Natl Cancer Inst 86:1785-1789, 1994!

Background. The incidence of estrogen receptor (ER)-positive breast cancer apparently is increasing. It remains unclear whether this increase is due to an improvement in receptor assay sensitivity, a change in patient characteristics, or a change in tumor biology. Methods. The distribution of ER, tumor size, and patient age for 11,195 tumor specimens gathered from patients nationwide from 1973 to 1992 were analyzed. All assays were performed in a single laboratory. A single-label, dextran-coated charcoal (DCC) method was used from 1973 to 1984, and a dual-label, DCC method, which allows the determination of both ER and progesterone receptor levels in the same assay, was used from 1985 to 1992. Results. The median level of ER has increased steadily from 14 fmol per milligram of protein in 1973 to 58 fmol per milligram of protein in 1992 (P < 0.0001). The percentage of ER-positive tumors also rose from 73-78% during the same period (P < 0.008). When the assay method was modified from single to dual label, no abrupt or stepwise increase occurred. Tumor size decreased over the same period (P < 0.0001). From 1973 to 1977, 48% of tumors were larger than 2 cm, and 15% were larger than 5 cm, compared to 60% and 90%, respectively, from 1988 to 1992. The percentage of women older than 50 years of age remained relatively constant over time. After adjusting for tumor size, age, number of positive lymph nodes, and change in assay method, a sustained rise in ER level remained. In a multivariate analysis that included age, age group, year of biopsy, tumor size, and number of positive nodes, the year of biopsy still was independently predictive of ER level (P < 0.0001). Conclusion. The measured level of ER in primary breast cancers has increased during the last 2 decades. It is unlikely that technical improvements or changes in tumor size, age, or nodal status fully explain this in crease. The rising level of ER may reflect a change in breast cancer biology and in hormonal events that influence breast cancer genesis and growth.

Background: Topotecan [(S)-9-dimethylaminomethyl(10-hydroxy-camptothecin), NSC 609699, SK&F 104864A], a semisynthetic analogue of the natural product camptothecin, is a cell cycle-specific drug that exerts antineoplastic activity through inhibition of topoisomerase I. Currently, topotecan is undergoing phase I and early phase II clinical trials. The dose-limiting toxicity for topotecan is myelosuppression. Purpose: Our purpose was to determine plasma concentrations and exposure times necessary for optimal clinical activity and tumor types that may be responsive in phase II clinical studies of topotecan. Methods: A soft-agar cloning system assay was used to determine the in vitro effects of topotecan against cells from biopsy specimens of colorectal, breast, lung, ovarian, renal cell, and gastric cancers and cancers of unknown primary origin. We studied 141 freshly explanted tumor specimens, using 1-hour exposure to topotecan, and 80 were studied using continuous exposure. A decrease in tumor colony formation resulting from drug exposure was considered an in vitro response if survival of colonies was up to 50% of that in controls. Results: With 1-hour exposure, in vitro responses were seen in 10% and 25% of assessable tumor specimens at final topotecan concentrations of 1.0 and 10.0 [mu]g/mL, respectively. With continuous exposures at concentrations of 0.1 and 1.0 [mu]g/mL, in vitro response rates were 34% and 76%, respectively. Specific activity was seen against colorectal, breast, non-small-cell lung, ovarian, and renal cell cancers, with responses observed in 27 %, 25%, 32%, 39%, and 83%, respectively, of assessable tumor specimens after continuous exposure to 0.1 [mu]g/mL topotecan. A subset of tumor specimens resistant to doxorubicin or fluorouracil was sensitive to topotecan, and the difference in sensitivity was statistically significant. In addition, some of the tumor specimens resistant to cyclophosphamide and etoposide were also sensitive to topotecan. Conclusions: Topotecan appears to be active in vitro against a variety of human tumors, including a subgroup resistant in vitro to standard antineoplastic agents. If plasma levels of 0.1 [mu]g/mL can be achieved for prolonged periods of time in ongoing clinical trials, topotecan should have substantial clinical activity. Implications: Further clinical development of topotecan is warranted. [J Natl Cancer Inst 84:181 1820, 1992]

Background: Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. Methods: Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun [NH.sub.2]-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. Results: Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P = .004) and GST (twofold; P = .004) activity and statistically significantly reduced glutathione levels (greater than twofold; P [is less than] .001) and HMS activity (10-fold; P [is less than] .001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P = .04) and phosphorylated c-Jun and JNK levels (P [is less than] .001) were statistically significantly increased in the tamoxifen-resistant tumors. Conclusion: Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth. [J Natl Cancer Inst 2000;92: 1926-34]

Background: Although the emerging complementary DNA (cDNA) array technology holds great promise to discern complex patterns of gene expression, its novelty means that there are no well-established standards to guide analysis and interpretation of the data that it produces. We have used preliminary data generated with the CLON-TECH Atlas[TM] human cDNA array to develop a practical approach to the statistical analysis of these data by studying changes in gene expression during the development of acquired tamoxifen resistance in breast cancer. Methods: For hybridization to the array, we prepared RNA from MCF-7 human breast cell tumors, isolated from our athymic nude mouse xenograft model of acquired tamoxifen resistance during estrogen-stimulated, tamoxifen-sensitive, and tamoxifen-resistant growth. Principal components analysis was used to identify genes with altered expression. Results and Conclusions: Principal components analysis yielded three principal components that are interpreted as 1) the average level of gene expression, 2) the difference between estrogen-stimulated gene expression and the average of tamoxifen-sensitive and tamoxifen-resistant gene expression, and 3) the difference between tamoxifen-sensitive and tamoxifen-resistant gene expression. A bivariate (second and third principal components) 99% prediction region was used to identify outlier genes that exhibit altered expression. Two representative outlier genes, erk-2 and HSF-1 (heat shock transcription factor-1), were chosen for confirmatory study, and their predicted relative expression levels were confirmed in western blot analysis, suggesting that semiquantitative estimates are possible with array technology. Implications: Principal components analysis provides a useful and practical method to analyze gene expression data from a cDNA array. The method can identify broad patterns of expression alteration and, based on a small simulation study, will likely provide reasonable power to detect moderate-sized alterations in clinically relevant genes. [J Natl Cancer Inst 1999; 91:453-9]