A combinatorial biomarker predicts pathologic complete response to neoadjuvant lapatinib and trastuzumab without chemotherapy in patients with HER2+ breast cancer.

BACKGROUND: HER2-positive (+) breast cancers, defined by HER2 overexpression and/or amplification, are often addicted to HER2 to maintain their malignant phenotype. Yet, some HER2+ tumors do not benefit from anti-HER2 therapy. We hypothesize that HER2 amplification levels and PI3K pathway activation are key determinants of response to HER2-targeted treatments without chemotherapy. PATIENTS AND METHODS: Baseline HER2+ tumors from patients treated with neoadjuvant lapatinib plus trastuzumab [with endocrine therapy for estrogen receptor (ER)+ tumors] in TBCRC006 (NCT00548184) were evaluated in a central laboratory for HER2 amplification by fluorescence in situ hybridization (FISH) (n = 56). HER2 copy number (CN) and FISH ratios, and PI3K pathway status, defined by PIK3CA mutations or PTEN levels by immunohistochemistry were available for 41 tumors. Results were correlated with pathologic complete response (pCR; no residual invasive tumor in breast). RESULTS: Thirteen of the 56 patients (23%) achieved pCR. None of the 11 patients with HER2 ratio <4 and/or CN <10 achieved pCR, whereas 13/45 patients (29%) with HER2 ratio ≥4 and/or CN ≥10 attained pCR (P = 0.0513). Of the 18 patients with tumors expressing high PTEN or wild-type (WT) PIK3CA (intact PI3K pathway), 7 (39%) achieved pCR, compared with 1/23 (4%) with PI3K pathway alterations (P = 0.0133). Seven of the 16 patients (44%) with HER2 ratio ≥4 and intact PI3K pathway achieved pCR, whereas only 1/25 (4%) patients not meeting these criteria achieved pCR (P = 0.0031). CONCLUSIONS: Our findings suggest that there is a clinical subtype in breast cancer with high HER2 amplification and intact PI3K pathway that is especially sensitive to HER2-targeted therapies without chemotherapy. A combination of HER2 FISH ratio and PI3K pathway status warrants validation to identify patients who may be treated with HER2-targeted therapy without chemotherapy.

Hyperprolactinemia-inducing antipsychotics increase breast cancer risk by activating JAK-STAT5 in precancerous lesions.

BACKGROUND: Psychiatric medications are widely prescribed in the USA. Many antipsychotics cause serum hyperprolactinemia as an adverse side effect; prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling both induces cell differentiation and suppresses apoptosis. It is controversial whether these antipsychotics increase breast cancer risk. METHODS: We investigated the impact of several antipsychotics on mammary tumorigenesis initiated by retrovirus-mediated delivery of either ErbB2 or HRas or by transgenic expression of Wnt-1. RESULTS: We found that the two hyperprolactinemia-inducing antipsychotics, risperidone and pimozide, prompted precancerous lesions to progress to cancer while aripiprazole, which did not cause hyperprolactinemia, did not. We observed that risperidone and pimozide (but not aripiprazole) caused precancerous cells to activate STAT5 and suppress apoptosis while exerting no impact on proliferation. Importantly, we demonstrated that these effects of antipsychotics on early lesions required the STAT5 gene function. Furthermore, we showed that only two-week treatment of mice with ruxolitinib, a JAK1/2 inhibitor, blocked STAT5 activation, restored apoptosis, and prevented early lesion progression. CONCLUSIONS: Hyperprolactinemia-inducing antipsychotics instigate precancerous cells to progress to cancer via JAK/STAT5 to suppress the apoptosis anticancer barrier, and these cancer-promoting effects can be prevented by prophylactic anti-JAK/STAT5 treatment. This preclinical work exposes a potential breast cancer risk from hyperprolactinemia-inducing antipsychotics in certain patients and suggests a chemoprevention regime that is relatively easy to implement compared to the standard 5-year anti-estrogenic treatment in women who have or likely have already developed precancerous lesions while also requiring hyperprolactinemia-inducing antipsychotics.

CYP2D6 genotype is not associated with survival in breast cancer patients treated with tamoxifen: results from a population-based study.

PURPOSE: A number of studies have tested the hypothesis that breast cancer patients with low-activity CYP2D6 genotypes achieve inferior benefit from tamoxifen treatment, putatively due to lack of metabolic activation to endoxifen. Studies have provided conflicting data, and meta-analyses suggest a small but significant increase in cancer recurrence, necessitating additional studies to allow for accurate effect assessment. We conducted a retrospective pharmacogenomic analysis of a prospectively collected community-based cohort of patients with estrogen receptor-positive breast cancer to test for associations between low-activity CYP2D6 genotype and disease outcome in 500 patients treated with adjuvant tamoxifen monotherapy and 500 who did not receive any systemic adjuvant therapy. METHODS: Tumor-derived DNA was genotyped for common, functionally consequential CYP2D6 polymorphisms (*2, *3, *4, *6, *10, *41, and copy number variants) and assigned a CYP2D6 activity score (AS) ranging from none (0) to full (2). Patients with poor metabolizer (AS = 0) phenotype were compared to patients with AS > 0 and in secondary analyses AS was analyzed quantitatively. Clinical outcome of interest was recurrence free survival (RFS) and analyses using long-rank test were adjusted for relevant clinical covariates (nodal status, tumor size, etc.). RESULTS: CYP2D6 AS was not associated with RFS in tamoxifen treated patients in univariate analyses (p > 0.2). In adjusted analyses, increasing AS was associated with inferior RFS (Hazard ratio 1.43, 95% confidence interval 1.00-2.04, p = 0.05). In patients that did not receive tamoxifen treatment, increasing CYP2D6 AS, and AS > 0, were associated with superior RFS (each p = 0.0015). CONCLUSIONS: This population-based study does not support the hypothesis that patients with diminished CYP2D6 activity achieve inferior tamoxifen benefit. These contradictory findings suggest that the association between CYP2D6 genotype and tamoxifen treatment efficacy is null or near null, and unlikely to be useful in clinical practice.

Balancing the risks and benefits of genomic data sharing: genome research participants' perspectives.

BACKGROUND: Technological advancements are rapidly propelling the field of genome research forward, while lawmakers attempt to keep apace with the risks these advances bear. Balancing normative concerns of maximizing data utility and protecting human subjects, whose privacy is at risk due to the identifiability of DNA data, are central to policy decisions. Research on genome research participants making real-time data sharing decisions is limited; yet, these perspectives could provide critical information to ongoing deliberations. METHODS: We conducted a randomized trial of 3 consent types affording varying levels of control over data release decisions. After debriefing participants about the randomization process, we invited them to a follow-up interview to assess their attitudes toward genetic research, privacy and data sharing. RESULTS: Participants were more restrictive in their reported data sharing preferences than in their actual data sharing decisions. They saw both benefits and risks associated with sharing their genomic data, but risks were seen as less concrete or happening in the future, and were largely outweighed by purported benefits. CONCLUSION: Policymakers must respect that participants' assessment of the risks and benefits of data sharing and their privacy-utility determinations, which are associated with their final data release decisions, vary. In order to advance the ethical conduct of genome research, proposed policy changes should carefully consider these stakeholder perspectives.

Keratin 6a marks mammary bipotential progenitor cells that can give rise to a unique tumor model resembling human normal-like breast cancer.

Progenitor cells are considered an important cell of origin of human malignancies. However, there has not been any single gene that can define mammary bipotential progenitor cells, and as such it has not been possible to use genetic methods to introduce oncogenic alterations into these cells in vivo to study tumorigenesis from them. Keratin 6a is expressed in a subset of mammary luminal epithelial cells and body cells of terminal end buds. By generating transgenic mice using the Keratin 6a (K6a) gene promoter to express tumor virus A (tva), which encodes the receptor for avian leukosis virus subgroup A (ALV/A), we provide direct evidence that K6a(+) cells are bipotential progenitor cells, and the first demonstration of a non-basal location for some biopotential progenitor cells. These K6a(+) cells were readily induced to form mammary tumors by intraductal injection of RCAS (an ALV/A-derived vector) carrying the gene encoding the polyoma middle T antigen. Tumors in this K6a-tva line were papillary and resembled the normal breast-like subtype of human breast cancer. This is the first model of this subtype of human tumors and thus may be useful for preclinical testing of targeted therapy for patients with normal-like breast cancer. These observations also provide direct in vivo evidence for the hypothesis that the cell of origin affects mammary tumor phenotypes.

DNA repair signature is associated with anthracycline response in triple negative breast cancer patients.

We hypothesized that a subset of sporadic triple negative (TN) breast cancer patients whose tumors have defective DNA repair similar to BRCA1-associated tumors are more likely to exhibit up-regulation of DNA repair-related genes, anthracycline-sensitivity, and taxane-resistance. We derived a defective DNA repair gene expression signature of 334 genes by applying a previously published BRCA1-associated expression pattern to three datasets of sporadic TN breast cancers. We confirmed a subset of 69 of the most differentially expressed genes by quantitative RT-PCR, using a low density custom array (LDA). Next, we tested the association of this DNA repair microarray signature expression with pathologic response in neoadjuvant anthracycline trials of FEC (n = 50) and AC (n = 16), or taxane-based TET chemotherapy (n = 39). Finally, we collected paraffin-fixed, formalin-embedded biopsies from TN patients who had received neoadjuvant AC (n = 28), and tested the utility of the LDA to discriminate response. Correlation between RNA expression measured by the microarrays and 69-gene LDA was ascertained. This defective DNA repair microarray gene expression pattern was significantly associated with anthracycline response and taxane resistance, with the area under the ordinary receiver operating characteristic curve (AUC) of 0.61 (95% CI = 0.45-0.77), and 0.65 (95% CI = 0.46-0.85), respectively. From the FFPE samples, the 69-gene LDA could discriminate AC responders, with AUC of 0.79 (95% CI = 0.59-0.98). In conclusion, a promising defective DNA repair gene expression signature appears to differentiate TN breast cancers that are sensitive to anthracyclines and resistant to taxane-based chemotherapy, and should be tested in clinical trials with other DNA-damaging agents and PARP-1 inhibitors.

The efficacy of sertraline for controlling hot flashes in women with or at high risk of developing breast cancer.

The aim of the study is to evaluate the efficacy of sertraline for controlling hot flashes in women with or at high risk of breast cancer. This was a randomized, double-blind, placebo-controlled study. All participants were asked to complete hot flash diaries. Participants reporting weekly hot flash scores >15 during baseline week underwent a 1-week single-blind placebo run-in. Those reporting hot flash score reductions >50% following placebo run-in were excluded. The remaining women received an assigned treatment for 4 weeks. Both groups' demographic and clinical characteristics were similar with a greater decline, but not statistically significant, in hot flash frequencies and scores in the sertraline-treated group compared with the placebo (P = 0.13 and P = 0.15, respectively). Emotional well-being improved significantly in the sertraline group (P = 0.041). The study failed to demonstrate effectiveness of sertraline in attenuating hot flashes in women with or at high risk of developing breast cancer who were not recommended to take hormone replacement therapy.

Hormone receptor status of a contralateral breast cancer is independent of the receptor status of the first primary in patients not receiving adjuvant tamoxifen.

PURPOSE: To determine whether the hormone receptor status of the primary breast cancer (PBC) is predictive of the hormone receptor status of the subsequent contralateral breast cancer (CBC). PATIENTS AND METHODS: We identified patients in our database with known estrogen receptor (ER; n = 193) and/or progesterone receptor (PgR; n = 178) status in their PBC and in their subsequent CBC. One hundred twenty-six of these patients had received no adjuvant therapy, 34 had received adjuvant tamoxifen, and 33 had received adjuvant chemotherapy alone. The median interval between the first diagnosis of PBC and the development of the subsequent CBC was 3 years. ER and PgR assays were assessed biochemically in two central reference laboratories using identical quality-controlled ligand-binding methods. RESULTS: Among systemically untreated patients (n = 126), 88% of patients with ER-positive PBC and 75% of patients with ER-negative PBC developed an ER-positive CBC (P = .11). Among the tamoxifen-treated patients, those with an ER-positive PBC were almost equally likely to develop an ER-positive (47%) or ER-negative (53%) CBC (P = .99). PgR status was similar. In the untreated group (n = 112), 59% of patients with a PgR-positive PBC and 66% with a PgR-negative PBC developed a PgR-positive CBC (P = .48). Among tamoxifen-treated patients (n = 33), 50% of patients with a PgR-positive PBC versus 27% of patients with a PgR-negative PBC developed a PgR-positive CBC (P = .28). CONCLUSION: ER and PgR status of the primary tumor does not predict the hormone receptor status of the subsequent CBC in the absence of selective pressure of adjuvant therapy. Thus, other reasons should be considered to clarify the failure of tamoxifen to reduce the incidence of CBC in patients with a receptor-negative PBC.

Genomic approaches in the management and treatment of breast cancer.

Breast cancer is the most common malignancy afflicting women from Western cultures. It has been estimated that approximately 211 000 women will be diagnosed with breast cancer in 2003 in the United States alone, and each year over 40 000 women will die of this disease. Developments in breast cancer molecular and cellular biology research have brought us closer to understanding the genetic basis of this disease. Unfortunately, this information has not yet been incorporated into the routine diagnosis and treatment of breast cancer in the clinic. Recent advancements in microarray technology hold the promise of further increasing our understanding of the complexity and heterogeneity of this disease, and providing new avenues for the prognostication and prediction of breast cancer outcomes. The most recent application of microarray genomic technologies to studying breast cancer will be the focus of this review.

Early exposure of the rat mammary gland to estrogen and progesterone blocks co-localization of estrogen receptor expression and proliferation.

An early single full-term pregnancy induces a long-lasting protective effect against mammary tumor development in humans and rodents. This protective effect can be mimicked in rats by short-term administration of estrogen and progesterone hormones prior to carcinogen administration. The hormones of pregnancy are able to induce a proliferative block upon carcinogen challenge that is not observed in the age-matched virgin. We wished to determine whether carcinogen is needed to induce a paracrine-to-autocrine shift of proliferation in steroid receptor positive cells or if such a cell population already exists in the age-matched virgin mammary gland. Here we show that estrogen receptor positive (ER+) proliferating cells are rare in the developing mammary gland of the virgin rat but represent the majority of the proliferating cells in the mature (96-day-old) mammary gland of the virgin rat. As the majority of the proliferating cells before carcinogen challenge were ER positive, the ER+ proliferating cells in the mature mammary gland may represent the target cells for carcinogen-induced transformation. Importantly, prior exposure of the mammary gland to pregnancy levels of estrogen/progesterone blocked this positive association. This ability to block the proliferation of the ER+ cells may be one factor by which pregnancy induces protection against breast cancer.

Pathological features and BRCA1 mutation screening in premenopausal breast cancer patients.

PURPOSE: Risk calculations for carrying BRCA1/BRCA2 mutations are based on family history and the age of onset of cancers. However, women may carry these deleterious mutations without a strong family history. Additional criteria for risk estimation would be of value. It has been recently established that BRCA1-associated breast cancers are associated with poor tumor differentiation (TD3) and estrogen receptor (ER) negativity. The aim of this study is to determine whether morphological features of breast cancers in premenopausal patients (age < 45 years) could determine additional women who may benefit from BRCA1 screening. EXPERIMENTAL DESIGN: In a prospective, systematic study of 76 consecutive breast cancer patients (age < 45 years), genomic DNA was obtained from peripheral blood, and eight mutations in BRCA1 (10.5%) were found. Archival paraffin-embedded breast cancer specimens were then analyzed for tumor differentiation and ER status. RESULTS: In patients < 45 years of age, 25% (6 of 24) of ER-negative and TD3 breast cancers were found to harbor mutations in BRCA1. Only 5.6% (2 of 36) of BRCA1-associated breast cancers did not have this morphological profile, compared with 94.4% (34 of 36) patients without BRCA1 mutations, giving an odds ratio of 5.67 (95% confidence interval, 1.04-32; P = 0.05). Finally, only one patient with BRCA1 mutations had a significant family history. CONCLUSIONS: In patients with early-onset breast cancer, the use of morphological criteria provides an additional strategy to determine those patients who might benefit from genetic testing.

AP-1 blockade inhibits the growth of normal and malignant breast cells.

We have previously demonstrated that basal AP-1 transcriptional activity is high in normal human mammary epithelial cells, intermediate in immortal breast cells, and relatively low in breast cancer cells. In this study we investigated whether differences in AP-1 transcriptional activity reflect differences in breast cells' dependence on AP-1 for proliferation. The cJun dominant negative, TAM-67, was used to determine the effect of AP-1 blockade on the growth of normal, immortal and malignant breast cells. We first showed that TAM-67 inhibits AP-1 activity in normal and malignant breast cells. We then determined whether this AP-1 inhibitor affected colony forming efficiency of the immortalized and malignant breast cells. The AP-1 inhibitor reduced colony formation of immortal breast cells by over 50% (by 58% in 184B5 cells and 62% in MCF10A cells), and reduced colony formation in the breast cancer cell line MCF7 by 43%, but did not reduce colony formation in the other breast cancer cell lines (T47D, MDA MB231 and MDA MB 435). We also determined the effect of AP-1 blockade on the growth of normal breast cells using a single cell proliferation assay. Using this assay, the growth of normal breast cells was extremely sensitive to AP-1 blockade, while immortal breast cells were moderately sensitive. We next directly tested the effect of TAM-67 expression on the growth of MCF7 breast cancer cells, using cells stably transfected with TAM-67 under the control of a doxycycline-inducible promoter. Upon induction, TAM-67 was expressed and AP-1 activity was inhibited in these cells. We then measured the growth of these cells in the presence or absence of TAM-67. The results of these studies show that the growth of MCF7 cells was suppressed by the AP-1 inhibitor, TAM-67. These results demonstrate that normal and immortalized breast cells, and some breast cancer cells (such as MCF7), require AP-1 to transduce proliferative signals, while other breast cancer cells (such as T47D, MDA MB 231 and MDA MB 435) do not. These studies suggest that the AP-1 transcription factor is a potential target for future agents for the prevention or treatment of breast cancer.

Array-based gene expression profiling to study aging.

With recent sequencing of the genome and development of high-density array technology, it is now possible to assess global gene expression in cells/tissues by a technique that is sensitive, quantitative, and rapid. Gene expression array technology is extremely useful in studying a complex, multigenetic process, such as aging, where one needs to understand the interaction of a large number of genes. Although the technology holds great promise, it is novel and not yet well-established and there are no widely-accepted standards to guide investigators in the analysis and interpretation of the data obtained. Gene expression array analysis requires strong biostatistical support to minimize false-positives and maximize true-positives in candidate gene identification. It also requires independent validation of the array measurements using other detection methods. Confirmation that differentially expressed (transcribed) genes are reflected by differential expression at the protein level will ultimately be an important measurement. In this review, we focus on the three steps necessary for aging studies when using the gene expression array technology: (1) array hybridization; (2) biostatistical analysis; and (3) array result confirmation. Genes identified by several investigators for their age-associated change using the gene expression array systems are also discussed.

Hsp27-induced MMP-9 expression is influenced by the Src tyrosine protein kinase yes.

The small heat shock protein hsp27 is associated with aggressive tumor behavior in certain subsets of breast cancer patients. Previously we demonstrated that hsp27 overexpression in breast cancer cells increased in vitro and in vivo invasiveness, suggesting that hsp27 influences the metastatic process. To investigate this role for hsp27, we have utilized MDA-MB-231 breast cancer cells that overexpress hsp27 and cDNA expression array technology. We demonstrate that hsp27 overexpression up-regulates MMP-9 expression and activity and down-regulates Yes expression. Furthermore, our results suggest that Yes may be involved in regulating MMP-9 expression, as well as in vitro invasion. Reconstitution of Yes expression by transfection into hsp27-overexpressing cells decreased MMP-9 expression, and increased in vitro invasiveness, abrogating the phenotype conferred by hsp27 overexpression. Therefore, our results provide a new potential mechanism by which hsp27 affects the metastatic cascade-through regulation of MMP-9 and Yes expression.

Mechanisms of hormonal prevention of breast cancer.

Reproductive history is a consistent risk factor for human breast cancer. Epidemiological studies have repeatedly demonstrated that early age of first pregnancy is a strong protective factor against breast cancer and provides a physiologically operative model to achieve a practical mode of prevention. In rodents, the effects of full-term pregnancy can be mimicked by a three-week exposure to low doses of estrogen and progesterone. Neither hormone alone is sufficient to induce protection. The cellular and molecular mechanisms that underlie hormone-induced refractoriness are largely unresolved. Our recent studies have demonstrated that an early cellular response that is altered in hormone-treated mammary cells is the initial proliferative burst induced by the chemical carcinogen methylnitrosourea. The decrease in proliferation is also accompanied by a decrease in the ability of estrogen receptor-positive cells to proliferate. RNA expression of several mammary cell-cycle-related genes is not altered in hormone-treated mice; however, immunohistochemical assays demonstrate that the protein level and nuclear compartmentalization of the p53 tumor suppressor gene are markedly upregulated as a consequence of hormone treatment. These results support the hypothesis that hormone stimulation, at a critical period in mammary development, results in cells with persistent changes in the intracellular regulatory loops governing proliferation and response to DNA damage. A corollary to this hypothesis is that the genes affected by estrogen and progesterone are independent of alveolar differentiation-specific genes. Suppressive subtractive hybridization-PCR methods have identified several genes that are differentially expressed as a consequence of prior estrogen and progesterone treatment. Future experiments are aimed at determining the mechanisms of hormone-induced upregulation of p53 protein expression as part of the overall goal of identifying and functionally characterizing the genes responsible for the refractory phenotype.

Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.

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(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<.001) and HMS activity (10-fold; P<.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<.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.

Enhanced antitumour activity of 6-hydroxymethylacylfulvene in combination with topotecan or paclitaxel in the MV522 lung carcinoma xenograft model.

6-Hydroxymethylacylfulvene (HMAF; MGI 114; Irofulven) is a semisynthetic analogue of the toxin illudin S, which is a product of the Omphalotus mushroom. MGI 114 induces cytotoxicity against a broad range of solid tumours in vivo, including the drug-refractory MV522 human lung cancer xenograft. In this study, the potential application of MGI 114 in the treatment of lung cancer was explored by evaluating the activity of MGI 114 in combination with either topotecan (TPT) or paclitaxel. Groups of eight nude mice bearing MV522 xenografts were treated with MGI 114, TPT or paclitaxel as single agents and with MGI 114 in combination with TPT or paclitaxel. MGI 114 was administered at doses of 2.5 and 5.0 mg/kg intraperitoneally (i.p.) daily on days 1-5, while TPT and paclitaxel were administered at doses of 0.5 or 1.0 mg/kg and 20 mg/kg, respectively, i.p. on days 1-5. In the single-agent studies, MGI 114, TPT and paclitaxel all resulted in decreased final tumour weights compared with vehicle-treated controls. As single agents, TPT, at the 0.5 mg/kg dose level, and paclitaxel, at the 20 mg/kg dose level, produced partial shrinkages (PSs). All combinations of MGI 114, and either TPT or paclitaxel, produced decrements in final tumour weights compared with monotherapy with the same doses of MGI 114, TPT and paclitaxel. Although all animals treated with the combination of MGI 114 and paclitaxel experienced PSs or complete shrinkages (CSs) (or died), analysis of the time to tumour doubling revealed that the combination of MGI 114 and TPT at 2.5 and 0.5 mg/kg, respectively, was synergistic. These results suggest that cytotoxic activity is enhanced when MGI 114 is combined with either TPT or paclitaxel, and clinical trials to further evaluate these combination regimens are warranted.

9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice.

Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.

cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by calorie restriction.

Calorie restriction (CR) extends life span and retards many age-related cellular and molecular changes in laboratory rodents. However, neither the breadth of its effects, its underlying mechanisms, nor the limits of its action is fully understood. Expression levels of 588 genes in livers from 3- and 24-month-old ad libitum-fed (AL), and 24-month-old CR (60% of AL intake) male C57BL/6J mice (four per group) were measured. Six genes met the statistical criteria for differential expression in old AL compared to young AL mice. Only one of these age-related changes was attenuated by CR. Four additional gene products, that did not change with age in AL mice, were differentially expressed in old CR compared to old AL mice. Northern and RT-PCR analyses confirmed differential expression of four of the six candidate genes identified by the array results. Many of the identified genes have not previously been reported to be affected by CR or aging. Some of the age-related changes in gene expression are consistent with an increased vulnerability of the aged liver to carcinogenic or other insults, with only partial protection against insult by CR. Incomplete reversal by CR of age-related changes in gene expression provides a potentially important path for probing the limits of CR action. These results also show the importance of independent confirmation in expression array profiling of age-related changes in gene expression.

Laparoscopic cholecystectomy and cirrhosis: a case-control study of outcomes.

The incidence of gallstone disease in patients with cirrhosis is greater than that in healthy patients. Previous surgical literature reported greater morbidity and mortality in patients with cirrhosis with both open and laparoscopic cholecystectomy (LC). We compared our recent experience with LC in patients with cirrhosis and controls. A retrospective review was performed using the search terms, "cirrhosis" and "laparoscopic cholecystectomy." Forty-eight patients with cirrhosis were identified and randomly matched with healthy controls by age and sex. Four controls were assigned per patient with cirrhosis. Outcomes assessed included mortality, duration of surgery, length of hospital stay, blood transfusion requirement, postoperative complications, and need for conversion to open cholecystectomy. Forty-eight patients with cirrhosis and 187 healthy controls underwent LC. Child-Pugh classification of severity of liver disease was as follows: Child's class A, 38 of 48 patients; Child's class B, 10 of 48 patients; and Child's class C, 0 of 48 patients. Patients with cirrhosis had statistically significantly lower albumin levels (P =.0001) and prolonged prothrombin times (P =. 05). Average duration of surgery for patients with cirrhosis was 1. 71 versus 1.57 hours (P =.57) for controls. Average length of hospital stay for patients with cirrhosis was 6.47 versus 4.77 days (P =.152) for controls. Average number of units of blood transfused in patients with cirrhosis was 0.156 versus 0.0 units (P =.025) in controls. Complications occurred in 6 of 48 patients with cirrhosis (12.5%) and 8 of 187 controls (4.2%; P <.05). No child's class C patient underwent LC. Four patients with cirrhosis (8.3%) and no controls were converted to open cholecystectomy. No postoperative infections were noted. There was no mortality in either group. LC in patients with Child's class A and B cirrhosis is reasonably safe and shows no increase in morbidity or mortality or worsening of outcome. Further studies are required to evaluate the management of acute gallbladder disease in Child's class C patients.