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BACKGROUND There have been many randomised trials of adjuvant tamoxifen among women with early breast cancer,and an updated overview of their results is presented. METHODS In 1995,information was sought on each woman in any randomised trial that began before 1990 of adjuvant tamoxifen versus no tamoxifen before recurrence. Information was obtained and analysed centrally on each of 37000 women in 55 such trials,comprising about 87% of the worldwide evidence. Compared with the previous such overview,this approximately doubles the amount of evidence from trials of about 5 years of tamoxifen and,taking all trials together,on events occurring more than 5 years after randomisation. FINDINGS Nearly 8000 of the women had a low,or zero,level of the oestrogen-receptor protein (ER) measured in their primary tumour. Among them,the overall effects of tamoxifen appeared to be small,and subsequent analyses of recurrence and total mortality are restricted to the remaining women (18000 with ER-positive tumours,plus nearly 12000 more with untested tumours,of which an estimated 8000 would have been ER-positive). For trials of 1 year,2 years,and about 5 years of adjuvant tamoxifen,the proportional recurrence reductions produced among these 30000 women during about 10 years of follow-up were 21% (SD 3),29% (SD 2),and 47% (SD 3),respectively,with a highly significant trend towards greater effect with longer treatment (chi2(1)=52.0,2ptextless0.00001). The corresponding proportional mortality reductions were 12% (SD 3),17% (SD 3),and 26% (SD 4),respectively,and again the test for trend was significant (chi2(1) = 8.8,2p=0.003). The absolute improvement in recurrence was greater during the first 5 years,whereas the improvement in survival grew steadily larger throughout the first 10 years. The proportional mortality reductions were similar for women with node-positive and node-negative disease,but the absolute mortality reductions were greater in node-positive women. In the trials of about 5 years of adjuvant tamoxifen the absolute improvements in 10-year survival were 10.9% (SD 2.5) for node-positive (61.4% vs 50.5% survival,2ptextless0.00001) and 5.6% (SD 1.3) for node-negative (78.9% vs 73.3% survival,2ptextless0.00001). These benefits appeared to be largely irrespective of age,menopausal status,daily tamoxifen dose (which was generally 20 mg),and of whether chemotherapy had been given to both groups. In terms of other outcomes among all women studied (ie,including those with ER-poor" tumours)� View Publication

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3),IL-6,granulocyte colony-stimulating factor,Flt3-ligand,and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells,colony-forming cells (CFC),or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period,and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However,the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant,17% +/- 3% and 17% +/- 8%,G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected,the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM),or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17,respectively),whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a textgreater/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols. View Publication

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore,one stem cell might easily account for the renewal of mammary epithelium over several transplant generations. View Publication

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold,P textless 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11,flt3-ligand,and Steel factor. Moreover,the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures,long-term culture-initiating cells increased 7- +/- 2-fold,myeloid colony-forming cells increased 140- +/- 36-fold,and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension,suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult. View Publication

CGP 57148 is a compound of the 2-phenylaminopyrimidine class that selectively inhibits the tyrosine kinase activity of the ABL and the platelet-derived growth factor receptor (PDGFR) protein tyrosine kinases. We previously showed that CGP 57148 selectively kills p210BCR-ABL-expressing cells. To extend these observations,we evaluated the ability of CGP 57148 to inhibit other activated ABL tyrosine kinases,including p185BCR-ABL and TEL-ABL. In cell-based assays of ABL tyrosine phosphorylation,inhibition of ABL kinase activity was observed at concentrations similar to that reported for p210BCR-ABL. Consistent with the in vitro profile of this compound,the growth of cells expressing activated ABL protein tyrosine kinases was inhibited in the absence of exogenous growth factor. Growth inhibition was also observed with a p185BCR-ABL-positive acute lymphocytic leukemia (ALL) cell line generated from a Philadelphia chromosome-positive ALL patient. As CGP 57148 inhibits the PDGFR kinase,we also showed that cells expressing an activated PDGFR tyrosine kinase,TEL-PDGFR,are sensitive to this compound. Thus,this compound may be useful for the treatment of a variety of BCR-ABL-positive leukemias and for treatment of the subset of chronic myelomonocytic leukemia patients with a TEL-PDGFR fusion protein. View Publication

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil,stem cell factor,and interleukin-3,88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost,but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure,most of the progenitors displayed a CD34+,CD38-,multidrug-resistance (MDR+),Rhodamine 123 low,Hoechst 33342 low phenotype,and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals,the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts. View Publication

We compared glucocorticoid receptor binding characteristics and glucocorticoid responsiveness of human mononuclear leukocytes (HML) from hypertensive patients and matched normotensive volunteers. We also considered associations of these variables with plasma renin activity,aldosterone,cortisol,corticotropin,and electrolyte concentrations. We calculated binding affinity (Kd; nmol/L) and capacity (Bmax; sites/cell) for dexamethasone and cortisol from homologous and heterologous competition curves for specific [3H]dexamethasone binding sites on HML isolated from the blood of normotensive volunteers and subjects with essential hypertension. Glucocorticoid responsiveness of HML was evaluated as IC50 values (nmol/L) for dexamethasone and cortisol for the inhibition of lysozyme release. We measured plasma hormones by radioimmunoassay. Kd values (mean+/-SE) for cortisol in HML of hypertensive patients were higher than in control subjects (24.6+/-2.4 versus 17.5+/-1.7 nmol/L,Ptextless.04). Binding capacity (4978+/-391 versus 4131+/-321 sites/cell),Kd values for dexamethasone (6.7+/-0.5 versus 5.7+/-0.3 nmol/L),and IC50 values for dexamethasone (3.4+/-0.3 versus 3.1+/-0.2 nmol/L) and cortisol (12.2+/-1.6 versus 9.5+/-0.3 nmol/L) were not significantly different. Patients with renin values less than 0.13 ng angiotensin I/L per second were markedly less sensitive to cortisol than those with higher values. Both Kd (30.3+/-2.5 versus 19.2+/-2.4 nmol/L) and IC50 values (15.5+/-1.8 versus 8.9+/-1.2 nmol/L) for cortisol were significantly higher in patients with lower renin values (Ptextless.03). Other variables,including plasma hormone and electrolyte values and binding characteristics for dexamethasone,were not different. These data suggest that cortisol binding to glucocorticoid receptor is slightly impaired in patients with essential hypertension. In vivo,this could lead to inappropriate binding of cortisol to mineralocorticoid receptors. Hence,decreased sensitivity to cortisol is associated with renin suppression. This hypothesis is supported by evidence of hypertension and low renin activity,which others have described in patients with primary glucocorticoid resistance due to mutations of the glucocorticoid receptor. View Publication

The effect of 9-cis-retinoic acid (9-cis-RA) on the growth of eight gastric cancer cell lines was related to their transcription levels of mRNAs for retinoid receptors. Northern blot analysis showed that seven (TMK-1,MKN-1,-28,-45,-74,HSC-39,KATO-III) out of eight gastric cancer cell lines synthesized mRNAs for retinoic acid receptors (RARs) and retinoid X receptor-alpha (RXR-alpha). MKN-7 cells did not transcribe either RARs or RXR-alpha at the mRNA level although they appeared to have no alterations at the gene level. The growth of all of the cell lines except for MKN-7 cells was inhibited by 1 x 10(-6) M 9-cis-RA. Cell cycle distribution analysis revealed that G0-G1 arrest was not induced by exposure to 9-cis-RA in the sensitive TMK-1 and KATO-III cells or the resistant MKN-7 cells. Interestingly,9-cis-RA temporarily increased the amount of the cyclin dependent kinase (cdk) inhibitor,Waf1/Cip1/Sdi1/p21 protein,and also reduced the amount of cdk-7,epidermal growth factor receptor (EGFR) and cyclin D1 proteins,followed by reduction in phosphorylation of the product of the retinoblastoma tumor suppressor gene (Rb) in the sensitive TMK-1 cells,but not in the resistant MKN-7 cells. These results suggest that 9-cis-RA has a cytostatic effect on gastric cancer cells that synthesize the receptor molecules through cell cycle regulatory machinery. View Publication

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential,but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand,Steel factor,interleukin 3,interleukin 6,and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells,a 4-fold expansion of LTC-IC,and a 2-fold (but significant,P textless 0.02) increase in CRU. The culture-derived CRU,like the original CB CRU,generated pluripotent,erythroid,granulopoietic,megakaryopoietic,and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition,their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal. View Publication

Retinoid induction of epidermal hyperplasia was investigated in hairless mice with synthetic ligands for the retinoic acid (RAR) and retinoid X (RXR) nuclear receptors. Induction of hyperplasia by all-trans retinoic acid and the RAR-specific retinoids TTNPB,tazarotene and AGN 190121 varied over a wide range (ED50 = 0.2-100 nmol/animal in three daily applications). Potency of induction was not directly correlated to receptor-binding affinity,but specificity of action could be demonstrated by inhibition with the high-affinity antagonist of the RARs,AGN 193109. Although RAR is functionally complexed with RXR in vivo,RXR-selective compounds have only weak potency in induction of hyperplasia. The ED50 value of the RXR-selective AGN 191701 was 600 nmol/animal compared with an ED50 value of 0.2 nmol for the structurally similar RAR-selective TTNPB. SR11237 and SR11217,also RXR-selective,each have an ED50 value of textgreater1000 nmol. Unlike RAR-specific retinoids,RXR-selective retinoids cause only very mild skin flaking at high doses. Relative potencies for cumulative topical irritation (flaking and abrasion) of both RAR and RXR ligands were well correlated with epidermal hyperplasia. These data are consistent with RXR as a silent partner in the RAR-RXR heterodimer in skin. View Publication

A variety of factors produced by stromal fibroblasts,including Flt3-ligand (FL),interleukin-11 (IL-11),Steel factor (SF),and IL-7,have been implicated in stimulating the production of pre-B cells and myeloid cells from primitive hematopoietic precursors. To investigate their relative roles in this process,either as single-acting or synergistic agents,we compared the yield and types of cells produced after 2 weeks from small numbers of Sca-1+ Lin- (i.e.,B220-,Ly-1-,Gr-1-,and Ter-119-) day 14.5 murine fetal liver cells placed in stromal cell-free cultures containing all possible combinations of FL,SF,IL-7,and IL-11. None of these factors alone supported the production (or survival) of any cells beyond 1 week: only pairs of factors consisting of either FL or SF plus either IL-11 or IL-7 were effective in this regard,with FL plus IL-11 being the most potent pair (approximately 7 x 10(4) cells obtained per 100 Sca-1+ Lin- input cells). The maximum numbers of cells were produced in the presence of FL,IL-11,and IL-7: these included both B220+ and Mac-1+/Gr-1+ cells (approximately 10(6) and approximately 2 x 10(5),respectively,per 100 Sca-1+ Lin- input cells). Both of these lineages were also obtained with each of the other possible three-factor combinations,albeit with variable effectiveness. Omission of either FL or IL-7 caused the greatest reduction in the yield of B220+ cells (approximately 130-fold and approximately 80-fold,respectively). Omission of IL-11 and,to a lesser extent,FL caused the greatest reduction in the yield of Mac-1+/Gr-1+ cells (approximately 90-fold and approximately 3-fold,respectively). When fetal calf serum was replaced with a defined serum substitute,the out put of B220+ cells remained the same but myelopoiesis was consistently enhanced (approximately 5- to 20-fold). These findings support a model involving factor redundancy in the extracellular signals required to stimulate the production and amplification of both lymphoid and myeloid cells from early Sca-1+ Lin- cells. They also reveal quantitative differences in the abilities of different competent factor combinations to promote this process,which may be further modulated by the presence of undefined serum components. View Publication

Ex vivo culture of human hematopoietic cells is a crucial component of many therapeutic applications. Although current culture conditions have been optimized using quantitative in vitro progenitor assays,knowledge of the conditions that permit maintenance of primitive human repopulating cells is lacking. We report that primitive human cells capable of repopulating nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice (SCID-repopulating cells; SRC) can be maintained and/or modestly increased after culture of CD34+CD38- cord blood cells in serum-free conditions. Quantitative analysis demonstrated a 4- and 10-fold increase in the number of CD34+CD38- cells and colony-forming cells,respectively,as well as a 2- to 4-fold increase in SRC after 4 d of culture. However,after 9 d of culture,all SRC were lost,despite further increases in total cells,CFC content,and CD34+ cells. These studies indicate that caution must be exercised in extending the duration of ex vivo cultures used for transplantation,and demonstrate the importance of the SRC assay in the development of culture conditions that support primitive cells. View Publication