Genotoxicity of tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s

The clastogenicity of tamoxifen and toremifene was tested insix human lymphoblastoid cell lines each expressing increasedmonooxygenase activity associated with a specific transfectedhuman cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 orCYP3A4). The chemicals were also tested in a cell line (MCL-5)expressing elevated native CYP1A1 and containing transfectedCYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, andin a cell line containing only the viral vector (Ho1). Dose-relatedincreases in micronuclel were observed when cells expressing2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. Thepositive responses in the cell lines were in the order MCL-5> 2E1 > 3A4 > 2D6. Toremifene also gave positive resultswith 2E1, 3A4 and MCL-5 cells, although the responses were lessmarked and the positive effects required higher doses than withtamoxifen. A synthesized epoxide of tamoxifen was also testedin these cell lines and produced similar increases in the incidencesof micronucleated cells. The increases in the responses observedwith the epoxide were greater than with tamoxifen or toremifene.The P450 isoenzyme activities in these cells were in a rangesimilar to those of human tumour-derived cell lines. Microsomes(1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells allmetabolized tamoxifen. The major metabolite detected by HPLCwas N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detectedin cells with cytochrome P450 2E1 and 2D6. These results areconsistent with the following conclusions. (1) Tamoxifen requiresmetabolic activation to DNA-reactive species by specific CYPmonooxygenases in order to exert its genotoxic effects. (2)The positive clastogenic effects elicited in lymphoblastoidcells by tamoxifen epoxide suggest that the genotoxic (and possiblythe carcinogenic) effects of tamoxifen may be due to one ormore epoxide metabolites that are generated intracellularly,probably in close proximity to the nudeus. (3) Tamoxifen ismore genotoxic than toremifene.     

Down-regulation of nitric oxide production by droloxifene and toremifene in human breast cancer cells

We investigated the effect of tamoxifen, 4-OH tamoxifen, toremifene droloxifene, interferon-alpha2a, interferon-alpha2b and interferon-alpha2c, singly and in combination, for their effect on nitric oxide production by MCF-7 and ZR-75-1 human breast cancer cells. Tamoxifen and 4-OH tamoxifen singly had no effect on nitric oxide production by either cell line. However, treatment with droloxifene or toremifene significantly reduced nitric oxide production by both MCF-7 and ZR-75-1 human breast cancer cell lines. Combination treatment with anti-estrogens and interferon-alpha2a interferon-alpha2b or interferon-alpha2c had no synergistic or additive effect compared to each drug singly.     

Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells.

Chronic administration of tamoxifen to female rats causes hepatocellular carcinomas. We have investigated damage to liver DNA caused by the administration of tamoxifen to female Fischer F344/N rats or C57B1/6 or DBA/2 mice using 32P-postlabelling. Following the administration of tamoxifen for 7 days (45 mg/kg/day) and extraction of hepatic DNA, up to 7 radiolabelled adduct spots could be detected after PEI-cellulose chromatography of the 32P-labelled DNA digests. Tamoxifen caused a time-dependent increase in the level of adduct detected up to a value of at least 1 adduct/10(6) nucleotides after 7 days dosing. A dose response relationship was demonstrated over the range of 5-45 mg/kg/day (0.013-0.12 mmol/kg/day). On cessation of dosing there was a loss of adducts from the liver DNA. These adducts were not detected in DNA from vehicle-dosed controls or in DNA from kidney, lung, spleen, uterus or peripheral lymphocytes. Pyrrolidinotamoxifen caused a similar level of adduct formation as tamoxifen. In contrast, no significant adduct formation could be detected in liver DNA from rats given droloxifene or toremifene. Mice given tamoxifen (45 mg/kg/day for 4 days) showed levels of adducts in the liver which were 30-40% of those present in rats. Exposure of rat hepatocytes to tamoxifen in vitro, resulted in induction of unscheduled DNA synthesis, when preparations from rats which had been pretreated with tamoxifen in vivo were used. No such increase could be detected in hepatocytes from control rats, suggesting tamoxifen may induce enzymes responsible for its own activation. Tamoxifen induced a significant increase in micronucleus formation in a dose dependent manner in cultures of MCL-5 cells, a human cell line that expresses 5 different human cytochrome P450 isoenzymes, as well as epoxide hydrolase.     

Pharmacokinetics of toremifene and its metabolites in patients with advanced breast cancer

Summary A multicenter phase I pharmacokineticstudy of a new triphenylethylene antiestrogen, toremifene, was examined in 70 patients with advanced breast cancer. Patients were randomized to receive single daily oral doses of either 10, 20, 40, 60, 200, or 400 mg for 8 weeks. Plasma toremifene and its major metabolites,N-desmethyltoremifene and 4-hydroxytoremifene, were determined weekly during therapy and at 0, 7, 14, and 21 days after the discontinuation of therapy. The time to reach steady-state plasma concentrations was between 1 and 5 weeks, with steady-state being achieved earlier (1–2 weeks) at daily doses of 200 and 400 mg. The time to peak concentration following oral doses of toremifene ranged from 1.5 to 4.5 h. The terminal half-life of elimination was 5.0, 6.0, and 5.0 days for toremifene, desmethyltoremifene, and 4-hydroxytoremifene, respectively. Plasma concentrations of 4-hydroxytoremifene were detectable only at high doses (200 and 400 mg/day) of toremifene. The results of this phase I pharmacokinetic study show that toremifene has metabolic and kinetic patterns that are similar to those previously reported with tamoxifen.This work was supported by a grant from Adria Laboratories     

Induction of hepatic aneuploidy in vivo by tamoxifen, toremifene and idoxifene in female Sprague-Dawley rats

Since tamoxifen is efficacious for the prevention of secondprimary breast neoplasms in humans and has a low reported incidenceof acute side effects, several structurally related compoundshave been developed for the treatment of breast cancer includingtoremifene and idoxifene. We have compared the karyotypic alterationsthat occur after a single per os administration of 35 mg/kgof tamoxifen, toremifene or idoxifene to female Sprague-Dawleyrats. One day following treatment, the rats were sacrificedand the hepatocytes isolated and cultured. After 47 h in culture,colcemid was added for 3 h prior to harvest of the hepatocytesfor karyotypic evaluation. At least 100 metaphase spreads wereexamined for each of five rats per treatment. Toremifene resultedin aneuploidy in 50±7% of the cells examined and idoxifeneinduced a 57±4% aneuploidy compared with the 85±7%level induced by tamoxifen. Since the level of aneuploidy insolvent-treated rats was 3±3%, the induction of aneuploidyin at least 50% of the cells from rats treated with tamoxifen,toremifene or idoxifene was highly significant Analysis of electronmicrographs of cultures treated with these antiestrogens demonstrateda range of phenotypes including multipolar spindles in toremifene-treatedrats and condensed chromosomes in the presence of an intactnuclear envelope in occasional idoxifene-treated rat hepatocytes.The exclusion of chromosomes from the spindle apparatus andthe lagging of some chromosomes on the metaphase plate correlatewith the high rate of induction of aneuploidy in the rat liveras determined by karyotypic analysis of hepatocytes from ratstreated with these triphenylethylenes.     

Droloxifene (3-hydroxytamoxifen) has membrane antioxidant ability: potential relevance to its mechanism of therapeutic action in breast cancer.

Droloxifene (3-hydroxytamoxifen), is a triphenylethylene derivative recently developed for the treatment of breast cancer. Droloxifene was found to exhibit a membrane antioxidant ability in that it inhibited Fe(III)-ascorbate dependent lipid peroxidation in rat liver microsomes and ox-brain phospholipid liposomes. It also inhibited microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH. Droloxifene was a better inhibitor of lipid peroxidation than tamoxifen, but was less effective than 17 beta-oestradiol in the two microsomal systems and in the preformed liposomal system. When introduced into ox-brain phospholipid liposomes, droloxifene inhibited Fe(III)-ascorbate induced lipid peroxidation to approximately the same extent as similarly introduced cholesterol and tamoxifen, although to a lesser extent than 17 beta-oestradiol. This inhibition of lipid peroxidation by droloxifene may result from a membrane stabilization that could be associated in cancer cells with decreased plasma membrane fluidity. This mechanism may be related to the clinically important antiproliferative action of droloxifene on cancer cells.     

Identification of human CYP forms involved in the activation of tamoxifen and irreversible binding to DNA

This study investigates which CYP forms are responsible for the conversion of tamoxifen to its putative active metabolite alpha-hydroxytamoxifen and irreversible binding to DNA. We have used eight different baculovirus expressed recombinant human CYP forms and liquid chromatography-mass spectrometry to show that only CYP3A4 is responsible for the NADPH-dependent alpha-hydroxylation of tamoxifen. Surprisingly, this CYP did not catalyse the formation of 4-hydroxytamoxifen. We demonstrate for the first time, by means of accelerator mass spectrometry, that CYP3A4 also catalysed the activation of [(14)C]tamoxifen to intermediates that irreversibly bind to exogenous DNA. Incubation of [(14)C]tamoxifen (20.6 kBq, 100 micro M) with CYP3A4, in the presence of NADPH for 60 min led to levels of DNA binding of 39.0+/-9.0 adducts/10(8) nucleotides (mean +/- SE, n = 6). While CYP3A4 converted tamoxifen to N-desmethyltamoxifen (38.3 +/- 7.20 pmol/20 min/pmol CYP, n = 4), the polymorphic CYP2D6 showed the highest activity for producing this metabolite (48.6+/-1.52pmol/20 min/pmol CYP). CYP2D6 was also the most active in catalysing 4-hydroxylation of tamoxifen, although an order of magnitude lower level was also detected with CYP2C19. With tamoxifen as substrate, no 3,4-dihydroxytamoxifen could be detected with any CYP form. CYP2B6 did not catalyse the metabolism or the binding of tamoxifen to DNA. It is concluded that CYP3A4 is the only P450 of those tested that converts tamoxifen to alpha-hydroxytamoxifen and the only one that results in appreciable levels of irreversible binding of tamoxifen to DNA.     

Comparison of the effects of tamoxifen and toremifene on liver and kidney tumor promotion in female rats

Female rats were subjected to a 70% partial hepatectomy andadministered either diethylnitrosamine (10 mg/kg) or the solvent,trioctanoin. After a 2 day recovery from the surgery, the ratswere placed on basal diet alone or containing phenobarbital(500 mg/kg diet), mestranol (0.2 mg/kg diet), tamoxifen (250or 500 mg/kg diet) or toremifene (250, 500 or 750 mg/kg diet)for 6 or 18 months prior to killing. The livers and kidneyswere prepared for pathological diagnoses. In addition, sectionsof liver from the 6 month killing were frozen and serially sectioned.The sections were stained for expression of the placental isozymeof glutathione S-transferase (GST), gamma glutamyl transpeptidase(GGT), canalicular ATPase (ATP) and glucose 6-phosphatase (G6P)and scored by quantitative stereology for number and volumefraction of liver occupied by altered hepatic foci (AHF) withalterations in these markers individually and combined (ANY).Each of the agents increased the volume fraction of liver occupiedby AHF when the ANY category was used. Statistical increasesin both the GGT-positive and G6P-deficient AHF populations wereobserved in the spontaneously as well as DEN-initiated groupstreated with tamoxifen or toremifene. After 18 months of administration,the highest concentration of tamoxifen increased the incidenceof malignant hepatic neoplasms in non-DEN-initiated rats. Toremifene,at the highest tested dose, increased the incidence of hepatocellularcarcinomas in the DEN-initiated groups to a level one-thirdthat observed with tamoxifen administration to DEN-initiatedrats. Both tamoxifen and toremifene increased the incidenceof hypernephromas in previously DEN-initiated rats. While bothtamoxifen and toremifene are effective promoting agents forDEN-initiated lesions, tamoxifen is more potent than toremifenein the induction of rat hepatocarcinogenesis.     

Antiestrogenic action of 3-hydroxytamoxifen in the human breast cancer cell line MCF-7

The antiestrogenic action of 3-hydroxytamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1-(3-hydroxyphenyl)-2 -phenylbut-1-ene] was characterized in vitro and compared with that of tamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. The relative binding affinities of 3-hydroxytamoxifen to estrogen receptor were 3.3% in cytosol of MCF-7 cells and 1.5% in human mammary carcinoma cytosol compared to values of 0.2 and 0.3% for tamoxifen (the affinity of 17 beta-estradiol considered to be 100%). The concentration of 3-hydroxytamoxifen necessary to suppress the 17 beta-estradiol-induced growth stimulation of MCF-7 cells was about tenfold lower than that for tamoxifen. The induction of progesterone receptor in MCF-7 cells by 17 beta-estradiol was inhibited by 3-hydroxytamoxifen. In the absence of 17 beta-estradiol, 3-hydroxytamoxifen gave rise to a moderate increase in the progesterone receptor levels, which demonstrates the partially estrogenic character of hydroxytamoxifen.     

Initiating activity of the anti-estrogen tamoxifen, but not toremifene in rat liver

A striking difference between two structurally related anti-estrogen medicines is that tamoxifen is strongly hepatocarcinogenic in the rat, whereas toremifene lacks such activity. To study the basis for this difference, the initiating potential of tamoxifen and toremifene were studied by measurement of rapid induction of hepatocellular altered foci (HAF) that express placental-type glutathione S-transferase in the livers of female Sprague-Dawley (S-D) rats and female Fischer 344 (F344) rats. Both agents were administered by gavage at equimolar doses up to a dose that produced marked weight gain suppression. In rats given the high dose of 40 mg/kg per day tamoxifen continuously for 36 weeks, 75% of S-D rats developed liver neoplasms, in contrast to only 10% of F344 rats. In the S-D strain, tamoxifen produced a tendency to increased HAF at 2 weeks at the dose of 40 mg/kg per day and by 12 weeks, a dose-related increase was evident. In contrast, toremifene induced no HAF even at the equimolar high dose of 42.4 mg/kg per day for 12 weeks. The induction of HAF by tamoxifen was less in the F344 rats. Neither agent elicited increases in hepatocellular proliferation in S-D or F344 rats. When phenobarbital was administered for 24 weeks as a promoting agent after the anti-estrogens, S-D rats given tamoxifen at 20 mg/kg per day for 12 weeks, developed liver neoplasms, but not F344 rats or rats of either strain given even a higher dose (42.4 mg/kg) of toremifene. Thus, tamoxifen has initiating activity in these rat strains whereas toremifene does not.      

托瑞米芬和他莫昔芬对血脂影响的对比研究

目的　探讨托瑞米芬 (TOR)在乳腺癌术后辅助治疗中对血脂的影响程度。方法　 10 3例乳腺癌患者分为治疗组和对照组。治疗组包括TOR组和TAM组 ,TOR组口服TOR 60mg/天 ,TAM组口服TAM 2 0mg/天。在内分泌治疗前 ,治疗 3、6、9和 12个月后 ,分别晨取患者空腹静脉血进行血脂检测。结果　TOR治疗组血总胆固醇平均水平下降 11.0 % ,S LDL平均水平下降 11.1% ,血甘油三酯平均水平下降 10 .7% ;TAM治疗组血总胆固醇平均水平下降 9.0 % ,S LDL平均水平下降 17.4% ,而血甘油三酯水平无明显变化 (P =0 .491)。结论　TOR对血脂的影响程度与TAM相近 ,2组血总胆固醇和S LDL平均水平均较治疗前下降。不同的是 ,TOR治疗组血甘油三酯水平下降 ,而TAM治疗组无明显变化。提示TOR可影响与冠心病发病相关的血脂水平。     

Alterations of drug metabolizing and antioxidant enzyme activities during tamoxifen-induced hepatocarcinogenesis in the rat

The triphenylethylene drug tamoxifen is a hepatocarcinogen inrats, has genotoxic potential and may produce carcinoma of theendometrium in humans, while the structurally closely relatedtoremifene has no carcinogenic or genotoxic potential. We haveinvestigated the effects of long-term treatment with tamoxifenand toremifene on the activities of drug metabolizing and antioxidantenzymes in rat liver. Female Sprague-Dawley rats were dosedwith equimolar doses of tamoxifen (11.3 and 45 mg/kg) and toremifene(12 and 48 mg/kg) for 12 months and were killed after 2 days,5 weeks, 3, 6 and 12 months of treatment. After 12 months mostrats treated with the high dose of tamoxifen had hyperplasticnodules and hepatocellular carcinomas, while in rats given toremifeneor the low dose of tamoxifen, only foci were observed. A strikingobservation was strong inhibition of the hexose monophosphateshunt (HMS) by tamoxifen and toremifene, which, except in thegroup given the high dose of tamoxifen, lasted throughout thetreatment period. Both antiestrogens induced susceptibilityto oxidative stress, as indicated by decreased hepatic contentsof reduced glutathione and by increased peroxidation potentialof microsomal preparations. The activity of glutathione S-transferasewas permanently induced by the high dose of tamoxifen from 5weeks onwards and was greater in tamoxifen-induced liver tumorsthan in corresponding macroscopically normal tissue. Similarly,the activity of HMS was elevated by the high dose of tamoxifenat the latest time points, and a further elevation was seenin tamoxifen-induced liver tumors. No such alteration in glutathioneS-transferase or HMS activity was seen in animals treated withtoremifene or with the low dose of tamoxifen. In conclusion,tamoxifen and toremifene differ markedly with respect to productionof liver tumors, and this difference in hepatocarcinogenic potentialis reflected in differential effects on glutathione-S-transferaseand HMS activities in rat liver.     

CYP2D6基因多态性对他莫昔芬及托瑞米芬代谢和疗效影响的研究进展

他莫昔芬和托瑞米芬均为选择性的雌激素受体调节剂,通过竞争性结合雌激素受体发挥抗癌作用,是绝经前乳腺癌最常用的辅助内分泌治疗药物,能够显著降低乳腺癌患者的复发和死亡风险。多项研究发现CYP2D6*10基因的突变会影响他莫昔芬的代谢导致预后不良。而托瑞米芬的代谢受CYP2D6基因多态性的影响较小,治疗乳腺癌的疗效与他莫昔芬相当,甚至优于他莫昔芬。本文就CYP2D6基因多态性与两种内分泌药物代谢和疗效的相关性研究作系统阐述。     

Regulation of ornithine decarboxylase gene expression in MCF-7 breast cancer cells by antiestrogens

Ornithine decarboxylase (ODC) is an enzyme intimately related to cell growth regulation. The metabolic products of ODC, the polyamines, are known to play a vital role in the structure and function of biological macromolecules including nucleic acids and proteins. The activity of ODC is stimulated by estrogens in their target cells. In order to gain insight into the molecular mechanism of action of antiestrogens in human breast cancer, we studied the effect of tamoxifen and 4-hydroxytamoxifen on the concentration of ODC mRNA, ODC activity, and the polyamine levels in a hormone-responsive breast cancer cell line, MCF-7. ODC mRNA concentration was reduced to 40% of the controls after 6 h of treatment of the cells with 100 nM 4-hydroxytamoxifen, but tamoxifen had no significant effect on ODC mRNA after treating with even 1 microM concentration for 36 h. ODC activity was, however, reduced to 40 and 75% of the controls after 24 h of treatment with 4-hydroxytamoxifen and tamoxifen, respectively. There was a significant reduction in the concentration of putrescine to 63% of control in tamoxifen-treated cells, but spermidine and spermine levels were not affected. With 4-hydroxytamoxifen, putrescine, spermidine, and spermine levels were reduced to 41, 62, and 79% of the control, respectively. In addition, exogenous putrescine was able to reverse the growth inhibitory effects of 4-hydroxytamoxifen. Overall, these results indicate that ODC and polyamine levels in MCF-7 cells are controlled by antiestrogens, and that suppression of polyamine biosynthesis plays a critical role in the growth inhibitory effects of antiestrogens.     

17β-Estradiol,diethylstilbestrol, tamoxifen,toremifene and ICI 164,384 induce morphological transformation and aneuploidy in cultured Syrian hamster embryo cells

To examine the ability of estrogens and anti-estrogens to induce cellular transformation and genetic effects, Syrian hamster embryo (SHE) cells were treated with estrogens, 17β-estradiol (E₂) or diethylstilbestrol (DES), or with anti-estrogens, tamoxifen (TAM), toremifene (TOR) or ICI 164,384. Treatment with each substance for 1–3 days suppressed cellular growth in a dose-dependent manner. Colony-forming efficiency (CFE) increased following treatment of cells with E₂ or DES for 48 hr at 3 or 10 μM but decreased at 20 or 30 μM. In contrast, CFE was increased by treatment with TAM, TOR or ICI 164,348 over the concentration range examined (1–30 μM). Treatment with each chemical at 1–30 μM for 48 hr caused morphological transformation of SHE cells in a dose-related fashion. The highest frequency was exhibited in SHE cells treated with DES at 20 μM and was 2 times higher than that induced by treatment with benzo[α]pyrene (B[α]P) at 4 μM. Transformation frequencies induced by other substances (E₂, TAM, TOR and ICI 164,348) did not exceed that induced by the B[α]P treatment. TOR showed a higher transforming ability over all concentrations examined when compared to the other anti-estrogens (TAM and ICI 164,348). No significant increases in the frequencies of chromosomal aberrations were observed in SHE cells that were treated with any of the chemicals. However, treatment of SHE cells with each chemical induced a dose-dependent increase of aneuploid cells in the near diploid range. Our results indicate that the ability of the estrogens and anti-estrogens to induce numerical chromosomal abnormality may be involved in their cell transformation activity and potential carcinogenicity. Int. J. Cancer, 70: 188–193, 1997. © 1997 Wiley-Liss, Inc.     

Dose-adjustment study of tamoxifen based on <Emphasis Type="Italic">CYP2D6</Emphasis> genotypes in Japanese breast cancer patients

CYP2D6 is a key enzyme responsible for the metabolism of tamoxifen to active metabolites, endoxifen, and 4-hydroxytamoxifen. The breast cancer patients who are heterozygous and homozygous for decreased-function and null alleles of CYP2D6 showed lower plasma concentrations of endoxifen and 4-hydroxytamoxifen compared to patients with homozygous-wild-type allele, resulting in worse clinical outcome in tamoxifen therapy. We recruited 98 Japanese breast cancer patients, who had been taking 20 mg of tamoxifen daily as adjuvant setting. For the patients who have one or no normal allele of CYP2D6, dosages of tamoxifen were increased to 30 and 40 mg/day, respectively. The plasma concentrations of tamoxifen and its metabolites were measured at 8 weeks after dose-adjustment using liquid chromatography–tandem mass spectrometry. Association between tamoxifen dose and the incidence of adverse events during the tamoxifen treatment was investigated. In the patients with CYP2D6*1/*10 and CYP2D6*10/*10, the mean plasma endoxifen levels after dose increase were 1.4- and 1.7-fold higher, respectively, than those before the increase (P < 0.001). These plasma concentrations of endoxifen achieved similar level of those in the CYP2D6*1/*1 patients receiving 20 mg/day of tamoxifen. Plasma 4-hydroxytamoxifen concentrations in the patients with CYP2D6*1/*10 and CYP2D6*10/*10 were also significantly increased to the similar levels of the CYP2D6*1/*1 patients according to the increasing tamoxifen dosages (P < 0.001). The incidence of adverse events was not significantly different between before and after dose adjustment. This study provides the evidence that dose adjustment is useful for the patients carrying CYP2D6*10 allele to maintain the effective endoxifen level.     

Chromosomal instability and ATR amplification gene in patients with persistent and polyclonal B-cell lymphocytosis (PPBL)

Forty-three patients with persistent and polyclonal B-cell lymphocytosis (PPBL) were studied. The PPBL diagnosis was based on the presence of a polyclonal lymphocytosis and the detection of binucleated lymphoid cells on peripheral blood examination. In order to define the cytogenetic profile in these patients, conventional cytogenetic analysis and fluorescence in situ hybridization were performed at diagnosis in all patients and also at follow-up in 10 patients. When excluding + i(3q) and PCC, chromosomal instability is a common occurrence in PPBL and is characterized by other independent clonal abnormalities, del(6q), + der(8) or + 8, polyploid karyotype, structural changes, aneuploidy and/or non clonal chromosomal aberrations with either loss or more frequently gain of chromosomes. These data show the presence of a chromosomal instability in 67.5% of PPBL patients. Finally, ATR amplification was detected by hybridization with BAC probe 26217 in + i(3q) positive metaphase cells. No ATR deletion was observed in the + i(3q) negative B-cells. As the natural history of PPBL remains unclear, it is necessary to diagnose PPBL patients and useful to recommend a careful and continued long follow-up in all PPBL patients.     

Efficacy and tolerability of toremifene and tamoxifen therapy in premenopausal patients with operable breast cancer: a retrospective analysis

Background

Given the use of tamoxifen as standard treatment for hormone receptor–positive breast cancer, the use of toremifene as an adjuvant endocrine therapy has not been widely examined. The present retrospective study compared the efficacy and safety of toremifene and tamoxifen in the treatment of operable hormone receptor–positive breast cancer in premenopausal women.

Methods

Premenopausal patients with hormone receptor– positive operable breast cancer were eligible. Enrolled patients (n = 1847) received either 60 mg toremifene (n = 396) or 20 mg tamoxifen (n = 1451) daily for a minimum of 5 years after surgery. Disease-free survival (dfs) was the primary endpoint. Overall survival (os) and time to distant recurrence were secondary endpoints.

Results

Treatment with toremifene and tamoxifen resulted in no between-group differences in dfs (p = 0.659) or os (p = 0.364). Mean dfs was 10.3 years for both groups. Mean os was 11.2 years for the toremifene group and 11.1 years for tamoxifen group. The 5-year dfs rate was 87.0% in the toremifene group and 85.0% in the tamoxifen group. The 5-year survival rate was 94.3% in the toremifene group and 93.5% in the tamoxifen group. Adverse events rates were similar in the two groups, with the exception of irregular menses, which occurred at a higher rate in the tamoxifen group than in the toremifene group (10.0% vs. 6.3%, p = 0.025).

Conclusions

In this retrospective study, the efficacy and safety profiles of toremifene and tamoxifen for the treatment of operable hormone receptor–positive breast cancer in premenopausal women were similar.     

Comparison of the DNA adducts formed by tamoxifen and 4-hydroxytamoxifen in vivo

Tamoxifen is a liver carcinogen in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports of DNA adducts in leukocyte and endometrial samples from women treated with tamoxifen suggest that it may be genotoxic to humans. One of the proposed pathways for the metabolic activation of tamoxifen involves oxidation to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. In the present study, we compared the extent of DNA adduct formation in female Sprague-Dawley rats treated by gavage with seven daily doses of 54 micromol/kg tamoxifen or 4-hydroxytamoxifen and killed 24 h after the last dose. Liver weights and microsomal rates of ethoxyresorufin O-deethylation, 4-dimethylaminopyrine N-demethylation and p-nitrophenol oxidation were not altered by tamoxifen or 4-hydroxytamoxifen treatment. Uterine weights were decreased significantly and uterine peroxidase activity was decreased marginally in treated as compared with control rats. DNA adducts were assayed by 32P-post-labeling in combination with HPLC. Two major DNA adducts were detected in liver DNA from rats administered tamoxifen. These adducts had retention times comparable with those obtained from in vitro reactions of alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide with DNA. Hepatic DNA adduct levels in rats administered 4-hydroxytamoxifen did not differ from those observed in control rats. Likewise, adduct levels in uterus DNA from rats treated with tamoxifen or 4-hydroxytamoxifen were not different from those detected in control rats. These data suggest that a metabolic pathway involving 4-hydroxytamoxifen is not a major pathway in the activation of tamoxifen to a DNA-binding derivative in Sprague-Dawley rats.     

Tamoxifen dose and serum concentrations of tamoxifen and six of its metabolites in routine clinical outpatient care

A sensitive and selective HPLC–MS/MS assay was used to analyze steady-state serum concentrations of tamoxifen, N-desmethyltamoxifen (E)-endoxifen, (Z)-endoxifen, N-desmethyl-4′-hydroxytamoxifen, 4-hydroxytamoxifen, and 4′-hydroxytamoxifen to support therapeutic drug monitoring (TDM) in patients treated with tamoxifen according to standard of care. When the (Z)-endoxifen serum concentration was below the predefined therapeutic threshold concentration of 5.9 ng/mL, the clinician was advised to increase the tamoxifen dose and to collect another serum sample. Paired serum samples from patients at one dose level at different time points during the tamoxifen treatment were used to assess the intra-patient variability. A total of 251 serum samples were analyzed, obtained from 205 patients. Of these patients, 197 used 20 mg tamoxifen per day and 8 patients used 10 mg/day. There was wide variability in tamoxifen and metabolite concentrations within the dosing groups. The threshold concentration for (Z)-endoxifen was reached in one patient (12 %) in the 10 mg group, in 153 patients (78 %) in the 20 mg group, and in 26 (96 %) of the patients who received a dose increase to 30 or 40 mg/day. Dose increase from 20 to 30 or 40 mg per day resulted in a significant increase in the mean serum concentrations of all analytes (p < 0.001). The mean intra-patient variability was between 10 and 20 % for all analytes. These results support the suitability of TDM for optimizing the tamoxifen treatment. It is shown that tamoxifen dose is related to (Z)-endoxifen exposure and increasing this dose leads to a higher serum concentration of tamoxifen and its metabolites. The low intra-patient variability suggests that only one serum sample is needed for TDM, making this a relatively noninvasive way to optimize the patient’s treatment.