in western countries , breast cancer is a major concern and its incidence and mortality rates have been recently influenced by new therapeutic strategies . our knowledge on cancer precursors , risk biomarkers , and cancer genetics has considerably increased , and prevention strategies are being successfully explored . unfortunately , over the last decade , breast cancer prevention has been mainly focused on endocrine therapies using selective estrogen receptors modulators ( serms ) and aromatase inhibitors ( ais ) . available preventive strategies for nonhormonal breast malignancies , more frequently expressed in brca mutation carriers and , in general , in high - risk population , are needed . for these reasons , a great number of novel chemopreventative agents are currently under investigation in order to evaluate their efficacy in this particular cohort of patients . in accordance with their recognized role in the regulation of cell growth , differentiation , apoptosis , and their recognized inhibitory effect on cell growth in er positive and negative breast cancer cells , retinoids ( either natural or synthetic compounds structurally related to vitamin a ) have long been studied for their chemotherapeutic effect and for their chemopreventive potential in breast cancer setting . only recently , retinoids have also been applied in this unaffected high - risk population and they have demonstrated to be able to suppress tumor promotion and modify some properties of fully transformed malignant cells by activating and/or repressing specific genes . retinoids initiate ligand - induced dimerization of retinoid acid receptors ( rar ,  , and  ) and retinoid x receptors ( rxr ,  , and  ) . subsequently , receptors bind to retinoid response elements on dna , and they initiate transactivation of retinoids response target genes . retinoid receptors are expressed in both normal and malignant breast epithelial cells and are critical for normal development . the mechanism by which retinoids inhibit breast cell growth has not been completely elucidated yet . given the role played by rar- in the carcinogenesis of different tumors , its regulation by retinoids has also been advocated as a putative mechanism of action of these agents . however , they have been shown to affect multiple signal transduction pathways , including igf , tgf , and ap-1-dependent pathways [ 48 ] , as showed in figure 1 . several preclinical models suggest that retinoids inhibit mammary carcinogenesis in carcinogen - treated rats and in transgenic mice [ 911 ] . recently , in order to reduce retinoids ' side effects , rxr - selective retinoids , commonly known as rexinoids , have been studied as cancer preventive agents . in particular , preclinical studies have demonstrated that these compounds maintain retinoids ' chemopreventive effect , but have greatly reduced toxicity . 9cra , a retinoid binding both rar and rxr , has significantly delayed the er - negative tumor development in sv40 tag mice and mnu - treated rats , although it induced significant cutaneous toxicity . in contrast , a rxr - selective retinoid , lgd1069 , or bexarotene ( targretin ) , has suppressed both er - positive and er - negative tumor development with minimal toxicity [ 14 , 15 ] . one of the most promising retinoids to be used in chemoprevention trials is the synthetic amide of retinoic acid fenretinide , n-4-hydroxyphenyl retinamide ( 4-hpr ) ( figure 2 ) . its biologic activity was assayed by sporn et al . , who also showed the preferential accumulation of this drug in the breast , instead of in the liver . the inhibition of chemically induced mammary carcinoma in rats by fenretinide was first described in 1979 . since then , promising in vitro data and a favorable toxicity profile compared with that of other retinoids have led to the extensive study of fenretinide in chemoprevention trials targeting different organs . fenretinide has been found to have significant chemopreventive action in a large variety of in vitro and in vivo systems . both fenretinide and its major metabolite , 4-metoxyphenyl retinamide ( mpr ) , selectively accumulate in the human breast . it should be noticed that some fenretinide - based toxicities could be due also to its hydrolysis so that it returns to retinoic acid in vivo . high - dose fenretinide is cytotoxic for a variety of different tumor cells in preclinical studies [ 3537 ] , although its accurate mechanism of action is not yet completely understood . however , it has been proposed that it might exert its inhibitory effects by means of both receptor - dependent and -independent mechanism ( table 1 , [ 1618 ] ) . although rar influence on fenretinide action is highly debated , recent evidence would support the hypothesis according to which a mechanism does not require such relationship . in particular , we would stress the importance of the studies carried out by giandomenico et al . , explaining the role of the stable expression of the dominant negative rar ; by anding et al . , showing that the use of a rar panantagonist influences an unhydrolyzable analogue of 4-hpr by inducing apoptosis with an independent rar signaling pathway ; by delia et al . , assuming resistance to differential responsiveness is present in different cell lines thus indicating that fenretinide may act through different receptor types . fenretinide characteristic feature is the ability to inhibit cell growth through the induction of apoptosis rather than differentiation , an effect that is strikingly different from that of the all - trans retinoic acid . moreover , 4-hpr - mediated apoptosis seems to be tissue - specific , so that multiple mechanisms might operate within specific tissues . for example , in ovarian carcinoma cell lines , retinoids may induce apoptosis through the depolarization of the mitochondrial membrane and activation of caspase pathway [ 23 , 24 ] , while in the breast and in others cell lines apoptosis seems to be related with a direct molecular interaction with tubulin . moreover , reactive oxygen species ( ros ) , such as hydrogen peroxide and superoxide , seem to be critical in mediating apoptosis in different cancer cell types [ 2628 ] . the ability to increase ros levels , in particular nitric oxide ( no ) by no synthases ( nos ) over the elevation of sphingolipid ceramide levels , has been suggested as an explanation of the apoptotic effect of fenretinide . recently , fenretinide has been shown to be able to induce no - mediated apoptosis in breast cancer ( brca-1)-mutated breast cancer cells . additional mechanisms are under investigations , such as the ability to inhibit cell growth by reducing the expression of growth - stimulating factors or by inducing the expression of growth - inhibitory factors . a recent proposed surrogate biomarker of fenretinide efficacy is circulating insulin - like growth factor 1 ( igf1 ) . the igf system plays a pivotal role in cell proliferation of both epithelial and mesenchymal tissues by stimulating mitosis , protecting cells from apoptosis , and maintaining transformed phenotype . large prospective studies have shown that high circulating levels of igf1 and lower levels of its major binding protein ( igfbp-3 ) are associated with a higher risk of developing subsequent premenopausal breast cancer and prostate , lung , and colorectal cancer [ 4447 ] . this indicates that circulating igf1 is a key regulator of cell and tumor proliferation for the vast majority of human epithelial cancer . fenretinide has been shown to inhibit igf1-stimulated growth of breast cancer cell lines ( bccls ) and to downregulate the igf system in both er - positive and er - negative bccl . the expression of her2 has been recently observed to reduce fenretinide ability to induce apoptosis in breast cancer cells . moreover , researchers found that her2 uses active human protein kinase ( akt ) to induce cyclooxygenase ( cox-2 ) expression and that inhibition of akt or cox-2 increased 4-hpr induces apoptosis mediated by no production . thus , a combination of 4-hpr with cox-2 inhibitors might be a new strategy to further investigate breast cancer chemoprevention . the 5-year administration of the milan study ( see below ) provided a vast corpus of information on the long - term safety and tolerability of this retinoid . as a major side effect , it induced a dose - related linear decrease of plasma retinol , associated with diminished retinal adaptation to darkness . in order to minimize this side effect , a 3-day treatment interruption at the end of each month was introduced to increase plasma retinol concentrations , allowing the partial recovery of retinal storage . however , an accurate and complete evaluation of toxicity was hampered by the lack of a placebo control group . dermatological , gastrointestinal , visual , and ophthalmologic events were relatively frequent , but were mostly mild . in a recent analysis of the phase iii trial , the most common adverse events were diminished dark adaptation ( cumulative incidence , 19% ) and dermatologic disorders ( 18.6% ) , such as skin and mucosal dryness , pruritus , and urticaria . less common events were gastrointestinal symptoms ( 13% ) and alterations of the ocular surface ( 10.9% ) . women in the control group complained of diminished dark adaptation , dermatologic disorders , gastrointestinal symptoms , and alterations of the ocular surface in 2.9% , 2.9% , 5.4% , and 3.2% of the cases , respectively . interestingly , most side effects decreased with time and were significantly more frequent in postmenopausal women . importantly , in contrast to other retinoids , prolonged administration of fenretinide is not associated with significant alterations of bone mineral density of the forearm . however , a trend towards an increase in bone resorption markers suggests the need for further assessment at different skeletal sites . after the completion of a phase i dose - ranging study , the 200  mg daily dose was chosen as the safest dose for prevention , as one case of a pathological electroretinogram after a 24-week administration was observed with the 300  mg / day dose . higher doses , up to 400  mg , have been used in women with metastatic cancer in combination with tamoxifen , with no evident toxicity on liver and lipid profile , but with an increased incidence of nyctalopia [ 53 , 54 ] . peak levels of 4-hpr occur at approximately 6  h in adults with terminal half - life of approx . since both 4-hpr and 4-mpr are selectively accumulated in the breast , evaluation of fenretinide as a chemopreventive agent in breast cancer has been particularly attractive . the most important clinical trials with fenretinide are mentioned in table 2 . as in the therapeutic setting , where drugs combinations are superior to monochemotherapy , the concept of combining agents with different mechanisms of action in the attempt to increase efficacy while minimizing side effects is a rational approach in chemoprevention . in preclinical models , combined administration of fenretinide and tamoxifen has proven additive and synergistic in both growth inhibition of mcf-7 cells and prevention of mnu - induced mammary carcinomas . moreover , the activity of 9-cis - retinoic acid against mnu - induced mammary tumors in sprague - dawley rats is enhanced by the combination with tamoxifen or raloxifene . the safety and the tolerability of the combination of fenretinide and tamoxifen have been investigated in clinical trials in metastatic breast cancer patients   and in women at increased risk . the concept of combining agents with different mechanisms of action in the attempt to increase efficacy on complementary molecular targets , while minimizing side effects is increasingly being pursued in breast cancer chemoprevention . a clinical randomized , double - blind , placebo - controlled phase iib trial with a 2  2 factorial design to test this interaction ( fenretinide and low - dose tamoxifen ) was conducted at the european institute of oncology . in spite of the favorable effects on plasma igf - i levels and mammographic density , this combination did not reduce breast neoplastic events compared to placebo , whereas both single agents , particularly fenretinide , showed numerical reduction in annual odds of breast neoplasms . fenretinide ( in combination with hrt ) was also studied by our group in 226 postmenopausal healthy women , randomized in a two - by - two factorial design to either oral cee 0.625  mg / day or transdermal e2 , 50  microg / day and to fenretinide 100  mg / twice a day or placebo for 12 months . oral cee showed more favorable changes than transdermal e2 on circulating breast cancer risk biomarkers , while fenretinide exerted little modulation on most biomarkers . the most important study where 4-hpr was administrated as a single agent is a multicentric phase iii randomized trial , coordinated by the istituto nazionale dei tumori in milan , started in 1987 . stage i ( t1 - 2 n0 ) breast cancer patients , aged 3370 years , who had been operated on for breast cancer within the previous 10 years and had received no systemic adjuvant therapy were eligible . women were randomly assigned to receive either no treatment or fenretinide given orally at a dose of 200  mg / day for 5 years . a placebo - control arm was not included in the study design because of the large capsule size and the objective nature of the main outcome measure . a 3-day drug stoppage at the end of each month was recommended in order to allow retinol recovery and to minimize dark adaptation impairment . the main outcome measure was the occurrence of contralateral breast cancer as first malignant event . the secondary endpoint was the incidence of ipsilateral breast cancer reappearance , defined as local recurrence in the same quadrant or the occurrence of a second breast malignancy in different quadrants from the primary tumor . fenretinide showed no effect on contralateral breast cancer occurrence and a nonsignificant 17% reduction in ipsilateral breast tumor reappearance . however , a different effect was noted when the analysis was stratified by menopausal status , with a beneficial trend in premenopausal women on both contralateral and ipsilateral breast cancer ( 38% ) and a reversed trend on contralateral breast cancer in postmenopausal women , as highlighted in figure 3 . importantly , the protective effect persisted for up to 15 years ( i.e. , 10 years after retinoid cessation ) . most notably , the younger the women were , the greater the benefit of fenretinide . such benefit was associated with a remarkable 50% risk reduction in women aged 40 years or younger , whereas the benefit disappeared after 55 years of age . interestingly , the incidence of ovarian cancer during the 5-year intervention period was significantly lower in the treatment arm . this effect has been confirmed in an important update . this phase iii trial suggested a possible role of fenretinide as a preventive agent acting at different levels of breast carcinogenesis . admittedly , the results obtained during the phase iii trial on our subgroups had not been foreseen when the study was planned . while there are plausible biological explanations for this selective effect , our findings are hypothesis generating and do not have immediate practical clinical implications , although they do provide the rationale for testing the drug 's efficacy in premenopausal women . moreover , this protective effect was suggested in women with a high probability of carrying a brca-1 mutation . indeed , fenretinide was highly effective in inhibiting the growth of brca-1 mutated breast cancer cell lines . when considering the protective activity of fenretinide on second breast cancer in young women and a similar trend on ovarian cancer , at least during intervention , it appears that women with germline brca-1 and brca-2 mutations may be ideal candidates for further investigation of this drug . all the collected data as well as fenretinide characteristics make this drug an excellent candidate for chemoprevention of the highlighted subgroup , that is , young healthy women with a high susceptibility to early - onset breast and ovarian cancer , such as brca1/2 mutation carriers or women with a significant familial risk . since the drug activities are probably not strictly influenced by hormonal responsiveness , it may affect also hormone nonresponsive cancers several drugs used in prevention settings are usually the same as those used for treatment ( adjusting dosage and/or route of administration ) . this is possible because their mechanism of actions is also active on early - phase carcinogenesis and not only on the inhibition of the tumor growth . this is confirmed by the reduction of second breast cancer found in the reported studies of veronesi et al . [ 40 , 41 ] . these data make 4-hpr a surrogate marker of primary prevention and a favorable effect of the drug would provide a strong rationale for a primary prevention trial in unaffected women at high risk for breast cancer . moreover , because its action does not seem to be influenced by the hormonal status , fenretinide might be active in hormone nonresponsive cancer prevention ( as occurs in brca-1 mutation carriers ) . although we obviously need to verify this hypothesis , we think it is an intriguing scenario that could be important in order to identify new pathways related to the efficacy of this drug . our division of cancer prevention and genetics at european institute of oncology to milan has already activated a new phase iii prevention trial addressed to this particular cohort of women . a total of 764 healthy women at increased breast cancer risk will be randomized to 4-hpr 200  mg / day versus placebo for 5 years . the subjects will be stratified by participating center and breast cancer risk ( brca1 mutation versus brca2 versus high risk subjects ) . the accrual estimated time is five years . the design of the study is explained in figure 4 .   the aim of the proposed trial is to assess the efficacy of fenretinide , in reducing the incidence of breast cancer in healthy young premenopausal women at increased familial / genetic risk for breast cancer ; the primary endpoint is to assess the incidence of histologically diagnosed invasive breast cancer and ductal intraepithelial neoplasia . secondary endpoints are the incidence of other noninvasive breast disorders ( i.e. , intraepithelial lobular neoplasia and atypical hyperplasia ) , ovarian cancer and other cancers . moreover , we propose an interdisciplinary research study to further investigate the mechanisms of action of fenretinide in preventing breast cancer . early intermediate biomarkers of efficacy after 12 , 36 , and 60 months of treatment , genetic interactions with breast cancer risk modifiers will be explored with the primary goal to identify molecular biomarkers of response prediction . in particular , we will evaluate the percentage change in circulating biomarkers of the igf system , androgens , retinol binding protein ( rbp-4 ) , insulin , blood glucose , and vegf after 12 , 36 , and 60 months of treatment . in a subgroup of participants , fine needle aspirate breast biopsy or cells obtained from breast ductal lavage will be drawn at baseline and after a 12-month treatment and the percentage change in rar expression correlated with apoptosis ( caspase-3 ) and proliferation ( ki-67 ) . genotyping of single - nucleotide polymorphisms ( snps ) linked to breast cancer risk ( mthfr , comt , gh , igfbp-3 , ar , and tgf- genes ) , degree of methylation of rassaf1 and rar  , and circulating progranulin will be assessed . the results will be correlated with mammographic instrumental measurements , plasma and tissue biomarkers after 1-year treatment . should the results of this trial confirm that fenretinide is effective in reducing breast and ovarian cancer incidence in this very high risk population , that this effect lasts for many years after treatment , and that the tolerability profile is good , we will have a further preventive chance and a new risk reduction strategy .
the incidence and mortality of breast cancer have been recently influenced by several new therapeutic strategies . 
 in particular our knowledge on cancer precursors , risk biomarkers , and genetics has considerably increased , and prevention strategies are being successfully explored . since their discovery , retinoids , the natural and synthetic derivatives of vitamin a , have been known to play a crucial role in cell and tissue differentiation and their ability to inhibit carcinogenesis has made them the ideal chemopreventive agents studied in several preclinical and clinical trials . 
 fenretinide ( 4-hpr ) is the most studied retinoid in breast cancer chemoprevention clinical trials due to its selective accumulation in breast tissue and its favorable toxicological profile . 
 this agent showed a significative reduction of the incidence of second breast tumors in premenopausal women confirmed after 15-year followups . 
 considering fenretinide protective action , a similar trend on ovarian cancer , this drug warrants reevaluations as a preventive agent for high - risk young women , such as brca-1 and 2 mutation carriers or with a high familial risk . 
 this favorable effect therefore provides a strong rationale for a primary prevention trial in these unaffected cohort of women .