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Merja Mets-Heikkil
(formerly Marttunen)



Merja Mets-Heikkil
(formerly Marttunen)

Department of Obstetrics and Gynecology Helsinki University Central Hospital University of Helsinki, Finland

ACADEMIC DISSERTATION To be presented for public examination by permission of the Medical Faculty of the University of Helsinki, in the Auditorium of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Haartmaninkatu 2, Helsinki, on May 23rd 2001, at 12 noon.

SUPERVISED BY Professor Olavi Ylikorkala, M.D., Ph.D. Department of Obstetrics and Gynecology Helsinki University Central Hospital University of Helsinki

REVIEWED BY Emeritus Professor Antti Kauppila, M.D., Ph.D. Department of Obstetrics and Gynecology Oulu University Hospital University of Oulu Docent Guillermo Blanco, M.D., Ph.D. Department of Oncology Oulu University Hospital University of Oulu

OFFICIAL OPPONENT Docent Tuula Salmi, M.D., Ph.D. Department of Obstetrics and Gynecology Turku University Central Hospital University of Turku

Graphic design Hanna Mets-Heikkil Multiprint Oy Helsinki 2001 ISBN 952-91-3428-2 ISBN 951-45-9969-1 (pdf)

To my family


LIST OF ORIGINAL COMMUNICATIONS ABBREVIATIONS I INTRODUCTION II REVIEW OF THE LITERATURE 1 BREAST CANCER 1.1 Incidence 1.2 Risk factors 1.3 Treatment 1.4 Prognosis and prognostic factors 2 ESTROGEN REPLACEMENT THERAPY IN POSTMENOPAUSAL WOMEN 2.1 Postmenopausal health and estrogen 2.2 Benefits of estrogen replacement therapy 2.2.1 Quality of life 2.2.2 Cardiovascular diseases Mechanisms of vascular protection by estrogens 2.2.3 Postmenopausal bone loss 2.3 Hazards of estrogen replacement therapy 2.3.1 Breast cancer 2.3.2 Endometrial cancer 2.3.3 Venous thromboembolism 2.4 Estrogen replacement therapy in women with previous breast cancer 3 TAMOXIFEN AND TOREMIFENE 3.1 Pharmacological and biological characteristics 3.2 Clinical use 3.3 Gynecological consequences 3.4 Cardiovascular organs 3.5 Bone III AIMS OF THE PRESENT STUDY IV PATIENTS AND METHODS 1 PATIENTS 2 STUDY TREATMENTS

7 8 9 10 10 10 12 14 15

17 17 18 18 20 22 24 26 26 27 28 29 32 32 33 34 36 37 38 39 39 42

3 METHODS 3.1 Clinical examinations and follow-up 3.2 Transvaginal sonography and endometrial sampling 3.3 Laboratory measurements 3.4 Bone density measurement 3.5 Statistical analyses V RESULTS 1 ESTROGEN REPLACEMENT THERAPY IN BREAST CANCER PATIENTS 2 TAMOXIFEN AND TOREMIFENE 2.1 Endometrial findings 2.2 Vascular effects 2.2.1 Uterine artery resistance 2.2.2 Prostacyclin and thromboxane 2.2.3 Endothelin-1 and nitric oxide 2.3 Bone VI DISCUSSION VII SUMMARY AND CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

43 43 44 45 47 47 48

48 50 51 53 53 53 54 55 59 65 67 69



I Merja B. Marttunen, Pivi Hietanen, Seppo Pyrhnen, Aila Tiitinen, Olavi Ylikorkala. A prospective study on women with a history of breast cancer and with or without oestrogen replacement therapy. Maturitas. In press. II Merja B. Marttunen, Bruno Cacciatore, Pivi Hietanen, Seppo Pyrhnen, Aila Tiitinen, Torsten Wahlstrm, Olavi Ylikorkala. Prospective study on gynecological effects of two antiestrogens tamoxifen and toremifene in postmenopausal women. Br J Cancer 84:897-902, 2001. III Merja B. Marttunen, Seppo Pyrhnen, Aila Tiitinen, Lasse Viinikka, Olavi Ylikorkala. Effect of antiestrogen regimen on prostacyclin and thromboxane A2 in postmenopausal patients with breast cancer: evidence of significance of hypertension, smoking or previous use of estrogen therapy. Prostaglandins 52:317-326, 1996. IV Merja B. Marttunen, Pivi Hietanen, Aila Tiitinen, Olavi Ylikorkala. Antiestrogens reduce plasma levels of endothelin-1 without affecting nitrate levels in breast cancer patients. Gynecol Endocrinol 14:55-59, 2000. V Merja B. Marttunen, Pivi Hietanen, Aila Tiitinen, Olavi Ylikorkala. Comparison of effects of tamoxifen and toremifene on bone biochemistry and bone mineral density in postmenopausal breast cancer patients. J Clin Endocrinol Metab 83:1158-1162, 1998. VI Merja B. Marttunen, Pivi Hietanen, Aila Tiitinen, HansJrgen Roth, Lasse Viinikka, Olavi Ylikorkala. Effects of tamoxifen and toremifene on urinary excretion of pyridinoline and deoxypyridinoline and bone density in postmenopausal patients with breast cancer. Calcif Tissue Int 65:365-368, 1999.


ANOVA analysis of variance BMD bone mineral density BMI body mass index BRCA 1 and 2 breast cancer gene mutations CA 15-3 tumor marker of breast cancer DEXA dual-energy x-ray absorptiometry DNA deoxyribonucleic acid Dpyr deoxypyridinoline ER estrogen receptor ERA The estrogen replacement and atherosclerosis trial ERT estrogen replacement therapy ERT/HRT hormone replacement therapy ET-1 endothelin-1 FSH follicle stimulating hormone HDL high-density lipoprotein cholesterol HERS The heart and estrogen/progestin replacement study HOP hydroxyproline HRT estrogen+progestin replacement therapy ICTP carboxyterminal cross-linked telopeptide Ki-67 proliferation marker of breast cancer cells LDL low-density lipoprotein cholesterol LH luteinizing hormone NO nitric oxide NOx nitrate+nitrite NTx aminoterminal cross-linked telopeptide OCC oral combined contraceptives PI pulsatility index PICP carboxyterminal propeptide of type I procollagen PINP aminoterminal propeptide of type I procollagen PMS premenstrual syndrome Pyr pyridinoline RIA radioimmuno assay RP-HPLC ion-pair reversed-phase high performance liquid chromatography RR relative risk SD standard deviation SE standard error SERM selective estrogen receptor modulator Tam tamoxifen TFA trifluoroacetetic acid Tor toremifene TxA2 thromboxane A2 TxB2 thromboxane B2


Breast cancer is the most common cancer of women in western countries, and its incidence is increasing (Garfinkel et al. 1994; Broeders and Verbeek 1997). Overall, 10-15% of all women will develop breast cancer during their lifetime (Broeders and Verbeek 1997; Lpez-Otn and Diamandis 1998). In Finland, each year are diagnosed approximately 3300 new cases of breast cancer (Finnish Cancer Registry 1997). Yet, in spite of the increasing incidence, the mortality for breast cancer has not increased during the last years. This apparent discrepancy can perhaps be explained by the earlier diagnosis (Garfinkel et al. 1994; Garne et al. 1997; UK Trial of Early Detection of Breast Cancer Group 2000), by the appearance of less malignant forms of the disease (SEER Cancer Statistics 1997), and also by the progress in treatment (Early Breast Cancer Trialists Collaborative Group 1992; 2000). The ethiopathogenesis of breast cancer is still unknown, but estrogen plays a major role (Mettlin 1992; Broeders and Verbeek 1997; Lpez-Otn and Diamandis 1998). This is supported for example by the fact that breast cancer occurs almost exclusively in women. Observational epidemiological studies also show increased risk of breast cancer after the long-term use of estrogen (ERT) or estrogen plus progestin (HRT) replacement therapy (Collaborative Group on Hormonal Factors in Breast Cancer 1997). Therefore ERT/ HRT has not been recommended for postmenopausal women with a history of breast cancer (Marchant 1994; Colditz 1997; Consensus Statement 1998). Antiestrogens are an interesting group of drugs owing both antiestrogenic and estrogenic activity (Buckley and Goa 1989; Wiseman and Goa 1997). They are used in the treatment (Early Breast Cancer Trialists Collaborative Group 1998; Pyrhnen et al. 1999) and also in the prevention of breast cancer (Fisher et al. 1998). At present two antiestrogens, tamoxifen and toremifene, which are structurally related compete for favor in clinical routine (Pyrhnen et al.1999). These drugs have a number of beneficial estrogenic effects (Neven et al. 1993) but on the other hand, antiestrogens do not alleviate vasomotor symptoms but may even aggravate them (Love et al. 1991). The present study was designed firstly to explore the usefulness and safety of ERT/HRT in women with a history of breast cancer and secondly, to compare the effects of tamoxifen and toremifene on female reproductive organs, vascular endothelium, and bone.


1.1 Incidence
It has repeatedly been established that the incidence of breast cancer has increased approximately by 1% per year in Finland (Finnish Cancer Registry 1997). Breast cancer is most common in North America and in Western Europe and most infrequent in Asia, e.g. in Japan (Figure 1). In Finland, the incidence of breast cancer has almost doubled since the 1970s (Figure 2) (Finnish Cancer Registry 1997, Statistics Centrum of Finland). Breast cancer accounts for approximately 30% of all malignant tumors in Finnish women. Due to the mammographic screening programs, the detection of early breast cancer has increased most drastically. This was seen, for example, in the United States where the proportions of stage I and of in situ cancer have increased markedly, whereas the

Spain France Finland Germany UK Sweden Denmark Netherlands USA 0 20 40 60 80 100 120 Incidence rate per 100 000 Figure 1. The age adjusted incidences of breast cancer in 1990 at ages 0-74 years in different countries



proportions of stage II-IV breast cancer have decreased (Figure 3) (SEER Cancer Statistics 1997). A similar phenomenon has also taken place in Finland (Klemi et al.1992). This could explain, at least partly, why the increased incidence of this cancer has not led to an increase in mortality, but in contrast, the mortality rate due to breast cancer may even be decreasing, at least in Finland (Figure 2) and also in Europe (Peto et al. 2000).
80 70

Incidence per 100 000

60 50 40 30 20 10 0






Figure 2. The incidence of breast cancer (grey bars) and deaths (white bars) for breast cancer in Finland (Finnish Cancer Registry, Statistics Centrum of Finland)

% 300 200


105.2% 100 34.1% 0 -14.2% -11.2% -17.4% -100 All In situ Stage I Stage II Stage III-IV Unknown

Figure 3. Percentual change in incidence of in situ and St I-IV breast cancer from 1983 to 1995 in the United States



1.2 Risk factors

Although the pathogenesis of breast cancer is not understood, several inherent and/or clinical factors can indicate an increased risk (Table 1) (Mettlin 1992; Collaborative Group on Hormonal Factors in Breast Cancer 1996, 1997; Broeders and Verbeek 1997; Lpez-Otn and Diamandis 1998, Colditz and Rosner 2000). One of the well established risk factors is age because the incidence of breast cancer, as that of almost any cancer, increases with advancing age. Family history of breast cancer denotes a clearly increased risk, and genetic predisposition is detectable in approximately 5-10% of all cases with breast cancer (Broeders and Verbeek 1997). Two major gene mutations related to breast cancer have been identified. BRCA1 gene is located on the long arm of chromosome 17 and BRCA2 on the long arm of chromosome 13 (Lpez-Otn and Diamandis 1998). Furthermore, the locus of the third candidate BRCA gene has been identified also on the long arm of chromosome 13 (Kainu et al. 2000). Estrogen-related risk factors are for example female gender, young age at menarche, high age at menopause, and postmenopausal obesity (Kelsey et al. 1993; Broeders and Verbeek 1997; Lpez-Otn and Diamandis 1998). The effect of parity has been also widely studied. Women with a first pregnancy after 35 years of age or nulliparous women have a higher risk for breast cancer, whereas an age below 21 years at the first full-term pregnancy seems to protect against breast cancer (Kelsey et al. 1993; Pathak et al. 2000). Also long-term postmenopausal use of ERT/HRT has been linked to the increased risk of breast cancer (Collaborative Group on Hormonal Factors in Breast Cancer 1997; Colditz and Rosner 2000). However, it is noteworthy that a quarter of breast cancer patients have none of these risk factors and all risk factors combined could account for less than 30% of the breast cancer incidence (Mettlin 1992).



Table 1. Major risk factors for breast cancer

Factor Female gender Age Socioeconomic group Diet Increased/decreased risk 100 fold compared to males Doubles every 10 years until menopause Highest incidence in high economical standard (I and II) High intake of unsaturated fat increases the risk High intake of soyproducts decreases the risk Postmenopausal obesity (BMI >35 kg/m2) doubles the risk Daily use of alcohol increases the risk A first degree relative with breast cancer before the age of 50 doubles the risk 3 to 5-fold cancer risk in contralateral breast 4 to 5-fold risk 1.5 to 2-fold risk Age below 12 years increases the risk Late age increases the risk Increases the risk High age at first delivery increases the risk Lactation over 24 months decreases the risk Current use of OCCs increases the risk, the risk disappears 10 years after cessation of OCCs Over 5 years of use increases the risk, excess risk disappears 5 years after cessation of estrogen Adding progestin to estrogen still increases the risk

Obesity Alcohol consumption Family history of breast cancer

Cancer in one breast Previous atypical hyperplasia Endometrial and ovarian cancer Age at menarche Age at menopause Nulliparity Age at first full term pregnancy Lactation Oral contraceptives (OCC)

Menopausal hormone use



1.3 Treatment
Surgery is the cornerstone in the treatment of breast cancer. It can be conservative (=local excision or segmental resection) or radical (=mastectomy). The type of operation depends on the size and location of the tumor and also on the patients opinion. Small (diameter below 4 cm), not centrally located tumors are almost always suitable for breast saving surgery, whereas multifocal and central lesions, as well as large tumors usually require mastectomy. Dissection of axillary lymph nodes is commonly combined to surgery, because the nodal status is the most powerful prognostic factor and guides the use of adjuvant therapy (Carter et al. 1989; Hortobagyi 1998). Because the dissection of axillary lymph nodes is related to a number of postoperative sequelae (lymphoedema, limited movements of shoulder, pain), recently has been introduced the removal of the sentinel node to avoid the risk of later complications. The sentinel node is most likely to drain the primary tumor, and the status of the sentinel node accurately predicts the nodal status of the whole axilla (Krag et al. 1998). Postoperative radiotherapy is given to reduce the risk of local recurrences which may occur after conservative surgery (Hortobagyi 1998; Early Breast Cancer Trialists Collaborative group 2000). After mastectomy, radiotherapy is given only to patients with T3 tumor (diameter >5 cm) or to patients with positive axillary nodes because they still have a risk of local recurrence (Early Breast Cancer Trialists Collaborative Group 2000). Postoperative adjuvant chemotherapy and/or hormonal therapy is given if the cancer has spread to axillary nodes or if patients have other clinically relevant risk factors for cancer recurrence (Early Breast Cancer Trialists Collaborative Group 1992; 1998). Adjuvant chemotherapy is given at three weeks interval by using a combination of cyclophosphamide and doxorubicin (CA) or a combination of cyclophosphamide, fluorouracil, and methotrexate or epirubicin (CMF or CEF). CMF regimen improved the 10-year survival by 7% in node-negative and by 11% in node-positive patients (Early Breast Cancer Trialists Collaborative Group 1992). Also the rate of local recurrences decreased by 35% (Early Breast Cancer Trialists Collaborative Group 1992). Postoperative antiestrogen treatment, which has been available from the seventies (Buckley and Goa 1989), is primarily given to postmenopausal women with estrogen and/or pro-



gesterone receptor positive tumors (Early Breast Cancer Trialists Collaborative Group 1992; 1998). Tamoxifen (20-40 mg/day) was the first drug to enter clinical use, and it reduced the risk of recurrence by 21% after 1 year of use, by 29% after 2 years of use, and by 47% after 5 years of use (Early Breast Cancer Trialists Collaborative Group 1998). Correspondingly, mortality decreased by 12%, 17%, and 26%, respectively (Early Breast Cancer Trialists Collaborative Group 1998). Tamoxifen treatment also prevented the development of cancer in the contralateral breast by 47% after 5 years of use (Early Breast Cancer Trialists Collaborative Group 1998). Toremifene, developed in the 1980s, is an antiestrogen which has been clinically used since 1989. Until now it has been used primarily in the treatment of advanced breast cancer (Wiseman and Goa 1997). Toremifene at the dose of 4060 mg has an antitumor activity comparable to that of tamoxifen at the dose of 20 mg (Homesley et al. 1993; Pyrhnen et al. 1999). The trial on toremifene as an adjuvant treatment of breast cancer is still continuing (Holli 1998), and according to the preliminary results tamoxifen and toremifene appear to have equal clinical efficacy (Holli et al. 2000).

1.4 Prognosis and prognostic factors

Prognosis of breast cancer depends on many different factors (Table 2) (Joensuu and Toikkanen 1992). The size of tumor and its possible spread to axillary lymph nodes are the major prognostic factors (Miller et al. 1994; Abner et al. 1998; Page et al. 1998; Tabr et al. 1999). Patients in whom cancer has spread beyond the axillary nodes have a much worse survival rate than patients whose tumor is limited to the breast (Carter et al. 1989; Page et al 1998). Biological factors, such as the estrogen and progesterone receptor status, histological grading, and proliferation markers, predicts the behavior of the tumor and helps in choosing suitable treatments for individual patients (Alanko et al. 1984; Blanco et al. 1984; Elston and Ellis 1991; Miller et al. 1994; Page et al. 1998). Histological grading on the scale from I to III is based on glandular formation, nuclear pleomorphism, and



frequency of mitoses (Elston and Ellis 1991). Markers of tumor cell proliferation reflect growth potential, and, for instance, the number of cells in S-phase fraction detected by flow cytometry, the DNA content, the presence of Ki-67 antigen are most often used in clinics (Kallioniemi et al 1987; Miller et al. 1994; Page et al 1998). The overexpression of cellular oncogens, such as c-erbB2, c-myc and ras has been associated with poor prognosis (Miller et al. 1994; Robertson and Evans 1997). Furthermore, high serum levels of the type I procollagen propeptides PINP and PICP and low PICP:PINP ratio have been shown to associate with a higly aggressive nature of breast cancer (Jukkola et al. 1997). The patients age at diagnosis is also a prognostic marker; younger women have a poorer prognosis than older patients with an equivalent stage of cancer (Tabr et al. 1999). It is noteworthy that an earlier detection of cancer, for example by screening mammogram programs, has improved the prognosis of breast cancer during the last few decades (Garfinkel et al. 1994; Garne et al. 1997; Tabr et al. 1999, UK Trial of Early Detection of Breast Cancer group 1999). However, breast cancer affects the rest of the patients life because it may relapse even 10-20 years after primary diagnosis (Joensuu et al. 1999).

Table 2. Impact of different prognostic factors on the 5-year survival of breast cancer patients (Joensuu and Toikkanen 1992)
Prognostic factor Tumor size 5-year survival (%) <2 cm (T1) 25 cm (T2) >5 cm (T3) or T4 Gradus I Gradus II Gradus III negative positive positive negative positive negative <4% >4% diploid non-diploid 92 75 58 97 79 59 93 62 88 65 90 69 93 70 89 75

Histologic differentiation

Axillary nodes Estrogen receptors Progesterone receptors S-phase fraction DNA ploidy




2.1 Postmenopausal health and estrogen
The menopause is accompanied by a number of hormonal changes reflected in typical climacteric symptoms (Table 3) (Brincat and Studd 1988; Goldani von Mhlen et al. 1995; Porter et al. 1996; Dennerstein et al. 2000). At least 80% of women suffer from one or more menopausal symptoms during the first years after menopause. Furthermore, the signs of urogenital atrophy caused by estrogen deficiency increase with advanced age, and the risk of urinary tract infections increases.

Table 3. The presentation (%) of the most important climacteric symptoms


Brincat and Studd 1988 68 51 71 44 48 92 78 88 64 20 20

Goldani von Mhlen et al. 1995 74 28

Porter et al. 1996 55-57 66 60 37 67

Dennerstein et al. 2000 41-52 38-45 32-42 6-11 45-57 26-31 26-32 38-42 20-28 25-47 14-26

Hot flushes, sweating Insomnia / sleeping disorders Headache Palpitation Muscle / joint pain Anxiety / irritability Depression Tiredness / lack of initiative Difficulty in concentrating Vaginal dryness Decreased libido Urogenital symptoms

25 20 32

58-72 51 64 34

24 48



Postmenopausal hypoestrogenism affects also the cardiovascular system. The risk of cardiovascular diseases increases after menopause, and women tend to reach the incidence in that of men (Sullivan and Fowlkes 1996). Indeed, a half of the women in the western world will get coronary artery disease and one-third will die from it (Grady et al. 1992). Therefore, it is understandable that the prevention of cardiovascular disease has been, and still is, one of the priorities in public health programs. The peak bone mass, which is reached normally at 25-30 years of age, is 30-50% lower in women than in men (Christiansen 1995). Neverthless, bone is affected by estrogen. This is supported by the finding that estrogen receptors are present in osteoblasts and osteoclasts, the cells responsible for bone formation and bone resorption, respectively (Oursler et al. 1993). In menopause, estrogen deficiency leads to increased osteoclast formation and activity, and thereby it enhances bone resorption both in trabecular and cortical bone (Roodman 1996). This may lead to osteoporosis if a woman lives long enough (Marcus 1996). The World Health Organisation has defined osteoporosis as a bone mineral density (BMD), measured by dual-energy x-ray absorptiometry, which is 2.5 standard deviation (SD) or more below the mean peak value in young adults. Immediately after menopause, bone mass decreases by 1-5% per year, and even after the age of 65, the decrease is 0.5-1% per year (Riis 1996). It has been estimated that 21% of postmenopausal women in the western countries will eventually develop osteoporosis (Looker et al. 1995). Postmenopausal women have a 15% lifetime probability of suffering a hip fracture and a 1.5% probability of dying from it (Grady et al. 1992). Also the risk of vertebral fractures increases, and although these fractures are not associated with increased mortality, they may cause significant morbidity (Lindsay et al. 1980; Grady et al. 1992). Thus, the prevention of osteoporosis is one of the major challenges in the health care system in western countries.

2.2 Benefits of estrogen replacement therapy 2.2.1 Quality of life

Menopausal symptoms, such as hot flushes, insomnia, depressive mood and tiredness, are the most common causes for the initiation of ERT/HRT in clinical routine. Estrogen

replacement therapy eliminates, or at least alleviates rather effectively these symptoms (Wiklund et al. 1992) and improves the quality of life, as measured by different standardized well-being and health questionnaires (Wiklund et al. 1993; Daly et al 1993) (Figure 4). The benefit in alleviation of menopausal symptoms has increased the popularity of ERT/HRT use in the western world, and for instance in Finland 44% of 55-year old women and 5% of 75-year old women used ERT/HRT in 1997 (Finnish Statistics on Medicines 1997). It is conspicuous that women with higher education are more likely to start the use of ERT/HRT, and for instance in Sweden 88% of postmenopausal female gynecologists use ERT/HRT, whereas the overall frequency of the use of ERT/ HRT among 55-56 years old women was 35% (Andersson et al. 1998).

Somatic symptoms


13 12 11 10 9 8 7 6 5 4 3 2 1 0
Vasomotor symptoms

WHQ score

Cognitive function


Figure 4. Improvement of menopausal symptoms as assessed by womens health questionnaire (WHQ) before and after 3 months of estrogen therapy (n=110, p<0.0001 in all changes) (Wiklund et al. 1992) (grey bars=before therapy, white bars=after therapy)

Sex life


2.2.2 Cardiovascular diseases

Several observational studies show a lower risk of cardiovascular diseases and deaths in women taking estrogen alone or in combination with progestin as compared to non-users (Table 4). In a recent meta-analysis, the relative risk of cardiovascular diseases among women who had used ERT/HRT compared to those who had never used ERT/HRT was 0.70 (Barret-Connor and Grady 1998). The largest single study, Nurses Health Study, in which 59 337 women were followed up to 16 years, showed that the relative risk of major coronary disease was 0.60 in current users of ERT and 0.39 in current users of HRT, and 0.85 in former hormone users (Grodstein et al. 1996). In this study, ERT/HRT did not prevent the risk of a brain ischemic stroke (Grodstein et al.
Table 4. The effect of estrogen replacement therapy on primary (prim) or secondary (sec) prevention of cardiovascular morbidity and/or mortality in observational studies

Author Bush et al. 1987 Petitti et al. 1987

No of study population 2270 6093 8841 2268 4544 8853 48470 1944 23174 59337 1098 337 726 7944

Study design sec prim&sec prim&sec sec prim&sec prim&sec prim prim&sec prim&sec prim sec sec sec prim&sec

Event Cardiovascular death Cardiovascular death Cardiovascular death Myocardial infarction Cardiovascular death Cardiovascular death Cardiovascular death Cardiovascular event Cardiovascular death Myocardial infarction Cardiovascular event Cardiovascular death Cardiovascular event Myocardial infarction

Risk ratio 0.37 0.60 0.59 0.54 0.16 0.37 0.69 0.56 0.66 0.81 0.39 (HRT) 0.60 (ERT) 0.80 0.38 0.64 (current) 0.90 (past)

Henderson et al. 1988 Sullivan et al. 1990 Hunt et al. 1990 Henderson et al. 1991 Stampfer et al 1991 Wolf et al. 1991 Falkeborn et al. 1992 Grodstein et al. 1996 Sullivan et al. 1997 OKeefe et al. 1997 Newton et al. 1997 Sourander et al. 1998

Cardiovascular disease 1.1 Cardiovascular death 0.21 (current) 0.75 (past)



1996). However, all these observational studies have been criticized because healthier women may have been more prone to start ERT/HRT (selection bias): the ERT users have had fewer cardiovascular risk factors, such as hypertension, hypercholesterolemia, or obesity before the start of ERT/ HRT (Matthews et al.1996; Rdstrm et al. 1999). The clinical data on the cardiovascular benefits of ERT/ HRT in women with ischemic heart disease (secondary prevention) are controversial. Previous observational studies have shown decreased risk of recurrent cardiovascular events in estrogen users (Bush et al 1987; Sullivan et al. 1990; Henderson et al. 1991; Sullivan et al. 1997; OKeefe et al. 1997; Newton et al. 1997) but the only randomized, placebo-controlled trial (HERS study) failed to show any benefit at least in older (an average of 67 years) women (Hulley et al. 1998). In this study, placebo or 0.625 mg of conjugated equine estrogen in combination with 2.5 mg of medroxyprogesterone acetate were given continuously to 2763 women for an average of 4.1 years (Hulley et al. 1998). During the first year of use, the risk of cardiac recurrence was increased (RR 1.52) in hormone users, whereas in 3-5 years, a clear trend (RR 0.87-0.67) for the favor of HRT was seen (Hulley et al. 1998). In another study, the effect of ERT/HRT on the progression of coronary atherosclerosis was studied in a randomized, placebo-controlled setting (ERA trial) (Herrington et al. 2000). In 309 women with angiographically verified coronary disease, conjugated estrogen with or without progestin, did not prevent the progression of coronary occlusion during a mean of 3.2 years follow-up, even if ERT/HRT had favorable effects on LDL and HDL cholesterol levels (Herrington et al. 2000). In future, the benefit of estrogen in the primary prevention of cardiovascular diseases should be ascertained by randomized, placebo-controlled studies, and some studies are presently conducted (The Womens Health Initiative Study Group 1998). Before these results are available there is no definite truth about the role of ERT/HRT in the primary prevention of coronary heart disease. Yet, it can be questioned whether truly randomized and placebo-controlled trials in menopausal research are possible (Ylikorkala 2000).


21 Mechanisms of vascular protection by estrogens

Estrogen has several favorable vascular effects, which may explain its cardiovascular protective efficacy (Table 5). It has been estimated that the favorable effect of estrogen on plasma lipids can explain approximately 20-30% of cardiovascular benefits of ERT/HRT (Wild 1996; Tikkanen 1999). Besides high lipid levels, also high plasma homocysteine levels may indicate a risk of atherosclerosis (Welch and Loscalzo 1998). In a Dutch study, estrogen has been shown to decrease these levels especially when the baseline levels were high (Mijatovic et al. 1998), whereas this was not seen in Finnish women with normal baseline homocysteine levels (Evi et al. 2000). Estrogen has also a favorable effect on glucose metabolism by enhancing tissue insulin sensitivity (Barrett-Connor and Laakso 1990; Lindheim et al. 1994; Raudaskoski et al. 1999). It is likely that these biochemical findings may require a more prolonged use of ERT before they become clinically apparent. Estrogen has also the direct and immediate effects on the vascular wall. This was first supported by the data which showed that estrogen binds to the estrogen receptors of vascular endothelial and smooth muscle cells (Mendelsohn and Karas 1999). Endothelial cells produce a number of agents with vasodilatory or vasoconstricive activity. One of the best studied agents is vasodilatory prostacyclin, which is an important mediator in several occlusive and hypertensive disorders (Ylikorkala et al. 1986; Cinotti and Pugliese 1989; Vane et al. 1990). It is stimulated by estrogen, at least in cultured endothelial cells (Mikkola et al. 1995). Endothelial cells also secrete free radical gas, nitric oxide (NO), which causes vasodilatation and inhibits platelet aggregation (Moncada et al. 1991). Nitric oxide production is diminished in conditions characterized by endothelial damage, such as atherosclerosis, hypertension, or diabetes (Moncada et al. 1991). Endothelin-1 (ET-1) is a peptide hormone which is also produced by endothelial and smooth muscle cells (Masaki 1993). Endothelin-1 is the most potent vasoconstrictor yet discovered (Masaki 1993), and indeed, elevated plasma ET-1 levels have been detected in several vasoconstrictive diseases, such as in myocardial infarction, pulmonary hypertension, and atherosclerosis (Masaki 1993). Estrogen administration in postmenopausal women increases the circulating levels of NO (Cicinel-



Table 5. Some plausible biological explanations for the beneficial effect of estrogen on vascular health

Risk Factor Lipids and lipoproteins Total cholesterol LDL cholesterol HDL cholesterol Triglycerides Lp(a) Endothelial cell function Prostacyclin Nitric oxide Endothelin-I Endothelium-independent vascular resistance Homocysteine Insulin sensitivity





li et al. 1997; Imthurn et al 1997) and decreases the level of ET-1 (Ylikorkala et al. 1995; Wilcox et al. 1997). We should also keep in mind the vasoconstrictive thromboxane A2 (TxA2), which is produced by platelets and serves as an endogenous antagonist of prostacyclin. A shift in the balance between prostacyclin and thromboxane A2 to the prostacyclin dominance may protect against cardiovascular diseases (Mendelsohn and Karas 1994). Estrogen also acts as a calcium antagonist (Collins et al. 1993) and reduces the levels of angiotensin-1 convertingenzyme in blood (Proudler et al. 1995). Thus estrogen may cause vasodilatation by mechanisms which operate outside the vascular endothelium. The vasodilatory effect of estrogen on blood vessels can be directly detected by Doppler ultrasound or with a straingauge plethysmography. Indeed, estrogen administration increases blood flow in aorta (Pines et al. 1991), radial (Lau et al. 1998; Vehkavaara et al. 2000), carotid and cerebral arteries (Penotti et al. 1996). It is conspicuous that oral or transdermal route of administration of estrogen causes similar vasodilatatory effect in the uterine and carotic arteries (Cacciatore et al. 1998). It is also noteworthy that both oral and transdermal HRT decrease systolic daytime blood pressure in normotensive women but have no effect on night time blood pressure (Cacciatore et al. 2001).



The biology of vascular endothelium is a complex entity and presently a target for hectic research. In the above I have very briefly outlined a few aspects of endothelial research, which have been most clearly linked to the use of ERT/HRT. For those readers who wish to learn more about vascular endothelial physiology and cardiovascular diseases, I refer to two current reviews (Vogel 1997; Lind et al. 2000).

2.2.3 Postmenopausal bone loss

Estrogen replacement therapy reduces the rate of bone loss mainly by decreasing bone resorption (Turner et al. 1994). In this regard, daily oral doses of 2 mg of estradiol or 0.626 mg of conjugated estrogens daily seem to be equally effective as daily 50 g of estradiol from a patch or 1.5 mg estradiol from a gel (Compston 1997). Recent studies indicate that also smaller doses of estrogen may protect bone (Weiss et al. 1999). Progestin added to ERT has been shown to potentiate the protective effect of estrogen on bone in some (The Writing Group for the PEPI Trial 1996), but not in all studies (Adachi et al. 1997). Data from observational studies show that ERT decreases the risk of hip fracture by at least 25% (Grady et al. 1992) and vertebral fractures by 50% to 80% (Lindsay et al. 1980; Maxim et al. 1995). In a large population-based case-controlled study the odds ratio for hip fracture was 0.35 in current users and 0.76 in past users (Michalsson et al. 1998). Furthermore, the bone protective effect of ERT increases with the duration of use (Moore et al. 1990; Grady et al. 1992), and the initiation of estrogen in late menopause also reduces the risk of hip fracture (Lindsay and Tohme 1990). The generally accepted method for bone mineral density (BMD) measurement is dual-energy x-ray absorptiometry (DEXA), and The World Health Organisation criteria for osteoporosis are based on this method. However, in order to detect the rate of bone turnover and to identify patients with increased bone loss, serial DEXA measurements are needed at one or two-year intervals. This calls for a need for markers that show changes in the rate of bone turnover within a few months. It has also been shown that high bone turnover is an independent predictor of increased fracture risk (Miller et al. 1999; Looker et al. 2000). Presently, there is a general

consensus that the most valuable biochemical markers of bone formation are serum bone specific alkaline phosphatase, osteocalcin, and carboxyterminal propeptide of type I procollagen (PICP), whereas urinary pyridinoline (Pyr), deoxypyridinoline (Dpyr), and carboxy- (ICTP) and aminoterminal (NTx) cross-linked telopeptides predict most reliably bone resorption (Table 6) (Calvo et al. 1996; Miller et al. 1999; Fink et al 2000; Looker et al. 2000). A large number of studies have reported significant associations between the levels of various bone biochemical markers and bone loss rates (Miller et al. 1999, Fink et al. 2000, Looker et al. 2000).
Table 6. The most often used bone turnover markers
Formation markers Serum osteocalcin of bone Characteristics Non-collagenous matrix protein of bone produced mainly by osteoblasts, specific marker of osteoblastic activity Isoenzyme produced by osteoblasts Propeptide products of type I collagen formation from procollagen; related to bone matrix formation

Serum bone-specific alkaline phosphatase Serum carboxyterminal propeptide of type I procollagen (PICP) Serum aminoterminal propeptide of type I procollagen (PINP) Resorption markers Urinary hydroxyproline (HOP) Urinary cross-linked aminoterminal telopeptide of type I collagen (NTx) Serum cross-linked carpoxyterminal teleopeptide of type I collagen (ICTP) Urinary pyridinoline (Pyr) Urinary deoxypyridinoline (Dpyr)

Aminoacid, derived from the breakdown of collagen Products of osteoclastic proteolysis of collagen

Collagen cross-links holding collagen molecules together; measuring collagen degradation

In conclusion, adequate calcium intake, physical exercice, and other health measures are naturally of primary importance in preventing of osteoporosis in postmenopausal women. ERT/HRT is the first choice of the drugs available for this purpose. Other options, such as selective estrogen receptor modulators (SERMs), bisphosphonates or calcitonin, can be reserved for more complicated cases.


2.3 Hazards of estrogen replacement therapy 2.3.1 Breast cancer

ERT/HRT has many health benefits as discussed above, but its use involves some side effects and hazards, the risk of breast cancer being the most frightful of those. The recent meta-analysis of 51 epidemiological studies on 52 705 breast cancer patients and 108 411 control women showed that the relative risk (RR) of getting breast cancer was 1.31 for current users who had used ERT for 5 to 9 years and 1.58 for those who had used ERT longer than 15 years (Table 7)

Duration of use Never <1 1-4 5-9 > 15 year years years years

RR 1.00 0.99 1.08 1.31 1.24 1.58

10-14 years

Table 7. The risk of breast cancer in women who use or have used ERT/HRT (current users and users whose last use < 5 years before diagnosis) (according to Collaborative Group on Hormonal Factors in Breast Cancer 1997)

(Collaborative Group on Hormonal Factors in Breast Cancer 1997). The risk of breast cancer increased by 2.3% per year in current ERT/HRT users. However, this risk of breast cancer vanished within five years after the cessation of HRT (Collaborative Group on Hormonal Factors in Breast Cancer 1997). Finnish Cancer Registry data from 1997 indicate that 2.8% of 50-year old women will develop breast cancer over the next 10-year period, and the current use of ERT/HRT increases this proportion to 3.4%. (Table 8). The good news is that the use of ERT/HRT by women with a familial history of breast cancer or with reproductive risk factors will not result in more than simple addition of risk of breast cancer (Kenemans and Scheele 1997; Scheele et al. 1999).

Duration 2 years 5 years 10 years

Number of patients 1/1000 3/1000 7/1000

Table 8. Excess of patients diagnosed with breast cancer in relation to the duration of ERT/HRT use in Finland



The mechanisms by which estrogen increases the risk of breast cancer are not known (Theriault 1996). Moreover, the impact of the progestin component of HRT on the breast cancer risk is not clear. Previous data demonstrated that progestin did not affect the risk of breast cancer caused by estrogen only (Collaborative Group on Hormonal Factors in Breast Cancer 1997), but recent data from Sweden and the United States showed that progestin increased this risk by 14-40% (Persson et al. 1999; Schairer et al. 2000; Ross et al. 2000; Colditz and Rosner 2000). In interpreting the data on the risk of breast cancer in hormone users, we must keep in mind that there are no placebo controlled, randomized trials on this topic. Thus all the epidemiological data discussed above are prone to a number of confounding factors and biases. First, high socioeconomic status is an independent risk factor of breast cancer (McPherson et al. 1994), and women belonging to this group more likely use ERT/HRT (Matthews et al. 1996; Rdstrm et al. 1999). Second, ERT/HRT users undergo breast examinations and mammography more frequently than nonusers. This can lead to detection bias; ERT/HRT users are more likely to be diagnosed with breast cancer than nonusers. The use of estrogen may also favor the development of cancers with less malignant potential (Gapstur et al. 1999), which might explain the better prognosis of breast cancer in patients with ERT/HRT use than in those without ERT/HRT use (Jernstrm et al. 1999).

2.3.2 Endometrial cancer

Endometrial cancer is the third commonest cancer in women in Finland; its incidence was 14.3 in 100 000 women in 1997 (Finnish Cancer Registry 1997). Endogen and exogen estrogen exposure is one of the risk factors of endometrial cancer (Franceschi 1989, Grady et al. 1995). Estrogen promotes the formation of estrogen receptors and proliferation of endometrium whereas progesterone down-regulates these receptors and causes secretory changes. This explains why estrogen without the use of progestin leads to an increase in the risk of endometrial hyperplasia (Kurman et al. 1985). This can progress to endometrial cancer, albeit only in a minor part of patients (Grady et al. 1995, Westhoff et al. 2000).


The meta-analysis of 29 epidemiological studies showed a 2.3-fold increase in the risk of endometrial cancer during the use of unopposed estrogen (Grady et al. 1995). This risk was higher with a prolonged duration of estrogen use. Less than one year of use caused a 1.4-fold risk, whereas more than 10 years of use increased the risk to 9.5-fold. Therefore, progestin has to be added to ERT in non-hysterectomized women. Progestin can be administered sequentially or continuously. The sequential addition of progestin has to be long enough; an addition of 10 days or more per month resulted in a RR of 0.9 of endometrial cancer (Voigt et al. 1991) and the interval between progestin phases should be short enough (Pukkala et al 2001). Thus, in regard to endometrial cancer, estrogen replacement therapy can be safely used if progestin is added concomintantly.

2.3.3 Venous thromboembolism

The balance between coagulation and fibrinolysis system determines the risk of thrombosis in a given individual. The effect of estrogen on this balance is complex. Although ERT/ HRT causes a reduction in the concentration of fibrinogen in plasma and activates fibrinolysis (Gebara et al. 1995; Conrad et al. 1997), it increases the risk of venous thromboembolism (Daly et al. 1996, Jick et al. 1996, Grodstein et al. 1996, Prez Gutthnn et al. 1997, Hulley et al. 1998, Varas-Lorenzo et al. 1998). This risk appears to be most pronounced during the first months of use, and there is no clear dose-dependence of ERT/HRT in this regard (Oger and Scarabin 1999). Moreover, progestin addition does not modify this risk (Oger and Scarabin 1999), finding which was confirmed also in the HERS study. The risk of venous thromboembolism was 2.3 in 1000 woman-years in a placebo group and 6.2 in 1000 woman-years in a hormone treated group (Grady et al. 2000). Fractures of the hip or other parts of the lower extremity causing immobilisation, the presence of cancer, and hospitalization increased this risk whereas intake of aspirin or lipid lowering statins decreased it (Grady et al. 2000). Antiestrogens, such as tamoxifen and raloxifene, have also increased the risk of thromboembolism from 2 to 5-fold in randomized trials (Fisher et al. 1998, Cummings et al. 1999), and in this regard antiestrogens behave similarly to ERT.



Furthermore, in the study comparing tamoxifen and toremifene, the incidence of thromboembolic events was slightly more common in the tamoxifen group (5.9%) than in the toremifene group (3.5%), but the difference was not significant (Holli et al. 2000). However, the absolute number of thromboembolic events in these studies remains low (in the order of 5 in 1000 women per year), and they are only seldom fatal. Therefore the risk of thromboembolism is not a serious concern during the use of ERT/HRT.

2.4 Estrogen replacement therapy in women with previous breast cancer

Due to the increased incidence and better prognosis of breast cancer, the number of breast cancer survivors has substantially increased in all western countries during the last two decades (Garfinkel et al. 1994). The general consensus has been that these patients should not use ERT/HRT, because theoretically such use can increase the risk of recurrence (Marchant 1994; Colditz 1997; Consensus Statement 1998). However, also these women can suffer from invalidating menopausal symptoms and certainly become exposed to similar postmenopausal health consequences related to estrogen deprivation as healthy postmenopausal women. Therefore, it there is justified reason to ask if a categoric refusal of ERT/HRT from these women will do more harm than good. Some non-randomized and mainly retrospective studies have been conducted on the use of ERT in patients with a history of breast cancer (Wile et al 1993; Powles et al.1993; DiSaia et al 1993; Eden et al. 1995; Vassilopoulou-Sellin et al. 1995; Natrajan et al. 1999; Ursic c aj and Bebar 1999) (Ta -Vrs ble 9). No excess risk for the recurrence of breast cancer has been found in these studies. The number of study subjects has ranged from 25 to 90 and the mean duration of ERT/ HRT from 15 to 66 months. Naturally, the data are far too limited to show the final risk or safety of using ERT/HRT in these patients. It is obvious that we need randomized, preferably placebo-controlled trials to assess the safety of ERT/HRT in breast cancer survivors. Conducting such trials is problematic. First, the randomization of breast cancer patients to hormone or placebo treatment may be ethically questionable at least if a



Table 9. Observational studies on the use of hormone replacement therapy in breast cancer survivors


No. of patients

Mean duration of use (months, range) 35 (6-78) 15 (1-44) 27 (1-233) 18 (4-144) 31 (24-142) 66 (6-384) 28 (3-72)

Recurrences (% / women -year) 3 (4%) 2 (5%) 7 (4%) 6 (4%) 1 (1%) 3 (1%) 4 (8%)

Wile et al. 1993 Powles et al. 1993 DiSaia et al. 1993 Eden et al. 1995 VassilopuolouSellin et al.1995 Natrajan et al. 1999
Urs ic -Vrs c aj et al. 1999

25 35 77 90 43 50 21

patient has invalidating hot flushes. This obstacle was elegantly demonstrated in one randomized study where after a 6-month pilot phase, 76% of women randomized to ERT/ HRT group wanted to continue ERT/HRT, whereas 50% of women randomized to placebo group wanted to swift to ERT/HRT group (Marsden et al. 2000). Second, alleviation of hot flushes and/or the occurrence of bleeding and other consequences of ERT/HRT reveals the code of the regimen in almost all patients, and thus one goal of randomized placebo-controlled trial, blinding, is lost. And third, the prognosis of breast cancer is presently so good that hundred thousands of such patients would be needed for sufficient statistical power, if the end point of the trial is the recurrence of cancer. Sequential or continuous progestin is routinely added to ERT in non-hysterectomized women for preventing endometrial hyperplasia. Recent studies have shown that progestin may still increase the risk of breast cancer associated with estrogen (Persson et al. 1999; Schairer et al. 2000; Ross et al. 2000; Colditz and Rosner 2000). The reason for that is unclear but the fact that in the breast tissue on the contrary to the endometrium progesterone does not down-regulate estrogen and progesterone receptors may contribute to this possible adverse effect (Hargreaves et al. 1998). In fertile age, the proliferation of breast tissue epithelial cells is higher



during the luteal phase which is characterised by high serum progesterone levels (Sdergvist 1998). It has also been shown that in postmenopausal women estrogen combined to continuous medroxyprogesterone acetate causes significantly higher proliferation rate in normal breast tissue than estrogen alone does (Hofseth et al. 1999). However, we still need more clinical data to answer the questions whether estrogen alone is more safe than estrogen + progestin, or whether there is any difference between sequential and continuous progestin in regard to the risk of breast cancer recurrence.




3.1 Pharmacological and biological characteristics

Tamoxifen and toremifene are non-steroidal triphenylethylene derivatives with antiestrogenic properties used in the treatment of breast cancer (Figure 5). Tamoxifen is the citrate salt of the trans isomer of a triphenylethylene compound, and toremifene is a chlorinated derivative of tamoxifen (Kangas 1990). They are almost completely absorbed after oral intake, and bioavailability is nearly 100%. Maximal plasma concentrations of parent compound and metabolites are reached within 4-7 hours (Buckley and Goa 1989; Wiseman and Goa 1997). Tamoxifen and toremifene are almost completly (99%) bound to plasma proteins, mainly to albumin. They are metabolized in the liver, and the majority of metabolites are excreted in the feces (Buckley and Goa 1989; Wiseman and Goa 1997).

OCH 2 CH 2 N

CH 3 CH 3


CH 3 CH 3

C=C CH 2 CH3 Tamoxifen

C=C CH2 CH 2 CI Toremifene

Figure 5. The chemical structures of tamoxifen and toremifene



Tamoxifen and toremifene which are also called selective estrogen receptor modulators (SERMs), possess both an estrogenic and antiestrogenic activity, depending upon the dose or target organ (Robinson and Jordan 1989; Wiseman and Goa 1997). These drugs exert an antitumor activity which is mediated principally through a specific and competitive inhibition of the binding of estrogen to estrogen receptor (Buckley and Goa 1989; Wiseman and Goa 1997). This activity is very similar for tamoxifen and toremifene (Kangas 1990). They both prevent the growth of estrogen sensitive cells of human breast cancer in vitro (Coezy et al 1982; Grenman et al 1991), but have no effect on the growth of estrogen receptor negative cell lines (Robinson and Jordan 1989). However, blocking of estrogen receptors is not the only mechanism of antitumor action, but antiestrogens may operate also through different growth factors, such as alfa and beta transforming growth factor (Dickson and Lippman 1987; Knabbe et al. 1987; Colletta et al. 1994) and epidermal growth factor (Freiss et al.1990). Furthermore, antiestrogens inhibit the activity of protein kinase C, which is a mediator of some tumor promoting factors (OBrian et al. 1985).

3.2 Clinical use of tamoxifen and toremifene

Tamoxifen was introduced more than 20 years ago for the palliative treatment of advanced breast cancer in postmenopausal women (Buckley and Goa 1989). Since then, tamoxifen has become the treatment of choice for patients at all stages of estrogen receptor (ER) positive breast cancer (Early Breast Cancer Trialists Collaborative Group 1998). Fifty-five randomized clinical studies on 37 000 patients with local ER positive breast cancer have shown that 5 years use of adjuvant tamoxifen reduces the recurrence rate by 47% and the mortality rate by 26% during the 10-year follow-up time (Early Breast Cancer Trialists Collaborative Group 1998). Although toremifene has been developed later, it competes for favor in the treatment of advanced breast cancer. Five studies have compared the efficacy of tamoxifen (20, 30, or 40 mg/day) and that of toremifene (40 or 60 mg/day) in postmenopausal patients with metastatic breast cancer (Pyrhnen et al. 1999). The overall response rates were 25.3% and 24.0% for tamoxifen and toremifene, respectively



(Pyrhnen et al 1999). A complete response was seen in 5.5% of patients treated with tamoxifen and in 7.0% of the patients treated with toremifene (Pyrhnen et al. 1999). Treatment failure occurred equally soon in the tamoxifen (5.5 months) compared to the toremifene group (4.9 months), (Pyrhnen et al 1999). These data suggest that tamoxifen 2040 mg/day and toremifene 60 mg are equally effective in the treatment of metastatic breast cancer (Homesley et al. 1993; Pyrhnen et al. 1997; Pyrhnen et al. 1999). However, more data on whether tamoxifen (20 mg/day) and toremifene (40 mg/day) are equally effective in the adjuvant treatment of breast cancer are beginning to appear and the first data on 899 patients with node-positive breast cancer have been recently published (Holli et al. 2000). According to this study breast cancer recurrence rate was 26.1% in the tamoxifen group and 23.1% in the toremifene group (p=0.31), and also the mean time to breast cancer recurrence and overall survival time was similar in both groups (Holli et al. 2000). Tamoxifen prevents also the occurrence of new cancers in the contralateral breast (Early Breast Cancer Trialists Collaborative Group 1992, 1998). This finding, combined with the proven efficacy of tamoxifen in the treatment of breast cancer (Hortobagyi 1998) has led to the rationale to use antiestrogens for breast cancer prevention (Chlebowski et al. 1993). Indeed, tamoxifen decreased the incidence of invasive breast cancer by 49% in one large, randomized, placebo-controlled study (Fisher et al. 1998) but not in two other studies with smaller number of participants (Powles et al. 1998, Veronesi et al. 1998). The newer SERMs, such as e.g. raloxifene, appear also effective in the prevention of breast cancer, because raloxifene decreased the risk of breast cancer by 76% during the three years of use in older postmenopausal women with osteoporosis (Cummings et al. 1999). All this implies that antiestrogenic compounds may have a place also in the prevention of breast cancer in future.

3.3 Gynecological consequenses of antiestrogens

Tamoxifen and toremifene have both antiestrogenic and estrogenic activity, which are briefly summarized in table 10. Therefore, the whole impact of their intake on woman body and life is complex. They cause and aggravate vasomotor



symptoms in postmenopausal women, although they reduce blood levels of FSH and LH (Jordan et al. 1987; Ellmn et al. 2000). Tamoxifen causes also maturation of the vaginal epithelial cells and increases their karyopyknotic index (Lahti et al. 1994). Both tamoxifen and toremifene cause the proliferation of the endometrium and increase the risk of endometrial polyps and hyperplasia (Tomas et al. 1995), and these changes are dependent on the duration of tamoxifen use (Cohen et al. 1999). The worst complication of tamoxifen use can be endometrial cancer. Tamoxifen treatment has been associated with a 4.1 to 7.5-fold relative risk of developing endometrial carcinoma in two extensive studies (Fisher et al. 1994; Rutqvist et al. 1995). These data were confirmed in a meta-analysis on 55 adjuvant tamoxifen trials with altogether 37 000 breast cancer patients; the risk of endometrial cancer was doubled by 1 or 2 years use of tamoxifen, and approximately 4-fold after the use of 5 years of tamoxifen (Early Breast Cancer Trialists Collaborative Group 1998). There are also some data which link tamoxifen to the formation of ovarian cysts and endometriomas, but this evidence is not so strong that it could be regarded as well established so far (Neven et al. 1993; Cohen 1993).

Table 10. Antiestrogenic and estrogenic effects of tamoxifen and toremifene in postmenopausal women

Antiestrogenic Gonadotropins Gynecologic organs Vasomotor symptoms

Estrogenic FSH LH Karyopycnotic index Endometrial hyperplasia Endometrial polyps Endometrial cancer

Breast tissue Cardiovascular system

ER-positive breast cancer tissue proliferation Risk of myocardial infarction Total cholesterol LDL cholesterol HDL cholesterol /Lipoprotein (a) Vasodilatation Risk of venous thrombosis Rate of bone loss




In view of rather similar pharmacology and clinical profile of tamoxifen and toremifene, it is likely that toremifene causes similar estrogenic effects in the female body as does tamoxifen, although some authors have questioned it (Wiseman and Goa 1997). In some animal studies toremifene has caused fewer tumours in the liver than tamoxifen (Hirsimki et al 1993), reflecting perhaps a weaker estrogenic effect. It is not known if the estrogenic activities of tamoxifen and toremifene are similar in human, and therefore more clinical comparative data are needed.

3.4 Antiestrogens and cardiovascular organs

Cardiovascular system seems to be a target tissue for estrogen and an important issue is to clear up the role of antiestrogen in the cardiovascular organs. In a Scottish adjuvant trial tamoxifen reduced the occurrence of myocardial infarction by 50% in 5 years (McDonald et al. 1995). This benefit may be mediated in part through the falls in total cholesterol and low-density lipoprotein (LDL) cholesterol (Love et al. 1994; Grey et al. 1995). Also toremifene reduces total cholesterol and LDL cholesterol levels (Gylling et al. 1995; Saarto et al. 1996). As regards the cardioprotective high-density lipoprotein (HDL) cholesterol, tamoxifen reduced it by 5% whereas toremifene increased it by 14% (Saarto et al. 1996). Both tamoxifen and toremifene decreased the level of lipoprotein (a) (Saarto et al.1996) which is a cholesterol-independent risk factor of ischemic heart disease. Tamoxifen also reduced the level of fibrinogen (Love et al. 1994) and that of homocysteine (Anker et al. 1995), which effects may also contribute to cardiovascular protection. All these effects may be related to the estrogenic activity of tamoxifen and toremifene. In contrast no data exist so far on whether antiestrogens could trigger a direct dilatation in the arteries or affect the endothelial factors, such as prostaglandins, NO, or ET-1.



3.5 Antiestrogens and bone

Because antiestrogens are given often for 5 or more years in postmenopausal women as an adjuvant therapy of breast cancer, it is important to know also their effect on bone integrity which, on the other hand, is already affected by postmenopausal hypoestrogenism. Tamoxifen has been shown to increase bone mineral density in postmenopausal women by 1-2% per year (Grey et al. 1995; Powles et al. 1996; Chang et al. 1996). However, so far we do not have any epidemiological data on the risk of hip and other fractures during tamoxifen use. Tamoxifen has been shown to reduce bone resorption as measured by the output of hydroxyproline (Ward et al.1993), pyridinoline, and deoxypyridinoline (Kenny et al. 1995), and also bone formation measured by bone-specific alkaline phosphatase and osteocalcin (Kenny et al. 1995). The data on the effect of toremifene on bone are scanty. Yet in one comparison tamoxifen (20 mg/day) and toremifene (60 mg/day) used for two years did not decrease BMD, whereas the addition of bisphosphonate clodronate to antiestrogens increased BMD by 2-4% (Saarto et al. 1997).




The objectives of the present study were to 1. evaluate the effects and safety of estrogen replacement in postmenopausal women with a history of breast cancer (publication I) assess and compare the gynecological consequences of tamoxifen and toremifene (publication II) explore some biological mechanisms dealing preferably with endothelial cells during the use of tamoxifen and toremifene (publications III and IV) compare the effects of tamoxifen and toremifene on bone metabolism and density (publications V and VI)

2. 3.





The study population which was collected with the permission of the local ethics committee, consisted of two patient groups: first, 131 women with a history breast cancer seeking a relief for menopausal symptoms (=ERT group) and second, 167 women who started either tamoxifen or toremifene as an adjuvant treatment for stage II-III breast cancer (=Tam/Tor group) (Table 11). All patients were postmenopausal as seen from the passage of more than 6 months from last menstruation and/or from serum FSH level being higher than 30 IU/L.
Table 11. Patient series in publications I-VI

I No. of patients (treatment) Age (years, meanSD) BMI (kg/m2, meanSD) Follow-up time Main parameters studied 131 (88 ERT, 43 control) 555.8 24.22.8 2.5 yrs Endometrial safety, relief of climacteric symptoms, recurrences

II 167 (84 tam, 83 tor) 62.88.4 27.20.3 2.3 yrs Recurrences, climacteric symptoms, endometrial findings

III 38 (17 tam, 19 tor) 60.38.4 26.24.4 6 months

IV 44 (25 tam, 19 tor) 61.77.1 25.93.0 12 months

V 30 (16 tam, 14 tor) 59.78.6 25.93.8 12 months BMD hydroxyproline ICTP, NTx, bone alkaline phosphatase, osteocalcin PINP, PICP

VI 30 (15 tam, 15 tor) 61.52.6 26.34.0 12 months BMD, pyridinoline deoxypyridinoline

Urinary Plasma prostacyclin endothelin-I and and nitrate thromboxane

(ERT=estrogen replacement therapy, tam=tamoxifen, tor=toremifene, BMD=bone mineral density, ICTP=cross-linked carboxyterminal telopeptide of type I collagen, NTx=cross-linked aminoterminal telopeptide of type I collagen, PINP=aminoterminal propeptide of type I procollagen, PICP=carboxyterminal propeptide of type I procollagen)



The patients in the ERT group were submitted by oncologists, surgeons, general practitioners, or gynecologists for gynecological consultation because of incapacitating climacteric symptoms (Table 11, Table 12). They had been operated on for a mean of 4.2 years (range 1 month to 20 years) before, when still 54 women (41%) had been at premenopausal age. Sixty-two patients (47%) had experienced radical mastectomy and 69 patients a breast-sparing operation. Axillary nodes were dissected in all except 6 women. One hundred patients had received postoperative radiotherapy

Table 12. Clinical characteristics of breast cancer patients scheduled to start estrogen alone (ERT) or combined with progestin (HRT) or who were followed as controls

Variable Number of patients Age at diagnosis (meanSD) Classification of tumor spread Size Ductal in situ < 2 cm 2-5 cm < 5 cm Axillary nodes N0 N1 Not analyzed Estrogen receptors Detectable Undetectable Not analyzed Progesterone receptors Detectable Undetectable Not analyzed Histologic grade Ductal in situ Gr I Gr II Gr III Not known

Women with ERT/HRT 88 496.8

Controls 43 487.4



NS 3 (3.4%) 67 (76.1%) 17 (19.3%) 1 (1.1%) 72 (81.8%) 10 (11.4%) 6 (6.8%) 57 (64.8%) 15 (17.0%) 16 (23.9%) 54 (61.4%) 13 (14.8%) 21 (23.9%) 3 (3.4%) 37 (42.0%) 28 (31.8%) 12 (13.6%) 8 (9.1%) 1 (2.3%) 29 (67.4%) 11 (25.6%) 2 (4.7%) <0.05 30 (69.8%) 13 (30.2%) 0 (0%) NS 29 (67.4%) 9 (20.9%) 5 (11.6%) NS 30 (69.8%) 7 (16.3%) 6 (13.9%) NS 1 (2.3%) 12 (27.9%) 15 (34.9%) 7 (16.3%) 8 (18.6%)



and 19 patients adjuvant chemotherapy. Five patients had used antiestrogens but stopped it 3 to 5 years before the initiation of the trial. Forty-three women were hysterectomised. Before the start of ERT/HRT, all patients underwent gynecological examinations (e.g. mammography, pelvic examination, Pap smear collection), and the benefits and risks of ERT/HRT were explained thoroughly orally and in written form, and all patients gave their written consent. The patients of Tam/Tor group were referred to gynecological follow-up after they had been randomized at the department of oncology to receive either tamoxifen (20 mg/ day) or toremifene (40 mg/day) for three years (Table 11). They were participants in the Finnish Breast Cancer Group Adjuvant Toremifene versus Tamoxifen Trial, and randomization was done by Finnish Cancer Register. Fifty-six patients had experienced conservative surgery and 111 patients mastectomy 6 to 8 weeks before the start of antiestrogens, and axillary nodes were dissected in all women. In addition, all women had received postoperative local radiotherapy. Thirty-eight patients were hysterectomized, 53 used antihypertensive drugs, and 27 smoked.




In the study I, 88 of 131 women (67%) admitted because of climacteric symptoms finally started the use of ERT/HRT (Figure 6). Hysterectomized patients (n=33) used 2 mg of oral (Progynova, Schering, Berlin, Germany) (n=31) or 1.5 mg transdermal estrogen (Estrogel, Leiras, Turku, Finland) (n=3). Non-hysterectomized patients received, in addition of oral (44 patients) or transdermal estrogen (10 patients), sequential medroxyprogesterone acetate (10 mg/day, for 10 days in each month, Provera, Pharmacia & Upjohn, Kalamazoo, Michigan) (Figure 6).

Visit 1:

Recruitment (n=131) - clinical examination, FSH measurement - oral and written information Patients decision on the start of ERT/HRT

Visit 2:

Yes (n=88) - oral estrogen (n=31) - transdermal estrogen (n=3) - oral estrogen+sequential oral progestin (n=44) - transdermal estrogen+sequential oral progestin (n= 10)

No (n=43) Reasons for refusal - premenopausal (n=6) - only mild climacteric symptoms, no risks of cardiovascular diseases or osteoporosis (n=17) - fear of recurrence (n=20)

Visits 3 -> :

Follow-up visits - measurement of weight, height, blood pressure - pelvic examination - Pap smear collection - ultrasound examination of uterus, endometrium and ovaries - rating of menopausal symptoms

Figure 6. Design of the trial to assess the value of hormone replacement (ERT/HRT) in patients with breast cancer (Study I)



In the study II, 84 patients used tamoxifen (20 mg/day, Tadex, Orion, Turku, Finland) and 83 used toremifene (40 mg/day, Fareston, Orion, Turku, Finland). The follow-up times of both study group are presented in Table 13.

Table 13. Follow-up times completed by patients using either estrogen or estrogen + sequential progestin (ERT/HRT), or tamoxifen (Tam) or toremifene (Tor)

Time < 1 year 1-2 years 2-3 years 3 years or more

ERT/HRT (n=88) 12 20 20 36

Tam (n=84) 23 19 42

Tor (n=83) 14 17 52

3.1 Clinical examinations and follow-up
Clinical examinations including measurements of weight, height, and blood pressure, as well as pelvic examination and Pap smear collection were performed in all patients before the start of hormone treatment. The same examinations were repeated 6 and 12 months later and then annually. Blood and urinary samples were collected after the overnight fast before the start of hormone regimens and at 6 and 12 months of treatment. Serum, plasma, and urinary samples were stored at -22C until assayed. Before the sampling, patients were instructed to avoid nitrate-rich (meat, fish, green vegetables, malt beverages and wine) and gelatin containing (meat, fish, poultry, yoghurt and pudding) food for 48 hours. The patients recorded menopausal symptoms (hot flushes, sweating, sleep disturbance, nervousness, depression, vertigo, fatigue, arthralgia, headache, tachycardia and vaginal dryness) on the modified Kupperman scale at each visit



(Wiklund et al. 1992). The severity of each symptom was graded form 0 to 3, and the severity score for hot flushes was multiplied by 4 and that for sweating, sleep disturbance, and nervousness by 2, and the scores were summed up. The patients were followed at the oncological or surgical departments in regard to the status of breast cancer. The breasts were carefully palpated at each visit, and mammography was performed annually. In addition, the serum level of CA 15-3, reflecting a possible growth or recurrence of breast cancer, was assessed at each visit.

3.2 Transvaginal sonography and endometrial sampling

Transvaginal sonography (Aloka SSD 500) was performed at each visit to assess gynecological organs including the endometrial thickness. Sonographic examination was complemented by saline sonohysterography (Widrich et al. 1996), if endometrial polyp was suspected (study I and II) or endometrial thickness exceeded 8 mm (study II). In patients with endometrial polyp diagnosed by saline sonohysterography, polyp was removed with electroresector through hysteroscopy. To assess the blood flow in the uterine artery, pulsatility index (PI) was measured by using doppler ultrasound (Hitachi 515A, Tokyo, Japan, 6.5 MHz endovaginal transducer) before the start of medication and at 6 and 12 months of trial in 30 patients using either tamoxifen (n=15) or toremifene (n=15) (Study II). Endometrial biopsy was collected with Pipelle endometrial aspirator (Unimar, Wilmington, CN) (Chambers and Chambers, 1992) from patients with intact uterus, in the study I if endometrial thickness exceeded 5 mm at entry or 10 mm on a follow-up visit, or if there was abnormal uterine bleeding before recruitment or during the trial. Endometrial biopsy was collected at each visit in the trial II. The endometrium was classified as atrophic (small simple tubular glands lined by cuboidal epithelium, the nuclei centrally located, no mitoses) or mildly proliferative (small tubular glands lined by cuboidal to columnar epithelium, ovoid nuclei basally or centrally located, some mitoses). The same experienced pathologist (Dr. T. Wahlstm) studied all histological samples.



3.3 Laboratory measurments

The serum concentration of FSH was measured by time-resolved fluoroimmunoassay (DELFIA; Wallac, Turku, Finland) according to the instructions of the manufacturer. Tumor marker CA 15-3 was measured by immunoradiometric assay (OHanlon et al. 1995). Prostacyclin and thromboxane output were assessed by their stable urinary metabolites 2,3-dinor-6-keto prostaglandin F1 and 2,3-dinor-thromboxane B2, respectively, by radioimmunoassays after solid-phase extraction and purification of the sample by high-performance liquid chromatography (Ylikorkala et al. 1986, Tulppala et al. 1991). To avoid the effect of differences in the dilution of urine, excretions were expressed against creatinine, which was measured by a routine laboratory method. The capacity of platelets to produce TxA2 was assayed by measuring the serum concentration of TxB2, a metabolite of TxA2, by specific radioimmunoassay (Viinikka and Ylikorkala 1980). Before measurement, blood samples were allowed to clot at +37C for 30 minutes and serum was then separated. The intra-assay coefficient of variation in measurements of prostacyclin and thromboxane metabolites was less than 8%, and the interassay coefficient of variation was between 10.4-14.1%. Plasma endothelin-1 was analyzed by radioimmunoassay (Ylikorkala et al. 1995). Acidified 3 ml plasma samples were extracted with Sep-Pak-Vac tC18 cartidges. After washes with 0.1% trifluoroacetetic acid (TFA) and 45% methanol-0.1% TFA, ET-1 was eluted with 90% methanol-0.1% TFA. The evaporated samples were dissolved in 0.45 ml of assay buffer (50 mmol/l phosphate buffer, pH 7.4, 150 mmol/l NaCl 1% bovine serum albumin). The radioimmunoassay was performed in tubes coated first with goat anti-rabbit immunoglobulin and then with diluted ET-1 antibody raised in rabbits. The samples were incubated for 24 hours and then with 10000 dpm of 125I-ET-1 for 48 hours. The bound radioactivity remaining in tubes after washing was counted, and the results calculated on the basis of a dilution series of authentic ET-1. The intra-assay coefficient of variation for measurement was 5.7%. Nitric oxide production was evaluated on the basis of the concentration of nitrate+nitrite (NOx). Nitrate was reduced to nitrite by nitrate reductase, the sample deproteinized with ZnSO4, and the concentration of nitrite was measured by the



spectrophotometrically based Griess reaction (Ylikorkala et al. 1998). The intra-assay coefficient of variation of NOx was 1.7% and 2.2% in the lower and higher concentration range, respectively. Bone resorption markers Urinary hydroxyproline (HOP) was measured with high-performance liquid chromatography (Turpeinen and Pomoell 1985). The intra-assay coefficient of variation of this method was 8.9%. Urinary cross-linked aminoterminal telopeptide of type I collagen (NTx) was measured with an enzymelinked immunosorbent assay using monoclonal antibody directed against th N-telopeptide of type I collagen isolated from human urine (Hanson et al. 1992). The intra-assay variation of this method was 6.2%. To avoid errors caused by differences in urine dilution, both HOP and NTx data were expressed against creatinine assessed by a routine laboratory method. Serum cross-linked carboxyterminal telopeptide of type I collagen (ICTP) was determined by radioimmunoassay (Telopeptide ICTP, Orion Diagnostica, Espoo, Finland) (Risteli et al. 1993), and the intra-assay coefficient of variation for this measurement ranged from 3% to 9%. Urinary excretion of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) was measured by ion-pair reversed-phase high performance liquid chromatoraphy (RP-HPLC) (Garnero et al. 1994). To avoid any effect of different dilutions of urine, Pyr and Dpyr data are expressed against grams of creatinine. The intra-assay coefficient of variation is less than 10% for Pyr and less than 15% for Dpyr. Bone formation markers Bone-specific alkaline phosphatase in serum was measured by immunoradiometric assay (Tandem-R Ostase, Hybritec Europe, Liege, Belgium) (Epstein 1988). The intra-assay variation in this method was below 7%. Serum osteocalcin was measured by an immunoradiometric assay using antibodies against human osteocalcin (Osteocalcin FEIA, Farmacia CAP System, Uppsala, Sweden) (Epstein 1988). The intra-assay coefficient of variation was 7%. Serum aminoterminal (PINP) and carboxyterminal (PICP) propeptide of type I procollagen were determined by RIA (Procollagen Intact PINP RIA Kit, Procollagen PICP RIA Kit, Orion Diagnostica) (Melkko



et al. 1996). The intra-assay coefficient of variation of the measurement of PINP was 5-8% and for measurement of PICP 3%.

3.4 Bone density measurement

Bone mineral density (BMD) in the lumbar spine (LI-IV) and in different sites of the proximal femur was measured by dual-energy x-ray absorptiometry (DEXA) (Hologic QDR1000, Waltham, MA) (De Boer et al. 1994). Data were given as density against area (g/cm2). The intra-assay coefficient of variation in our department was 0.5% in lumbar spine and 1% in femoral neck.

3.5 Statistical analyses

All data are expressed as a mean standard deviation (SD) (Studies I-III ) or standard error (SE) (Studies IV and VI). The significance of differences between the groups with numerical variables was determined with paired and unpaired Students t-test. When repeated measures were obtained, the two-way analysis of variance (ANOVA) was used. The comparison of two proportions in case of paired samples was performed by McNemars test in study II. Chi-square test was used to compare the distribution of categorical variables of two independent samples (Studies I and II). Pearson coefficient (study IV) or Spearman nonparametric correlation analysis (study V, VI) were used for the determination of correlation between the two variables.





The mean follow-up time with estrogen regimen was 2.51.5 years (range 1 month to 5.2 years), which corresponds to 216 woman-years. Forty-three women were followed as control population without estrogen regimen for a mean of 2.51.3 years (range 1 month to 4.6 years) (= 111 woman-years). Ten of the 88 patients discontinued estrogen treatment within the 12 to 39 months; four because of lack of climacteric symptoms and six because of recurrence of disease or new breast cancer. One woman with contralateral breast cancer wanted to continue ERT/HRT. Five patients needed to increase the dose of oral estradiol to 3 mg between 6 and 12 months for sufficient alleviation of symptoms, and three women reduced the dose from 2 to 1 mg between 1 and 4 months because of breast tenderness. Three women with sequential regimen experienced PMSlike symptoms during progestin phase, and these women subsequently took progestin courses at two-month intervals. ERT/HRT abolished or alleviated vasomotor or other climacteric symptoms (Figure 7). Forty-seven patients with intact uterus had regular withdrawal bleeding, seven patients had spotting, and seven patients did not bleed al all. None discontinued the treatment because of adverse effects. Two women had transient simple endometrial hyperplasia after one and two years of the regimen. Five of 88 patients using ERT/HRT experienced the recurrence of the cancer after a mean of 19 months (range from 12 to 36 months) use of ERT/HRT, and two developed contralateral breast cancer after 14 and 24 months use of ERT/ HRT, respectively. All the recurrences or contralateral breast cancers were detected as a consequence of clinical symptoms, Ca 15-3 levels was of no help to predict these cases.

In control population, four women had a relapse of cancer and one woman got contralateral breast cancer. The recurrence rates (%/woman-year) in ERT-users and non-users were 3.0% and 4.0%, respectively.

40 35
Kupperman index

30 25 20 15 10 5 0 Before At 6 months At 12 months a a

Figure 7. Kupperman indexes (mean+SD) before and at 6 and 12 months of estrogen replacement in 76 patients with a history of breast cancer a) p<0.0001 compared to initial




A total of 167 patients completed a mean of 2.30.8 years of follow-up. Thirty-seven patients discontinued the follow-up, 17 (11 in the tamoxifen group, six in the toremifene group) because of the failure to report at research center according to the protocol, five patients (three in the tamoxifen group, two in the toremifene group) because of adverse effects, and 15 patients (seven in the tamoxifen group, eight in the toremifene group) because of recurrence of breast cancer. Vasomotor symptoms and vaginal irritation were the most common adverse events during antiestrogen use, and in this regard tamoxifen and toremifene did not differ from each other. (Figure 8). Vaginal bleeding occurred in 13 patients (five in the tamoxifen group, eight in the toremifene group). In these patients, endometrial polyps were detected in four patients, endometrial proliferation in eight patients, and one patient had atrophic endometrium. Small uterine fibroids were detected in 28 patients, but neither tamoxifen nor toremifene caused any growth of these tumors or appearance of new fibroids. Simple ovarian cysts were detected in four women starting toremifene, but they

% 70 60 50 40 30 20 10 0 Before At one year Before At one year

Vasomotor symptoms

Vaginal symptoms

Figure 8. The rate of vasomotor and vaginal symptoms before and during first year use of tamoxifen (grey bars) or toremifene (white bars) in 167 patients with a history of breast cancer 50

disappeared spontaneously during the first 6 months. One ovarian thecoma diagnosed after one-year use of tamoxifen was removed laparoscopically. Before the start of antiestrogens, five patients suffered from symptoms of partially prolapsed uterus, and vaginal hysterectomy was performed in these patients during the trial. Pap smears showed no premalignant lesions before or during the trial in any patient.

2.1 Endometrial findings (Study II)

Endometrial thickness increased significantly during both regimens already during the first six months, and no further thickening was seen after one year (Figure 9). Before the start of antiestrogen regimen, an adequate endometrial sample was obtained in 94 of 129 (73%) patients with intact uterus. The sampling did not succeed in 25 women (19%) due to cervical stenosis and in 10 women (8%) due to intolerable pain. Endometrium was atrophic in 71 patients, whereas 19 patients showed proliferative endometrium; 16 of these had used estrogen replacement before the diagnosis of breast cancer. Four polyps (1 in tamoxifen group, 3 in toremifene group) were detected before the start of antiestrogen regimen.

Endometrial thickness (mm)

9 8 7 6 5 4 3 2 Before

a a

b a a

a a

a a

At 6 months

12 months

24 months

36 months

Figure 9. Endometrial thickness (mean+SD) in breast cancer patients using either tamoxifen (grey bars) or toremifene (white bars) a) p<0.001 compared to initial b) p<0.05 between 6 and 12 months


% 80 70 60

c b a b c

50 40 30 20 10 0

% 80 70 60

a c b c

50 40 30 20 10 0

% 80 70 60 50

40 30 20 10 0 Before At 6 At 12 At 24 At 36 months c c

Figure 10. Endometrial findings during the first 3 years use of tamoxifen (grey bars) or toremifene (white bars). In addition, one patient had endometrial carcinoma at one year of toremifene use and one patient had endometrial breast cancer metastasis at two years of tamoxifen use. a) p<0.001 b) p<0.005 c) p<0.05 compared to initial 52

During the control visits, endometrial sampling succeeded in 81% of patients. Proliferative endometrium was seen in 46.8% of samples in tamoxifen users and in 32.2% of samples in toremifene users (p<0.0001 between groups) (Figure 10). From the 24 polyps diagnosed during the antiestrogen regimen, 17 existed in tamoxifen users and seven in toremifene users (p<0.05). All polyps detected before and during the antiestrogen regimen were removed hysteroscopically. One patient on toremifene developed endometrial carcinoma at 12 months and she also had a metastasis of breast cancer in her ovary. One patient got breast cancer metastasis on her endometrium after 24 months of tamoxifen use.

2.2 Vascular effects 2.2.1 Uterine artery resistance (Study II)

Tamoxifen did not have any effect on uterine artery resistance, but toremifene reduced it significantly (p<0.05) by 12 months. The changes in PI or endometrial thickness did not correlate with each other in the whole series or in either subgroup.

2.2.2 Prostacyclin and thromboxane

Neither tamoxifen (n=15) nor toremifene (n=19) caused any change in the production of prostacyclin or thromboxane A2 (Figure 11). Before the start of the antiestrogen regimen, patients using antihypertensive drugs had lower urinary prostacyclin metabolite (dinor-6-keto) level (18.56.1 vs 35.518.5 ng/mmol, p<0.05) and also lower platelet capacity to produce thromboxane A2 (62.667.8 vs 134.675.6 ng/mL, p<0.05) than did the normotensive patients.



60 50
ng/mmol creatinine

40 30 20 10 0 Before At 6 months Before At 6 monts Dinor-6-keto Dinor-TxB2

Figure 11. Urinary excretion of the metabolites of prostacycylin (2,3-dinor-6ketoprostaglandin F1, dinor-6-keto) and thromboxane A2 (2,3dinor-thromboxane, dinor-TxB2) before and at 6 months of regimen with tamoxifen (n=17, grey bars) or toremifene (n=21, white bars)

2.2.3 Endothelin-1 and nitrite/nitrate (Study IV)

Toremifene reduced the level of endothelin-1 (ET-1) by 12.9% at 6 months (p<0.01) and a similar fall was also seen at 12 months, whereas tamoxifen did not cause any change in ET1 level (Figure 12). The changes in ET-1 at 12 months during the antiestrogen regimen were dependent on baseline ET-1 level (r=0.49, p=0.0008). Antiestrogens failed to affect the levels of plasma nitrite/nitrate, but the ratio between nitrite/ nitrate and ET-1 rose by 31.6% (p<0.05) at 6 months and by 35.6% (p<0.05) at 12 months of the use of antiestrogens.
5 4,5
Endothelin-1 (ng/L)

4 3,5 3 2,5 2 1,5 Before At 6 months At 12 months a b

Figure 12. Plasma levels of endothelin-1 before and at 6 and at 12 months of treatment with tamoxifen (n=25, grey bars) or toremifene (n=19, white bars) a) p<0.01 b) p=0.06 compared to initial 54

2.3 Bone (Studies V and VI)

Bone resorption Both tamoxifen and toremifene reduced urinary output of NTx at 6 and 12 months (Figure 13). Urinary Pyr and Dpyr decreased significantly at 6 months during both regimens, but after 12 months of treatment Pyr and Dpyr were significantly decreased only in women using tamoxifen (Figure 13). No significant changes were seen in the other variables for bone resorption.
10 0

Change in NTx (%)

-10 -20 -30 -40 b -50 -60 10 0 a a b

Change in Pyr (%)

-10 -20 -30 -40 -50 10 0 a b a

Change in Dpyr (%)

-10 -20 -30 a -40 -50 a At 6 months a At 12 months

Figure 13. Changes in bone resorption markers during tamoxifen (grey bars) or toremifene (white bars) use, a)p<0.01, b)p<0.05 in patients with breast cancer


Bone formation Tamoxifen caused a significant fall in osteocalcin, PINP, and PICP, whereas toremifene did not cause any change in these (Figure 14). Neither tamoxifen nor toremifene caused any changes in bone-specific alkaline phosphatase or ICTP.
150 Change in Osteocalcin (%) 125 100 75 50 25 0 -25 -50 -75 40 30 Change in PINP (%) 20 10 0 -10 -20 -30 -40 -50 20 15 Change in PICP (%) 10 5 0 -5 -10 -15 -20 -25 -30 At 6 months b At 12 months a b a

Figure 14. Changes in bone formation markers during tamoxifen (grey bars) or toremifene (white bars) regimen a) p<0.01 b) p<0.05 56

Bone mineral density Tamoxifen was accompanied by increases in BMD in the lumbar spine (0.4-2.0%), in femoral neck (0.7-1.0%), and in Wards triangle (2.8-5.0%). Toremifene did not cause any change in BMD or tended to slightly decrease it (Figure 15).

a 8 6
Change in BMD (%)

4 2 0 -2 -4

Lumbar spine

Femoral neck

Throchanteric region

Wards triangle

Figure 15. Changes in BMD during one year with tamoxifen (grey bars) or toremifene (white bars) in 30 patients with a history of breast cancer (Study V) a) p<0.05 compared to initial



Correlation between changes in bone biochemistry and in BMD The fall in NTx predicted most accurately the change in BMD; the falls in urinary NTx at 6 months correlated significantly with changes in the lumbar spine BMD (r=-0.58, p<0.001) in the whole patient series (Figure 16).

60 40
Change in NTx

20 0 -20 -40 -60 -80 -100 -,06 -,04 -,02 0 ,02

r= 0.58 p<0.001




Change in BMD
Figure 16. Correlation between the changes in cross-linked aminoterminal telopeptide of type I collagen (NTx) (at 6 months) and in bone mineral density (BMD) (at 1 year) in 30 patients with a history of breast cancer and with current use of antiestrogens




One in ten Finnish women will be diagnosed with breast cancer during her lifetime (Finnish Cancer Registry 1997). Nowadays breast cancer is diagnosed in an earlier phase perhaps as a consequence of mammographic screening programs (Garfinkel et al. 1994; Garne et al. 1997; UK Trial of Early Detection of Breast Cancer Group 2000), and also the treatment of breast cancer has improved (Early Breast Cancer Trialists Collaborative Group 1992, 2000). Therefore, in spite of the 100% rise in incidence from the beginning of 1970ies, the absolute breast cancer mortality has not increased (Finnish Cancer Registry 1997, Statistics Centrum of Finland; Peto et al. 2000). Therefore clinicians see nowadays increasing numbers of women who have survived their breast cancer. Because of the possible role of estrogen in the etiology of breast cancer, it has been thought that ERT/HRT can trigger the recurrence of breast cancer, and therefore a history of breast cancer has been regarded as a rather absolute contraindication for ERT/HRT (Marchant 1994; Colditz 1997; Consensus Statement 1998). However, the rationale of this denial has been questioned (Cobleigh et al. 1994; Braendle 1998) because the prognosis of breast cancer is presently so good that most women with a history of breast cancer may die from other diseases than breast cancer. One factor contributing to improved prognosis of breast cancer is the use of antiestrogens (Early Breast Cancer Trialists Collaborative Group 1992, 1998). This effect must be mediated through antiestrogenic effect in the breast cancer tissue (Buckley and Goa 1989; Wiseman and Goa 1997), but in some other tissues and organs antiestrogens, for example tamoxifen, have estrogenic effects (McDonald et al. 1995; Grey et al. 1995; Powles et al. 1996; Chang et al 1996). Furthermore, the risk of endometrial cancer has been reported to increase during the long-term use of tamoxifen (Early Breast Cancer Trialists Collaborative Group 1998) which can also be seen as one sign of estrogenic effect. On the other hand, tamoxifen causes or worsens typical menopausal vasomotor symptoms (Buckley and Goa 1989).Toremifene is a newer antiestrogenic compound which has similar antiestrogenic activity on breast cancer tissue to tamoxifen (Pyrhnen et al. 1999),



but so far little has been known about the estrogenic consequences of toremifene. In this study, it has been evaluated the usefulness and safety of ERT/HRT and compared the gynecologic and other consequences of tamoxifen and toremifene in postmenopausal breast cancer patients. I studied 131 postmenopausal patients with breast cancer seeking help for menopausal symptoms. From those, 108 women had a sound indication for using ERT/HRT, and 88 patients finally started ERT/HRT. Forty-three women who did not start ERT/HRT were followed as a control group. All patients were carefully examined at the oncological department before being included in the trial to ensure that none had any recurrence at recruitment. Similar oncological examinations (mammography, tumor marker CA 15-3 measurement) were annually repeated during the trial to detect any possible recurrences as soon as possible. The history of breast cancer denotes a 1.5- to 2-fold risk of endometrial cancer in these women (Adami et al. 1997). Therefore endometrium was assessed by serial histological samples and ultrasonography. The use of estrogen effectively alleviated climacteric symptoms in these patients. This was expected because no data imply that this effect of ERT/HRT should be different in these patients. A high continuation rate (88.4%) of ERT/HRT provides an additional proof of the good efficacy of ERT/HRT in the control of vasomotor symptoms, although the chance to see always the same gynecologist, often after a very short notice, may have contributed to the high compliance. The overall risk of recurrence or contralateral breast cancer was similar in patients with (3%) or without ERT/HRT (4%). This result is in line with previous data showing no excess risk of relapse during 2-3 years of use of ERT/HRT (Table 14). Furthermore, although breast cancer per se indicates an increased risk of endometrial cancer (Adami et al. 1997), no primary endometrial cancer developed during the follow-up. Even if our patient series was small and not randomized or placebo-controlled, and control patients had a higher risk of cancer recurrece than ERT/HRT patients, our data on breast safety provides an important piece of information to clinicians.Yet I would like to emphasize that to prove the final safety of ERT/HRT in breast cancer patients, much larger prospective and preferably placebo-controlled trials are needed, and the follow-up time must be at least in



Table 14. Previous data on the use of estrogen replacement therapy following breast cancer as compared with the the present data (NA=data not available)

Variable No. of patients

Wile 1993 25

Powles 1993 35

DiSaia 1993 77

Eden 1995 90

V-Sellin Natrajan 1995 1999 43 50

U-Vrs caj 1999 21

Present study 88 NA

Stage of tumor, no. of patients (%) Ca in situ I II III NA 2 (7.7) 13 (52) 7 (28) 1 (4) 2 (7.7) 6 (7.8) 43 (55.8) 17 (22.1) 5 (6.5) 6 (7.8) 2 (4.7) 22 (51.2) 19 (44.2) 0 0 1 (2) 46 (92) 3 (6) 0 0

3 (3.4) 59 (67) 22 (25) 1 (1.1) 3 (3.4)

Size of tumor, no. of patients (%) T1 T2 T3 12 (34.3) 14 (40) 9 (25.7) 70 (79.5) 17 (19.3) 1 (1.1)

Axillary nodes, no. of patients (%) Negative Positive NA 12 (34.3) 10 (28.6) 13 (37.1) 58 (75.3) 13 (16.9) 6 (7.8) 72 (80) 18 (20) 28 (65.1) 8 (18.6) 7 (16.3) 46 (92) 3 (6) 1 (2) 14 (66.7) 7 (33.3) 72 (81.8) 10 (11.4) 6 (6.8)

25 (100)

Estrogen reseptors,no. of patients (%) Positive Negative NA 28 (36.4) 12 (13.3) 12 (15.6) 10 (11.1) 37 (48) 68 (75.6) 27 (1-233) 7 4 18 (4-144) 6 4 7 (16.3) 20 (46.5) 16 (37.2) 31 (24-142) 1 1 12 (24) 7 (14) 31 (62) 66 (6-384) 3 1 5 (23.8) 16 (76.2) 57 (64.8) 15 (17) 16 (18.2) 29 (1-62) 7 3

25 (100)

35 (100) 15 (1-44) 2 5

Duration of ERT use (months) 35 (range) (6-78) No. of recurrences Recurrence %/ woman-year 3 4

28 (3-72) 4 8



the order of decades. In practice such studies are difficult to arrange and perhaps even unethical. Amelioration of climacteric symptoms during ERT/HRT reveals the code of treatment, and then it is no longer genuinely placebo-controlled. Moreover, how can a physician offer a placebo to a symptomatic climacteric patient wishing to receive estrogen. We can hope that newer SERMs will be useful and safe and thus replace the use of ERT/HRT in breast cancer survivors. Tamoxifen and toremifene showed both antiestrogenic and estrogenic effects in our patients. They similarly worsened vasomotor climacteric symptoms, but fortunately these symptoms were tolerable and/or alleviated by time during the trial, and caused the discontinuation of antiestrogens only in five patients (3%). Breast cancer patients have a 1.6-fold risk of endometrial cancer compared to healthy women (Adami et al 1997), and previous data imply that tamoxifen use still increases this risk to 2-4-fold (Early Breast Cancer Trialists Collaborative Group 1998). No similar epidemiological evidence exists for the carcinogenic effect of toremifene. Tamoxifen has been shown to increase the endometrial thickness and to cause the proliferation or hyperplasia of endometrium, as well as endometrial polyps (Table 15) (Lahti et al. 1993; Kedar et al. 1994; Gibson et al. 1996; Cohen et al. 1997; Marconi et al. 1997; Ozsener et al. 1998; Seoud et al. 1999; Cohen et al. 1999; Barakat et al. 2000; Gerberet et al. 2000). In our study tamoxifen and toremifene caused a similar thickening in the endometrium but this did not correlate with the histology of the endometrium. The incidence of proliferative endometrium, which was before the start of regimens 20.4% and 20.0% in tamoxifen and toremifene users,
Table 15. Studies reporting on the incidences of tamoxifenassociated endometrial pathologies
Author No of study Population 51 61 75 175 101 104 67 261 111 247 Polyps % 33.3 8.2 13.3 8.0 43.6 4.8 10.4 11.5 4.5 5.7 Hyperplasia % 3.9 16.4 1.3 15.4 8.9 16.3 4.5 8.4 2.7 3.2 Carcinoma % 2.0 0 8.0 0 2.0 3.8 1.5 0.4 0 1.2

Lahti et al. 1993 Kedar et al. 1994 Gibson et al. 1996 Cohen et al. 1997 Marconi et al. 1997 Ozsener et al. 1998 Seoud et al. 1999 Cohen et al. 1999 Barakat et al. 2000 Gerberet et al. 2000



respectively, increased more markedly in women using tamoxifen (47.8%) than in those using toremifene (32.2%). Furthermore, the incidence of endometrial polyps was higher in patients using tamoxifen. These findings are in contradiction to the results of the previous prospective comparison between tamoxifen and toremifene showing the equal number of endometrial polyps and proliferative endometrial samples in the both groups (Tomas et al. 1995). One possible explanation to this contradiction might be the shorter follow-up time and also the smaller number of study participants in the study of Tomas et al. In our study, yet only one patient using toremifene developed endometrial carcinoma, which was detected at 12 months follow-up visit. Thus the incidence of endometrial cancer in antiestrogen users (3/10 000 women-years) was lower than that in the Finnish general population of the same age (6/10 000 women-years; Finnish Cancer Registry 1997). Despite the limited number of patients and short follow-up time, data in this study show the safety of antiestrogens towards endometrium at least if the duration of exposure does not exceed three years. Yet a word of caution is needed because a 2-4-fold risk of endometrial cancer in tamoxifen users has been seen in large epidemiological studies (Fisher et al. 1994; Early Breast Cancer Trialists Collaborative Group 1998). Therefore a close endometrial surveillance has been recommended for the longterm users of antiestrogens (Barakat 1996). The results of this study suggest that such a surveillance is not needed during the first 2-3 years of treatment. Based on animal studies, toremifene may be less estrogenic than tamoxifen (Hirsimki et al. 1993). This may be reflected in women as well, because toremifene had a weaker estrogenic effect in the endometrium than did tamoxifen. At the present it could be stated that large and long-term epidemiological data are needed for the assessment of endometrial safety in the long-term use of toremifene. Tamoxifen has been shown to protect against myocardial infarction (McDonald et al. 1995). The mechanism of this action of antiestrogens is poorly understood, although beneficial changes in blood lipids are likely (Love et al. 1994; Grey et al. 1995; Gylling et al. 1995; Saarto et al. 1996). It is also known that the vascular wall and endothelium are of importance for vascular health (Ylikorkala et al. 1986; Mikkola et al. 1995), but no data existed on the effect of tamoxifen or toremifene on these tissues.



Therefore, an important finding of this study was that neither tamoxifen nor toremifene affected the production of prostacyclin, TxA2, or NO. In contrast, toremifene, but not tamoxifen, decreased the release of ET-1. The vascular function was also assessed directly by Doppler equipment. Toremifene significantly reduced the pulsatility index (PI) in the uterine artery, reflecting a vasodilatory effect of toremifene in this artery, whereas this effect was not seen with tamoxifen. In principle the vasodilatory effect of toremifene resembles that of estrogens (Cacciatore et al. 1998). The toremifeneinduced vasodilatation at least in the uterine artery may have been caused, in part, by the fall in ET-1. If a similar vasodilatation would appear to be systemic affecting the vascular bed, toremifene may prove to have in that way cardiovascular protective properties; yet long-term detailed data on vascular physiology during antiestrogen use are still needed. Bone is also one target organ for the action of estrogen and antiestrogens, and this is supported by the presence of both - and -estrogen receptors in bone (Gustafsson 1999). In our patients tamoxifen increased BMD, whereas toremifene only prevented the menopause-induced bone loss. This finding of the favorable effect of tamoxifen on bone is in line with some previous data (Grey et al. 1995; Powles et al 1998; Chang et al. 1996), but the bone-preserving effect of toremifene is new. This study shows that by measuring urinary NTx, it is possible to predict the subsequent change in BMD. This data does not allow to deduce the reasons for the difference in the bone effects of tamoxifen and toremifene but, naturally, one can ask if the dosages used were really equipotent and if equipotency is uniform. It can be possible that estrogen receptors in bone cells are stimulated more by tamoxifen than by toremifene. Furthermore, it can be possible that tamoxifen and toremifene have similar effect on bone if used for longer periods, but the first year data of this study do not allow further conclusion. Summing up our comparative data I may conclude that tamoxifen appears slightly more estrogenic than toremifene on the endometrium and bone, whereas toremifene has stronger estrogenic vasodilatory effect. Our results collected during relatively short follow-up time do not allow us to deduce if these differences persist during more prolonged use of antiestrogens and if they lead to clinically significant changes in patients outcomes.




One hundred and thirty-one postmenopausal women with a history of breast cancer were studied. Eighty-eight patients started ERT/HRT and 43 patients served as controls; the initiation of ERT/HRT was not randomized and the study groups were not strictly comparable. The patients were followed a mean of 2.5 years. In 88 women starting ERT/HRT, five recurrences and two contralateral breast cancers were detected. In the control group (n=43), four patients developed recurrence and one a contralateral breast cancer. The risk per year in ERT/HRT users was 7/216 woman-years (3%) and in control group 5/111 woman-years (4%). Thus ERT/ HRT did not increase the risk of breast cancer recurrence during this rather short period of time. I also followed 167 women who started either tamoxifen (20 mg/day, n=84) or toremifene (40 mg/day, n=83) as an adjuvant therapy for st II-III breast cancer. The mean followup time was 2.3 years. Tamoxifen and toremifene caused similar vasomotor symptoms in postmenopausal breast cancer patients. During the follow-up, only one endometrial cancer developed in a woman using toremifene. As a sign of estrogenic effect, the rate of proliferative endometrium increased from 20.4% to 47.8% in tamoxifen users, whereas the rise from 20.0% to 32.2% in toremifene users was smaller (p<0.0001). Also the number of endometrial polyps during the antiestrogen use was greater in the tamoxifen group (17 polyps) than in the toremifene group (7 polyps) (p<0.05). Prostacyclin and TxA2 may be mediators responsible for the favorable cardiovascular effect of estrogen. Neither tamoxifen nor toremifene caused any change in the production of these vasoactive agents. Toremifene decreased the level of vasoconstrictive ET-1 by 12.9% at 6 months (p=0.01), and by 9.2% at 12 months (p=0.06), whereas tamoxifen did not have any effect on ET-1. Both tamoxifen and toremifene prevented postmenopausal bone loss, as assessed by bone mineral density (BMD) measurement. Tamoxifen increased BMD by 0.4-5%, whereas toremifene did not increase it. Bone preserving effect could



also be verified by measuring various bone markers reflecting bone turn-over. Both tamoxifen and toremifene caused a fall in urinary aminoterminal cross-linked telopeptide (NTx), pyridinoline, and deoxypyridinoline, and tamoxifen caused a fall in serum osteocalcin, aminoterminal and carboxyterminal propeptide of type I procollagen. Yet only NTx showed correlations to the subsequent changes in BMD.

Based on the findings it is possible to make the following conclusions:

1. Short lasting hormone replacement therapy in women with a history of breast cancer is not related to a detrimental effect on the number of recurrences due to this disease, but follow-up of this population was short and the number of patients was small. 2. Tamoxifen and toremifene do not cause premalignant or malignant endometrial lesions during the first 2-3 years of their use. Thus close endometrial surveillance, as has often been advocated recently, is not be warranted during the first three years of antiestrogen treatment providing that patients are asymptomatic. 3. The probable cardiovascular protection provided by antiestrogens is unlikely to be associated with any changes in prostacyclin or TxA2. However, toremifene reduces ET-1 and arterial resistance at least in the uterine artery. 4. Both tamoxifen and toremifene protects against bone loss in postmenopausal breast cancer patients.




This study was carried out at the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, University of Helsinki, Finland during the years 1993-1999. I am very grateful to Professor Olavi Ylikorkala, M.D., Ph.D. and Professor Markku Seppl, M.D., Ph.D., Heads of the Department of Obstetrics and Gynecology, Helsinki University Central Hospital, for providing me the excellent facilities to carry out this study. I would also like to thank the personnel of the department and my colleagues for their support.

I gratefully acknowledge: Professor Olavi Ylikorkala, my supervisor, for guiding me with enthusiasm and unending patience in this work through the years. He always found time for me and encouraged me in many arduous days. Without his help and guidance I would never have completed this thesis. Docent Aila Tiitinen, M.D., Ph.D., for introducing me to the practice of clinical research. I appreciate very highly her experience in clinical work which has been an irreplaceable example for me. I also express my deepest gratitude for supporting and helping me in many study problems as well as in personal life. Professor Antti Kauppila, M.D., Ph.D. and Docent Guillermo Blanco, M.D., Ph.D., the official reviewers, for their interest and careful review of the final manuscript. Their valuable comments and constructive criticism greatly improved the text. Professor Seppo Pyrhnen, M.D., Ph.D. and Docent Pivi Hietanen, M.D., Ph.D., for their collaboration during the different phases of this study. Their viewpoints, comments, and kindly support have been necessary for this work. Docent Bruno Cacciatore, M.D., Ph.D., Docent Torsten Wahlstrm, M.D., Ph.D., Docent Lasse Viinikka, M.D., Ph.D. and Mr. Hans-Jrgen Roth, Head of Laboratory Group, for their excellent scientific work as collaborators and co-authors of the published articles.



Ms. Jaana Matero, Ms. Teija Karkkulainen, Ms. Marja-Leena Pekonen, Mrs. Eira Halenius, and Ms. Marjatta Vallas, for their excellent assistance. Dr. Martti Trnwall, M.D., Ph.D., Medical Director of Leiras Oy and Dr. Juhani Elo, M.D., Ph.D., Director of Medical Unit and Health Economics of Leiras Oy for positive and encouraging attitude to this work, without which it would never have been possible to finalise this thesis. Ms. Carol Norris and Mrs. Auni Aflatuni, for the language revision of the manuscripts and this doctoral thesis. Ms. Raili Alanne, Mrs. Kirsi Siivonen, Mrs. Eila Niemi and Mr. Mika Jrvi, for providing me library services. Mrs. Laila Selkinen and Mrs. Pirjo Peippo-Lavikka, for their kind help in many practical matters. Ms. Hanna Mets-Heikkil, for the graphic design of this thesis. My mother Niina, and my father Aarne, for their support during my whole life. They have offered me resources for a good life and happiness. My husband Tauno, for his love and patience during the last years of this study. He never questioned the rationale of this work, and his help in every day life has been irreplaceable. My two sons, Mikko and Markus, for all the love and happiness they have brought. They are the best that has happened to me during my life. This study was financially supported by Helsinki University Central Hospital Research Funds.
Helsinki, May 2001




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