Rachel S Leeuwaarde

MD, PhD Martinus A Vrede

MD Floyd Henar

MD Rob Does

MD Philip Issa

MD Emmett Burke

MD Otto Visser

MSc, PhD Frank Rijmen

MD, PhD Anneke M Westermann

MD Rachel S Van Leeuwaarde

A nationwide analysis of incidence and outcome of breast cancer in the country of Surinam, during 1994-2003

come

Introduction

BC is the most common malignancy among women worldwide. However, the incidence is highest in developed countries, and relatively low in the developing world [START_REF] Brinton | Ethnicity and variation in breast cancer incidence[END_REF].

The republic of Surinam is a former Dutch colony situated in South America, that gained independence in 1975. Its history of colonization, slave labor and contract laborers has led to great ethnic diversity in its population of almost 500,000 [2]. The major ethnic groups are of Indian descent (the so called "Hindustani", 27.4%), or mixed African descent (the so called "Creoles", 17.7%), Marroons (descendants of runaway slaves,14.7%) and Javanese (descendants of Indonesian contract laborers,14.6%) [START_REF]Algemeen bureau voor de statistiek: Census kantoor[END_REF]. In the 1970s and 1980s, after independence and during a period of civil unrest, mass emigration to the Netherlands took place, so that the Netherlands now has a population of almost 350,000 originally from Surinam [4].

In the year 2002 the Gross Domestic Product (GDP) per capita was US$ 4, 447. The annual per capita health expenditure was US$ 385, corresponding to an expenditure of 8.6% of the GDP [5]. The equivalent numbers for the USA in 2002 were US$ 36, 056 for GDP, and US $5,274 or 14.6% of the GDP for health [6].

There are five hospitals in Surinam, four of which are in the capital of Paramaribo.

Medical specialists have all been trained, at least partly, in first world hospitals, mostly in the Netherlands. Surinam has one pathology laboratory in Paramaribo, where all biopsies and surgical specimens obtained anywhere in the country are sent.

Radiotherapy was not available in Surinam during the study period, so that patients who were eligible for radiotherapy had to go out of the country to receive it. A substantial part of the population has limited health care coverage and many patients have to contribute to the cost of health care out of private funds. There is at present no cancer registry. Feb 12, 2007 RLE 4 Because of the paucity of data on BC in Surinam, the aim of this study was to retrospectively investigate the epidemiology, treatment and outcome of BC over the last decade in order to establish the baseline situation for future health care planning and evaluation. We compared these data to BC statistics of first generation immigrants from Surinam to the Netherlands.

Methods

Between June 1, 2005, and September 30, 2005, data were collected for all patients with a pathological diagnosis of invasive BC between January 1, 1994, and December 31, 2003.

After retrieval from the pathology laboratory archives, the patient charts in the different hospitals were collected and hand-searched for the following data: patient demographics; tumor characteristics (including TNM-stage, grade, and hormone receptor status); treatment characteristics and follow-up data. The last date of follow-up was the last recorded hospital contact, or death of patient. During the study period, there were no national guidelines for treatment of breast cancer.

This study was approved by the Ministry of Health, the National Medical Ethics Board, the boards of directors and the medical staffs of all hospitals in Surinam. The medical ethics committee of Surinam granted a waiver for informed consent for this archival study. Data on Surinamese women with breast cancer in the Netherlands were retrieved from the Netherlands Cancer Registry. We used data for the provinces of North-Holland and Flevoland, which host 35% of the Surinamese population in the Netherlands.

Analysis

A database was made in Access © to store all data. The interval between the different therapies was calculated in this program. Kaplan-Meyer curves and survival times were calculated in SPSS © . The age standardized incidence rate was calculated using the

Results

Incidence

From January 1, 1994, until December 31, 2003, 421 patients were diagnosed with BC in Suriname, two were male. The median incidence was 38,5 per year with a peak of 70 patients in the year 2000. The crude incidence rate in the study period was 15,7/100,000 per year. The age adjusted incidence rate was 28 per 100,000 per year (Table 1). The age adjusted incidence rate for first generation Surinamese women in the Netherlands in the same period of time was 65 per 100,000 per year (Table 1).

Patient characteristics

The median age at BC diagnosis was 55 years, with a range of 26 to 90. BC incidence is higher in Surinamese women in the Netherlands than in Surinamese women in Surinam, and this seems to be especially true for women over 55 (Figure 1). BC occurs more often in Creoles (35,7/100,000/year), than in Hindustani (18,2/100,000/year) and Javanese women (20,8/100,000/year).

Data on parity (n=173) and age at first pregnancy (n=61) were not available for the majority of patients. Data on lactation and duration of lactation were so scarce that they

Tumor characteristics

In Surinam, ductal cancers were most common (94%), with a minority of lobular tumors (1.7%) and tumors of unknown origin (4.3%). (Table 3) More than 60% of all tumors were T2 (>20mm and <50mm) and larger, while less than a quarter were T1 (<20mm). In almost 15% of cases, tumor size was not known, either because the patients never came back after the diagnostic biopsy for further treatment, or because they were not eligible for surgery due to tumor spread at presentation. The lymph node status was known for 323 patients, with almost equal numbers with tumor-positive (N+) and tumor-negative (N0) axillary nodes. The 96 patients with an unknown axillary node status were either patients who only had a diagnostic biopsy and did not return for further treatment or had an advanced stage at presentation and were not eligible for surgery.

The hormone receptor status was known for 69 patients only (Table 3), 40% of whom had a positive estrogen receptor (ER) and/or progestagen receptor (PgR). All but two of these hormone receptor tests were done after 2000.

At time of presentation a small number of patients (5.7%) had distant metastases. The majority of tumors for which the histological grade was known, was poorly differentiated according to the Bloom-Richardson classification. The mitotic activity index (MAI), which was determined in Surinam from the year 2000, was known for 95 tumors, of which 53.7% had a MAI ≤10 mitotic figures/10 hpf and 46.3% had a MAI ›10 mitotic figures/10 hpf.

Treatment

A large majority (79.7%) of patients underwent surgery. Modified radical mastectomy was the procedure most often performed, with some form of breast conserving therapy for a minority of patients (Table 4). The 20.3% of patients who did not receive surgery, were diagnosed with BC, but either never came back for further treatment or had an advanced stage of BC and were thus not eligible for surgery.

Hormone therapy was prescribed for 216 (51.6%) patients. The lymph node status was known for 186 of these patients, and 62% of N+ patients and 52% of N0 patients received hormone therapy.

In the 78 N0 patients who received hormone therapy, the indications for hormone therapy were T1 tumor with BR 3 (n=4), T3 or T4 tumor (n=9), T2 with a BR2 or 3 (n=27). For 38 patients the indication for hormone therapy was not specified. 18 of 26 patients who had positive hormone receptors received hormone therapy. The 8 patients with a positive hormone receptor who did not receive hormone therapy died or were lost to follow-up soon after diagnosis.

A fifth of all patients received adjuvant chemotherapy. This was not appreciably influenced by the size of the primary tumor. Chemotherapy was given to 35.1% of all node positive (N+) patients. Fifty-one percent of N+ women <50 years and 20% of N+ women > 50 years received chemotherapy. Seventeen percent of N0 women <50 years and 4% of N0 women >50 years received chemotherapy.

Thirty percent of all patients who were eligible for chemotherapy according to the 2005 Surinam guidelines. (obviously not available during the study period) actually received it in our cohort.

Thirty-three patients (of 42 who had breast conserving surgery) were irradiated abroad after lumpectomy. Twenty-six patients received radiotherapy after radical mastectomy.

The indication for adjuvant radiotherapy was irradical resection in five patients, T3 tumor (n=3), T4 tumor (n=3), age < 35 years (n=2), ER-/PgR-(n=2), and grossly positive lymph nodes (>N2 in 7 patients). For 4 patients who were treated with adjuvant radiotherapy after mastectomy, the indication could not be retrieved from the available records.

Treatment duration

The median time from the date of diagnosis until the date of surgery in Surinam was 17 days, with not much variation between the different hospitals in Paramaribo. The median time between breast conserving surgery in Surinam and radiotherapy abroad was 11 weeks. For the 26 patients who received radiotherapy after mastectomy, the median time between mastectomy and radiotherapy was 11.5 weeks. The median time between surgery and second treatment modality, either chemotherapy or radiotherapy was 9 weeks. The median time from the end of the first to the start of the second adjuvant treatment modality was 12 weeks.

Outcome

Follow-up data of 384 (91.7%) patients were available, but median follow-up was only 23 months. Five-year follow-up was available for 73 patients. The five year overall survival for these patients was 79%, with a median of 110 months (Figure 2).

During follow-up, 45 patients were diagnosed with distant metastases, 47 with a locoregional relapse and 27 with both distant spread and locoregional relapse. Patients who received breast conserving therapy developed locoregional relapse in 9.1% of cases, while in patients who underwent mastectomy the locoregional relapse rate was 20.5%.

The mean disease free survival was 100 months (Figure 2), and highly correlated to T stage, with 111 months for T1 tumors, 94 months for T2 tumors, 77 months for T3 tumors and for T4 tumors 44 months. For patients with negative lymph node status mean survival was 116 months and for positive lymph node status 78 months (Figure 3).

Discussion

In this retrospective population-based survey the incidence of BC in Surinam was studied over a 10-year period. The strength of this study lies in the fact that all pathology-confirmed diagnoses in the country were included in the analysis, and all hospitals participated. The main weakness is the paucity of follow-up data with a median The crude cancer incidence rate in Surinam increased from 9.5/100 000 in 1964-1976 [START_REF] Brathwaite | Breast Cancer in Surinam[END_REF] to 16.7/100 000 in 1980-2000 [START_REF] Mans | Cancer incidence in Suriname from 1980 through 2000 a descriptive study[END_REF], which is similar to our results. The rise in BC incidence over the past decades is seen worldwide [START_REF] Kelsey | The epidemiology of breast cancer[END_REF][START_REF] Key | Epidemiology of breast cancer[END_REF] and has been mostly correlated with changes in reproductive factors. This may to a large degree account for the lower BC incidence in developing countries vs. the western world [11;12]. Although increasing, the mean age at first birth in Surinam is still low at 21 years[5], compared to over 28 years in most western European countries [START_REF] Snick | An update on the age of subfertile couples in Walcheren: age at registration mirrors increasing age at first birth[END_REF]. In addition, mean parity in Surinam was 2.7 children per women in 2000 [START_REF]Algemeen bureau voor de statistiek: Census kantoor[END_REF], while in the Netherlands it was only 1.7 (2001-2003) [START_REF] Snick | An update on the age of subfertile couples in Walcheren: age at registration mirrors increasing age at first birth[END_REF].

Interestingly, the incidence of BC in Surinamese women in Surinam was considerably lower than the incidence of first generation Surinamese women in the Netherlands (Figure 1). The main possible explanation is thought to lie in demographic changes, with Surinamese women in the Netherlands adapting to Dutch reproductive statistics with less children at a higher age, although increased detection due to public awareness and screening mammography programs may also play a part.

The median age at BC diagnosis was lower than the median age in the western world, albeit fairly high compared to other low-income countries, underlining the in-between state of development of Surinam society [11;12;14]. A confounding factor could be the relatively low life expectancy of women in Surinam (69 years at birth[5], versus 80 in the USA[6]), especially in view of the fact that 43% of new BC cases in the USA are diagnosed in women older than 65 years. The most important competing risk for death in Surinam is cardiovascular disease, which has a high mortality because (preventive) care is not always optimized. The fact that the proportion of Creole BC patients is higher than would be expected by the distribution of ethnicities in the population, may both be explained by genetic factors comparable to the incidence data for e.g. African-Americans in the USA, but also because cardiovascular disease is much more prevalent in Hindustanis, the largest ethnic group.

Women presented with late stage BC more often than in the western world, e.g. 41.6% of patients had lymph node involvement at time of diagnosis, whereas during the same time period this was only 30% in the USA and 31% in the Netherlands [START_REF]Ref Type: Internet Communication[END_REF]. More advanced stage at diagnosis is commonly found in developing countries [START_REF] Harirchi | Breast cancer in Iran: results of a multi-center study, Asian Pac[END_REF], and is thought to be related to lack of universal coverage, lack of screening programs, and fear of cancer in general. Tumor size and stage decreased in the western world since the introduction of mammography in the Netherlands in the last decades of the 20 th century [START_REF] Aubard | Impact of screening on breast cancer detection. Retrospective comparative study of two periods ten years apart[END_REF][START_REF] Van Dijck | Alterations of stage distribution for breast cancer since the implementation of national screening program in the Netherlands during 1989-1995[END_REF][START_REF] Fracheboud | Decreased rates of advanced breast cancer due to mammography screening in The Netherlands[END_REF], and this went hand in hand with increased public awareness and reduced taboo status of the disease [19;20]. In Surinam, the vivid culture of traditional medicine and taboo status of BC, almost certainly contribute to a more advanced stage at diagnosis. However, the convergence of both incidence and stage of BC in first generation immigrants from Surinam to the native Dutch population challenges the size of the effect of different attitude and beliefs. It underscores both the relationship between poor socioeconomic status and access to health care, and the positive effect of BC campaigns to enhance BC awareness and possibly of screening [START_REF] Stirbu | Cancer mortality rates among first and second generation migrants in the Netherlands: Convergence toward the rates of the native Dutch population[END_REF].

Although data on tumor size and lymph node status were generally available from the pathology lab, other prognostic and predictive factors were harder to interpret. We found a discrepancy between the Mitotic Activity Index (MAI) and Bloom-Richardson grading.

The majority of tumors had low MAI (under 10 hpf) but this was paired with high tumor grades according to Bloom-Richardson. Grading is generally considered more subjective and less reproducible than MAI. According to several authors MAI is a strong, widely available, easily assessable, inexpensive, well-reproducible prognosticator, if the protocol is carefully followed. Fixation delay for as long as 24 hours after extirpation of the tumor does not decrease the accuracy of the MAI, in contrast to BR evaluation [START_REF] Baak | Prospective multicenter validation of the independent prognostic value of the mitotic activity index in lymph node-negative breast cancer patients younger than 55 years[END_REF]. In a country with limited resources, MAI could prove to be more reliable than grading, but short of central pathology revision, there is no way of knowing what was most accurate or consistent in Surinam.

Because of the absence of immunohistochemistry (IHC) technology in the pathology laboratory in Surinam to determine hormone receptor status, paraffin-embedded archival tissues were sent to the Netherlands to determine the receptor status of the tumor whenever deemed necessary for treatment decisions. Unfortunately, only a minority of patients had tissue sent to the Netherlands, and most of them had a negative receptor status. Is this an accurate estimation? False negatives of hormone receptor are not uncommon, especially with increasing time delay before tissue fixation, increased duration of fixation and type of fixative, all of which may have been the case in our patient cohort. False negatives increase especially in BC with a low receptor positivity such as is typically seen in patients of African descent [START_REF] Chen | Histological characteristics of breast carcinoma in blacks and whites[END_REF][START_REF] Furberg | Tumor characteristics in African American and white women[END_REF][START_REF] Rhodes | Reliability of immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems[END_REF][START_REF] Taylor | Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques[END_REF]. At present it is impossible to say whether this played a part in our study population, but the lack of IHC to determine predictive markers is bound to hamper treatment decision making, and possibly outcome. Thus, since hormone receptor status was known for only 69 patients, prescription of adjuvant hormone therapy was mostly blinded to ER-status. This seems not unreasonable, in view of the uncertainty about the quality of the assessments, and the mild cost and systemic effects of hormone therapy.

The indications for adjuvant systemic therapy for BC have broadened over the past decades. Although regional differences exist, in general adjuvant systemic therapy is prescribed for larger tumors, N+ tumors and small tumors that are poorly differentiated.

No guideline existed in Surinam during the study period, but in 2005 the National Oncology Committee developed a BC treatment guideline based on both Dutch and American (USA) guidelines [27;28], although in some cases the suggested drug regimens differed to suit Surinam's economical situation [5]. Indications for adjuvant systemic therapy according to this government-approved guideline were compared to the proportion of patients receiving such therapy in the study cohort. It should be noted that over the study time period, in western countries adjuvant chemotherapy practice was also considerably more conservative than it is now. The aim of the comparison was A minority of patients who would be eligible for adjuvant chemotherapy according to the guideline actually received it (30%). Based on western experience after the introduction of national guidelines [29], this number could go up to at least 70% for chemotherapy, which means that the number of patients that will receive adjuvant chemotherapy may double or even triple.

The treatment interval between the different treatment modalities is a predictor for outcome, with a long treatment interval as an unfavorable factor. Compared to the Netherlands, the median interval between diagnosis and surgery was shorter (17 days vs. around 28 days), which points to the absence of long waiting lists for surgery so common in the Netherlands. However, treatment intervals between surgery and chemotherapy compared unfavorably to the Netherlands (9 vs. 4 weeks). We speculate that the lack of availability of cytotoxic drugs in Surinam may in some case account for the delay, but lack of a sense of urgency in both doctors and patients may also play a part.

The time between surgery and radiotherapy was approximately 11 weeks. In the Netherlands an 8 week interval is considered the upper limit of what is acceptable, with a preference for 4-6 weeks. Because of the absence of radiotherapy facilities within the country, a longer interval may have been unavoidable in this group. In view of this, it may be worth considering advising radical mastectomy instead of breast conserving therapy for all patients, until radiotherapy will be available in the country.

For a relatively large part of our cohort (20-25%), no data on lymph node status or local treatment were available. Some of these patients may not have received local treatment because of advanced stage of disease at time of diagnosis, but we speculate that others never returned for further treatment. This is consistent with the relatively short length of follow-up, in spite of our concerted efforts to track down the disease course of all patients. We were unable to determine whether patients in general did not return for follow-up because disease was uncontrolled, or because they were asymptomatic. In either case, this may have biased the survival data, which raises doubt about their Feb 12, 2007 RLE 13 validity, even though obvious trends such as worse outcome for higher stages could be confirmed.

Conclusion

In this study we have shown the feasibility of conducting a nationwide epidemiologic survey in Surinam. Most of our findings could be related to the demographics of Surinam and the low-resource status of the economy. The introduction of the Surinam BC treatment guideline, the planned introduction of immunohistochemistry, and the anticipated introduction of radiotherapy in late 2010 are expected to ameliorate diagnosis and treatment of BC patients in Surinam. The projected start of a national cancer registry, combined with a recently improved continued medical education program will further enhance these goals. Improvement in BC awareness and possible implementation of BC screening programs will take much longer.

[29] A.Wockel, C.Kurzeder, V.Geyer, I.Novasphenny, R.Wolters, M.Wischnewsky, R.Kreienberg, D.Varga. Effects of guideline adherence in primary breast cancer-a 5-year multi-center cohort study of 3976 patients, Breast, 19, (2010) 120-127.

Feb 12

 12

Feb 12

 12 of this report.

Feb 12

 12 , 2007 RLE 8

Feb 12

 12 of less than 2 years, which impedes discussion of outcome. Although all pathologically confirmed BC diagnoses in Surinam during the study period were included in the analyses, it is conceivable that additional women were diagnosed with advanced BC during the study period without tumor biopsies or surgery. The absence of a national cancer registry in the country makes it impossible to estimate or correct for this in any way. However, this is the first population-based study to look at individual demographic, tumor and treatment data.

Feb 12

 12

Feb 12

 12

Feb 12

 12 in future health care planning.

Figure 1 :

 1 Figure 1: Age specific breast cancer incidence in Surinam and in Surinamese women in the Netherlands Figure 2: Overall & disease free survival of breast cancer patients in Surinam Figure 3: Overall cumulative survival of breast cancer patients with N0/N+ in Surinam

Figure 1 :Figure 3 :

 13 Figure 1: Age specific breast cancer incidence in Surinam and in Surinamese women in the Netherlands

Table 1 :

 1 Characteristics of breast cancer patients in Surinam andSurinamese women in the provinces North-Holland and Flevoland in the

	Netherlands, 1994-2003
		Surinam	The Netherlands
		Number of patients	Number of patients
		%	%
		(ASR*)	(ASR*)
	Total	419 (28)	196 (65)

Table 3 : Tumor characteristics of breast cancer patients in Surinam 5 6

 3

		Number of
	Strata		Percentage
		patients
	Total	419	100%
	Type of tumor
	Ductal	394	94
	Lobular	7	1.7
	Unknown	18	4.3
	T-size
	T1 (≤20 mm)	98	23.4
	T2 (>20, ≤50 mm)	157	37.5
	>T3 (>50 mm)	96	22.9
	Unknown	68	16.2
	Number of involved axillary lymph nodes
	N0	149	35.6
	N1(1-3)	93	22.2
	N2(4-9)	51	12.2
	N3(≥10)	30	7.2
	Unknown	96	23.2

Table 4 : Therapy characteristics of breast cancer patients in Surinam 10 25

 4

		n	%	Lymph node	Lymph node
				positive	negative
	Total	419	100
	Surgery	334	79,7
	-Mastectomy	292	69,7	150	127
	-Lumpectomy	42	10,0	14	20
	Radiotherapy	59	13,8	33	24
	-after lumpectomy	33	7,9	20	16
	-after mastectomy	26	5,9	13	8
	Chemotherapy	86	20,5
	-CMF	45	10,5	35	4
	-Anthracycline-containing	31	7,9	21	7
	-Unknown	10	1,9
	Hormone therapy

Acknowledgements

We are very grateful for the participation and assistance of the following persons and institutions in this study: Surinam: Boards of hospitals & medical staffs Ministry of health: M. Eersel, MSc Administrative staff of all participating hospitals Paramaribo, Surinam: Academisch Ziekenhuis Surgery: R.B. Girjasing, MD. S. Nannan Panday, MD Internal medicine: H.D. Chee, MD C.H. Adhin, MD, I.J. Lie Kiauw, MD J. Loor, MD, J.E. Anijs, MD Diakonessenhuis Surgery: A.E. Bergen MD


Ferring Pharmaceuticals

La Rosette

Davis Iii

Kold Olesen

Efficacy and safety of androgen deprivation therapy after switching from monthly leuprolide to monthly degarelix in patients with prostate cancer

Efficacy and safety of androgen deprivation therapy after switching from monthly leuprolide to

• In a randomised 1-year trial, the GnRH antagonist, degarelix administered to prostate cancer patients resulted in a more rapid suppression of testosterone and PSA without testosterone surge during the first month compared to the GnRH agonist, leuprolide.

• Although these findings show major differences in the kinetics of testosterone and PSA suppression during the first month, there is currently no information available in the literature when switching from a GnRH agonist to a GnRH antagonist.

What's new

• Our trial demonstrates that prostate cancer patients can be successfully switched from leuprolide to degarelix treatment.

• In a planned analysis at 3 months, it was shown that patients can be safely switched from leuprolide to degarelix with sustained efficacy as measured by biochemical markers (testosterone and PSA).

METHODS

Participants were134 patients with histologically confirmed PCa who had completed 1 year of treatment with leuprolide 7.5 mg monthly before being switched to degarelix. These patients were re-randomised for the extension trial to receive a starting dose of 240 mg degarelix followed by monthly maintenance doses of either 80 (n=69) or 160 mg (n=65). For efficacy assessment, serum testosterone, prostate-specific antigen (PSA), luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels measured at days 3, 7, 14, 28, 56 and 84 assessed whether treatment efficacy is sustained. Safety and tolerability assessments included adverse events (AEs), physical examinations, electrocardiograms and clinically significant changes in laboratory safety parameters.

RESULTS

Serum testosterone, LH, and PSA levels were all sustained in both treatment arms during the observation period. Interestingly, FSH levels were further decreased by 30% following the switch to degarelix. With the exception of injection site reactions, the overall prevalence and pattern of AEs during the first 3 months after the switch was comparable to that during the last 3 months leuprolide treatment in the main trial. There were 5 (4%) patients discontinued to treatment-related AEs including injection site pain (n=3) and fatigue (n=2).

CONCLUSIONS

This 3-month analysis indicates that patients with prostate cancer can be safely GnRH receptor agonists effectively reduce serum testosterone levels to castrate levels (≤0.5 ng/mL) in 90-100% of patients, but only after 7-21 days [START_REF] Conn | Gonadotrophin-releasing hormone and its analogues[END_REF][START_REF] Lepor | Comparison of single-agent androgen suppression for advanced prostate cancer[END_REF]. In addition, the agonists initially activate the GnRH receptors leading to a surge in serum LH and testosterone which might cause delayed therapeutic effects and exacerbation of symptoms such as spinal cord compression, bone pain and urethral obstruction [START_REF] Waxman | Importance of early tumour exacerbation in patients treated with long acting analogues of gonadotrophin releasing hormone for advanced prostatic cancer[END_REF][START_REF] Thompson | Sudden death due to disease flare with luteinizing hormone-releasing hormone agonist therapy for carcinoma of the prostate[END_REF][START_REF] Boccon-Gibod | Cyproterone acetate lead-in prevents initial rise of serum testosterone induced by LHRH analogs in the treatment of metastatic carcinoma of the prostate[END_REF].

In contrast, the GnRH antagonist degarelix quickly binds to and blocks the GnRH receptors in the anterior pituitary gland which in turn results in rapid decreases in LH and FSH and ultimately in testosterone reaching castrate levels (≤0.5 ng/mL) within 1-3 days of subcutaneous (s.c.) administration [START_REF] Van Poppel | Degarelix: a novel gonadotrophinreleasing hormone (GnRH) blocker -results from a one-year, multicentre, randomised, phase 2 dose-finding study in the treatment of prostate cancer[END_REF][START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF][START_REF] Gittelman | A 1-year, open label, randomized phase II dose finding study of degarelix for the treatment of prostate cancer in North America[END_REF].

More recently, GnRH antagonists also became available for physicians both in North America and Europe. In the pivotal, 1-year phase III trial, degarelix administered subcutaneous (s.c.) at a starting dose of 240 mg followed by monthly (every 28 days) maintenance doses of 80 mg (240/80 mg regimen) or 160 mg (240/160 mg regimen) was as effective as monthly intramuscular (i.m.) doses of 7.5 mg leuprolide in achieving and maintaining testosterone suppression at castrate level (≤0.5 ng/mL) with response rates ranging from 96.4% to 98.3% [START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF]. In leuprolidetreated patients, testosterone surge occurred in 80% of the patients whilst none of the patients experienced this with degarelix. In line with the testosterone changes, PSA levels also declined more rapidly in the degarelix treatment groups during the first 28 days. While these findings highlight major differences in the kinetics of This clinical situation was addressed as part of an ongoing long-term extension trial to the aforementioned pivotal trial. In this extension trial, patients previously on degarelix continued with the same monthly degarelix maintenance doses, whereas those previously treated with leuprolide were re-randomised to receive degarelix at a starting dose of 240 mg followed by monthly maintenance doses of either 80 mg or 160 mg. Patients switched to degarelix were monitored closely during the initial 3month period. A pre-planned interim analysis of the data was scheduled when all patients had completed 3-months treatment at which time-point leuprolide plasma levels would also be negligible [START_REF]LUPRON DEPOT 7.5 mg Package Insert[END_REF].

PATIENTS AND METHODS

Trial entry criteria in the main trial

The study design and the patient population participating in the study have been described previously in detail [START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF]. In brief, patients were required to be ≥18 years with histologically confirmed adenocarcinoma of the prostate (all stages) for whom endocrine treatment (except for neoadjuvant hormonal therapy) was indicated. In addition, patients were required to have a screening testosterone level >1.5 ng/mL and a PSA level ≥2 ng/mL. The trial was performed in accordance with the Declaration of Helsinki as well as Good Clinical Practice Guidelines.

In the original pivotal trial, prostate cancer patients received 1-year treatment with either monthly injections of leuprolide 7.5 mg i.m. or with degarelix at a starting dose of 240 mg s.c. followed by monthly maintenance doses of either 80 or 160 mg.

Design of extension trial

All patients who completed the initial comparative phase were eligible to enter the extension trial. Patients treated with degarelix continued with the same monthly maintenance doses while those patients who previously received treatment with leuprolide 7.5 mg were re-randomised to one of the two degarelix treatment regimens to receive a starting dose of 240 mg for one month followed by monthly maintenance doses of either 80 mg or 160 mg, respectively. Following the switch, patients were monitored closely and an interim analysis was performed according to trial protocol after all patients in the extension trial had been exposed to degarelix for at least 3 months. The results reported in this manuscript focus on the group of patients switched from leuprolide to degarelix and treated with these treatment regimens for 3 months.

Demographics and baseline characteristics

The baseline and disease characteristics are shown in Table 1. Baseline median testosterone, PSA, LH and FSH levels are presented for both prior to the main trial and at the time of the switch (after one-year treatment with leuprolide). Baseline and disease characteristics for patients switching from leuprolide to degarelix treatment were comparable and representative of the initial population treated with leuprolide.

Testosterone levels

The median testosterone levels over time from the start (baseline) of the main trial and for those patients continuing in the extension trial, up to 84 days after the start of the extension trial are displayed in Figure 2A. In the extension phase, serum testosterone levels were ≤0.5 ng/mL from day 3 to day 84. Testosterone levels remained suppressed and during the first 84 days after the switch from leuprolide to degarelix treatment the median testosterone levels were 0.1 ng/mL. One patient with a serum testosterone level 1.4 ng/mL at the time of the switch, had serum

LH and FSH levels

The median percent reduction in LH over time from the start of the main trial (baseline) to Day 84 in the extension trial is displayed in Figure 2B. There was a similar pattern of changes in LH levels as with testosterone. The approximate 99% reduction in median LH levels was maintained after the switch from leuprolide to degarelix treatment.

The median percent reduction in FSH over time from the start of the main trial (baseline) to Day 84 in the extension trial is displayed in Figure 2C. FSH levels decreased rapidly with approximate maximum reductions of 90% at 28 days after the initial degarelix doses and after one-year treatment FSH levels were reduced to about 82% relative to baseline. For leuprolide, there was an initial increase of FSH level followed by a decrease of about 75% at day 28. After one-year treatment with leuprolide FSH levels were reduced to about 55% relative to baseline. Interestingly, after the treatment switch, FSH levels were further reduced to about 80% relative to baseline.

PSA levels

The median percent reduction in PSA over time from the start (baseline) is displayed in Figure 3D. PSA levels were reduced more rapidly during the first 28 days of treatment in the degarelix treatment groups with median reductions of about 65% and 83% at Day 14 and Day 28, respectively compared to corresponding median Thereafter PSA levels were reduced to a similar extent in the degarelix and leuprolide treatment groups and after 1 year treatment there was ≥95% reduction in PSA from baseline in all treatment groups. The ≥95% median reduction of PSA levels was maintained after switching from leuprolide to degarelix treatment, and during the first 84 days of treatment with degarelix. The median PSA levels were 0.5 ng/mL or less.

Safety and Tolerability

The prevalence of treatment-emergent AEs (at least 3%) for the first and last 3 months treatment with leuprolide 7.5 mg in the main trial and during the first 3 months after the switch from leuprolide to degarelix is displayed in Table 2. Adverse events are grouped according to domains of local tolerability, ADT-related, diseaserelated and 'other' AEs. Except for local tolerability injection site reactions, the overall prevalence and pattern of AEs during the last 3 months of leuprolide treatment in the main trial was comparable to those after the switch from leuprolide to degarelix.

Injection site reactions were the most frequently reported AEs associated with the switch. The local reactions occurred primarily after the first injection with 28% of 134 starting dose injections reported to be associated with injection site reactions.

However, only 6% of 125 (leuprolide/degarelix 240/160 mg) and 3% of 127 (leuprolide/degarelix 240/80 mg) maintenance dose injections during the first 3 months were reported to be associated with injection site reactions. The most common types of injection site reactions were pain and erythema.

After the switch from leuprolide to degarelix, 5 (7% and 8%) patients in both dose groups had serious adverse events (SAEs) in the first three months. These SAEs also included 2 (3%) patients with prostate cancer (PCa) progression in the leuprolide 7.5 mg /degarelix 240/160 mg group. Importantly, none of the SAEs was considered as related to degarelix treatment. Two patients died due to PCa progression and PSA levels were already increasing during the last 3-6 months on leuprolide. There were 7 (5%) patients discontinued from the extension trial within 3 months (up to 91 days) after switching leuprolide to degarelix due to AEs. These included 3 patients with injection site pain and 2 patients with fatigue (started during treatment with leuprolide in main trial) which were assessed as possibly or probably related to degarelix. The remaining 2 patients were discontinued from the trial due to prostate cancer progression (not treatment-related).

Overall 5 (4%) patients had prostate cancer progression reported as a new AE during the first 3 months after the switch from leuprolide to degarelix treatment. Four of the patients with prostate cancer progression had metastatic prostate cancer and one patient had locally advanced prostate cancer and PSA levels prior to starting leuprolide treatment ranged from 619 ng/mL to 10,952 ng/mL. In addition, PSA levels were increasing in these 5 patients during the last 3-6 months of leuprolide therapy.

Following the switch, PSA levels continued to increase during degarelix treatment in these patients.

There were a few patients with laboratory abnormalities including 1 additional (2%) patient in the leuprolide 7.5 mg/degarelix 240/80 mg treatment groups who had alanine aminotransferase (ALT) levels of more than three times the upper limit of normal range (ULN) during the first 3 months of the extension trial. The increase in ALT was reversible. In addition to the laboratory liver function test abnormalities, there was 1 patient in the leuprolide 7.5 mg /degarelix 240/80 mg group with a serum urea There is currently no information available in the literature when switching from a GnRH agonist to a GnRH antagonist and this was investigated as part of an ongoing long-term extension trial. An interim analysis of the data was scheduled when all patients had completed 3 months treatment and the main purpose of this analysis was to confirm that there was no unexpected safety or lack of efficacy issues associated with switching patients from leuprolide to degarelix treatment.

The dosing regimens were selected for optimal treatment for patients switched from leuprolide to degarelix and included a starting dose of 240 mg degarelix followed by monthly maintenance doses of either 80 or 160 mg. It has been established in previous dose-finding trials [START_REF] Van Poppel | Degarelix: a novel gonadotrophinreleasing hormone (GnRH) blocker -results from a one-year, multicentre, randomised, phase 2 dose-finding study in the treatment of prostate cancer[END_REF][START_REF] Iversen | Determining the optimal initiation and maintenance doses of degarelix for hormone therapy of prostate cancer patients[END_REF] that an initial starting dose of 240 mg degarelix is required in order to achieve sufficient suppression of testosterone to castrate levels (≤0.5 ng/mL) in 95% of patients for at least 28 days. In addition, subsequent to the starting dose, lower monthly maintenance doses are effective in producing sustained testosterone suppression [START_REF] Van Poppel | Degarelix: a novel gonadotrophinreleasing hormone (GnRH) blocker -results from a one-year, multicentre, randomised, phase 2 dose-finding study in the treatment of prostate cancer[END_REF][START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF][START_REF] Gittelman | A 1-year, open label, randomized phase II dose finding study of degarelix for the treatment of prostate cancer in North America[END_REF]. The principle of using a high loading dose followed by low maintenance doses to effectively suppress testosterone, LH and FSH has also been demonstrated in healthy males who were treated with the GnRH antagonist, cetrorelix [START_REF] Behre | High loading and low maintenance doses of a gonadotropin-releasing hormone antagonist effectively suppress luteinizing hormone, follicle-stimulating hormone and testosterone in normal men[END_REF].

When switching from leuprolide to degarelix, testosterone suppression was maintained at castrate levels from day 3-84 in all patients and the median testosterone level was 0.1 ng/mL. One of the leuprolide-treated patients had a serum testosterone level of 1.4 ng/mL, which was rapidly suppressed to <0.5 ng/mL by degarelix, suggesting that a switch can potentially be beneficial in those with insufficient testosterone suppression on the agonist.

FSH levels that had decreased by 50% from baseline after 1 year of treatment with leuprolide were further decreased to 80% reduction from baseline following the decreased more rapidly and to a greater extent with degarelix [START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF] and another GnRH antagonist, abarelix [START_REF] Mcleod | A phase 3, multicenter, open-label, randomized study of abarelix versus leuprolide acetate in men with prostate cancer[END_REF][START_REF] Trachtenberg | A phase 3, multicenter, open label, randomized study of abarelix versus leuprolide plus daily antiandrogen in men with prostate cancer[END_REF] than with leuprolide. In addition to its role in the maintenance of spermatogenesis [START_REF] Sofikitis | Hormonal regulation of spermatogenesis and spermiogenesis[END_REF] in adult males, there is some evidence that FSH may be a regulator of bone resorption [START_REF] Sun | FSH directly regulates bone mass[END_REF] and hence increases in FSH could drive bone loss. In a recent clinical trial in postmenopausal women [START_REF] Rendina | FSHR gene polymorphisms influence bone mineral density and bone turnover in postmenopausal women[END_REF], it was demonstrated that FSH receptor polymorphisms influence bone mineral density (BMD) and bone turnover. Thus, the stronger suppression of FSH by the antagonist suggests a potential safety benefit, which requires further exploration in a larger comparative study addressing BMD changes and fracture risk. Suppression of PSA levels was maintained after the treatment switch. During the first 84 days of degarelix treatment, the median PSA levels were ≤0.5 ng/mL. There were 5 (4%) patients with prostate cancer progression reported as a new AE. This was not unexpected since PSA levels were already rising during the last 3-6 months of leuprolide treatment. Four of the patients with prostate cancer progression had metastatic prostate cancer and one patient had locally advanced disease with high baseline PSA levels ranging from 619 to 10,952 ng/ml. Thus, these results suggest that while degarelix keeps PSA suppression sustained in patients with hormone-sensitive tumours, it may not be able to provide added benefits in those with very progressive cancer disease or already castration-resistant tumours [START_REF] Miller | open label, exploratory study of degarelix as second-line hormonal therapy in patients with prostate cancer (CS27)[END_REF].

After switching from leuprolide to degarelix treatment, degarelix was welltolerated. The pattern of AEs included those related to ADT therapy and AEs occurring as a consequence of the primary disease as expected in this patient population. The prevalence rates of these events during the first 3 months after the switch to degarelix were comparable with those during the last 3 months on leuprolide. The most frequently reported local tolerability reactions which occurred after the switch to degarelix were injection site pain and erythema. The injection site reactions occurred typically after the loading injection with a much lower incidence after subsequent dosing. This observation is consistent with results in the main 1-year trial [START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF] and is probably due to the higher dose/volume given for the first time (two injections as opposed to one injection for subsequent doses). During the first 3 months of degarelix treatment, only 2% of patients discontinued due injection site pain, indicating that overall benefits outweigh the relative inconvenience of an injection site reaction. The two other treatment-related discontinuations were due to fatigue which had however already started during the initial treatment with leuprolide.

With the exception of injection site reactions, the overall pattern and prevalence of adverse events during the first 3 months after the switch from leuprolide to degarelix was comparable to that reported during the last 3 months of leuprolide treatment in the main trial.

CONCLUSIONS

This 3-month analysis indicates that patients with prostate cancer can be safely switched from leuprolide to degarelix treatment. After switching to a starting dose of 240 mg degarelix followed by monthly administration of degarelix, at doses of 80 mg or 160 mg, effective suppression of LH and testosterone (at castrate levels) and PSA are all maintained accompanied by further decreases in FSH levels. Data from currently ongoing long-term studies are awaited to further explore the clinical significance of these changes in biochemical markers.

3 OBJECTIVES

 3 To evaluate whether switching prostate cancer (PCa) patients from leuprolide to degarelix is associated with any change in the efficacy of testosterone suppression or safety profile during the first 3 months.

INTRODUCTION

  to degarelix treatment with sustained efficacy as measured by biochemical markers.Androgen deprivation therapy (ADT) is commonly used in the management of advanced prostate cancer. Gonadotrophin-releasing hormone (GnRH) receptor agonists have been the treatment of choice for most patients for a number of years.

  suppression with degarelix compared to leuprolide, there is currently no clinical information available in the literature describing switching patient medication from a GnRH agonist to a GnRH antagonist. The critical questions for the treating physician is whether the switch has any unexpected change in the pattern of efficacy of androgen deprivation therapy (ADT) and whether it is associated with any safety issues.

  Trial parameters and data analysisEfficacy assessments included changes in serum hormone (testosterone, LH and FSH) and PSA levels which were measured by a central laboratory (Esoterix Inc).Serum testosterone levels were determined using a validated liquid chromatography with tandem mass spectrometry assay with a lower limit of quantitation of 0.03 ng/mL. Testosterone was measured in triplicates and the median value is reported. PSA analyses were performed using a validated immunoassay. LH and FSH analyses used validated immunochemiluminometric methods. Safety and tolerability assessments included laboratory values (biochemistry, haematology, and urinalysis), clinical parameters (adverse events [AEs], ECGs, physical examination and vital signs). Global Central Labs PPD performed the analyses of all clinical chemistry, haematology and urinalysis parameters. Baseline characteristics were summarized by descriptive statistics. Serum levels of testosterone, PSA, LH, and FSH at baseline of the main and the extension trials are shown with median values and interquartile ranges. Changes of the biomarkers are illustrated by median levels over time (testosterone) and median percentage changes from baseline (LH, FSH and PSA). Prevalence rates of AEs (at least 3%) were summarized during the first and the last 3 months of the main trial and the first 3 months of the extension trial for comparative purposes.

9 RESULTS

 9 The extension trial was initiated in March 2007 and by February 2008 all patients had been treated with degarelix for at least 3 months. Of the 172 completers of the main trial in the leuprolide arm, 135 patients were re-randomised and 134 patients were treated with degarelix in the extension trial. During the first three months of treatment with degarelix, 13 (10%) patients discontinued the trial [8 (12%) degarelix 240/80 mg and 5 (8%) degarelix 240/160 mg]. The patient flow and reasons for discontinuation are displayed by treatment group in Figure 1.

  levels ≤0.5 ng/mL from day 3-84 during degarelix treatment. At the time of database cut-off, 1 patient in the leuprolide 7.5 mg / degarelix 240/80 mg group had testosterone levels >0.5 ng/mL on consecutive occasions after day 84.

  of approximately 20% and 68% in the leuprolide treatment arm.

  7 mmol/L, 2 patients in both the leuprolide 7.5 mg /degarelix 240/160 mg and 240/80 mg treatment groups with a low haematocrit ratio of ≤0.37 and 1 patient in the leuprolide 7.5 mg/ degarelix 240/160 mg with a low haemoglobin level (≤115 g/L). used in the management of prostate cancer and there may be situations where the treating physician and/or patient want to try an alternative form of androgen deprivation therapy. For example if a patient is chronically bothered by adverse effects commonly associated with a GnRH agonist, it could be an option to consider switching to a GnRH antagonist instead. In the pivotal Phase 3 trial[START_REF] Klotz | The efficacy and safety of degarelix: a 12month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer[END_REF], there was a significantly lower incidence of musculoskeletal (arthralgia) adverse events (4% vs. 9%) and urinary tract infection (3% vs. 9%) with degarelix as compared with leuprolide. Furthermore, the GnRH blocker could be considered if a patient does not achieve and/or sustain testosterone suppression at castrate levels on the agonist (failure rate is ~10%[START_REF] Sarsody | Endocrine effects, efficacy and tolerability of 10.8-mg depot formulation of goserelin acetate administered every 13 weeks to patients with advanced prostate cancer[END_REF][START_REF] Oefelein | Failure to achieve castrate levels of testosterone during luteinizing hormone releasing hormone agonist therapy: the case for monitoring serum testosterone and a treatment decision algorithm[END_REF][START_REF] Morote | Failure to maintain a suppressed level of serum testosterone during long-acting depot luteinizing hormone-releasing hormone agonist therapy in patients with advanced prostate cancer[END_REF]). Insufficient testosterone suppression may have safety implications for the management of persisting pain, urinary symptoms or even for disease progression[START_REF] Yri | Failure to Achieve Castration Levels in Patients Using Leuprolide Acetate in Locally Advanced Prostate Cancer[END_REF][START_REF] Perachino | Testosterone levels in patients with metastatic prostate cancer treated with luteinizing hormone-releasing hormone therapy: prognostic significance?[END_REF], but this may also be an issue when the therapy targets rapid shrinkage of the prostate before high-dose radiotherapy[START_REF] Yri | Failure to Achieve Castration Levels in Patients Using Leuprolide Acetate in Locally Advanced Prostate Cancer[END_REF]. The most recent safety concern related to GnRH agonists is the cardiovascular risk (myocardial infarction, stroke, sudden death), which had to be added to the Warnings and Precautions section of the drug labels upon request by FDA in October 2010 [16]. This warning does not yet apply to the GnRH antagonists and switching patients with established cardiovascular disease -who are at the greatest risk of complications [17] -could be considered. Regarding the financial implications of the switch, the average sales price (ASP) to Medicare in the US for a year supply of degarelix [(FIRMAGON) starting dose 240 mg, maintenance dose 80 mg/month] is 2659 US dollars which is comparable to the ASP for leuprolide [(LUPRON) 7.5 mg/month] of 2357 US dollars.

  to degarelix. It has been shown in previous studies that FSH levels are

Figure 1 .Figure 2 .

 12 Figure 1. Patient flow during the first 3 months of treatment with degarelix in the


Inger Phd Ab

MD Hernell Olle

PhD b , Hörnell Agneta Phd

MD Lind Torbjörn

Inger Öhlund
email: inger.ohlund@kost.umu.se

Serum lipid and apolipoprotein levels in four-year-old children are associated with parental levels and track over time

Keywords: Serum lipids, children, parental, dietary fat, tracking

Background/Objectives: There are only a few studies linking dietary fat intake to serum lipid levels in young children. Our objective was to prospectively evaluate serum lipid levels from infancy to early childhood, and to explore their possible association with dietary, growth, and parental factors.

Methods: Children (n = 127) followed from early infancy were examined for serum lipid levels, anthropometry, and dietary intake at 4 y of age. We also studied possible associations with parental anthropometric and blood biochemistry data collected from 122 mothers' and 118 fathers' when children were 4 y of age. Results: Serum concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC) and the apolipoprotein B/apolipoprotein A-1 ratio (apo B/apo A-1) showed significant tracking from infancy to 4 y. Furthermore, children's TC levels correlated with paternal TC level from 6 mo to 4 y, but with maternal only at 4 y. In girls, both LDLC and HDLC correlated with parental LDLC and HDLC. In all children, intake of saturated fatty acids (SAFA) was higher than recommended, and in 90% of the children, polyunsaturated fatty acid (PUFA) intake was lower than recommended.

Conclusions:

Serum lipid levels values tracked from infancy to 4 y and were associated with parental values. Higher serum lipid levels at 4 y compared to 6-18 mo of age may result from changes in the quality of dietary fat. We therefore suggest that intake of dietary fat in 4 y old children should be more focused on quality. Furthermore, as there were strong associations between the child and parental serum lipid levels this supports the view that family-based rather than individual intervention is preferable.

INTRODUCTION

The relationships between childhood dietary intake, serum lipid levels, obesity, and long-term risk for cardiovascular disease (CVD) are complex [START_REF] Berenson | Atherosclerosis: a nutritional disease of childhood[END_REF][START_REF] Burke | Family lifestyle and parental body mass index as predictors of body mass index in Australian children: a longitudinal study[END_REF][START_REF] Burke | Predictors of body mass index and associations with cardiovascular risk factors in Australian children: a prospective cohort study[END_REF]. Overweight children have lower levels of high-density lipoprotein cholesterol (HDLC) and higher levels of serum triglycerides (TG) compared to normal-weight children [START_REF] Freedman | The relation of overweight to cardiovascular risk factors among children and adolescents: the Bogalusa Heart Study[END_REF][START_REF] Burke | Predictors of body mass index and associations with cardiovascular risk factors in Australian children: a prospective cohort study[END_REF].

Serum lipid levels differ between girls and boys already in early childhood, and dietary fat intake seems to affect girls and boys differently [START_REF] Rogers | Fat content of the diet among preschool children in southwest Britain: II. relationship with growth, blood lipids, and iron status[END_REF][START_REF] Niinikoski | Impact of repeated dietary counseling between infancy and 14 years of age on dietary intakes and serum lipids and lipoproteins: the STRIP study[END_REF]Öhlund et al., 2008). Total dietary fat intake, particularly higher intake of saturated fatty acids (SAFA), are positively associated with total serum cholesterol (TC) levels [START_REF] Cowin | Associations between dietary intakes and blood cholesterol concentrations at 31 months[END_REF][START_REF] Rogers | Fat content of the diet among preschool children in southwest Britain: II. relationship with growth, blood lipids, and iron status[END_REF][START_REF] Niinikoski | Impact of repeated dietary counseling between infancy and 14 years of age on dietary intakes and serum lipids and lipoproteins: the STRIP study[END_REF]. Studies in Sweden [START_REF] Enghardt | Dietary habits and nutrient intake in Swedish children 4 year old and school children in grade 2 and 5 (Riksmaten -barn 2003) Uppsala[END_REF][START_REF] Garemo | Metabolic markers in relation to nutrition and growth in healthy 4-y-old children in Sweden[END_REF], as well as other countries [START_REF] Agostoni | Dietary fats and cholesterol in italian infants and children[END_REF][START_REF] Cowin | Associations between dietary intakes and blood cholesterol concentrations at 31 months[END_REF], report that intakes of SAFA are higher than the recommended ≤10% of total energy intake (10 E%), while intakes of polyunsaturated fatty acids (PUFA) are close to or below the recommended minimum intake of 5 E%. Both tracking (associations over time) from early infancy and heredity may be additional important predictors of serum lipid levels later in life. In a longitudinal study, we followed healthy Swedish infants from 6 mo of age and found that a higher intake of PUFA was associated with lower total serum cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) levels at 12 mo (Öhlund et al., 2008). In this longitudinally followed study group, we investigated whether these associations remained later in life and whether there was any association with parental serum lipid levels.

The main objectives in the present follow-up study were to evaluate serum lipid levels from infancy to early childhood, i.e., 4 y of age; determine possible associations with dietary, growth, and parental factors; and examine possible tracking of serum lipid levels from infancy to childhood.

METHODS

Study cohort

This was a follow-up of 127 healthy children (63 girls and 64 boys) at 4 y of age, who were prospectively followed from early infancy. Full details are published elsewhere [START_REF] Lind | Effects of weaning cereals with different phytate contents on hemoglobin, iron stores, and serum zinc: a randomized intervention in infants from 6 to 12 mo of age[END_REF]Öhlund et al.,2008).

Of the original 300 children 234 children (80%) completed monthly dietary and anthropometrical registrations from 6 to 18 mo of age. These children and their parents were invited to participate in the present follow-up study at 4 y of age starting in 2001. The parents of 127 children (54%) consented to renewed dietary, anthropometric, and biochemical measurements of the children. Of the parents, 240 (122 mothers and 118 fathers) consented to anthropometric and biochemical measurements when their child was 4 y. Mean maternal and paternal age was 33.7 y (4.5) and 35.3 y (5.5), respectively. The participating 4-y-olds were not different from the non-participating children with respect to weight and length at birth, 12 mo, and 18 mo; duration of breastfeeding; or with respect to dietary fat intake and serum lipid levels at 12 and 18 mo. However, a higher proportion of parents had university educations among the families who participated in the 4-y follow-up compared to those who did not (33% vs. 25%, p < 0.001). Of the 107 non-participating children, 12 were sick at the follow-up, 16 had moved from the area; of the remainder, the parents declined to participate without explanation.

The study was approved by the local Ethics Committee of the Faculty of medicine, Umeå University, Sweden.

Anthropometric data and biochemical analyses

Between 6-18 mo, the participating children were measured monthly. They were weighed to the nearest 0.01 kg using a Seca 835 digital baby scale, and recumbent length was measured to the nearest millimeter using a Harpenden infantometer (CMS Weighing Equipment, London, UK).

At 4 y, weight was measured to the nearest 0.1 kg using a Seca 835 digital adult scale (Seca, Hamburg, Germany) and height to the nearest millimeter was measured using a CMS stadiometer (CMS Weighing Equipment). Mid upper-arm circumference was measured using a plastic measuring tape. Subcutaneous fat was measured by subscapular and triceps skin-fold thickness using a Harpenden skinfold caliper (Baty International, West Sussex, UK).

Body mass index (BMI) was calculated and cut-off levels for overweight/obesity in children were set separately for girls and boys using the definition suggested by the International Obesity Task Force [START_REF] Cole | Establishing a standard definition for child overweight and obesity worldwide: international survey[END_REF]. These cut-off points for overweight and obesity (iso-BMI ≥ 25 and ≥ 30, respectively) were adjusted for age and gender and corresponded to the well-known thresholds of BMI ≥ 25 kg/m 2 and BMI ≥ 30 kg/m 2 for overweight and obesity, respectively, among adults.

Venous blood samples were taken at least 2 h after a meal from the children at 6, 12, and 18 mo, and at 4 y they were taken while the children and their parents were in a fasting state. Sera were stored at -20 °C until analysis. Serum lipids were analysed for TC, HDLC, apolipoprotein A-1 (apo A-1), apolipoprotein B (apo B), and TG by the Roche/Hitachi method, which is the routine analysis method used at the department of Clinical Chemistry at Umeå University hospital. LDLC was calculated using the Friedewald formula (Friedewald Levy and Fredrickson 1972).

Dietary assessment

We assessed dietary intake in the children using monthly 5-d food records from 6 to 18 mo and then once at the 4 y follow-up. Product names were used; for example, the names of spreads, ice creams, candies, yogurts, and meals at fast-food restaurants. The parents used a booklet with photos of serving sizes of meals, snacks, etc., but also used standard household measures for quantities as described earlier [START_REF] Lind | Effects of weaning cereals with different phytate contents on hemoglobin, iron stores, and serum zinc: a randomized intervention in infants from 6 to 12 mo of age[END_REF]. If the participating child attended day care, the dietitian visited the centre to inform the staff of how to record dietary intake. Data regarding breastfeeding were recorded prospectively, except for children who had stopped breastfeeding before inclusion in the study at 6 mo of age. Each intake of breast milk was recorded either as a "meal" or a "snack" estimated as 134 ml per meal up to 8 mo of age, then 102 g per meal beyond 8 mo, and as 25 g per snack at all ages [START_REF] Lind | Effects of weaning cereals with different phytate contents on hemoglobin, iron stores, and serum zinc: a randomized intervention in infants from 6 to 12 mo of age[END_REF]. Nutrient intakes from breast milk were calculated in accordance with [START_REF] Jensen | Handbook of milk composition[END_REF] and [START_REF] Tsang | Nutrition during infancy[END_REF]. Breastfeeding at 6 mo was recorded as exclusive if no other foods were introduced, and partial if foods or formula had been introduced. Breastfeeding duration was used for establishing associations between breastfeeding and serum lipid levels. Energy and nutrient intake were calculated with the MATs S406 software program (Rudans Lättdata, Västerås, Sweden), which uses the food composition database of the Swedish National Food Administration, as well as special products for children and other products not included in the original database. Dietary fat intakes were described as percent of total energy (E%) and PUFA, mono saturated fatty acids (MUFA), and SAFA were described as proportions (%) of total fat. To evaluate the accuracy of the reported energy intake, we calculated the ratios of energy intakes (EI) to basal metabolic rate (BMR) in girls and boys [START_REF] Livingstone | Markers of the validity of reported energy intake[END_REF].

Calculations and statistical analyses

Statistical analyses were performed using SPSS 15.0 (SPSS, Chicago, IL, USA). Mean and standard deviation (SD) were used to describe serum lipid and apolipoprotein levels and dietary fat intakes. Gender difference and changes in dietary fat intake over time were analysed by Student's t-test. The skinfold measurements were skewed and were therefore log transformed before analysis. Pearson's correlation was used to analyse associations of serum lipids over time (tracking) and their association with parental levels. Paired sample t-test was used for changes in variables over time. Data reduction of dietary fat intakes were used and presented as mean daily intakes during the 6-9 mo, 10-14 mo, 15-18 mo and 4 y periods. The relationships between serum lipids and apolipoprotein levels at 4 y and dietary fat intake from 6 m to 4 y, duration of breastfeeding, the child's BMI at 4 y, parental serum lipid levels, and parental BMI were investigated using univariate regression analysis. Factors significantly associated (p≤ 0.05) with the childrens serum lipids and ApoB/apoA-1 levels in the univariate analysis were included in a stepwise multivariate regression analyze.

RESULTS

Serum lipids and anthropometrics

Mean TC levels were stable during infancy. From 18 mo to 4 y, serum TC, LDLC, HDLC, ApoA-1, and ApoB levels increased (p ≤ 0.001) while TG levels decreased (p ≤ 0.001) (Table 1).

The TC, HDLC, LDLC, Apo A-1 and Apo B levels as well as the TC/HDLC and Apo B/Apo A-1 ratios showed significant tracking over time. Furthermore, the children's TC levels were correlated with their father's TC level from 6 mo-4 y, but with their mother's only at 4 y. At 4 y, girls' but not boys' HDLC was positively correlated with maternal HDLC. Furthermore girls', but not boys' LDLC levels were positively correlated with parental LDLC. (Table 2) At 4 y, 16% of the children were overweight (iso-BMI ≥ 25) with no gender difference. Among parents, overweight was more common among fathers compared to mothers (47% vs 28%, p < 0.05). BMI in children was positively correlated with mid-upper arm circumference (r = 0.80, p < 0.001), subscapularis skinfold (r = 0.58, p < 0.001), and triceps skinfold (r = 0.66, p < 0.001). No correlations were found between the children's serum lipid levels and growth rate, any of the other anthropometric parameters, or parental BMI (data not shown).

At 4 y, TC levels >5.2 mmol/l were observed in 22% (seven boys and 17 girls) of the children, in 49% of the fathers, and in 32% of the mothers. LDLC >3.34 mmol/l was observed in 24% of the children. The children with higher TC (>5.2 mmol/l) had significantly higher LDLC, HDLC, and apo B levels and apo B/apo A1-ratios, compared to children with TC <5.2 mmol/l (data not shown).

Dietary intakes

At 6 mo, five infants were exclusively breastfed, 97 were partially breastfed, and 24 no longer breastfed. The mean (SD) duration of breastfeeding was 8.7 (3.4) mo, and two children had not been breastfed at all. Mean (SD) daily energy intake at 4 y was 1462 (240) and 1382 (228) kcal per day in boys and girls, respectively; the calculated ratio of mean EI:BMR was 1.61 in both boys and girls.

Mean dietary fat intake decreased from 42 E% at 6 mo to 33 E% at 4 y (Table 3). However, as dietary fat (E%) decreased, the proportion of SAFA increased significantly (p ≤ 0.001), whereas the proportions of MUFA and PUFA decreased (p ≤ 0.001 and p ≤ 0.001) (Figure 1). The intake of SAFA was higher than recommended in all children and the intake of PUFA was lower in most of the children [START_REF] Nnr | Nordic Nutrition recommendations[END_REF]. At 4 y, none of the children had an intake of SAFA below the recommended upper level of 10 E% whereas only 10% of the children had a total PUFA intake above the recommended lower level of 5 E%. For most children, the intake of MUFA was within the recommended limits [START_REF] Nnr | Nordic Nutrition recommendations[END_REF] .

Associations between children's serum lipid levels and dietary fat intake, and parental serum lipids.

At 6 mo, children who were still partially or exclusively breastfed had higher TC levels compared to those who were not breastfed (p < 0.001), but the difference did not remain at 12 mo, 18 mo, or 4 y. Factors associated with children's serum lipid levels were analysed using regression analysis in two models (Table 4 and5). The serum levels of TC, LDLC, and HDLC, and the apo B/apo A-1 ratio were used as dependent variables. Independent variables with (p < 0.05) in the univariate model were included in a multivariate model. The child's serum lipid levels at 4 y were associated with both parents´ serum lipid levels in the univariate model and in the final multivariate model the child´s TC and LDLC were associated with paternal TC and LDLC levels and the child´s HDLC with maternal HDLC levels. Duration of breastfeeding was negatively associated with HDLC and positively associated with the apoB/apoA-1 ratio in the univarate model but this did not remain in the multivariate analysis. Higher intake of SAFA before 18 mo was associated with a higher apoB/apoA-1 ratio. In opposite, a higher intake of unsaturated fatty acids (MUFA at 10-14 mo and 15-18 mo, and PUFA at 10-14 mo) was associated with a lower apoB/apoA-1 ratio in the univariate model. In the final analysis, higher intake of SAFA 10-14 mo remained positively associated with the child´s apoB/apoA-1 ratio.

DISCUSSION

The main finding in the present study was the correlation between child and parental serum lipid levels. In the study group, which comprised children with a high intake of SAFA and a low intake of PUFA compared to recommended guidelines, TC levels above the cut-offs for abnormal TC (>5.2 mmol/l) [START_REF] Daniels | Lipid screening and cardiovascular health in childhood[END_REF] were present in 22% of the children, 49% of the fathers, and 32% of the mothers. Furthermore, 24% of the children had LDLC > 3.35 mmol/l, defined as a risk factor for adult dyslipidemia and associated cardiovascular disease (CVD) [START_REF] Nicklas | Tracking of serum lipids and lipoproteins from childhood to dyslipidemia in adults: the Bogalusa Heart Study[END_REF][START_REF] Jolliffe | Distribution of lipoproteins by age and gender in adolescents[END_REF]. This calculated risk factor in the present study group was 4-fold higher compared to the risk factor calculated for the Bogalusa Heart Study [START_REF] Nicklas | Tracking of serum lipids and lipoproteins from childhood to dyslipidemia in adults: the Bogalusa Heart Study[END_REF]. Over time, serum TC, LDLC, HDLC, ApoA-1, and ApoB levels increased significantly. In a previous paper, we reported that a higher intake of PUFA was associated with lower TC and LDLC levels at 12 mo (Öhlund et al., 2008). However, three years later, at 4 y of age, the associations between dietary fat intake and serum lipids and apolipoproteins were weak. Compared with Finnish children on a restricted, low-fat diet [START_REF] Niinikoski | Impact of repeated dietary counseling between infancy and 14 years of age on dietary intakes and serum lipids and lipoproteins: the STRIP study[END_REF], these children had more favourable lipid levels at 12 mo (Öhlund et al., 2008), but this did not remain until 4 y of age. The reason may be that during the period 6 mo to 4 y, there was a deterioration in the quality of dietary fat intake. Although total dietary fat intake decreased from 6 mo to 4 y, the proportion of SAFA (%) increased whereas the proportions of both MUFA and PUFA (%) decreased with decreasing dietary fat (E%) resulting in lower fat of quality by 4 y of age. Thus there was an inverse relationship, between decreases in the intakes of PUFA and MUFA and increases in the TC and LDLC levels. Moreover, quality of dietary fat intake at 10-18 mo was associated with the apo B/apoA-1 ratio. A higher intake of SAFA at 10-14 mo and 15-18 mo was associated with higher apo B/apoA-1 ratios, and a higher intake of MUFA and PUFA was associated with lower apo B/apoA-1 ratios at 4 y of age. To our knowledge, few studies have examined associations between dietary patterns and apo B/apoA-1 ratios [START_REF] Kurvinen | Serum lipid and apolipoprotein profiles in newborns and six-year-old children: the Tallinn Young Family Study[END_REF]; more surveys should be performed.

Another finding of our study was tracking of serum lipids from infancy to early childhood. TC and HDLC levels and apo B/apo A-1 ratios tracked from 6 mo to 4 y. Tracking of the apo B/apo A-1 ratio was strongest, and was stronger in boys than in girls. Apo B and apo A-1 levels and their ratio are possibly superior to TC, HDLC, and LDLC levels in terms of predicting CVD [START_REF] Estonius | How do conventional markers of lipid disorders compare with apolipoproteins?[END_REF][START_REF] Kallner | Measurement of apolipoproteins is better than routinely measured traditional lipids[END_REF]. Although there are limited data regarding the association between childhood apo B/apo A-1 ratios and long-term effects on CVD risk, they are suggested to have high predictive value in paediatric risk assessments [START_REF] Frontini | Usefulness of childhood non-high density lipoprotein cholesterol levels versus other lipoprotein measures in predicting adult subclinical atherosclerosis: the Bogalusa Heart Study[END_REF][START_REF] Juonala | Childhood levels of serum apolipoproteins B and A-I predict carotid intima-media thickness and brachial endothelial function in adulthood: the cardiovascular risk in young Finns study[END_REF]. Tracking of serum lipids in Swedish children has not been reported previously, but our results are in agreement with the Finnish STRIP study [START_REF] Rask-Nissila | Impact of dietary intervention, sex, and apolipoprotein E phenotype on tracking of serum lipids and apolipoproteins in 1-to 5-year-old children: the Special Turku Coronary Risk Factor Intervention Project (STRIP)[END_REF]. Further, tracking of serum lipids from childhood into adulthood was demonstrated by the Bogalusa Heart Study [START_REF] Nicklas | Tracking of serum lipids and lipoproteins from childhood to dyslipidemia in adults: the Bogalusa Heart Study[END_REF]. This supports the importance of early interventions in families with known raised serum lipid levels and known CVD risk [START_REF] Magnussen | Utility of currently recommended pediatric dyslipidemia classifications in predicting dyslipidemia in adulthood: evidence from the Childhood Determinants of Adult Health (CDAH) study, Cardiovascular Risk in Young Finns Study, and Bogalusa Heart Study[END_REF]. However, the cut-off levels for risk assessments in children are not yet clear, although guidelines have been developed by the United States National Heart, Lung and Blood Institute's Pediatric Cardiovascular Risk Reduction Initiative [START_REF] Kwiterovich | Cut points for lipids and lipoproteins in children and adolescents: should they be reassessed?[END_REF].

There were no correlations between the children's serum lipid levels, growth rate, BMI, or parental BMI. This is in accord with a British study in which central obesity, but not BMI, was negatively associated with HDLC levels in boys and positively associated with LDLC levels in girls [START_REF] Cowin | Cholesterol and triglyceride concentrations, birthweight and central obesity in pre-school children. ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood[END_REF].

A strength of our study is that it was based on prospective longitudinal data for anthropometric and biochemical variables and carefully repeated registrations of dietary intake of the children. A further strength of the study is the anthropometric and biochemical data collected from the parents when the child was 4 y. However, some limitations of this study should be noted. We have no data regarding the physical activity of the children and parents, and no data regarding the parents' diet and could not analyse whether the associations between the child´s and their parents are based on diet or genetic factors . Furthermore, we were unable to include more than 127 out of the original 234 children in the follow-up study. The by far most common reason for not participating in the follow-up study was lack of time/willingness to do another 5-day period of diet recording, which may lead to a selection bias. However there were no differences, except for the parents' education level, between the participating and non-participating children when compared at 12 and 18 mo. In addition, assessment of dental health at 4 y compared with population data suggested that there was no significant selection bias affecting the present followup (Öhlund et al., 2007). However, we cannot exclude that parents accepting to participate were more health concerned or had higher health awareness than those who did not. . In conclusion, serum lipid and apolipoprotein levels tracked from infancy to 4 y, and the strongest tracking was for the apo B/apo A-1 ratio. Higher serum lipid levels at 4 y compared to 6-18 mo of age may, at least partly, result from changes in the quality of dietary fat. We therefore suggest that intake of dietary fat in 4 y old children should be more focused on quality. Furthermore, as there were strong associations between the child and parental serum lipid levels this supports the view that family-based rather than individual intervention is preferable.

TABLES 1-4

Table 1. Mean serum lipid and apolipoprotein levels in children from 6 mo to 4 y and their parents. Data provided as mean (SD).

Abbreviations: TC, total cholesterol; HDLC, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol; apo A-1, apolipoprotein A-1 and apo B, apolipoprotein B 1 Parental serum lipids assessed when child was 4 y old, maternal mean age was 33.7 (4.5) y and paternal 35.3 (5.5) y. Apolipoprotein A-1(apoA-1) and apolipoproteinB (apoB) not available (n/a) for mothers' and fathers'. No significant differences in apoB/apoA1 between 4 y old boys and younger age groups.*p < 0.05 between gender at 6 mo. No significant differences between gender at 12 m, 18 m, and 4 y. ***All differences in serum lipids between 4 y of age and the younger ages are significant at p ≤ 0.001 unless otherwise stated.

Table 4 .

 4 Abbreviations: TC, total cholesterol; LDLC, low-density lipoprotein cholesterol; HDLC, high-

Table 3 .

 3 Changes in dietary fat intake as percent of energy (E%) and as proportion of total fat (%) from 6-9 mo to 4 y in Swedish children.

			6-9 mo	10-14 mo	15-18 mo	4 y
			(n = 126)	(n = 124)	(n = 107)	(n = 99)
		Total dietary fat (E%)	38.6 (5.4)	29.4 (3.6)	34.6 (3.7)	33.2 (4.0)
		SAFA (E%)	16.1 (3.1)	14.5 (2.2)	14.4 (2.0)	14.8 (2.2)
		(%)	41.3 (2.8)	40.9 (3.4)	41.6 (3.6)	44.6 (3.7)
		MUFA (E%)	15.7 (2.3)	14.0 (1.5)	13.6 (1.4)	12.1 (1.7)
		(%)	40.7 (1.8)	40.0 (2.4)	39.2 (2.2)	36.5 (2.5)
		PUFA (E%)	6.3 (1.2)	5.7 (1.1)	5.2 (1.6)	4.1 (1.0)
		(%)	16.5 (2.9)	16.2 (2.5)	14.9 (2.9)	12.3 (2.5)
		n-6 fatty acids (E%)	na 1	na 1	na 1	3.3 (0.92)
		n-3 fatty acids (E%)	na 1	na 1	na 1	0.86 (0.78)
		Abbreviations: SAFA, saturated fatty acids; PUFA, polyunsaturated fatty acid; MUFA,
		monounsaturated fatty acid.
	5	1 Data not available.
		Data provided as mean (SD).

Table 5 .

 5 Final multivariate analysis of factors associated with serum lipids and ratio of apolipoprotein levels in four-year-old children, adjusted for gender.45Abbreviations: TC, total cholesterol; LDLC, low-density lipoprotein cholesterol; HDLC, highdensity lipoprotein cholesterol; apo A-1, apolipoprotein A-1 and apo B, apolipoprotein B; SAFA, saturated fatty acids; PUFA, polyunsaturated fatty acid; MUFA, monounsaturated fatty acid. Independent variables entered into the analyses were variables with P<0.05 in the univariate regression analysis; dietary fat intake (E%); SAFA, MUFA, and PUFA as E% and as proportion (%); duration of breastfeeding (mo); and parental serum lipids (TC, LDLC; and HDLC), 1 parental apo B and apoA-1 not available. *p < 0.05, **p < 0.01, ***p < 0.001 Changes in dietary fat intake as a proportion of total fatty acids (TFA) from 6 mo to 4 y of age. The proportion of SAFA increased (mean 3.4%, p ≤ 0.001) as the proportions of PUFA and MUFA decreased (mean 4.3%, p ≤ 0.001 and mean 5.0%, p ≤ 0.001), respectively.

	Model 2	Multivariate regression analysis
	Dependent	Independent	R 2	B(s.e)
	TC	Paternal TC	0.11	0.28 (0.08)***
	LDLC	Paternal LDLC	0.07	0.23(0.09)**
	HDLC	Maternal HDLC	0.08	0.17 (0.62)**
	Apo B/apoA-1 1	SAFA 10-14 mo	0.92	0.02 (0.01)**

Acknowledgements

The authors are most grateful to all of the participating children and their parents, and to research 285 nurse Margareta Henriksson for help with collecting the data. We also thank associate professor Hans Stenlund, Department of Public Health and Clinical Medicine, Epidemiology and Public Health Sciences, Umeå University, for invaluable statistical support.

This study was supported by grants from the Goljes Memory Foundation, the Oskar Foundation, and the Mayflower Charity Foundation.

Conflicts of Interests 290

The authors declare no conflict of interest.