MD Mathias Warm
email: mathias.warm@uk-koeln.de

Ronald Kates

Friedrich Overkamp

Anke Thomas

Nadia Harbeck

Benefits of early and prolonged fulvestrant treatment in 848 postmenopausal advanced breast cancer patients

Keywords: advanced breast cancer, fulvestrant, progesterone receptor, therapy response

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Introduction

In advanced breast cancer, control of disease progression by endocrine rather than by cytotoxic agents induces fewer side effects and contributes to quality of life. However, in initially hormone-sensitive tumors, tamoxifen resistance [START_REF] Milano | What clinicians need to know about antioestrogen resistance in breast cancer therapy[END_REF][START_REF] Adamo | Overview and new strategies in metastatic breast cancer (MBC) for treatment of tamoxifen-resistant patients[END_REF][START_REF] Kurokawa | ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanism[END_REF] develops via estrogen-receptor (ER) agonistic effects, e.g. in bone or endometrium; increased tumor-cell sensitivity developing during long-term estrogen deprivation also promotes aromatase inhibitor (AI) resistance [START_REF] Howell | The future of fulvestrant ("Faslodex")[END_REF].

Fulvestrant (Faslodex ® , AstraZeneca) is an ER-antagonist with no known agonistic effects [START_REF] Wakeling | A potent specific pure antiestrogen with clinical potential[END_REF][START_REF] Wakeling | Similarities and distinctions in the mode of action of different classes of antiestrogens[END_REF].

Fulvestrant binds to the ER, blocks its function, causing rapid degradation of the ER, reduces subsequent progesterone receptor (PgR) expression [START_REF] Wakeling | A potent specific pure antiestrogen with clinical potential[END_REF], and inhibits ER-mediated gene transcription.

This distinct mode of action results in a reduced risk of cross-resistance with other endocrine agents [START_REF] Fawell | Inhibition of estrogen receptor-DNA binding by the ""pure"" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization[END_REF][START_REF] Dauvois | The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling[END_REF][START_REF] Nicholson | Responses to pure antiestrogens (ICI 164384, ICI 182780) in estrogen-sensitive and -resistant experimental and clinical breast cancer[END_REF][START_REF] Mcclelland | Short-term effects of pure anti-oestrogen ICI 182780 treatment on oestrogen receptor, epidermal growth factor receptor and transforming growth factor-alpha protein expression in human breast cancer[END_REF][START_REF] Pietras | Improved antitumor therapy with Herceptin and Faslodex for dual targeting of HER-2 and estrogen receptor signalling pathways in human breast cancers with overexpression of HER-2/neu gene[END_REF][START_REF] Howell | Pharmacokinetics, pharmacological and antitumour effects of the specific anti-oestrogen ICI 182780 in women with advanced breast cancer[END_REF]. The two registration phase-III trials in postmenopausal patients with advanced breast cancer (ABC) following antiestrogen therapy showed that response and progression-free survival (or time to progression, TTP) of fulvestrant and anastrozole were similar [START_REF] Osborne | Double-blind, randomized trial comparing the efficacy and tolerability of fulvestrant versus anastrozole in postmenopausal women with advanced 24[END_REF][14][15]. In ABC, fulvestrant was equally effective when compared to the steroidal AI exemestane after non-steroidal aromatase inhibitor therapy

[16] and also equally effective to tamoxifen in patients without prior endocrine therapy [17]. In vitro, fulvestrant inhibits growth of ER-positive breast cancer cells [18], reduces ER levels [START_REF] Mcclelland | Short-term effects of pure anti-oestrogen ICI 182780 treatment on oestrogen receptor, epidermal growth factor receptor and transforming growth factor-alpha protein expression in human breast cancer[END_REF], and inhibits estrogen-regulated proteins such as PgR, pS2, and Cathepsin D more strongly than tamoxifen [19].

This paper evaluates response to fulvestrant and survival (EFS and OS) in postmenopausal patients with advanced breast cancer receiving fulvestrant within an "In Practice Evaluation Program" (IPEP).

The large (n=848) and clinically representative collective provides sufficient statistical power to model "predictive" marker-treatment interactions and detect uncommon serious adverse events (SAE). The IPEP framework also allows estimation of the frequency of delayed fulvestrant responses (beyond 2 nd examination/later than three months) and the probable consequences of prolonging/discontinuing therapy (based on course of response). These are central issues of clinical concern that have not been adequately addressed and which do not necessarily require a randomized study design.

Methods

Patients and treatment

Postmenopausal patients (n=848) with locally advanced or metastatic breast cancer received fulvestrant (Faslodex ® , AstraZeneca) 250mg via once-monthly intramuscular injections. The study was prospectively documented from

Assessments and analyses

Baseline (1) and follow-up examinations [START_REF] Adamo | Overview and new strategies in metastatic breast cancer (MBC) for treatment of tamoxifen-resistant patients[END_REF][START_REF] Kurokawa | ErbB (HER) receptors can abrogate antiestrogen action in human breast cancer by multiple signaling mechanism[END_REF][START_REF] Howell | The future of fulvestrant ("Faslodex")[END_REF] were scheduled at 3-month intervals for 9 months; within the IPEP, longer surveillance times also occurred. Response was categorized by individual physicians according to current guidelines (www.ago-online.de). AEs and SAEs were collected at each examination and coded according to MedDRA version 9.0. Multiple AEs occurring in a given patient within one organ system were counted once.

Baseline disease characteristics and detailed therapeutic history were documented; number and type of prior endocrine regimens, chemotherapy regimens, total (endocrine plus chemotherapy) regimens, and further information were computed from this data; numbers include adjuvant plus palliative therapies unless otherwise stated.

Concomitant medications counted as one line of treatment; interrupted regimens also counted only once. Binary (1/0) indicators were defined for "any past AI", "any past tamoxifen", and "past AI with no tamoxifen". Binary indicators were also defined describing presence or absence of metastatic disease at the following sites: bone, visceral, soft tissue, distant without bone, only bone, any distant, locally advanced and "other" (unknowns classified absent). The total number of fulvestrant injections received was denoted "fulvestrant cycles"; a binary indicator was defined using the media number of cycles (>7 vs. 7 cycles, representing 6 months of treatment versus shorter).

Statistical analyses

Endpoints were overall survival, event-free survival, and best response to fulvestrant. Duration of overall survival (OS) and event-free survival (EFS) were defined from the time of first fulvestrant injection. To calculate the event-free survival interval, "events" refer to premature termination due to SAE, disease progression (PD), or death. Patients terminating for other reasons were considered rightcensored. Response proportions are given with respect to the entire study population (n=848).

The impact of various factors on EFS and OS was determined using Cox proportional hazards models (stepwise forward unless otherwise stated); OS was estimated using the product-limit method (log-rank test). Weibull distributions were also fitted to estimate median OS and EFS when most patients were censored before median survival was reached in Kaplan-Meier estimates.

Chi-square or Fisher"s exact test were used to analyze relationships between categorical variables.

The T-test (or the Mann-Whitney U-test) was used to assess relationships between binary and metric variables. Spearman (rank) correlations are denoted R S . Responses were coded on an ordinal scale: 0=unknown, 1=PD, 2=stable disease (SD), 3=partial response (PR), 4=complete response (CR).

Binary SAS and SPSS were used to perform these statistical analyses; 95% confidence intervals were reported; all p-values are two-sided.

Results

Patient characteristics and treatment

Baseline characteristics are shown in Table 1

Initial predictors of response

Chemotherapy pretreatment was unfavorably associated with best response to fulvestrant (p = 0.017; Fisher), with higher progression (18.6% : 11.8%) and lower CR (7.2% : 13.7%) rates; patients with prior endocrine therapy had higher response (PR+CR) rates than those without (34.5% : 17.1%; p = 0.002).

A response during last treatment significantly predicted response to fulvestrant: OR = 3.01 (2.15-4.23 , p <0.001); R S = 0.3 (p <0.001) (Table 2).

Course of response

Best response was delayed (beyond 2 nd examination) in 115 patients (see Table 2). Remarkably, 18%

(84/459) of patients with stabilization at 2 nd examination subsequently achieved partial or even complete remission; of those with >7 cycles (about 64%), 26% improved, compared to 4.8% of the others (p <0.001).

Association between number of fulvestrant cycles and response

Number of fulvestrant cycles were strongly associated with benefit [PR, CR, SD], as one would expect.

Treating response as an ordinal variable, number of fulvestrant cycles also correlated with response (R S = 0.42, p <0.001). Dichotomizing the treatment variable (>7 vs. <=7 cycles) yielded log(OR) = 3.8

(3.1-4.5); fractionally ranking fulvestrant cycles yielded log(OR) = 9.8 (8.4-11.2), implying, e.g., OR >100 for "benefit" in a patient receiving nine fulvestrant cycles compared to four. An ROC curve for "benefit" as a function of cycles had AUC = 0.93 (0.90-0.95).

Obviously, this association partially reflects the fact that patients with favorable outcome will be more likely to continue fulvestrant. However, it is truly noteworthy that "fulvestrant cycles" strongly predicted subsequent remission [log(OR) = 4.1 (2.9-5.4)] in the key subgroup (n=459) of patients with SD at 2 nd examination.

Overall survival

Overall, 84 deaths were recorded, 66 within 9 months. Estimated 9-month OS (including censoring) was 89.1% (86.5%-91.8%); median OS was estimated at 26 months (Weibull fit). OS was strongly associated with best response to fulvestrant [HR=0.3 (0.2-0.45) per ordinal level] (Figure 1).

In univariate models, higher number of fulvestrant cycles, fewer previous therapy lines, better response to last therapy, and higher BMI were favorable for OS; locally advanced disease, visceral or exclusively bone metastases were unfavorable (Table 3, left).

In multivariate analysis, fulvestrant cycles dominated all other factors in the whole collective (Table 3, top right) and in subgroups (SD at second examination, CB by second examination). Patients with SD at 2 nd examination who received >7 fulvestrant cycles had an HR of 0.120 (0.054 -0.267) for OS, with visceral metastases having an unfavorable effect on outcome.

As the number of fulvestrant cycles was determined not by randomization, but by course of response, Table 3 (bottom right) also presents multivariate analyses of OS, restricted to factors known initially (i.e., excluding fulvestrant cycles). All significant univariate factors were independent multivariate predictors for OS in this analysis.

Disease progression and event-free survival

Overall, 532/848 (uncensored) patients had CB, i.e., were known to be progression-free for 24 weeks; 167 progressed (one with subsequent response) or died within 24 weeks. Events were recorded in 265/848 patients. Median EFS was 12 months (Weibull and Kaplan-Meier estimators).

In addition to number of fulvestrant cycles, earlier therapy line and negative PgR status appeared to be independently favorable for EFS (Table 4). There were no significant associations between EFS and BMI, locally advanced disease, bone metastasis (only), line of endocrine therapy, line of AI therapy, soft tissue metastasis, breast cancer survival, HER2 status or ER status. Since PgR status entered the multivariate model despite lacking univariate significance, receptor-treatment interactions were tested for: an interaction between PgR status and number of fulvestrant cycles was detected (HR = 0.22).

Noting HR = 2.85 for PgR status and HR = 0.06 for number of fulvestrant cycles, and comparing with the other multivariate EFS model, the implication is that while EFS generally improved with prolonged fulvestrant treatment, PgR-positive patients benefited most. The PgR-positive and PgR-negative groups were well-balanced in terms of the number of fulvestrant cycles received; qualitatively similar findings were observed grouping "unknown" with positive PgR status. No interactions were detected involving either ER or HER2 status.

In the subgroup of patients with SD by 2 nd examination (3 months), patients receiving >7 cycles had a HR of 0.479 (0.294 -0.782) for EFS, with visceral metastases having an unfavorable impact.

Safety and Tolerability

Seven SAEs were recorded, none of which were judged to be attributable to fulvestrant. Two SAEs resulted in subsequent death (pulmonary embolism, pneumonia); one non-fatal stroke and one nonfatal heart attack also occurred. Treatment tolerability was assessed by patients and physicians for both the patient"s last breast cancer therapy and for fulvestrant (Table 6). Patient and physician ratings were strongly correlated (R S > 0.87; p <0.001), with slightly higher physician-rated tolerability. Very good tolerability was reported in 41.4%

to 47.5% of patients receiving fulvestrant (46.7% to 51.6% of physicians) compared to 20.0% for last therapy (18.0% of physicians). The percentages of patients (or physicians) reporting "insufficient" tolerability of fulvestrant was 1.5% and the percentages reporting "good" or "very good" exceeded 90%.

Almost half of all patients reported an improvement in tolerability of fulvestrant compared with their last therapy (Table 7). Tolerability at 2 nd examination was also positively associated with EFS: Combining the "good" and "very good" categories, the HR associated with each degree of improvement (e.g., "insufficient"  "satisfactory" etc.) was 0.570 (0.425 -0.765, P <0.001). This association may be partly attributable to the correlation (R S = 0.14, P <.001) between tolerability (ordinal scale) and number of fulvestrant cycles; in the metastatic setting, patient-reported tolerability may also reflect palliative benefits and thus quality of life.

Discussion

Due to its efficacy even after failure of other endocrine agents, fulvestrant represents a valuable treatment option for patients with advanced breast cancer whose disease is not immediately lifethreatening [START_REF] Fawell | Inhibition of estrogen receptor-DNA binding by the ""pure"" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization[END_REF][START_REF] Dauvois | The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling[END_REF][START_REF] Nicholson | Responses to pure antiestrogens (ICI 164384, ICI 182780) in estrogen-sensitive and -resistant experimental and clinical breast cancer[END_REF][START_REF] Mcclelland | Short-term effects of pure anti-oestrogen ICI 182780 treatment on oestrogen receptor, epidermal growth factor receptor and transforming growth factor-alpha protein expression in human breast cancer[END_REF][START_REF] Pietras | Improved antitumor therapy with Herceptin and Faslodex for dual targeting of HER-2 and estrogen receptor signalling pathways in human breast cancers with overexpression of HER-2/neu gene[END_REF][START_REF] Howell | Pharmacokinetics, pharmacological and antitumour effects of the specific anti-oestrogen ICI 182780 in women with advanced breast cancer[END_REF]. Previous results [21] demonstrated the importance of allowing sufficient time (>2 months) for evaluating response to fulvestrantfor example, a patient may still respond despite intermittent rises in CA15.3. In phase III trials, time to response was 3.1 months for Fulvestrant (range 0.9 -33.1 months) [22]. The present single-arm study comprises the largest available data set for studying fulvestrant in advanced breast cancer, providing more power for subgroup and multivariate interaction analyses.

Analysis of the degree to which response and survival are positively influenced by fulvestrant therapy requires careful consideration of the present study design: Since this is an observational study, a "feedback" process is possible, i.e., duration of fulvestrant therapy is influenced by response, so that a cause-and-effect or dose-response relationship cannot be inferred. The "best" responders (PR or CR within 3 months) tended to prolong fulvestrant treatment, receive more cycles, and benefit most.

Patients without benefit by 3 months tended to discontinue fulvestrant and almost never improved even if they had subsequent fulvestrant treatment.

However, in "intermediate" responders -the 459 patients with SD within three months, but not yet partial or complete response -the decision to prolong fulvestrant therapy was split (64% received >7 cycles). It truly noteworthy that in this subgroup, number of fulvestrant cycles (i.e., continuation of fulvestrant after SD at 3 months) was strongly associated with subsequent response (OR 60). For example, a remission occurred in approximately one-quarter of intermediate responders receiving >7 fulvestrant cycles, compared with about one-twentieth of those receiving 7 cycles. Moreover, intermediate responders receiving >7 cycles had a substantial advantage in OS and EFS. The survival benefit of receiving more fulvestrant cycles was almost as strong in patients with (only) SD by 3 months as it was in the overall population. Summarizing, in the subgroup of "intermediate responders," the inference of a positive dose-response relationship does appear to be the most likely explanation of the data.

Considering the substantial pre-treatment and other patient characteristics, the estimated 9-month OS of 89% (median OS 26 months) compares favorably with the 24-month median OS considered typical for patients metastatic breast cancer. Estimated median EFS was 12 monthswhich also gives a conservative estimate of median TTP. These estimates underline the benefits of fulvestrant for patients with hormone-sensitive, metastatic breast cancer.

Seven SAEs were recorded, none of which were attributable to fulvestrant; and no new or unexpected safety issues arose. An advantage of the intramuscular mode of administration of fulvestrant is that compliance is not an issue. Over 90% of both patients and physicians rated fulvestrant tolerability as "very good" or "good" throughout the study. Moreover, almost half of all patients reported improved tolerability of fulvestrant compared with their last therapy. It is also noteworthy that tolerability was predictive of improved EFS. This relationship could be explained by the likelihood of receiving more cycles, which was strongly associated with improved EFS and OS.

A patient"s response to fulvestrant reflects multiple dynamically interacting and competing biological processes regulating cancer cell growth in that individual. Previous trials have shown that fulvestrant is active in patients with visceral metastases [23][24][START_REF] Mlineritsch | Fulvestrant ('Faslodex') in heavily pretreated postmenopausal patients with advanced breast cancer: single centre clinical experience from the compassionate use programme[END_REF][START_REF] Mauriac | Activity of fulvestrant versus exemestane in advanced breast cancer patients with or without visceral metastases: data from the EFECT trial[END_REF]. However, direct indicators and surrogates for disease aggressiveness would still be expected to exert some negative influence on response and survival. Thus, presence of visceral metastases had negative impact on OS and EFS (in all patients and in the subgroup with SD at 3 months). The (positive) association of "fewer lines of previous therapy" with EFS and OS and the (negative) association of "previous chemotherapy" with fulvestrant response could also be attributable to their association with disease aggressiveness, as evidenced by the correlation of visceral metastases with chemotherapy. In contrast, "best response to previous therapy" was a strong predictor of response to fulvestrant, EFS and OS, but was not strongly associated with disease aggressiveness markers. This observation is consistent with results from the EFECT trial [16,[START_REF] Chia | Fulvestrant: expanding the endocrine treatment options for patients with hormone receptor-positive advanced breast cancer[END_REF]. Further research is required to permit a better understanding of the relationship between response to previous therapy and response to fulvestrant.

Here, for the first time, a predictive interaction between PgR status and number of fulvestrant cycles was identified in the multivariate analysis of EFS. A previous report [20] based on a study of 155 metastatic breast cancer patients did not find an interaction of this type. However, the effect size observed here would not have been significant in a 155-patient study, because the confidence interval for log(HR) would have been about 2.3 times wider, thus overlapping zero; hence the apparent difference could be attributable to increased power. Interpretation of the predictive interaction requires consideration of the multivariate model (Table 4) as a whole: In patients receiving fewer fulvestrant cycles, EFS was worse in PgR-positive than in PgR-negative patients (controlling for line of therapy and presence of visceral metastases). In the whole collective, EFS improved with number of fulvestrant cycles, but the observed benefit was greater in PgR-positive patients -more than enough to compensate for the "disadvantage" of PgR-positivity.

Considering the biological role of PgR regulation in fulvestrant efficacy, the possible increased benefit in patients with positive PgR status could provide an interesting hypothesis for future controlled studies (with centralized receptor determination). In view of the association of molecular types with clinical outcomes [START_REF] Sorlie | Repeated observation of breast tumor subtypes in independent gene expression data sets[END_REF], we note that the predictive value of presence or absence of PgR could also hint at a fundamental difference between luminal A vs. luminal B type tumors even in the metastatic context.

While factors and surrogates for disease aggressiveness (e.g., visceral metastases, previous chemotherapy, etc.) were negatively associated with response, patients responding at all (stabilization or better) by three months had substantial survival benefit from continued fulvestrant treatment. This statement applies to patients with previous chemotherapy, aromatase inhibitor therapy (whether pretreated by tamoxifen or not). Notwithstanding, our data suggest that it would be prudent to plan therapy strategies in advanced and metastatic breast cancer by providing fulvestrant as an additional endocrine therapy line to patients at a sufficiently early stage so as to improve the chances of responding. This strategy is also supported by recent health economic data [START_REF] Lux | Cost-utility analysis for advanced breast cancer therapy in Germany: results of the fulvestrant sequencing model[END_REF].

In this IPEP, fulvestrant therapy was usually terminated in patients with PD at three months and continued in patients with remission (CR or PR) at this time. Current clinical practice is supported by our data in these patient groups. However, in patients achieving "only" SD by three months (who constituted the majority) fulvestrant therapy was terminated in over 30% of cases. The present results

suggest that continuation of fulvestrant therapy in all patients achieving SD at three months is likely to benefit these patients in terms of (possibly delayed) remission, quality of life, and survival. It would be valuable to validate these findings in the ongoing clinical fulvestrant trials [START_REF] Freedman | Predicting benefit from fulvestrant in pretreated metastatic breast cancer patients[END_REF][START_REF] Mello | Prolonged time to progression with fulvestrant for metastatic breast cancer[END_REF]. Moreover, recent trial results with a higher dose of fulvestrant (500 mg) suggest that the observed effects in responders may even be greater when fulvestrant is administered at the higher dose [START_REF] Robertson | Activity of fulvestrant 500 mg versus anastrozole 1 mg as first-line treatment for advanced breast cancer: results from the FIRST study[END_REF][START_REF] Leo | Results of the CONFIRM Phase III Trial Comparing Fulvestrant 250 mg With Fulvestrant 500 mg in Postmenopausal Women With Estrogen Receptor-Positive Advanced Breast Cancer[END_REF].

breast cancer progressing on prior endocrine therapy: Results of a North American trial. J Clin

Figure

Figure 1 OS by best response to fulvestrant; 9-month OS in each subgroup was estimated using the product limit method (p<0.001): response unknown (50%), PD (76%), SD (92%) PR (98%), CR (100%).

PD unknown PD unknown

  A, Robertson JF, Quaresma Albano J et al (2002) Fulvestrant, formerly ICI 182,780, is as effective as anastrozole in postmenopausal women with advanced breast cancer progressing after prior endocrine treatment. J Clin Oncol 20:3396-3403 15. Robertson JF, Osborne CK, Howell A et al (2003) Fulvestrant versus anastrozole for the treatment of advanced breast cancinoma in postmenopausal women: a prospective combined analysis of two multicenter trials. Cancer 98:229-238 16. Chia S, Gradishar W, Mauriac L et al (2008) Double-blind, randomized placebo controlled trial of fulvestrant compared with exemestane after prior nonsteroidal aromatase inhibitor therapy in postmenopausal women with hormone receptor-positive, advanced breast cancer: results from EFECT. J Clin Oncol 26:1664-1670 17. Howell A, Robertson JF, Abram P et al (2004) Comparison of fulvestrant versus tamoxifen for the treatment of advanced breast cancer in postmenopausal women previously untreated with endocrine therapy: a multinational, double-blind, randomized trial. J Clin Oncol 22:1605-1613 18. Hu XF, Veroni M, De Luise M et al (1993) Circumvention of tamoxifen resistance by the pure antiestrogen ICI 182,780. Int J Cancer 55:873-876 19. Freddie CT, Christensen GL, Lykkesfeldt AE (2004) A new MCF-7 breast cancer cell line resistant to the arzoxifene metabolite desmethylarzoxifene. Mol Cell Endocrinol 220:97-107 20. Bartsch R, Wenzel C, Altorjai G et al (2007) Her2 and progesterone receptor status are not predictive of response to fulvestrant treatment. Clin Cancer Res 13:4435-4439 21. Robertson JF, A. Agrawal A, Gutteridge E et al (2006) Correlation of CA15.3 levels with clinical response in advanced breast cancer (ABC) patients receiving fulvestrant. J Clin Oncol 24:18S (abstr 641) 22. Dodwell D, Pippen J (2006) Time to response: Comparison of fulvestrant and oral endocrine agents. Clin Breast Cancer 7:244-247 23. Mauriac L, Pippen JE, Quaresma Albano J et al (2003) Fulvestrant (Faslodex) versus anastrozole for the second-line treatment of advanced breast cancer in subgroups of postmenopausal women with visceral and non-visceral metastases: combined results from two multicentre trials. Eur J Cancer 39:1228-1233

  Table 5 summarizes all non-serious AEs occurring among the 848 patients included in this study. No new or unexpected safety issues arose. Of the 84 patients who died during this study, heart failure/insufficiency was explicitly cited in the deaths of six patients; one of these six patients had suffered a previous AE attributed to fulvestrant (urticaria). In all, nine of the 84 patients who died had a documented AE; of these, the only AE attributed to fulvestrant was the

aforementioned urticaria. Breast cancer was the reported cause of 69/84 deaths; of the remaining 15 deaths, the reported causes were as follows: organ failure (heart/cardiovascular, kidney, liver, lungs/pneumonia, multiple) and general (general worsening, cachexia, unknown); there was no evidence that any of the deaths were attributable to fulvestrant.

Table 1

 1 Patient baseline characteristics: a) Distributions of metric variables; b) Tumor characteristics:

	Tables
	metastases and hormone receptors
	a)
	Metric variables				Total
			Mean	SD	N valid
	Age [years]			64.4	10.4	836
	Body Mass Index [kg/m^2]			26.4	4.6	824
	Survival (from original breast cancer diagnosis to study begin) [months]	93.4	68.4	788
	Duration of response to last therapy [months]		18.3	19.7	575
	Number of prior therapy regimens		4.0	2.1	848
	b)
	Sites of	Patients, n (%)
	metastases
	Category	Present	Absent or unknown
	Local advanced	324 (38.2)	524 (61.8)
	Any distant	796 (93.9)	52 (6.1)
	Visceral	321 (37.9)	527 (62.1)
	Bone	486 (57.3)	362 (42.7)
	Soft tissue	161 (19.0)	687 (81.0)
	Other distant
	metastases	133 (15.7)	715 (84.3)
	Only bone	215 (25.4)	633 (74.6)
	Distant without bone	252 (29.7)	596 (70.3)
	Receptor status	Patients, n (%)
	ER	Negative	27 (3.2)
		Unknown	23 (2.7)
		Positive	798 (94.1)
	PgR	Negative	116 (13.7)
		Unknown	40 (4.7)
		Positive	692 (81.6)
	HER2	Negative	345 (40.7)
		Unknown	333 (39.3)
		FISH +	56 (6.6)
		IHC+++	114 (13.4)
	ER = estrogen receptor; PgR = progesterone receptor; HER2 = human epidermal growth factor
	receptor 2

Table 2

 2 Best response (BR) to fulvestrant vs BR to last therapy, BR at second examination or BR at 3rd examination

				Best response to fulvestrant
			unknown	PD	SD	PR	CR	Total
		Unknown	36	18	59	24	13	150
		PD	6	11	22	6	1	46
	Best response to last therapy	SD	32	63	193	62	10	360
		PR	22	23	95	65	13	218
		CR	4	6	19	20	25	74
		Unknown	100	10	13	3	0	126
	Response	PD	0	111	0	1	0	112
	to fulvestrant (2 nd	SD	0	0	375	73	11	459
	examination)	PR	0	0	0	100	14	114
		CR	0	0	0	0	37	37
		Unknown	100	2	2	0	0	104
	Best response	PD	0	119	0	0	0	119
	to fulvestrant (up to 3 rd	SD	0	0	386	24	3	413
	examination)	PR	0	0	0	153	5	158
		CR	0	0	0	0	54	54
	Totals		100	121	388	177	62	848
	PD = disease progression; SD = stable disease; PR = partial response; CR = complete response

Table 3

 3 Proportional hazard models for OS. Entered but not significant: age, antihormonal therapy line, bone metastases, soft tissue metastases, time since breast cancer diagnosis, HER2, PgR, ER, interactions of fulvestrant cycles with PgR, ER, HER2. Left: univarate.

	Right: two multivariate models

Table 4

 4 Upper left: Univariate hazard ratios of factors for EFS in forward stepwise Cox regression; upper right: multivariate model excluding fulvestrant. Lower left: multivariate EFS model including fulvestrant; Lower right: multivariate receptor interaction model by backward stepwise Cox regression

						Multivariate
	EFS		Univariate			(excluding fulvestrant;
						other factors entered)
	Factors	P	HR (95% CI)	ln (HR)	P	HR (95% CI)	ln (HR)
	Fulvestrant therapy:
	number of cycles (fractionally ranked)	<0.001	0.015 (0 .009 -0.025 )	-4.20		not entered
	Line of therapy (number)	<0.001 1.14 ( 1.08 -1.20 )	0.13	<0.001 1.11 ( 1.05 -1.17 )	0.10
	Line of palliative hormonal therapy	0.003 1.18 ( 1.06 -1.31 )	0.16		not
	Palliative AI therapy	0.047 1.30 ( 1.00 -1.69 )	0.27		not significant
	Response to last therapy (scaled 1 to 4)	0.002 0.85 ( 0.77 -0.94 )	-0.16	0.002 0.84 ( 0.75 -0.94 )	-0.18
	Visceral metastases (present vs absent)	0.001 1.52 ( 1.19 -1.94 )	0.42	0.013 1.41 ( 1.08 -1.84 )	0.34
	Bone metastases (present vs absent)	0.014 1.38 ( 1.07 -1.78 )	0.32		not significant
	Binary PgR status
	(positive vs negative or		not significant			not significant
	unknown)
	EFS	Multivariate (fulvestrant & factors entered)	Multivariate (fulvestrant, factors & receptor interactions entered)
	Factors	P	HR (95% CI)	ln (HR)	P	HR (95% CI)	ln (HR)
	Fulvestrant therapy:
	number of cycles	<0.001 0.02 ( 0.01 -0.03 )	-4.18	<0.001 0.06 ( 0.02 -0.20 )	-2.90
	(fractionally ranked)
	Line of therapy (number)	0.012 1.07 ( 1.02 -1.13 )	0.07	0.040 1.06 ( 1.00 -1.12 )	0.06
	Visceral metastases (present vs absent)		not significant		0.080 1.27 ( 0.97 -1.66 )	0.24
	Binary PgR status
	(positive vs negative or	0.007 1.70 ( 1.16 -2.50 )	0.53	0.002 2.85 ( 1.49 -5.46 )	1.05
	unknown)
	Interaction: binary PgR
	status * fractionally ranked		not entered		0.040 0.22 ( 0.05 -0.94 )	-1.49
	fulvestrant cycles

Table 6

 6 Tolerability of previous therapy and fulvestrant as reported by patients and physicians

	Tolerability	Last therapy			Fulvestrant
				Examination 2	Examination 3	Examination 4
		Patient	Physician	Patient	Physician	Patient	Physician	Patient	Physician
	Very good	20.0%	18.0%	41.4%	46.7%	43.1%	47.3%	47.5%	51.6%
	Good	47.0%	42.2%	48.8%	47.1%	49.4%	47.5%	46.1%	44.2%
	Satisfactory	25.1%	27.5%	8.4%	5.4%	5.9%	4.4%	5.0%	3.3%
	Insufficient	8.0%	12.3%	1.4%	0.8%	1.5%	0.8%	1.4%	0.9%
	Number reporting	817	796	773	777	591	590	423	430

Table 7

 7 Improvement of tolerability reported by patients from last therapy to fulvestrant at 2

	nd

Acknowledgement

The authors would like to thank Silke Zaun MD, PhD from AstraZeneca for supplying the patient data, for very useful discussions, and for editing support. Dawn Batty, PhD from Complete Medical Communications provided editing support, funded by AstraZeneca. Approval of the final manuscript rested with the authors, who are responsible for all scientific content.

Renal and urinary disorders 4 0 Surgical and medical procedures 3 0

Immune system disorders 1 0

Reproductive system and breast disorders 1 1

Total 244


MD Berit Andersen

epidemiologist, Ph.D Irene Van Valkengoed

Ineta Sokolowski

MD Jens K Møller

MD Lars Østergaard

Professor (5 Frede Olesen

Impact of intensified testing for urogenital Chlamydia trachomatis infections: A randomized study with nine year of follow-up by

Keywords: Chlamydia trachomatis, pelvic inflammatory disease, epididymitis, random allocation, infertility, ectopic pregnancy

Background

DNA amplification assays are increasingly being used to facilitate testing of asymptomatic individuals for urogenital Chlamydia trachomatis. The long-term clinical benefit in terms of avoided infertility and ectopic pregnancy is unknown.

Methods

In 1997, a total of 15,459 women and 14,980 men aged 21-23 were living in Aarhus County, Denmark. A random sample of 4,000 women and 5,000 men were contacted by mail and offered the opportunity to be tested for C. trachomatis by means of a sample obtained at home and mailed directly to the laboratory. The remaining 11,459 women and 9,980 men received usual care and constituted the control population. All men and women were subsequently followed for nine years by use of Danish health registers. We collected data on pelvic inflammatory disease (PID), ectopic pregnancy (EP), infertility diagnoses, in-vitro fertilization (IVF) treatment, and births in women, and on epididymitis in men. The intervention and control groups were compared using Cox regression analyses and the intention-to-screen principle.

Results

Among women, we found no differences between the intervention group and the control group: hazard ratios (95% confidence interval) for: PID, 1.12 (0.70-1.79); EP, 0.97 (0.63-1.51); infertility, 0.87 (0.71-1.07); IVF treatment, 0.88 (0.62-1.26); and births, 1.02 (0.95-1.10). In men, the hazard ratio for epididymitis was 1.25 (0.70-2.24).

Conclusions

A population based offer to be tested for urogenital C. trachomatis infection by use of noninvasive samples and DNA-amplification did not reduce the long-term risk of reproductive complications in women or epididymitis in men.

Introduction

C. trachomatis can cause urogenital infections, and might cause pelvic inflammatory disease (PID), ectopic pregnancy (EP), and infertility in women. [START_REF] Westrom | Effect of acute pelvic inflammatory disease on fertility[END_REF][START_REF] Mardh | Chlamydia trachomatis infection in patients with acute salpingitis[END_REF][START_REF] Hillis | Delayed care of pelvic inflammatory disease as a risk factor for impaired fertility[END_REF] The organism is frequently asymptomatic, and the United States Centre for Disease Control and Prevention, [START_REF]Chlamydia screening among sexually active young female enrollees of health plans-United States, 1999-2001[END_REF] Australia, [START_REF] Hocking | Chlamydia screening -Australia should strive to achieve what others have not[END_REF] The Netherlands, [START_REF] Sheldon | Holland plans chlamydia screening in 300,000 young people[END_REF] and England 7 all recommend regular testing of young women using sensitive DNA amplification assays. However, only sparse scientific evidence supports the hypothesis that widespread testing of asymptomatic individuals for C. trachomatis DNA will reduce the risk of later reproductive complications [START_REF] Low | Effectiveness of chlamydia screening: systematic review[END_REF] Two randomized trials reported fewer PID cases after one year of follow-up in high-risk women and high-school women tested once for C. trachomatis. [START_REF] Scholes | Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection[END_REF][START_REF] Ostergaard | Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: a cluster-randomized 1-year follow-up study[END_REF] However, the benefit of widespread testing -in terms of avoidance of long-term reproductive complications such as EP and infertility -has not been studied in controlled intervention trials; nor has the benefit of widespread testing of men. The major obstacle for conducting such studies is that the damage to reproductive organs caused by a C. trachomatis, i.e. EP or infertility, may be noted only several years later, when the woman wants to conceive. This obstacle can be overcome by performing randomized studies in communities with unique personal identification numbers and high-validity registers of discharge diagnoses. The Danish health care system provides this scenario [START_REF] Andersen | The Danish National Hospital Register. A valuable source of data for modern health sciences[END_REF] .

In 1997 we performed a population-based intervention trial in which young men and women were randomized to either an offer to be tested for C. trachomatis by use of a home obtained and mailed sample or usual care [START_REF] Andersen | Population-Based Strategies for Outreach Screening of Urogenital Chlamydia trachomatis infections: A Randomised Controlled Trial[END_REF] . In the present paper we report the effect of this intervention after nine years of follow-up in terms of PID, EP or infertility in women and epididymitis in men.

Methods

Study population

The study was carried out in the county of Aarhus, which in 1997 had a population of approximately 650,000 inhabitants (12% of the Danish population). In Denmark, all inhabitants are registered in the county health service register by use of their personal identification number (CPR number). This number, which is unique to every Danish citizen, incorporates sex and date of birth, and permits accurate linkage of information between registers. In the county health service register, we identified the CPR numbers of all women and men who were born in 1974, 1975, or 1976 and were living in the County of Aarhus by 13 October 1997 (aged 21 to 24 at initiation of the study). This age-group was chosen as it had the highest prevalence of infection according to the local register at the Department of Microbiology, Aarhus University Hospital.A total of 15,459 women and 14,980 men fulfilled these inclusion criteria and were included in the study.

Intervention

The intervention was described in an earlier publication [START_REF] Andersen | Population-Based Strategies for Outreach Screening of Urogenital Chlamydia trachomatis infections: A Randomised Controlled Trial[END_REF] and is only briefly described here (Figure 1). In October 1997, a group of 4,000 women and 5,000 men were randomly selected from the study population. The 9,000 individuals were all offered a test for C. trachomatis by use of samples obtained at home. The remaining 11,459 women and 9,980 men constituted the control group. In the period between 31 October 1997 and 6 December 1997, all 9,000 participants in the intervention group received an invitation by direct mail to be tested for C. trachomatis by taking a sample at home and mailing it directly to the diagnostic laboratory. The intervention group was further subdivided into two randomly assigned groups (group 1 and group 2), each containing 2,000 women and 2,500 men. The difference between intervention groups 1 and 2 was that group 1 participants received the test package together with the invitation, whereas group 2 participants had to return a prestamped, preaddressed reply card to the study centre to receive the test package. The participation rates and number of detected infections due to our intervention are described in Figure 1. A total of 1,175 women (29.4%) and 1,033 men (20.7%) were tested as part of the intervention and 84 C. trachomatis infections in women and 60 C. trachomatis infections in men were identified (prevalence 7.1% and 5.8 %, respectively).

Usual care

Individuals in the intervention groups as well as those in the control group had the opportunity of usual care consisting of an endocervical and/or urethral swab sample taken by a physician in his/her office. Free testing is available in Denmark. In the study period of three months 9.4 % of women in the control group and 9.0 % of women in the intervention group were tested as part of usual care. For men the corresponding figures were 1.4 % and 1.5 % for the two groups, respectively (for details see reference 12).

Randomization

A computer-based randomization was performed by administration personnel of Aarhus County.

To check that the randomization of individuals was successful, we collected data on important social characteristics by use of the CPR numbers (both intervention groups and control group).

Data regarding living status, occupation, and country of origin were collected from the governmental agency Statistics Denmark.

Treatment of infected individuals

In the present study infected individuals should contact a general practitioner (GP) for medical treatment and partner notification. In a previous study [START_REF] Østergaard | Efficacy of home sampling for universal screening of Chlamydia trachomatis[END_REF] this procedure facilitated treatment of at least 95% of individuals (unpublished data). From a previous study we also know that more than 85% of C. trachomatis infected individuals seen by a GP in our area receive either medical treatment to current partner immediately (patient expedited partner therapy) or test of current partner is facilitated (by patient referral) [START_REF] Andersen | Urogenital Chlamydia trachomatis infections in general practice: diagnosis, treatment, follow-up and contact tracing[END_REF] .

In the present study all C. trachomatis positive individuals also received a second offer to be tested for the infection by use of a home-obtained and mailed sample 24 weeks after the initial test. A total of 59 women were tested and three were C. trachomatis positive for the second time. The corresponding number in males was that 32 submitted a second sample and one was C. trachomatis positive for the second time [START_REF] Andersen | Follow-up after asymptomatic urogential Chlamydia trachomatis infection. Comparative cohorte study[END_REF] .

Outcomes: PID and epididymitis

The entire study population (comprising individuals who accepted the test offer, those who did not, and the control group) was followed in central governmental registers during the first year after the test offer to assess the rates of PID (women) or epididymitis (men). In Denmark, these patients are treated either in hospitals or in general practices.

Treatment in hospital.

All patients admitted to a hospital in Denmark are registered by their CPR number in the Danish Hospital Discharge Registry and classified according to the Danish versions of the ICD-10 (since 1994). The discharge registry was evaluated previously, and it was found that 87% of the codes for diagnoses were in accordance with the hospital case records for paediatrics, general surgery and gynaecology/obstetrics [START_REF] Andersen | The Danish National Hospital Register. A valuable source of data for modern health sciences[END_REF] . In our study the codes used for PID were: N700, N709, N710, N719, N730, N733, N735, N738, O030, O035, O040, O045, O070, and the codes used for epididymitis were: N450 and N459.

Outpatient treatment -Patients receiving antibiotic treatment outside of hospitals in Denmark are registered by use of the CPR number in Statistics Denmark, according to the type of medication prescribed by the doctor. Outpatient treatment of PID and epididymitis caused by C. trachomatis is mainly handled in general practices. From a minor study performed in our community, we knew that one-tenth of the general practitioners (GPs) would refer a suspected case of PID or epididymitis for treatment in a hospital, and one-third of GPs would treat a PID in their practice by use of doxycycline (an antibiotic that is not used to treat many other conditions in Denmark) (B. Andersen, personal communication). Therefore, we collected data on the use of doxycycline from Statistics Denmark.

For the purposes of this study, we defined PID in women and epididymitis in men as being registered with one of the relevant ICD-10 codes in the Danish Hospital Discharge Registry or receiving doxycycline after having consulted a GP.

Outcomes: in vitro fertilization treatment

Women in Denmark receiving fertility treatment, whether it is in a public or private hospital, are registered in a central registry at the National Board of Health using their CPR number. In this registry, the type of treatment received and the date, place, and results of treatment are registered. All women were followed in this register until the end of 2004 (the latest year of complete data) or until they underwent an in vitro fertilization (IVF) procedure.

Outcomes: EP, female infertility, and births

All of the women in the study population were followed-up via the Danish Hospital Discharge Registry for nine years after randomization. The following outcomes were considered: discharge diagnosis of EP, infertility, or childbirth. The codes used were O00 (all sub-codes) for EP; N97 (all sub-codes) for female infertility; and O80, O81, O82, O83, or O84 (all sub-codes) for childbirth. Women were followed in this register until the end of 2006.

Power calculation

We used a power of 80% and an alpha of 5% to calculate the statistical power. We considered the risk of PID for women in the control group to be 3% [START_REF] Land | Epidemiology of Chlamydia trachomatis infection in women and the cost-effectiveness of screening[END_REF] , and we wanted to be able to detect a 1% minimum reduction in the number of PID cases in the intervention group compared with the control group (intention-to-screen analysis). These assumptions required inclusion of 3,607 women in the intervention group and 10,821 women in the control group, which was less than the total actual number included in our study.

Statistical analyses

For the intention-to-screen analyses, we compared the occurrence of outcomes between those who received no intervention (control group) at baseline and those who received the intervention (intervention group). For this purpose, data regarding the two types of approach strategies were merged. In the Cox regression analysis, individuals were followed from the time of randomization (October 31, 1997) to the first date of an event. If no event occurred, the followup ended on February 5, 1999 for PID and epididymitis (one year after the last test was performed as part of the intervention), by the end of 2004 for the outcome of IVF treatment, and by the end of 2006 for all other outcomes. First, we calculated the number of events per 100 person-years at risk for the total population and for individuals in the intervention group and the control group. The corresponding hazard ratio (control group compared with intervention group) and 95% confidence interval (CI) was calculated for each event. Second, we performed a subanalysis within the intervention group by calculating the events per 100 person-years at risk and the corresponding hazard ratios for participants and nonparticipants. Separate analyses were not conducted for C. trachomatis-positive test results compared with C. trachomatis-negative results, because the numbers of events in each subgroup were too low to allow calculation of hazard ratios.

Stata 9 was used for statistical analysis of data.

Approval

The study was approved by the local Ethical Committee in the County of Aarhus and by the Danish Data Protection Agency.

Results

As seen from Table 1, approximately one fourth of the study population lived alone, one fourth lived with a partner without being married, and one fifth were married. The remaining participants lived with their parents or had other arrangements. The majority were employed, while about 12% were students and 12% received welfare payments. Only 5% were immigrants. These characteristics were well balanced between the intervention and the control group after randomization. For the PID and epidymitis diagnoses, the follow-up time was one year. For IVF treatment, the follow-up time was seven years, and for all other outcomes, the follow-up time was nine years. Cox regression analyses were performed. HR, hazard ratio; IR, incidence rate per 100 person-years at risk.

A subanalysis of those who accepted the test offer and those who did not is shown in Table 3. No statistically significant differences were found for any of the outcomes among women or men. For the PID and epidymitis diagnoses, the follow-up time was one year. For IVF treatment, the follow-up time was seven years, and for all other outcomes, the follow-up time was nine years. Cox regression analyses were performed. HR, hazard ratio; IR, incidence rate per 100 person-years at risk.

Discussion

In this randomized study we included more than 30,000 individuals and offered 9,000 of these the opportunity to be tested for C. trachomatis by use of a non-invasive sample. We found no impact of this intervention on PID, EP, or fertility complications in women or epididymitis in men.

The three major strengths of this study were its randomized design, its long-term follow-up, and the unique Danish citizen numbers that allowed for complete follow-up of the entire study population in high validity national registers [START_REF] Andersen | The Danish National Hospital Register. A valuable source of data for modern health sciences[END_REF] . Population-based randomized studies with long follow up at the level of the individual level is a unique possibility in countries with a registration system as in the Scandinavian countries.

A weakness in our study was the lack of a precise diagnosis for PID. Most PID diagnoses are obtained by clinical examination only, rather than by laparoscopy. There is no doubt that we were not able to include all diagnoses in our study, not even all correct PID diagnoses because the proxy doxycycline was the only measure we had for this diagnosis in general practice. This is also reflected in the fact that an incidence of PID on 3% was not reached in the study as we expected from the power-calculation, which may wrongly result in rejection of the hypothesis that widespread testing prevents PID. However, as this was a randomized study, the impact of this possible lack of consistency probably had an equal effect in both groups. There is a tendency towards a slightly lower incidence of PID in the intervention group compared to the control-group but this was not significant and would not have been significant if the expected incidence was actually reached in the same proportion of individuals. In contrast, the outcome measures of EP, IVF treatment, and delivery were precise, but the relatively short follow-up period for these conditions may result in underestimation of the effect of the intervention .

Partner notification (PN) is important in STI control and lack of PN can possibly eliminate the effect of testing for C. trachomatis since index-patients can very quickly be reinfected. In our study PN was not measured, but only few infected individuals were C.

trachomatis positive six months later. This indicates that lack of PN was not a major problem in our study, but we cannot completely ignore a dilution of a positive effect of the intervention. This is the first study to report the effect of intensified testing for C. trachomatis on long-term reproductive complications EP, infertility, IVF-treatment and birth in women and epididymitis in men. Two earlier studies reported the effect of a single test offer on PID in women within one year after the intervention [START_REF] Scholes | Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection[END_REF][START_REF] Ostergaard | Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: a cluster-randomized 1-year follow-up study[END_REF][START_REF] Østergaard | Efficacy of home sampling for universal screening of Chlamydia trachomatis[END_REF] and both studies reported a reduction in PID incidence in contrary to our study. The study by Scholes et al [START_REF] Scholes | Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection[END_REF] was performed in a high-risk population which, therefore, does not permit its direct comparison with the present study. The study by Ostergaard et al [START_REF] Ostergaard | Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: a cluster-randomized 1-year follow-up study[END_REF] was in high-schools, which is also a low low-risk population, but this study experienced considerable drop-out before follow-up (a drop-out that differed between the intervention group and the control group), and the outcome measures were based on self-reports.

The use of a registry-based design allowed us to overcome these types of bias.

Negative results should always prompt considerations of study design. It our study such a consideration could be that the power of the study was not strong enough, but in comparison with comparable randomised studies [START_REF] Scholes | Prevention of pelvic inflammatory disease by screening for cervical chlamydial infection[END_REF][START_REF] Ostergaard | Home sampling versus conventional swab sampling for screening of Chlamydia trachomatis in women: a cluster-randomized 1-year follow-up study[END_REF] (645 tested women with 44 detected infections and 867 tested women with 43 detected infections), the strength of our study should be sufficient to show an effect for the outcome PID. It could also be argued that only one fourth of the invited individuals in our study participated in the intervention and as in cancer screening studies nonparticipants could be at a higher risk for a complication than participants. However, a comparison of different C. trachomatis screening studies shows that the prevalence tends to decline the higher the participation rate indicating that some kind of self-selection takes place [START_REF]Review of Chlamydia control activities in EU countries[END_REF] .

Also in our analysis of nonparticipants [START_REF] Andersen | Population-Based Strategies for Outreach Screening of Urogenital Chlamydia trachomatis infections: A Randomised Controlled Trial[END_REF] we found that those who declined the invitation to be tested probably had a lower infection risk than those who accepted the invitation. Testing of asymptomatic individuals may give rise to psychosocial problems for the tested individuals [START_REF] Kangas | Psychosocial impact of Chlamydia trachomatis testing in general practice[END_REF][START_REF] Duncan | Qualitative analysis of psychosocial impact of diagnosis of Chlamydia trachomatis: implications for screening[END_REF] and the positive predictive value of a test decreases as the prevalence decreases. Testing asymptomatic individuals for C. trachomatis, benefits and harms should be balanced.

Evidence for the natural history of asymptomatic C. trachomatis infections is clearly not strong [START_REF] Wallace | What is the excess risk of infertility in women after genital chlamydia infection? A systematic review of the evidence[END_REF] . Our knowledge of the association between C. trachomatis infections and PID, EP, and infertility stems from important landmark studies by Weström et al. [START_REF] Westrom | Effect of acute pelvic inflammatory disease on fertility[END_REF][START_REF] Westrom | Pelvic inflammatory disease and fertility. A cohort study of 1,844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results[END_REF] . These studies showed that PID was often followed by fertility complications later in life. However, the studied populations were not divided into patients with C. trachomatis infection and other urogenital tract infections. Subsequently, numerous seroepidemiological studies [START_REF] Hawes | Seroepidemiology of Chlamydia trachomatis infection in infertile women in Melbourne[END_REF][START_REF] Mehanna | Chlamydial serology among patients with tubal factor infertility and ectopic pregnancy in Alexandria, Egypt[END_REF][START_REF] Reniers | Chlamydial antibodies and tubal infertility[END_REF][START_REF] Thejls | Diagnosis and prevalence of persistent chlamydia infection in infertile women: tissue culture, direct antigen detection, and serology[END_REF][START_REF] Brunham | Chlamydia trachomatis-associated ectopic pregnancy: serologic and histologic correlates[END_REF][START_REF] Coste | Sexually transmitted diseases as major causes of ectopic pregnancy: results from a large case-control study in France[END_REF] have supported the idea that asymptomatic infections facilitate fertility complications. However, these studies have a retrospective design and are based on serology, which may have invited erroneous conclusions. [START_REF] Johnsen | Chlamydia antibody response in healthy volunteers immunized with nonchlamydial antigens: a randomized, double-blind, placebo-controlled study[END_REF] . On the contrary, in a small study by Morré et al, [START_REF] Morre | The natural course of asymptomatic Chlamydia trachomatis infections: 45% clearance and no development of clinical PID after one-year follow-up[END_REF] 30 women with an untreated, asymptomatic C. trachmatis infection were followed for a year and none of them developed PID.

In a very recent study by Oakeshott et al [START_REF] Oakeshott | Randomised controlled trial of screening for Chlamydia trachomatis to prevent pelvic inflammatory disease: the POPI (prevention of pelvic infection) trial[END_REF] 2529 women were randomised to either immediately test and treatment or no intervention. Within one year 9.5% of control women compared to 1.6% of women in the intervention group developed PID. This difference seemed apparent but was not statistically significant and the authors conclude that the effectiveness of a single Chlamydia test in preventing PID may have been overestimated. This conclusion corresponds with the findings of our study. It can be suggested that a major study will show an effect of an intervention, but the overall effect of C. trachomatis testing on the development of PID has earlier been overestimated. It may be that the natural history of C. trachomatis infection has changed over time or that the highly sensitive DNA-amplification methods to detect C. trachomatis also detects potentially harmless asymptomatic infections and even dead organisms.

In conclusion, our study found that a single round of testing for C. trachomatis by DNA amplification assay was not associated with clinical benefit in terms of fewer later reproductive complications. This may be so because the new DNA amplification tests are too sensitive and detect infections that would not lead to later complications, or because the natural history of C. trachomatis infections has changed. A major study or several rounds of screening may change the conclusion but based on existing evidence and the results of this study we suggest that the rationale for widespread C. trachomatis testing should be re-evaluated. Possible benefits should be weighed against harms. When screening interventions are considered a key question should be how to evaluate the intended outcomes in order to obtain more answers and add to the evidence base within this field.

Total word count: 3260 (excluding tables)

Key messages:

• A widespread offer in a Danish population aged 21 to 24 years to be tested for C. trachomatis infection by use of a home-obtained and mailed sample was successful in terms of number of tested individuals and detected infections.

• After one to nine years of follow-up the intervention could not be shown to have any impact on pelvic inflammatory disease, ectopic pregnancy or infertility in women or epididymitis in men.

• Long term effect of widespread testing for C. trachomatis is still unknown.

Table 1 :

 1 Living status, occupation and country of origin for all randomised individuals. Data are extracted from Danish registers.

		Intervention	Control	All participants
		n = 9,000	n = 21,439	n= 30,439
		n	%	n	%	N	%
	Living status *
	-Living alone	2,457	27.3%	5,718	26.7%	8,175	26.8%
	-Married	1,644	18.3%	3,759	17.7%	5,403	17.8%
	-Living with partner †	2,218	24.7%	5,493	25.6%	7,711	25.3%
	Occupation *
	-student	1,150	12.8%	2,896	13.5%	4,046	13.3%
	-employed	6,323	70.3%	14,935	69.6%	21,258	69.8%
	-unemployed	354	3.9%	831	3.8%	1,185	3.9%
	-welfare payment ‡	1,104	12.3%	2,647	12.3%	3,751	12.3%
	Origin *
	-Danish	8,411	93.5%	20,067	93.6%	28,478	93.5%
	-Immigrant	467	5.1%	1,122	5.2%	1,589	5.2%
	-Descendant	53	0.6%	120	0.6%	173	0.6%

Table 2

 2 Comparison of long-term effects of C. trachomatis testing in the intervention group vs. the control group (intention-to-screen analysis).

		Control	Intervention
		group	group
		n = 21,439	n = 9,000
		IR	n	IR	n	HR	95% Confidence
							intervals
	Women		11,459		4,000
	PID	0.51	74	0.45	23	1.12	0.70 to 1.79
	EP	0.072	75	0.074	27	0.97	0.63 to 1.51
	Infertility	0.31	322	0.36	129	0.87	0.71 to 1.07
	IVF treatment	0.13	109	0.15	43	0.88	0.62 to 1.26
	Births	3.03	2833	2.97	967	1.02	0.95 to 1.10
	Men		9,980		5,000
	Epididymitis	0.32	40	0.25	16	1.25	0.70 to 2.24

Table 3

 3 Comparison of long-term effects of C. trachomatis testing for participating and non participating individuals in the intervention group.

		Nonparticipants	Participants
		n = 6,792	n = 2,208
							95% Confidence
		IR	n	IR	N	HR	intervals
	Women		2,825		1,175
	PID	0.45	16	0.47	7	0.96	0.40 to 2.34
	EP	0.07	19	0.07	8	1.00	0.44 to 2.28
	Infertility	0.33	85	0.41	44	0.81	0.56 to 1.17
	IVF-treatment	0.15	31	0.14	12	1.09	0.56 to 2.12
	Births	2.87	659	3.19	308	0.90	0.79 to 1.03
	Men		3,967		1,033
	Epididymitis	0.20	10	0.46	6	0.44	0.14 to 1.20

* The numbers do not add up to 100% because of other possible categories and missing data. † Living with partner but not married.

‡

Not including students.

Among the entire population of 15,459 women, 97 had a diagnosis of PID within the first year (6.3 cases per 1,000 women). Twenty-three of these women were admitted to hospital and 74 were treated in general practice. During the entire nine-year study period, a total of 102 women were hospitalized with EP (0.73 cases per 1,000 women), 451 had a diagnosis of infertility (3.2 cases per 1,000 women), and 3,800 gave birth to at least one child (27.3 birth per 1,000 women). During the seven-years follow-up for IVF treatment, 152 received this procedure (1.4 per 1,000 women). During one year of follow-up for the 14,980 men, seven were discharged from a hospital with a diagnosis of epididymitis and 49 received treatment in general practice (3.9 per 1,000 men) (Figure 1).

The intention-to-treat analysis (Table 2) of women revealed no differences between the intervention group and the control group with regard to all outcome measures (hazard ratio [95% CI]): PID, 1.12 (0.70-1.79); EP: 0.97 (0.63-1.51); infertility, 0.87 (0.71-1.07); IVF treatment 0.88 (0.62-1.26); and births, 1.02 (0.95-1.10). In the analysis of men, we found no significant reduction in epididymitis, with a hazard ratio of 1.25 (95% CI: 0.70-2. 24).

Acknowledgements:

This study received financial support from the Danish Medical Research Council (grant no. 22-02-0540), the NOVO foundation and the Research Foundation in Aarhus County. The financial supporters did not have the possibility to influence any parts of the study and the researchers are independent from funders.

There are no conflicts of interest in this study.

Contributions of authors:

All authors participated in design of the study, interpretation of data and editing of the manuscript. BA also ran the intervention and wrote the paper, and IS also performed the statistical analyses.

Statement:

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Maria Conceição Fontes

Maria José Saavedra

Arturo Monera

Conceição Martins

Antonio Martínez-Murcia

Phylogenetic identification of Aeromonas simiae from a pig, first isolate since species description

Keywords: Aeromonas simiae, phylogeny, 16S rDNA, gyrB, rpoD

The species Aeromonas simiae was first described on the basis of two strains (CIP 107798 T and CIP 107797) isolated from faeces of healthy monkeys (M. fascicularis) from Mauritius, which were kept in quarantine in the Centre for Primatology, Louis Pasteur University, Strasbourg, France. In the present study, during a survey to determine the prevalence of Aeromonas at three different pig slaughterhouses at north of Portugal, a single strain MDC2374 (out of 703 isolates) was identified as Aeromonas simiae on the basis of 16S rDNA, gyrB and rpoD sequencing, confirming that traditional biochemical approaches are not resolute to identify rarely isolated Aeromonas species. To our knowledge, this is the first time since species description that A. simiae is newly isolated and identified. .

A c c e p t e d M a n u s c r i p t

Introduction

The taxonomy of the genus Aeromonas is complex and continually changes due to the addition of newly described species and the reclassification or extended description of existing taxa. The genus Aeromonas belongs to the family Aeromonadaceae [START_REF] Colwell | Proposal to Recognize the Family Aeromonadaceae fam. nov[END_REF] and its members are gram-negative, non-spore-forming bacilli or coccobacilli, oxidase and catalase-positive, facultatively anaerobic, resistant to the vibriostatic agent O/129 and generally motile by a polar flagella [START_REF] Martin-Carnahan | GenusI. Aeromonas Stanier 1943, 213 AL[END_REF]. Members of the genus Aeromonas are human pathogens, being associated with a wide spectrum of enteric and non-enteric diseases in [START_REF] Janda | Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions[END_REF]. Aeromonas species are common inhabitants of aquatic ecosystems. They have also been isolated in different kinds of animals and food [START_REF] Isonhood | Aeromonas Species in Foods[END_REF]. Furthermore, Aeromonas spp. have been isolated from pig carcasses and faeces and also from processing equipment [START_REF] Gill | Occurrence of Aeromonas, Listeria and Yersinia in carcass processing equipment at two slaughtering plants[END_REF][START_REF] Saavedra | Phylogenetic identification of Aeromonas strains isolated from carcasses of pig as new members of the species Aeromonas allosaccharophila[END_REF].

Aeromonas simiae was described on the basis of two strains (CIP 107798 T e CIP 107797) isolated from faeces of healthy monkeys (M. fascicularis) from Mauritius, that were kept in quarantine in the Centre for Primatology, Louis Pasteur University, Strasbourg, France. To our knowledge, since its description [START_REF] Harf-Monteil | Aeromonas simiae sp. nov., isolated from monkey faeces[END_REF], no more isolates of this species have been identified. Further studies showed identical gyrB, rpoD and 16S rDNA sequences [START_REF] Saavedra | Update phylogeny of the genus Aeromonas[END_REF] and the same genotyping pattern [START_REF] Figueras | Genotyping of isolates included in the description of a novel species should be mandatory[END_REF], revealing that they are two isolates of a single strain. The report of the ad-hoc committee for the re-evaluation of the species definition in bacteriology encourages microbiologists to base a species description on more than a single strain, recommends the analysis of housekeeping gene sequences and the application of DNA profiling methods [START_REF] Stackebrandt | Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology[END_REF]. Sequencing of gyrB and rpoD has demonstrated that both genes are excellent molecular chronometers for phylogenetic inference in the genus Aeromonas (Yañez et al., 2003;[START_REF] Soler | Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes[END_REF][START_REF] Saavedra | Update phylogeny of the genus Aeromonas[END_REF][START_REF] Küpfer | Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences[END_REF]. In the present study, based on gyrB, rpoD and 16s rDNA gene sequencing, we report the A c c e p t e d M a n u s c r i p t phylogenetic identification of one strain of A. simiae (MDC2374) from pig faeces, isolated in a slaughterhouse in Portugal.

Materials and Methods

Sample collection and isolation of Aeromonas species

A total of 134 samples (44 pig carcasses intended for human consumption, 25 diaphragm muscle, 66 pig faeces, 6 samples of processing equipment and 12 samples of water) were obtained from three different pig slaughterhouses and kept at 4ºC until laboratory examination.

The 66 samples of pig faeces consisted in small portions of ileum with faeces. At the laboratory 25 g of ileum with faeces (after being scalded) were weighted aseptically, homogenised for at least 2 minutes in stomacher bags containing 225 ml of alkaline peptone and incubated 24 h at 30ºC. After enrichment, the homogenate was streaked on glutamate starch phenol-red agar (GSP) agar (Merck) plates. After incubation at 30ºC for 24h, the yellow colonies were isolated and subcultured into tryptone soya agar (TSA) (Oxoid). Oxidase positive colonies were taken as presumptive Aeromonas isolates. Culture collection strains CIP 107798 T and CIP 107797 (MDC55) were purchased from the Spanish Collection of Type Cultures (CECT). Strains were maintained on TSA (Oxoid) plates at 4ºC and were stored in 10 % glycerol at -20ºC.

Biochemical Characterization

The biochemical tests for the strain MDC2374 were determined after overnight growth on TSA plates (Oxoid) and performed using the commercial Kits API 20 E and API 20 NE (BioMérieux) and the automated Vitek system (BioMérieux). The fermentation (acid production) of D-mannitol, L-arabinose, D-cellobiose, D-sucrose and salicin was determined in Andrade peptone water (Sigma). The pink colour of the medium was considered to be a positive reaction.

A c c e p t e d M a n u s c r i p t

DNA extraction and Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR )

DNA extraction was performed by resuspending a single colony from fresh cultures in 100 µl of TE buffer and 200 µl of Chelex (BioRad) were added. Tube were vortexed at high speed for 1 min, incubated at 96ºC for 10 min and kept at -20ºC for 10 min. This process was repeated three consecutive times. The tube was again vortexed and centrifuged for 5 min at 12 000 g. The supernatant was transferred to a fresh tube and stored at -20ºC.

All isolates from the same sample were subjected to ERIC-PCR in order to recognize identical strains. Isolates showing different ERIC-PCR patterns were subjected to further phylogenetic analysis. The oligonucleotides primers used for ERIC-PCR were ERIC 1R (5' -ATG TAA GCT CCT GGG GAT TCA C-3') and ERIC 2 (5' -AAG TAA GTG ACT GGG GTG AGC G-3') (Versalovic et al., 1991). PCR amplifications were performed in a total volume of 25 µl, containing 2,5 µl of 10x PCR buffer, 50 pmol of each primer, 400 µM of dNTP`s, 1 µl (1U) of taq DNA polymerase and 2,5 µl (50 ng) of template DNA. The reaction mixtures were subjected, on a MJ Research PTC100 thermal cycler, to a initial denaturation for 4 min at 94 ºC followed by 35 cycles, with a thermal cycling of 94 ºC for 15 s, 55 ºC for 30 s, 72 ºC for 45 s and a single final extension for 5 min at 72 ºC. Following amplification, 8 µl of the PCR products were electrophoresed on 1.2 % agarose gels in TAE buffer. The 1 kb DNA ladder was used as molecular size marker. Agarose gels were stained on ethidium bromide, visualized with an ultraviolet lamp and photographed in the GelDocMega cabinet.

PCR amplification and sequencing of gyrB, rpoD, and 16S rDNA

PCR amplification and procedures for sequencing of gyrB, rpoD and 16S rRNA genes were performed at the Molecular Diagnostics Center (MDC), as previous described (Martínez-Murcia et al. 1999;[START_REF] Soler | Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes[END_REF]), except for using the BigDye Terminator V3.1 Cycle A c c e p t e d M a n u s c r i p t

Phylogenetic data analysis

The nucleotide sequences were aligned by the Clustal X program version 1.8 [START_REF] Thompson | The CLUSTAL X window interface: flexible strategies for multiple sequences alignment aided by quality analysis tools[END_REF]. For alignments, previously published reference sequences (Martinez-Murcia et al. 1992a;Martínez-Murcia 1999;Yañez et al., 2003;[START_REF] Soler | Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes[END_REF][START_REF] Saavedra | Update phylogeny of the genus Aeromonas[END_REF] were used. Genetic distances were obtained by Kimura's 2 parameter model [START_REF] Kimura | A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]) and evolutionary tree was constructed by the Neighbour-Joining method (Saitou and Nei 1987) with the Mega program [START_REF] Kumar | MEGA2: Molecular Evolutionary Genetics Analysis software[END_REF].

Results and discussion

In this survey, Aeromonas spp. was detected in 104 out of 134 samples collected in the pig slaughterhouses. A total of 480 Aeromonas were isolated from pig carcasses, diaphragm muscle, dehairing equipment and water, but also 223 Aeromonas isolates were obtained from pig faeces (data not shown). To avoid redundancy, isolates were subjected to ERIC-PCR analysis and 66 strains, showing different genetic patterns from each other, were selected for phylogenetic classification by gyrB gene sequencing. The obtained gyrB phylogenetic analisys showed that strain MDC2374 clustered with the type strain of the species Aeromonas simiae. For this strain, nucleotide sequences of 16S rRNA, rpoD amplicons were also obtained. Experiments were repeated at least twice from single colonies of original cultures to confirm readings and to solve ambiguities. The derived rpoD sequences were concatenated with these of gyrB, aligned with sequences from reference strains of all Aeromonas species described to date, including the culture collection strains CIP 107798 T and CIP 107797, and a phylogenetic tree was constructed (Fig. 1). The gyrB and rpoD sequences obtained (GenBank accession numbers GQ860942 and A c c e p t e d M a n u s c r i p t GQ860943, respectively) from this isolate showed seventeen and two differences, respectively, with these of A. simiae reference strains (CIP 107798 T and CIP 107797), This result confirmed that isolated strain MDC2374 represents a new member of the species Aeromonas simiae.. The strain obtained in this study and the culture collection strains CIP 107798 T and CIP 107797 were also subjected to 16S rDNA sequencing (approx. 1400 bp) to check previously reported data and derived phylogenetic 16S rDNA tree (Fig. 2) showed this strain belongs to A. simiae. Sequence of strain MDC2374 (GenBank accession number GQ860944) presented a variation at 9 nucleotide positions with A. simiae reference strains (Table 1). The 16S rDNA sequences from strains CIP 107798 T and CIP 107797 were identical (GenBank accession number GQ860945), supporting data previously published [START_REF] Saavedra | Update phylogeny of the genus Aeromonas[END_REF].

Biochemically, MDC2374 differs from previously described mesophilic Aeromonas species, except for A. schubertii, by its inability to produce indole, gas from glucose and acid from D-mannitol, in agreement to Harf-Monteil and collaborators ( 2004) respect to type strain CIP 107798 T . Moreover, strain MDC2374 may be differentiated from A. schubertii by its ability to produce acid from D-cellobiose and D-sucrose, butalso its inability to produce acetoin from sodium pyruvate (Voges Proskauer test negative).

To our knowledge, it is the first time that A. simiae is newly identified since first description. The results demonstrate that housekeeping gene sequencing is invaluable in the finding of strains belonging to rarely isolated Aeromonas species such as A. simiae, and confirmed its potential as a rigorous frame for Aeromonas taxonomy. The finding of a new A. simiae strains would allow establishing a phylogenetic cluster of the species A. simiae more diverse and robust. Table 1

Figure 1 :

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 1 A

 1 Figure 1

Table 1 . Nucleotide differences in the 16S rDNA gene sequences of Aeromonas simiae strains.

 1 Following the E.coli numbering system. 1 Strain CIP 107797 showed identical sequence.

	Strain	Positions at 16S rDNA*
		250 456 459 461 469 471 472 473 476 647
	1 CIP 107798 T	T	T	G	A	A	T	G	C	G	C
	MDC2374	A	G	T	G	T	C	A	G	T	T

*

Acknowledgements

The MDC work has been supported by Grant IMIDTA/2007/68 from IMPIVA, Generalitat Valenciana, Spain. M.J. Saavedra and M.C. Fontes were the recipients of grants SFRH/BSAB/774/2008 and SFRH/BD/25415/2005, respectively, from theFundação para a Ciência e Tecnologia. Part of this work was also supported by funds from the European Commission for the HEALTHY WATER Project (FOOD-CT-2006-036306). However, the authors are solely responsible for the content of this publication and it does not represent the opinion of the European Commission. The European Commission is not responsible for any use that might be made of data appearing therein.