MD Despina D Briana

PhD Dimitrios Gourgiotis

MD Maria Boutsikou

MD Stavroula Baka

PhD Antonios Marmarinos

MD Sofia Liosi

MD Dimitrios Hassiakos

MD Ariadne Malamitsi-Puchner

Clara Cell Protein in Full-term Pregnancies: The Influence of Intrauterine Growth Restriction

Keywords: Clara cell protein, lung, intrauterine growth restriction, fetus, neonate Clara cell protein, lung, intrauterine growth restriction, fetus, neonate Clara cell protein, lung, intrauterine growth restriction, fetus, neonate

Introduction

Clara cell protein (known as CC16, CCSP, CC10 or uteroglobin), one of the major secretory proteins of the lungs, is produced by the nonciliated Clara cells, lining the bronchial and bronchiolar epithelium. 1 CC16 has been implicated in fetal lung growth 2 and has been ascribed many anti-inflammatory/immunomodulatory properties, including inhibition of pro-inflammatory cytokine production and neutrophil infiltration. 1,3,4 Thus, CC16 prevents and ameliorates lung injury in adult mice and humans 5,6 and protects the respiratory tract from life-threatening immunological reactions elicited by infectious agents. 3,6 CC16 concentrations in bronchoalveolar lavage fluid (BALF) depend on the number and integrity of Clara cells. 1 BALF CC16 concentrations are increased in patients with respiratory distress syndrome and decreased in patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. 4,7,8 On the other hand, serum CC16 concentrations reflect alveolo-capillary membrane permeability. 4 Lower serum CC16 concentrations have been detected in adults with chronic obstructive pulmonary disease, 9 in children with asthma 10 and preterm neonates with respiratory distress and bronchopulmonary dysplasia. 11,12 Furthermore, serum CC16 is considered a sensitive and specific marker of lung injury in adults. 1,4,9 Recent epidemiological and experimental evidence in humans and animals indicates that the lung can be detrimentally affected by an adverse intrauterine environment, resulting in persistent alterations in lung structure and impaired respiratory function during postnatal life. 13 In this respect, intrauterine growth restriction (IUGR) has been associated with respiratory compromise and higher incidence of lower respiratory tract infections and chronic obstructive airway disease in later life. [14][15][16] However, data

Materials and Methods

The Ethics Committee of our teaching hospital approved the study protocol. All included mothers provided signed informed consent before recruitment. Forty parturients giving consecutively birth, either to 20 AGA or 20 asymmetric IUGR fullterm singleton infants (birth weight ≤ 5 th customized centile) were included in the study. The Gestation Related Optimal Weight computer-generated program 21 was used to calculate the customized centile for each pregnancy, taking into consideration significant determinants of birth weight, such as maternal height and booking weight, ethnic group, parity, gestational age and gender. 21 Gestational age was estimated using the date of the last menstrual period and early antenatal ultrasound. Birth weight was measured with an electronic scale.

Ten of the 20 mothers with IUGR offspring were smoking >10 cigarettes/day during the whole duration of pregnancy. The remaining 10 mothers presented with preeclampsia 22 (6 cases), hypothyroidism (2 cases), iron-deficient anemia (1 case), and gestational diabetes mellitus (1 case). Doppler studies were performed in the IUGR group every 10-15 days, starting from the 32 nd gestational week. During each Doppler velocimetry evaluation, three consecutive measurements of the pulsatility index (PI) of the studied vessel (uterine, umbilical, cerebral arteries) were done and the mean value was recorded. Concerning uterine and umbilical arteries, 23,24 mean PI values were progressively found to be in the upper physiological limits for the corresponding gestational age in 10 cases (ranging between the 90 th and the 95 th percentile), while in the remaining 10 cases PI values showed increased impedance to flow, being above the 95 th percentile for gestational age. Regarding middle cerebral arteries, 25 Doppler studies showed resistance to be in the lower physiological limits for gestational age, indicating the For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed and was defined as diminished, if <2 cm. Placental weights were reduced 26 ranging from 240 to 450g.

In the AGA group, mothers were healthy and were either non-smokers or abstained from smoking during pregnancy. Placentas were normal in appearance and weight. 26 Tests for congenital infections were negative in all women of both groups, and their offspring had no symptoms of intrauterine infection or signs of genetic syndromes.

One-and five-minute Apgar scores were in all cases and controls ≥ 8. All neonates were breastfed. Demographic data of participating subjects are listed in Table 1.

Blood was collected in pyrogen-free tubes from: i) the mothers during the first stage of labor, or before receiving anesthesia in cases of elective caesarean section ii) the umbilical cords after double clamping, reflecting fetal state, and iii) the neonates before feeding on postpartum day 1 (N1) and 4 (N4), characterizing transition and stabilization to extrauterine life, respectively. Serum was separated by centrifugation after clotting and was kept frozen at -80 ºC until assay.

The determination of serum CC16 concentrations was performed by enzyme immunoassay (EIA) (Biovendor, GmbH, 69120 Heiderberg, Germany). The minimum detectable concentration, intra-and interassay coefficients of variation were 20 pg/mL, 5.7% and 4.1%, respectively.

Statistical analysis

CC16

Discussion

The results of this study indicate a lack of significant differences in serum CC16 concentrations between IUGR cases and AGA controls. Serum CC16 concentrations can be affected by lung CC16 production, as well as by CC16 effusion from the lungs across the alveolo-capillary membrane. 1 In human adults, BALF CC16 levels correlate well with serum concentrations, and, therefore, the latter have been suggested as a reliable indicator of the total amount of protein synthesized in the respiratory tract. 27 CC16 production seems to be vital in postnatal pulmonary adaptation. However, in animal models, there is no evidence to suggest that the postnatal absorption of lung fluid is dependent on pulmonary perfusion, indicating that lung liquid absorption is dependent on the pulmonary epithelium and not on the pulmonary vasculature. 28 Furthermore, CC16 plays an important role in the development of lung injury associated with lung maturity. 2,11 Thus, low cord blood CC16 concentrations in preterm infants 12 , due to reduced production, consequent to Clara cells immaturity, 29 independently predict the development of bronchopulmonary dysplasia, 12 while intratracheal administration of recombinant human CC16 in preterm infants with respiratory distress syndrome reduces the neutrophil counts, which is indicative of CC16 anti-inflammatory properties. 30 Furthermore, the importance of CC16 as an anti-inflammatory pneumoprotein places neonates with CC16 deficiency at possible risk of postnatal respiratory injury. 11 IUGR has been proposed to induce permanent changes in lung structure and pulmonary defense mechanisms. 13 In this respect, increased incidence of respiratory distress and lower respiratory tract infections has been documented in low birth weight, even full-term, infants. 14,31 On the other hand, it is well established that stress 32 Thus, IUGR-characterized by hypercortisolaemia 33 -has been previously suggested to be associated with enhanced fetal lung maturation. 33,34 However, this association remains controversial on the basis of human and animal observations, as well as histological data. 13,34 In this respect, increased amniotic fluid indices of lung maturation have been documented in cases with presumed placental insufficiency. 34 However, prolonged intrauterine exposure to glucocorticoids in IUGR could have an inhibitory effect on alveolar formation, resulting in an emphysematous state of the lung. 35 Moreover, in a series of studies, the lungs of IUGR fetuses appeared to be structurally impaired 13 with reduced weight, DNA and protein content, 36 and immature alveolar wall and airblood barrier. 37 Nevertheless, in our study, the lack of differences in CC16 concentrations between IUGR cases and AGA controls possibly assumes that the lung immaturity and later respiratory diseases associated with the former may not be related to early CC16 deficiency.

This study confirms the presence of a gestational influence on CC16 production, which has also been suggested by previous reports concerning amniotic fluid, 2,38 BALF of mechanically ventilated preterm neonates 39 and umbilical cord blood of both preterm and term infants. 11 CC16 is detectable in amniotic fluid from the 15 th week of gestation 2 and increases with advancing gestational age, possibly due to continuous fetal CC16 production, secondary to fetal airway growth and/or lung maturation. 2,38 Furthermore, in accordance with a previous report, 11 our results indicate that serum CC16 concentrations peak on the first day of life. This production surge at birth has been demonstrated to be more pronounced in term compared to preterm infants, conferring the former with superior extrauterine pulmonary protection. 11 Thus, term Additionally, our data suggest that neonatal CC16 concentrations are higher than maternal ones. Similarly, a recent study demonstrated increased CC16 concentrations in newborns compared to healthy adults. 11 Such concentrations may be necessary for postnatal lung adaptation. Furthermore, we were not able to confirm the reported circadian alterations of serum CC16, with on average a 20% decrease at the end of the day. 40 Finally, the absence of a gender effect in circulating CC16 concentrations is in line with previous reports in healthy adults. 41,42 In conclusion, circulating CC16 concentrations are similar between IUGR and AGA groups, possibly suggesting that the lung immaturity and later respiratory diseases associated with the former may not be related to early CC16 deficiency. Moreover, serum CC16 concentrations increase with increasing gestational age and peak on the first day of life, possibly indicating a vital role of this protein in fetal lung maturation and extrauterine pulmonary adaptation, respectively. Future research is necessary to elucidate the role of CC16 in fetal lung development and postnatal respiratory adaptation in both normal and complicated pregnancies.

F

Introduction

Clara cell protein (known as CC16, CCSP, CC10 or uteroglobin), one of the major secretory proteins of the lungs, is produced by the nonciliated Clara cells, lining the bronchial and bronchiolar epithelium. 1 CC16 has been implicated in fetal lung growth 2 and has been ascribed many anti-inflammatory/immunomodulatory properties, including inhibition of pro-inflammatory cytokine production and neutrophil infiltration. 1,3,4 Thus, CC16 prevents and ameliorates lung injury in adult mice and humans 5,6 and protects the respiratory tract from life-threatening immunological reactions elicited by infectious agents. 3,6 CC16 concentrations in bronchoalveolar lavage fluid (BALF) depend on the number and integrity of Clara cells. 1 BALF CC16 concentrations are increased in patients with respiratory distress syndrome and decreased in patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. 4,7,8 On the other hand, serum CC16 concentrations reflect alveolo-capillary membrane permeability. 4 Lower serum CC16 concentrations have been detected in adults with chronic obstructive pulmonary disease, 9 in children with asthma 10 and preterm neonates with respiratory distress and bronchopulmonary dysplasia. 11,12 Furthermore, serum CC16 is considered a sensitive and specific marker of lung injury in adults. 1,4,9 Recent epidemiological and experimental evidence in humans and animals indicates that the lung can be detrimentally affected by an adverse intrauterine environment, resulting in persistent alterations in lung structure and impaired respiratory function during postnatal life. 13 In this respect, intrauterine growth restriction (IUGR) has been associated with respiratory compromise and higher incidence of lower respiratory tract infections and chronic obstructive airway disease in later life. [14][15][16] However, data predominantly derive from animal experimentation. [17][18][19] This study was based on the hypothesis that circulating CC16 concentrations may differ between IUGR and appropriate for gestational age (AGA) fetuses and neonates, since the former may present with structural lung immaturity, impaired immunocompetence and increased risk for developing respiratory infections and chronic obstructive lung disease in later life. [14][15][16][17][18][19][20] For the evaluation of the amniotic fluid, the largest fluid column on the vertical plane was assessed and was defined as diminished, if <2 cm. Placental weights were reduced 26 ranging from 240 to 450g.

In the AGA group, mothers were healthy and were either non-smokers or abstained from smoking during pregnancy. Placentas were normal in appearance and weight. 26 Tests for congenital infections were negative in all women of both groups, and their offspring had no symptoms of intrauterine infection or signs of genetic syndromes.

One-and five-minute Apgar scores were in all cases and controls ≥ 8. All neonates were breastfed. Demographic data of participating subjects are listed in Table 1.

Blood was collected in pyrogen-free tubes from: i) the mothers during the first stage of labor, or before receiving anesthesia in cases of elective caesarean section ii) the umbilical cords after double clamping, reflecting fetal state, and iii) the neonates before feeding on postpartum day 1 (N1) and 4 (N4), characterizing transition and stabilization to extrauterine life, respectively. Serum was separated by centrifugation after clotting and was kept frozen at -80 ºC until assay.

The determination of serum CC16 concentrations was performed by enzyme immunoassay (EIA) (Biovendor, GmbH, 69120 Heiderberg, Germany). The minimum detectable concentration, intra-and interassay coefficients of variation were 20 pg/mL, 5.7% and 4.1%, respectively.

Statistical analysis

CC16

Discussion

The results of this study indicate a lack of significant differences in serum CC16 concentrations between IUGR cases and AGA controls. Serum CC16 concentrations can be affected by lung CC16 production, as well as by CC16 effusion from the lungs across the alveolo-capillary membrane. 1 In human adults, BALF CC16 levels correlate well with serum concentrations, and, therefore, the latter have been suggested as a reliable indicator of the total amount of protein synthesized in the respiratory tract. 27 CC16 production seems to be vital in postnatal pulmonary adaptation. However, in animal models, there is no evidence to suggest that the postnatal absorption of lung fluid is dependent on pulmonary perfusion, indicating that lung liquid absorption is dependent on the pulmonary epithelium and not on the pulmonary vasculature. 28 Furthermore, CC16 plays an important role in the development of lung injury associated with lung maturity. 2,11 Thus, low cord blood CC16 concentrations in preterm infants 12 , due to reduced production, consequent to Clara cells immaturity, 29 independently predict the development of bronchopulmonary dysplasia, 12 while intratracheal administration of recombinant human CC16 in preterm infants with respiratory distress syndrome reduces the neutrophil counts, which is indicative of CC16 anti-inflammatory properties. 30 Furthermore, the importance of CC16 as an anti-inflammatory pneumoprotein places neonates with CC16 deficiency at possible risk of postnatal respiratory injury. 11 IUGR has been proposed to induce permanent changes in lung structure and pulmonary defense mechanisms. 13 In this respect, increased incidence of respiratory distress and lower respiratory tract infections has been documented in low birth weight, even full-term, infants. 14,31 On the other hand, it is well established that stress can lead to maturation of the fetal lung, mediated, at least in part, by an increase in fetal cortisol. 32 Thus, IUGR-characterized by hypercortisolaemia 33 -has been previously suggested to be associated with enhanced fetal lung maturation. 33,34 However, this association remains controversial on the basis of human and animal observations, as well as histological data. 13,34 In this respect, increased amniotic fluid indices of lung maturation have been documented in cases with presumed placental insufficiency. 34 However, prolonged intrauterine exposure to glucocorticoids in IUGR could have an inhibitory effect on alveolar formation, resulting in an emphysematous state of the lung. 35 Moreover, in a series of studies, the lungs of IUGR fetuses appeared to be structurally impaired 13 with reduced weight, DNA and protein content, 36 and immature alveolar wall and airblood barrier. 37 Nevertheless, in our study, the lack of differences in CC16 concentrations between IUGR cases and AGA controls possibly assumes that the lung immaturity and later respiratory diseases associated with the former may not be related to early CC16 deficiency.

This study confirms the presence of a gestational influence on CC16 production, which has also been suggested by previous reports concerning amniotic fluid, 2,38 BALF of mechanically ventilated preterm neonates 39 and umbilical cord blood of both preterm and term infants. 11 CC16 is detectable in amniotic fluid from the 15 th week of gestation 2 and increases with advancing gestational age, possibly due to continuous fetal CC16 production, secondary to fetal airway growth and/or lung maturation. 2,38 Furthermore, in accordance with a previous report, 11 our results indicate that serum CC16 concentrations peak on the first day of life. This production surge at birth has been demonstrated to be more pronounced in term compared to preterm infants, conferring the former with superior extrauterine pulmonary protection. Furthermore, we were not able to confirm the reported circadian alterations of serum CC16, with on average a 20% decrease at the end of the day. 40 Finally, the absence of a gender effect in circulating CC16 concentrations is in line with previous reports in healthy adults. 41,42 In conclusion, circulating CC16 concentrations are similar between IUGR and AGA groups, possibly suggesting that the lung immaturity and later respiratory diseases associated with the former may not be related to early CC16 deficiency. Pediatric Pulmonology

  flow redistribution process, in order to spare vital organs (brain, heart and adrenals). Nevertheless, amniotic fluid was diminished in all IUGR cases.

  maturation of the fetal lung, mediated, at least in part, by an increase in fetal cortisol.

  better equipped for extrauterine life had highest serum and lung fluid CC16 on day 1, indicating that CC16 production at birth may be an important part of postnatal adaptation.11

  of IUGR on fetal lung development are limited and

Fig. 1 .

 1 Figure legend

Table 1 .

 1 Demographic data for AGA and IUGR neonates and their mothers

	o r
	P
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Table 2 .

 2 Mean±standard deviation (SD) values (ng/ml) of CC16 in maternal (MS), fetal (UC), postnatal day 1 (N1) and 4 (N4) serum in appropriate for gestational age (AGA) and intrauterine growth restricted (IUGR) groups.

	GROUP		MS	UC	N1	N4
	AGA	Mean±SD	2.48±0.53	2.67±0.58	8.76±5.98	4.43±1.97
	IUGR	Mean±SD	2.54±0.68	2.94±0.69	7.39±3.70	5.52±3.15

Page 21 of 47 John Wiley & Sons, Inc. for-gestational-age-(AGA) pregnancies. Methods: Serum CC16 concentrations were determined by EIA in 40 mothers and their 20 IUGR and 20 AGA singleton full-term fetuses-neonates on postnatal day 1-(N1) and 4-(N4). Results: No significant differences in CC16 concentrations were observed between IUGR and AGA groups.

protein in fetal lung maturation and extrauterine pulmonary adaptation.

Table 1 .

 1 Moreover, serum CC16 concentrations increase with increasing gestational age and peak on the first day of life, possibly indicating a vital role of this protein in fetal lung maturation and extrauterine pulmonary adaptation, respectively. Future research is necessary to elucidate the role of CC16 in fetal lung development and postnatal respiratory adaptation in both normal and complicated pregnancies. Demographic data for AGA and IUGR neonates and their mothers

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Mean±standard deviation (SD) values (ng/ml) of CC16 in maternal (MS), fetal (UC), postnatal day 1 (N1) and 4 (N4) serum in appropriate for gestational age (AGA) and intrauterine growth restricted (IUGR) groups.

	GROUP		MS	UC	N1	N4
	AGA	Mean±SD	2.48±0.53	2.67±0.58	8.76±5.98	4.43±1.97
	IUGR	Mean±SD	2.54±0.68	2.94±0.69	7.39±3.70	5.52±3.15

John Wiley & Sons, Inc.Pediatric Pulmonology

Acknowledgments

No conflict of interest is declared. Page 12 of 47 John Wiley & Sons, Inc. Pediatric Pulmonology Acknowledgments No conflict of interest is declared. Page 33 of 47 John Wiley & Sons, Inc. Pediatric Pulmonology


Silvia Carraro

MD Giorgio Piacentini

MD Marta Lusiani

Seda Zeynep

Marco Uyan

Maurizio Filippone

Schiavon

Loris Attilio

Eugenio Boner

Baraldi
email: baraldi@pediatria.unipd.it

MD Zeynep S Uyan

MD Marco Filippone

MD Maurizio Schiavon

MD Attilio L Boner

MD Eugenio Baraldi

John Wiley

MD Marco Filippone

EXHALED AIR TEMPERATURE IN CHILDREN WITH BRONCHOPULMONARY DYSPLASIA

Keywords: bronchopulmonary dysplasia, asthma, exhaled air temperature, biomarkers bronchopulmonary dysplasia, asthma, exhaled air temperature, biomarkers bronchopulmonary dysplasia, asthma, exhaled air temperature, biomarkers

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Bronchopulmonary dysplasia (BPD) is the most common form of chronic lung disease of prematurity. In childhood and adolescence, BPD survivors may have respiratory symptoms and lung function impairment that closely resemble those of asthmatic patients, 1 even if BPD patients present fewer acute exacerbations, poorer response to inhaled corticosteroids and less pronounced airway reversibility. A recent study reported that lowbirth-weight survivors have significantly higher odds of hospitalization for respiratory diseases (wheezing, respiratory infections and respiratory failure) as young adults than their normal-birth-weight counterparts. 2 Although there are no data to suggest an asthma-like inflammatory profile in the airways of BPD survivors, they are often empirically treated as if they were asthmatic (e.g. with glucocorticosteroids). 3,4 Very little is known, however, about the possible biochemical and inflammatory mechanisms involved in the pathogenesis of the symptoms and airway obstruction in BPD survivors, 3 and no data are available on the pathological features of these patients' lungs beyond infancy. 3 It is common knowledge that asthma is a chronic inflammatory disease of the airways and several non-invasive biomarkers have been proposed for indirectly assessing different patterns of airway inflammation. 5 Exhaled nitric oxide has been studied the most and it is considered a reliable marker of eosinophilic inflammation in the airways. 6,7 Since "calor" is one of the cardinal signs of inflammation, some authors recently recommended considering exhaled air temperature as a potential marker of inflammation. [8][9] Increased values of exhaled breath temperature have been reported in children and adults with asthma, [8][9][10][11] correlating significantly with the concentrations of exhaled nitric oxide. [8][9][10] To our knowledge, no studies have evaluated exhaled air temperature in individuals with BPD. The aim of the present cross-sectional study was to evaluate exhaled breath temperature (PLET, end-expiratory maneuver plateau temperature) in BPD survivors and to compare it with the temperature measured in age-matched asthmatic subjects and healthy controls.

MATERIALS AND METHODS

Study subjects

BPD survivors: 17 BPD survivors were considered (table), drawn from a population with BPD born at gestational ages below 31 weeks and with birth weights under 1500 g admitted to the Neonatal Intensive Care Unit between 1990 and 1994 and followed up routinely thereafter at the Pediatrics Department in Padova. BPD was defined as oxygen dependence persisting 28 days after birth in infants on mechanical ventilation (18 days on average). At birth, the mean gestational age of the sample was 28.3 weeks (SD 1.9) and the mean weight was 1011 g (SD 222).

All the BPD survivors were clinically stable at the time of recruitment, 5 of them were on low-to medium-dose inhaled steroid (ICS) treatment and two were being treated with theophylline. Two of the BPD survivors were sensitive to common airborne allergens (dust mites, dog and cat dander, grass and tree pollens).

Asthmatic subjects: 17 allergic asthmatic adolescents (table) who had been born at term were enrolled from among those regularly attending our outpatients clinic. Their asthma had been diagnosed by a pediatric respiratory physician based on their clinical history (cough, shortness of breath, recurrent wheezing, chest tightness) and an increase in FEV 1 after salbutamol (400 µg) >12%, according to international guidelines. 12 All the asthmatic patients were clinically stable at the time of recruitment, 6 of them were on low-to medium-dose inhaled steroid (ICS) treatment (100-200 mcg/day budesonide equivalent) and one of them was being treated with long-acting beta2 agonists too. ) with no history of pre-term birth, atopy or respiratory diseases were recruited as a control group. For all three groups, subjects who had suffered any airway infections in the previous 4 weeks were excluded from the study.

The Ethics Committee of our hospital reviewed and approved the protocol and all parents gave their informed consent.

At recruitment, all the adolescents underwent physical examination, exhaled air temperature measurement, FE NO measurement and spirometry. All the measurements were performed in the afternoon. FE NO measurement was not possible in 3 subjects (1 with asthma and 2 with BPD)

Exhaled breath temperature (PLET)

Exhaled breath temperature (end-expiratory maneuver plateau temperature, PLET) was measured as described elsewhere. 13 The environmental conditions were controlled in order to maintain a stable temperature of 23±1°C and a relative humidity of 40-50%.

The measurements were repeated three times. The inspiratory air stream temperature was recorded during a complete inhalation to total lung capacity (TLC). The exhaled airway temperature was measured during a slow exhalation to residual volume (RV) using the Medical Mass Flow Sensor of the Vmax Spectra 229 Pulmonary Function Laboratory (VIASYS Healthcare, Yorba Linda, CA, USA). Dynamic airway temperature and airflow were measured with the Mass Flow Sensor. The response time of the Mass Flow Sensor to a steep change in airway temperature was approximately 64 milliseconds, with a thermal resolution of 0.01ºC. Mouth pressure was measured at the airway opening using a differential pressure transducer. Exhaled flow was directed through an alternate pathway with a fixed resistor in place to control the expiratory air flow rate and maintain an airway pressure sufficient to keep the velum closed and prevent any contribution of warmed gas from nasal sinuses. A mouth pressure of > 5 cm H 2 O also helped the subject to stabilize pressure and exhalation flow rate, were transferred to a personal computer and analyzed using software specifically developed for this purpose (Vmax Temperature, VIASYS Healthcare, Yorba Linda, CA, USA). Real-time signal display consisted of inspiratory and expiratory airway temperature, respiratory flow rate and mouth (airway) pressure on a time axis. Flow and pressure limit indicators were presented on screen as a coaching aid.

At the end of the test, the software automatically calculated the PLET, which was the parameter used to compare the groups.

Fractional exhaled nitric oxide (FE NO ) and pulmonary function measurement

FE NO was measured using the NIOX system (Aerocrine, Stockholm, Sweden), according to the ERS/ATS guidelines for measuring FE NO in children. 14 Children inhaled NO-free air to total lung capacity (TLC) and exhaled through a dynamic flow restrictor with a target flow of 50 ml/sec for at least 6-7 seconds. No nose clip was used. The NIOX system was calibrated using a 200 ppb NO tank (Lindegas Hoek Loos Speciality Gases, Amsterdam, Netherlands) according to the manufacturer's instructions.

Lung function was measured by means of a 10-liter bell spirometer (Biomedin, Padova, Italy) and the best of three maneuvers was expressed as a percentage (%) of the predicted reference values, according to the ERS/ATS guidelines. 15

Statistical analysis

It has been calculated that a sample size of 17 subjects allows detecting a 1°C difference between groups of with a power of 80% at a 2-sided -level of .05.

Age and anthropometric data were expressed as the mean and standard deviation (SD) (table).

Since PLET, FE NO and lung function data were not normally distributed (as demonstrated by the Shapiro-Wilk test), they were expressed as medians and interquartile ranges (IQR), analyzed using the Kruskal-Wallis test, followed (where significant) by the Mann-Whitney U-test for comparisons between groups. Correlations were evaluated using Spearman's rank test. Statistical significance was set at a 2-sided alpha level of 0.05.

RESULTS

Exhaled breath temperature (PLET)

The Kruskal Wallis test revealed a significant difference in the PLET for the 3 groups (p<0.001) (figure 1A). The between-group comparison found a significantly lower PLET in BPD survivors (26.72°C [25.11-27.57]) than in asthmatic patients (29.60°C [29.20-30.02], p<0.001), while no significant difference emerged between BPD survivors and healthy controls (26.97°C [26.58-27.38] p=0.23). The asthmatic cases had a significantly higher PLET than the healthy controls (p<0.001).

Exhaled nitric oxide (FE NO )

The Kruskal Wallis test found a significant difference in FE NO for the 3 groups (p=0.003) (figure 1B). The between-group comparison revealed a significantly higher FE NO in asthmatic cases (29.9 ppb [15.0-49.2]) than in either healthy controls (11.7 ppb [8.5-16.8], p=0.008) or BPD survivors (10.6 ppb [7.0-13.2], p=0.002), with no significant difference between the latter two groups (p=0.28).

Lung function

The Kruskal Wallis test disclosed significant differences in all the spirometric parameters.

The between-group comparison showed that BPD children had lower FVC, FEV 1 , FEF 25-75 and FEV 1 /FVC than either asthmatic cases or healthy controls; and the asthmatic cases had lower FEV 1 , FEF 25-75 and FEV 1 /FVC than healthy controls (table, figure 1C and1D).

Correlations

Considering the whole sample, a significant correlation emerged between PLET and FE NO (R=0.42 p=0.004) (figure 2).

In BPD survivors there was a correlation approaching statistical significance between FVC and PLET (p=0.058 R=0.47), which was stronger considering the whole sample (R=0.28 p=0.036).

None of the other spirometric parameters correlated with PLET values in BPD survivors.

No significant correlations emerged between spirometric measurements and PLET in asthmatic cases or healthy controls.

DISCUSSION

This study shows that exhaled breath temperature is significantly lower in BPD survivors than in asthmatic patients.

BPD survivors have respiratory symptoms and lung function profiles that resemble those of asthmatic children 16 and this explains why they are often considered and empirically treated as asthmatic patients during their childhood. 3 Very little it is known about the processes underlying the obstructive lung function pattern in BPD survivors and there is no evidence to support any asthma-like inflammation in this condition. 4,17,18 Here we demonstrate that both PLET and FE NO are lower in BPD survivors than in asthmatic children, suggesting that the two conditions have a different pathogenesis (figure 1A and1B). In a previous study, we already showed that exhaled NO is lower in BPD survivors than in asthmatic cases; 19 the present study further supports the lack of any asthma-like airway inflammation in BPD survivors. A possible limit in the interpretation of our results is the potential effect of ICS treatment and atopy on PLET values. In fact while all asthmatic patients were allergic, only two BPD patients were sensitized. In addition some patients in both groups were treated with ICS. In keeping with previous studies conducted on asthmatic patients, [8][9] PLET and FE NO correlated significantly. It is worth noting that the scatter plot (figure 2) shows a clear separation between asthmatic cases and BPD survivors, again suggesting that different pathogenetic mechanisms underlie these two chronic obstructive lung diseases.

As previously reported, 8-10 asthmatic subjects have higher levels of both FE NO and PLET, which are thought to reflect eosinophilic airway inflammation. 6,[START_REF] Piacentini | Exhaled air temperature and eosinophil airway inflammation in allergic asthmatic children[END_REF] The higher exhaled breath temperature in asthmatic subjects also seems to suggest an increased vascularity and bronchial blood flow. 10 In BPD survivors, on the other hand, PLET and FE NO are significantly lower than in asthmatic patients and even tend to be lower than in healthy controls, although without reaching the statistical significance (figure 1A and1B). This trend could be explained by the anatomical alterations that characterize BPD lungs, namely alveolar simplification, variable degree of interstitial fibrosis 21,22 and dysmorphic vascular growth. 23 These anatomical alterations might presumably be responsible for an impaired heat exchange between the bronchial vessels and the airway lumen, giving rise to normal-to-low values of exhaled air temperature in BPD survivors.

As previously reported, 24 we confirm here that BPD survivors have a lung function pattern with airflow limitation (figure 1C). An autoptic study demonstrated thickening of the airway walls and an increase in smooth muscle, suggesting that these aspects may have a role in airway obstruction and hyper-responsiveness in BPD survivors. 25 Moreover BPD survivors have a significantly reduced forced vital capacity (figure 1D). This last features might be explained by the impaired growth of the BPD survivor's lungs, which would lead to reduced pulmonary volumes. Previous studies have already documented in BPD survivors an impaired lung function. 24,26,27 It may be that lung function impairment in BPD survivors is not just the static outcome of the initial damage, but due to an ongoing remodeling/damaging process. Although our findings go against any suggestion of an asthma-like eosinophilic inflammatory profile, other inflammatory pathways (e.g. BPD patients present fewer acute exacerbations, poorer response to inhaled corticosteroids and less pronounced airway reversibility though they do not share the same origins. A recent study reported that low-birth-weight survivors have significantly higher odds of hospitalization for respiratory diseases (wheezing, respiratory infections and respiratory failure) as young adults than their normal-birth-weight counterparts. 2 Although there are no data to suggest an asthma-like inflammatory profile in the airways of BPD survivors, they are often empirically treated as if they were asthmatic (e.g. with glucocorticosteroids). 3,4 Very little is known, however, about the possible biochemical and inflammatory mechanisms involved in the pathogenesis of the symptoms and airway obstruction in BPD survivors, 3 and no data are available on the pathological features of these patients' lungs beyond infancy. 3 It is common knowledge that asthma is a chronic inflammatory disease of the airways and several non-invasive biomarkers have been proposed for indirectly assessing different patterns of airway inflammation. 5 Exhaled nitric oxide has been studied the most and it is considered a reliable marker of eosinophilic inflammation in the airways. 6,7 Since "calor" is one of the cardinal signs of inflammation, some authors recently recommended considering exhaled air temperature as a potential marker of inflammation. [8][9] Increased values of exhaled breath temperature have been reported in children and adults with asthma, [8][9][10][11] correlating significantly with the concentrations of exhaled nitric oxide. [8][9][10] To our knowledge, no studies have evaluated exhaled air temperature in individuals with BPD. The aim of the present cross-sectional study was to evaluate exhaled breath temperature (PLET, end-expiratory maneuver plateau temperature) in BPD survivors and to compare it with the temperature measured in age-matched asthmatic subjects and healthy controls.

MATERIALS AND METHODS

Study subjects

BPD survivors: 17 BPD survivors were considered (table), drawn from a population with BPD born at gestational ages below 31 weeks and with birth weights under 1500 g admitted to the Neonatal Intensive Care Unit between 1990 and 1994 and followed up routinely thereafter at the Pediatrics Department in Padova. BPD was defined as oxygen dependence persisting 28 days after birth in infants on mechanical ventilation (18 days on average). At birth, the mean gestational age of the sample was 28.3 weeks (SD 1.9) and the mean weight was 1011 g (SD 222).

All the BPD survivors were clinically stable at the time of recruitment, 5 of them were on low-to medium-dose inhaled steroid treatment and two were being treated with theophylline. Two of the BPD survivors were sensitive to common airborne allergens (dust mites, dog and cat dander, grass and tree pollens). Asthmatic subjects: 17 allergic asthmatic adolescents (table) who had been born at term were enrolled from among those regularly attending our outpatients clinic. Their asthma had been diagnosed by a pediatric respiratory physician based on their clinical history (cough, shortness of breath, recurrent wheezing, chest tightness) and an increase in FEV 1 after salbutamol (400 µg) >12%, according to international guidelines. 12 All the asthmatic patients were clinically stable at the time of recruitment, 6 of them were on low-to medium-dose inhaled steroid treatment (100-200 mcg/day budesonide equivalent) and one of them was being treated with long-acting beta2 agonists too. ) with no history of pre-term birth, atopy or respiratory diseases were recruited as a control group. For all three groups, subjects who had suffered any airway infections in the previous 4 weeks were excluded from the study.

The Ethics Committee of our hospital reviewed and approved the protocol and all parents gave their informed consent.

At recruitment, all the adolescents underwent physical examination, exhaled air temperature measurement, FE NO measurement and spirometry. All the measurements were performed in the afternoon. FE NO measurement was not possible in 3 subjects (1 with asthma and 2 with BPD)

Exhaled breath temperature (PLET)

Exhaled breath temperature (end-expiratory maneuver plateau temperature, PLET) was measured as described elsewhere. 13 The environmental conditions were controlled in order to maintain a stable temperature of 23±1°C and a relative humidity of 40-50%. pressure and exhalation flow rate, were transferred to a personal computer and analyzed using software specifically developed for this purpose (Vmax Temperature, VIASYS Healthcare, Yorba Linda, CA, USA). Real-time signal display consisted of inspiratory and expiratory airway temperature, respiratory flow rate and mouth (airway) pressure on a time axis. Flow and pressure limit indicators were presented on screen as a coaching aid.

At the end of the test, the software automatically calculated the PLET, which was the parameter used to compare the groups.

Fractional exhaled nitric oxide (FE NO ) and pulmonary function measurement

FE NO was measured using the NIOX system (Aerocrine, Stockholm, Sweden), according to the ERS/ATS guidelines for measuring FE NO in children. 14 Children inhaled NO-free air to total lung capacity (TLC) and exhaled through a dynamic flow restrictor with a target flow of 50 ml/sec for at least 6-7 seconds. No nose clip was used. The NIOX system was calibrated using a 200 ppb NO tank (Lindegas Hoek Loos Speciality Gases, Amsterdam, Netherlands) according to the manufacturer's instructions.

Lung function was measured by means of a 10-liter bell spirometer (Biomedin, Padova, Italy) and the best of three maneuvers was expressed as a percentage (%) of the predicted reference values, according to the ERS/ATS guidelines. 15

Statistical analysis

It has been calculated that a sample size of 17 subjects allows detecting a 1°C difference between groups of with a power of 80% at a 2-sided -level of .05.

Age and anthropometric data were expressed as the mean and standard deviation (SD)

(table).

Since PLET, FE NO and lung function data were not normally distributed (as demonstrated by the Shapiro-Wilk test), they were expressed as medians and interquartile ranges (IQR), analyzed using the Kruskal-Wallis test, followed (where significant) by the Mann-Whitney U-test for comparisons between groups. Correlations were evaluated using Spearman's rank test. Statistical significance was set at a 2-sided α level of 0.05.

RESULTS

Exhaled breath temperature (PLET)

The Kruskal Wallis test revealed a significant difference in the PLET for the 3 groups (p<0.001) (figure 1A). The between-group comparison found a significantly lower PLET in BPD survivors (26.72°C [25.11-27.57]) than in asthmatic patients (29.60°C [29.20-30.02], p<0.001), while no significant difference emerged between BPD survivors and healthy controls (26.97°C [26.58-27.38] p=0.23). The asthmatic cases had a significantly higher PLET than the healthy controls (p<0.001).

Exhaled nitric oxide (FE NO )

The Kruskal Wallis test found a significant difference in FE NO for the 3 groups (p=0.003) (figure 1B). The between-group comparison revealed a significantly higher FE NO in asthmatic cases (29.9 ppb [15.0-49.2]) than in either healthy controls (11.7 ppb [8.5-16.8], p=0.008) or BPD survivors (10.6 ppb [7.0-13.2], p=0.002), with no significant difference between the latter two groups (p=0.28).

Lung function

The Kruskal Wallis test disclosed significant differences in all the spirometric parameters.

The between-group comparison showed that BPD children had lower FVC, FEV 1 , FEF 25-75 and FEV 1 /FVC than either asthmatic cases or healthy controls; and the asthmatic cases had lower FEV 1 , FEF 25-75 and FEV 1 /FVC than healthy controls (table, figure 1C and1D).

Correlations

Considering the whole sample, a significant correlation emerged between PLET and FE NO (R=0.42 p=0.004) (figure 2).

In BPD survivors there was a correlation approaching statistical significance between FVC and PLET (p=0.058 R=0.47), which was stronger considering the whole sample (R=0.28 p=0.036) whereas Nnone of the other spirometric parameters correlated with PLET values in BPD survivors.

No significant correlations emerged between spirometric measurements and PLET in asthmatic cases or healthy controls.

DISCUSSION

This study shows that exhaled breath temperature is significantly lower in BPD survivors than in asthmatic patients. BPD survivors have respiratory symptoms and lung function profiles that resemble those of asthmatic children 16 and this explains why they are often considered and empirically treated as asthmatic patients during their childhood. 3 Very little it is known about the processes underlying the obstructive lung function pattern in BPD survivors and there is no evidence to support any asthma-like inflammation in this condition. 4,17,18 Here we demonstrate that both PLET and FE NO are lower in BPD survivors than in asthmatic children, strongly suggesting that the two conditions have a different pathogenesis (figure 1A and1B). In a previous study, we already showed that exhaled NO is lower in BPD survivors than in asthmatic cases; 19 the present study further supports the lack of any asthma-like airway inflammation in BPD survivors.

A possible limit in the interpretation of our results is the potential effect of ICS treatment and atopy on PLET values. In fact while all asthmatic patients were allergic, only two BPD patients were sensitized. In addition some patients in both groups were treated with ICS. In keeping with previous studies conducted on asthmatic patients, [8][9] PLET and FE NO correlated significantly. It is worth noting that the scatter plot (figure 2) shows a clear separation between asthmatic cases and BPD survivors, again suggesting that different pathogenetic mechanisms underlie these two chronic obstructive lung diseases.

As previously reported, 8-10 asthmatic subjects have higher levels of both FE NO and PLET, which are thought to reflect eosinophilic airway inflammation. 6,[START_REF] Piacentini | Exhaled air temperature and eosinophil airway inflammation in allergic asthmatic children[END_REF] The higher exhaled breath temperature in asthmatic subjects also seems to suggest an increased vascularity and bronchial blood flow. 10 In BPD survivors, on the other hand, PLET and FE NO are significantly lower than in asthmatic patients and even tend to be lower than in healthy controls, although without reaching the statistical significance (figure 1A and1B). This trend could be explained by the anatomical alterations that characterize BPD lungs, namely alveolar simplification, variable degree of interstitial fibrosis 21,22 and dysmorphic vascular growth. 23 These anatomical alterations might presumably be responsible for an impaired heat exchange between the bronchial vessels and the airway lumen, giving rise to normal-to-low values of exhaled air temperature in BPD survivors.

As previously reported, 24 we confirm here that BPD survivors have a lung function pattern with airflow limitation (figure 1C). An autoptic study demonstrated thickening of the airway walls and an increase in smooth muscle, suggesting that these aspects may have a role in airway obstruction and hyper-responsiveness in BPD survivors. 25 Moreover BPD survivors have a significantly reduced forced vital capacity (figure 1D). This last features might be explained by the impaired growth of the BPD survivor's lungs, which would lead to reduced pulmonary volumes.

Previous studies have already documented an impaired lung function in BPD survivors an impaired lung function and characterized by an accelerated decline. 24,26,27 In a follow-up study, Doyle et al 28 reported significant airflow obstruction in BPD survivors at 18 years of age, with an accelerated decline in their FEV 1 /FVC ratio, suggesting an earlier

5

 5 Healthy controls: 17 age-matched healthy adolescents (table

  at 5-6 l/min. All the signals, including exhaled air temperature, mouth

  means of non-parametric tests. Differences between groups were

Figure 1 .

 1 FIGURE LEGENDSFigure 1. Panel A shows PLET values in the 3 groups; panel B shows FE NO values in the 3 groups; panel C shows FVC values in the 3 groups; panel D shows FEV1 values in the 3 groups. **p<0.001 and *p<0.01 vs healthy controls; #p<0.05 and §p<0.01 vs asthmatic subjects; γ p<0.001 vs BPD survivors

Figure 2 .

 2 Figure 2. Correlation between FE NO values and PLET values in BPD, asthmatic and healthy subjects (R=0.42 p=0.004).

INTRODUCTION

  Bronchopulmonary dysplasia (BPD) is the most common form of chronic lung disease of prematurity. In childhood and adolescence, BPD survivors may have respiratory symptoms and lung function impairment that closely resemble those of asthmatic patients, 1 even if

5

 5 Healthy controls: 17 age-matched healthy adolescents (table

  The measurements were repeated three times. The inspiratory air stream temperature was recorded during a complete inhalation to total lung capacity (TLC). The exhaled airway temperature was measured during a slow exhalation to residual volume (RV) using the Medical Mass Flow Sensor of the Vmax Spectra 229 Pulmonary Function Laboratory (VIASYS Healthcare, Yorba Linda, CA, USA). Dynamic airway temperature and airflow were measured with the Mass Flow Sensor. The response time of the Mass Flow Sensor to a steep change in airway temperature was approximately 64 milliseconds, with a thermal resolution of 0.01ºC. Mouth pressure was measured at the airway opening using a differential pressure transducer. Exhaled flow was directed through an alternate pathway with a fixed resistor in place to control the expiratory air flow rate and maintain an airway pressure sufficient to keep the velum closed and prevent any contribution of warmed gas from nasal sinuses. A mouth pressure of > 5 cm H 2 O also helped the subject to stabilize at 5-6 l/min. All the signals, including exhaled air temperature, mouth

  means of non-parametric tests. Differences between groups were

  respiratory health than in the general population. Lung function declines physiologically in adulthood and senescence, albeit without reaching levels associated with disability.29 This decline could be more of a problem in BPD survivors, whose maximal lung function is underdeveloped. It may be that lung function impairment in BPD survivors is not just the static outcome of the initial damage, but due to an ongoing remodeling/damaging process. Although our findings go against any suggestion of an asthma-like eosinophilic inflammatory profile, other inflammatory pathways (e.g. neutrophilic or lymphocytic) and/or remodeling mechanisms may be involved. Elucidating this pathophysiological substrate could have a great impact on the long-term management of BPD subjects.In conclusion, we showed that exhaled breath temperature (PLET) and exhaled nitric oxide (which are considered biomarkers of eosinophilic airway inflammation) are not increased in BPD survivors. Further studies are needed to assess the role of other noninvasive biomarkers, as measured for instance in exhaled breath condensate, to shed light on the pathogenic processes affecting the BPD survivor's lung and thus pave the way to the development of new, specifically targeted drugs.

Figure 1 .

 1 Figure 1. Panel A shows PLET values in the 3 groups; panel B shows FE NO values in the 3 groups; panel C shows FVC values in the 3 groups; panel D shows FEV1 values in the 3 groups. **p<0.001 and *p<0.01 vs healthy controls; #p<0.05 and §p<0.01 vs asthmatic subjects  p<0.001 vs BPD survivors

Figure 2 .

 2 Figure 2. Correlation between FE NO values and PLET values in BPD, asthmatic and healthy subjects (R=0.42 p=0.004).

Table .

 . Demographic characteristic and lung function measures of study subjects. Anthropometric data are expressed as mean (SD). Spirometric data are expressed as median and IQR. **p<0.001 and *p<0.01 vs healthy controls; #p<0.05 and §p<0.01 vs asthmatic subjects.

			Pediatric Pulmonology	Page 30 of 32
			Healthy	Asthma	BPD
	Number (males) 17 (11)	17 (10)	17 (7)
	Age (y)		14.4 (1.8)	13.3 (2.1)	14.5 (2.0)
	Height (cm)		163.6 (7.5)	159.5 (9.1) 161.5 (11)
	Weight (Kg)		56.5 (9.4)	57.9 (12.9) 53.9 (12.2)
	FVC (%pred)	106 [99-110] 101 [95-107] 85 [77-100]* #
	FEV 1 (%pred) FEF 25-75 (%pred) 121 [107-133] 75 [57-87]** 46 [40-67] # ** 104 [100-111] 88 [76-93]** 66 [58-82]** § F o
	FEV 1 /FVC	91 [88-93] r	78 [76-84]** 72 [67-80]**
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	Pediatric Pulmonology
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John Wiley & Sons, Inc.Pediatric Pulmonology

Acknowledgement

The authors thank VIASYS Healthcare, Yorba Linda, CA, USA, for providing hardware and software for exhaled air evaluation.

Acknowledgement

The authors thank VIASYS Healthcare, Yorba Linda, CA, USA, for providing hardware and software for exhaled air evaluation.


Anders W Jørgensen

Karsten Juhl Jørgensen

Peter C Gøtzsche

Juhl Karsten

Peter C Jørgensen

Gøtzsche

Unbalanced reporting of benefits and harms in abstracts on rofecoxib

Keywords: Rofecoxib, Cyclooxygenase-2 inhibitors, Abstracts, Bias, Thrombosis, Gastrointestinal bleeding

Purpose It was predicted from the mechanism of action that, compared to older non-steroidal anti-inflammatory drugs, rofecoxib (Vioxx) would reduce gastrointestinal bleeding, but also that it would increase the occurrence of cardiovascular thrombosis. From the patient's point of view, both effects are important and should be investigated and reported similarly. We studied how they have been reported over time. Methods We searched PubMed for abstracts on rofecoxib that commented on gastrointestinal bleeding or cardiovascular thrombosis or both. Two researchers, blinded to date of publication and authors, assessed the abstracts independently. We judged the authors' view on rofecoxib and comments on gastrointestinal bleeding and thrombosis as being favourable, neutral or unfavourable towards rofecoxib.

Results

We included 393 abstracts commenting on gastrointestinal bleeding (72%) and cardiovascular thrombosis (54%) or both. Before October 2000, all abstracts (n=27) mentioned only gastrointestinal bleeding and 89% were positive towards rofecoxib. The year before the withdrawal of rofecoxib (October 2003 to September 2004) (n=46), 59% of abstracts commented on gastrointestinal bleeding only, 17% on thrombosis only, 24% on both and 67% were still positive. From October 2006 to September 2007 (n=54), 13% mentioned gastrointestinal bleeding, 54% thrombosis, 33% mentioned both and only 11% were positive. Conclusions The reporting of benefits and harms was not balanced and changed markedly over time. Knowledge of increased risk of thrombosis existed early on, but the harms came into focus too late, when the drug was already withdrawn, and when tens of thousands of patients had been harmed unnecessarily.

Introduction

Rofecoxib was marketed in 1999 as first-line treatment of osteoarthritis with the claim that it reduced pain as effectively as conventional non-steroidal anti-inflammatory drugs (NSAIDs), but with less gastrointestinal bleeding. However, the drug also caused serious thrombotic cardiovascular events [START_REF]Merck announces voluntary worldwide withdrawal of VIOXX[END_REF][START_REF] Bresalier | Adenomatous Polyp Prevention on Vioxx (APPROVe) Trial Investigators (2005) Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial[END_REF]. On 30 September 2004, Merck, the manufacturer, withdrew it from the market. Rofecoxib has been estimated to have caused the death of tens of thousands of patients because of thromboses [3].

The part of a paper that is most often read is the abstract and sometimes clinical decisions are based solely on abstracts [START_REF] Barry | Family physicians' use of medical abstracts to guide decision making: style or substance?[END_REF][START_REF] Hopewell | CONSORT for reporting randomised trials in journal and conference abstracts[END_REF]. The recently published CONSORT guideline for abstracts states that any important adverse (or unexpected) effects of an intervention should be described in the abstract [START_REF] Hopewell | CONSORT for reporting randomised trials in journal and conference abstracts[END_REF]. We quantified how often benefits and harms in terms of gastrointestinal bleeding and cardiovascular thrombosis, respectively, were reported in abstracts on rofecoxib, how often the drug was described favourably, and how this pattern changed over time.

Methods

Search strategy and data extraction

One author searched PubMed (24 September 2007) using the search terms "Vioxx OR rofecoxib". All records with an abstract were assessed for inclusion by two observers independently. At the same time, they extracted the data using a pilot-tested data sheet. Any disagreements were settled by discussion. The observers were blinded to any information about authors and institutions and to the date of publication, and assessed only the title and the text of the abstract. The blinding was obtained by exporting the relevant parts of the PubMed records into Microsoft Excel.

Inclusion and exclusion criteria

We included abstracts that commented on the effect of rofecoxib on gastrointestinal bleeding or cardiovascular thrombosis or both, and contained a comment reflecting the authors' view on rofecoxib. We accepted abstracts that implicitly referred to these harms by using phrases such as "gastrointestinal adverse effect" and "cardiovascular risk". We also included abstracts that commented on the effect of cyclooxygenase 2 (COX-2) inhibitors and other NSAIDs in general when these included rofecoxib, albeit indirectly, e.g. "celecoxib had no tangible advantage in terms of serious gastrointestinal complications … overall mortality was higher with celecoxib than in the placebo group. The difference was similar to that observed in placebocontrolled trials of rofecoxib in Alzheimer's disease."

We excluded abstracts that only commented on harms that did not involve gastrointestinal bleeding or cardiovascular thrombosis, such as nausea or hypertension. We also excluded abstracts that did not have a comment reflecting the authors view on rofecoxib, e.g. "The manufacturer claims that in clinical studies rofecoxib inhibits COX-2 but not COX-1, has the power of high-dose NSAIDsdiclofenac and ibuprofen-and superior GI [gastrointestinal] safety profile compared to conventional NSAIDs." Abstracts of in vitro studies, animal studies, medical devices and pharmacokinetics were also excluded.

Evaluation of the authors' view on rofecoxib

We categorised the authors' view on rofecoxib as either favourable, neutral or unfavourable. If an active comparator was not used as a reference, we accepted placebo or statements that did not involve a comparator. The judgement was preferentially made using the conclusion of the abstract. If this was not possible, we used statements in the results section or elsewhere, e.g. "Because of its more favorable gastrointestinal toxicity profile compared with non-selective NSAIDs, rofecoxib is safer in patients …". We also judged the individual comments on gastrointestinal bleeding and cardiovascular thrombosis in the same way.

Analysis

We used graphs and descriptive statistics to assess how the reporting in abstracts changed over time. Because rofecoxib was withdrawn on 30 September 2004, our 1-year intervals are from October to September, e.g. year 2000 was defined as October 1999 to September 2000, both months included. For abstracts that only contained information about the year of publication, we used the date the citation was added to the PubMed database [EDAT].

Results

Our PubMed search identified 2,047 records and we included 393 abstracts. Most records were excluded because there was no abstract in PubMed or because the abstract did not contain a comment on gastrointestinal bleeding or cardiovascular thrombosis (Fig. 1). Twentynine of the excluded abstracts mentioned hypertension, but did not comment on cardiovascular thrombosis.

Reporting of harms over time

During the whole observation period, 181 of the included abstracts (46%) commented on gastrointestinal bleeding only, 110 (28%) on cardiovascular thrombosis only and 102 (26%) commented on both. Of the 283 (181+102) abstracts commenting on gastrointestinal bleeding, 141 (50%) used the explicit terms "ulcer", "gastrointestinal bleeding" or "perforation", or "serious gastrointestinal adverse effect". The remaining 142 abstracts (50%) used the less explicit terms "gastrointestinal risk", "gastrointestinal safety", or "gastrointestinal adverse effect". Of the 212 (110+102) abstracts commenting on cardiovascular thrombosis, 137 (65%) used the explicit terms "thrombosis", "thromboembolic" or "thrombotic effect", "myocardial infarction" or "stroke". The remaining 75 abstracts (35%) used the less explicit terms "cardiovascular risk", "cardiovascular safety" or "cardiovascular adverse effect".

Until and including September 2000, no abstracts commented on cardiovascular thrombosis (the first was published in November 2000), i.e. 100% (n=27) of the abstracts commented only on gastrointestinal bleeding (Fig. 2). The percentage of abstracts that only commented on gastrointestinal bleeding decreased to 59% in 2004 (n= 46), before rofecoxib was withdrawn, and to 13% in 2007 (n=54). The percentage of abstracts that commented only on cardiovascular thrombosis increased from 0 to 17% in 2004 and to 54% in 2007. The percentage commenting on both gastrointestinal bleeding and cardiovascular thrombosis increased from 0 to 24% in 2004, and to 33% in 2007. The greatest change in reporting was seen immediately after the withdrawal of rofecoxib in 2004 (Fig. 2).

Authors' general view on rofecoxib over time

Until and including September 2000 (n=27), the proportion of abstracts favouring rofecoxib was 89%. In 2004 (n=46), it was 67%. The greatest change was seen after the withdrawal of rofecoxib and in 2007 (n=54) where only 11% of the abstracts were positive (Fig. 3). We investigated the robustness of this result by including only those abstracts where our judgement was based on the conclusion section of the abstracts. This graph had a similar slope (dotted line in Fig. 3).

Authors' views on harms before and after the withdrawal of rofecoxib Two hundred and eleven abstracts (54%) were published before the withdrawal of rofecoxib in 2004 and 182 (46%) were published after. Before the withdrawal, 193 (91%) abstracts commented on gastrointestinal bleeding, and 168 (87%) of them were favourable towards rofecoxib, 19 (10%) were neutral and 6 (3%) unfavourable. Fifty-six (27%) abstracts commented on cardiovascular thrombosis, and 5 (9%) of those were favourable towards rofecoxib, 31 (55%) neutral and 20 (36%) unfavourable.

After the withdrawal, the effect on gastrointestinal bleeding was mentioned in 89 (49%) abstracts, and 67 (75%) of those were favourable towards rofecoxib, 14 (16%) neutral and 8 (9%) unfavourable. The thrombotic effect was mentioned in 156 (86%) abstracts and, none of them were favourable towards rofecoxib, 26 (17%) neutral and 130 (83%) unfavourable.

Discussion

We found that most abstracts on rofecoxib reported only on the beneficial effect regarding less gastrointestinal bleeding, and that they were generally in favour of rofecoxib, from the introduction of the drug in 1999 to its withdrawal in 2004. After the withdrawal, most abstracts reported on the harmful effects, cardiovascular thrombosis, and few were in favour of rofecoxib.

Such findings might be expected for drugs with important but rare harms that are unknown when the drugs are introduced on the market and only discovered later. However, this is not the only explanation for our findings. It has been documented that the company suppressed cardiovascular harms in the scientific literature [START_REF] Krumholz | What have we learned from Vioxx?[END_REF] and intimidated researchers and speakers who were critical of rofecoxib [START_REF] Krumholz | What have we learned from Vioxx?[END_REF][START_REF] Fries | Letter to Raymond Gilmartin re: physician intimidation[END_REF][START_REF] Rout | Vioxx maker Merck and Co drew up doctor hit list[END_REF].

Before its introduction, it was predicted from the mechanism of action that the drug should reduce the incidence of gastrointestinal bleeding [START_REF] Knijff-Dutmer | Effects of nabumetone compared with naproxen on platelet aggregation in patients with rheumatoid arthritis[END_REF] but also increase the incidence of thrombosis, compared with non-selective NSAIDs [START_REF] Schonbeck | Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions[END_REF][START_REF] Catella-Lawson | Effects of specific inhibition of cyclooxygenase-2 on sodium balance, hemodynamics, and vasoactive eicosanoids[END_REF][START_REF] Mcadam | Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX-2[END_REF]. Two trials conducted by Merck, 090 [3,13,[START_REF] Weaver | Treatment of patients with osteoarthritis with rofecoxib compared with nabumetone[END_REF] and VIGOR [START_REF] Bombardier | Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis[END_REF], both showed that rofecoxib increased the risk of cardiovascular events significantly. However, the first trial, which ended in 1999, was not published in a scientific journal until 2006 [START_REF] Weaver | Treatment of patients with osteoarthritis with rofecoxib compared with nabumetone[END_REF]. The second trial was published in the New England Journal of Medicine, but the increased risk of myocardial infarction was interpreted as a beneficial aspirin-like prophylactic effect of the control NSAID [START_REF] Bombardier | Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis[END_REF]. This interpretation was speculative and was later refuted. Furthermore, three cases of myocardial infarction in the rofecoxib arm had been omitted from the paper [START_REF] Curfman | Expression of concern: Bombardier et al, "Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis[END_REF].

In 2001, it was documented in a systematic review that COX-2 inhibitors increased the risk of cardiovascular events [START_REF] Mukherjee | Risk of cardiovascular events associated with selective COX-2 inhibitors[END_REF], and a cumulative meta-analysis of trials from 2004 showed that a clear relationship between rofecoxib and increased risk of myocardial infarction existed already by the end of 2000 [START_REF] Jüni | Risk of cardiovascular events and rofecoxib: cumulative meta-analysis[END_REF]. Two other meta-analyses did not find evidence of an increase in cardiovascular risk with rofecoxib, but they were conducted by employees of Merck [START_REF] Konstam | Cardiovascular thrombotic events in controlled, clinical trials of rofecoxib[END_REF][START_REF] Reicin | Comparison of cardiovascular thrombotic events in patients with osteoarthritis treated with rofecoxib versus nonselective nonsteroidal anti-inflammatory drugs (ibuprofen, diclofenac, and nabumetone)[END_REF]. Over the studied time period, there has been an increased focus on harms [START_REF] Ioannidis | Better reporting of harms in randomized trials: an extension of the CONSORT statement[END_REF][START_REF] Bernal-Delgado | Abstracts in high profile journals often fail to report harm[END_REF][START_REF] Kim | Rofecoxib, Merck, and the FDA[END_REF] and the quality of reporting trial results in abstracts [START_REF] Hopewell | CONSORT for reporting randomised trials in journal and conference abstracts[END_REF], which may have had an impact on our results. However, our sample of abstracts did not exclusively consist of trial abstracts, and the increased attention to harms does not explain the dramatic change in focus from beneficial to harmful effect when rofecoxib was withdrawn.

The safety data from trials on rofecoxib were far too positive compared to a real-world setting. None of the trials in the application for marketing approval were designed to evaluate the cardiovascular risk [START_REF] Krumholz | What have we learned from Vioxx?[END_REF]. In fact, they included patients that had an unusually low cardiovascular risk. Medicare patients in Tennessee, who were treated with rofecoxib in clinical practice, had a baseline risk of getting a myocardial infarction that was eight times higher than that for the patients in the trials [START_REF] Jüni | Risk of cardiovascular events and rofecoxib: cumulative meta-analysis[END_REF]. Patients at high risk of developing peptic ulcers were also often left out of trials on rofecoxib. In 2002, an analysis of cardiovascular adverse events was added to the protocols of three studies [START_REF] Kim | Rofecoxib, Merck, and the FDA[END_REF] including the one [START_REF] Bresalier | Adenomatous Polyp Prevention on Vioxx (APPROVe) Trial Investigators (2005) Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial[END_REF] that led to the withdrawal of rofecoxib [START_REF]Merck announces voluntary worldwide withdrawal of VIOXX[END_REF]. This was considered breaking news and is likely to have initiated the change in focus from beneficial effects to harms.

Publishing and disseminating scientific papers on medical interventions is an important marketing strategy for the pharmaceutical industry [START_REF] Steinman | Narrative review: the promotion of gabapentin: an analysis of internal industry documents[END_REF], and Merck's active role in the writing of journal articles is likely to have influenced how rofecoxib was portrayed and perceived by the clinicians. It is difficult to explore Merck's role in more detail in relation to our results. Merck's information control could have been clarified by looking at reporting in relation to type of financial support. We did not attempt to do this, as ghost authorship and other forms of support from drug companies are often not revealed in scientific papers [START_REF] Gøtzsche | Ghost authorship in industry-initiated randomised trials[END_REF], and Merck used guest and ghost authors for many of the papers on rofecoxib [START_REF] Ross | Guest authorship and ghostwriting in publications related to rofecoxib: a case study of industry documents from rofecoxib litigation[END_REF]. Merck also conducted a seeding trial [START_REF] Hill | The ADVANTAGE seeding trial: a review of internal documents[END_REF], the ADVANTAGE trial, published in Annals of Internal Medicine [START_REF] Lisse | Gastrointestinal tolerability and effectiveness of rofecoxib versus naproxen in the treatment of osteoarthritis: a randomized, controlled trial[END_REF], and sponsored the Australasian Journal of Bone and Joint Medicine, which looked like a peerreviewed medical journal but was only a marketing tool. Most of the articles in the journal presented data favourable to Merck products, including rofecoxib, without disclosing sponsorship [29].

A strategy to increase drug sales that has been used by Merck [START_REF]Warning letter from FDA's division of drug marketing and communications to Merck regarding aggrastat[END_REF] and many other drug companies is to stimulate off-label use [START_REF] Steinman | Narrative review: the promotion of gabapentin: an analysis of internal industry documents[END_REF][START_REF] Ratner | Pfizer settles largest ever fraud suit for off-label promotion[END_REF]. This may also be the case for rofecoxib [START_REF] Tesorieri | Federal grand jury probes Merck's handling of Vioxx[END_REF] and could explain why many abstracts mentioned or evaluated the effect of rofecoxib in relation to other conditions than arthritis (Fig. 4). After having assessed one-third of the abstracts (n=1,370), one observer decided to register the conditions (apart from arthritis) that rofecoxib was proposed for. These were mainly neurological disorders, cancer and pain related to minor surgery. The U.S. Food and Drug Administration approved rofecoxib for osteoarthritis, acute pain, primary dysmenorrhea and rheumatoid arthritis.

We searched for abstracts in PubMed only, as PubMed is the most widely used database for medical research. It is likely that more abstracts would have been included if we had searched additional databases, but we would not expect it to have led to any important changes in our results.

It has been suggested that increases in blood pressure related to rofecoxib are a mechanism for the increase in the risk of cardiovascular events [START_REF] Singh | Consequences of increased systolic blood pressure in patients with osteoarthritis and rheumatoid arthritis[END_REF]. We excluded 26 abstracts for the reason that they only commented on hypertension, but they would not have changed the results much as they were scattered over the years 2001 to 2007.

We believe that if the reporting of benefits and harms in abstracts is unbalanced, doctors will get a false perception of the drug's value. In particular, readers need information on deaths, and on harms that can be lethal, such as thromboses causes by COX-2 inhibitors. In the drug literature, there is plenty of evidence of flawed research [START_REF] Gøtzsche | Methodology and overt and hidden bias in reports of 196 double-blind trials of nonsteroidal, antiinflammatory drugs in rheumatoid arthritis[END_REF][START_REF] Bero | Influences on the quality of published drug studies[END_REF][START_REF] Safer | Design and reporting modifications in industrysponsored comparative psychopharmacology trials[END_REF][START_REF] Melander | Evidence b(i)ased medicine -selective reporting from studies sponsored by pharmaceutical industry: review of studies in new drug applications[END_REF][START_REF] Bero | Factors associated with findings of published trials of drug-drug comparisons: why some statins appear more efficacious than others[END_REF][START_REF] Hrachovec | Reporting of 6-month vs 12-month data in a clinical trial of celecoxib[END_REF][START_REF] Jüni | Are selective COX 2 inhibitors superior to traditional non steroidal anti-inflammatory drugs?[END_REF][START_REF] Brophy | Selling safety-lessons from muraglitazar[END_REF][START_REF] Nissen | Effect of muraglitazar on death and major adverse cardiovascular events in patients with type 2 diabetes mellitus[END_REF][START_REF] Vedula | Outcome reporting in industry-sponsored trials of gabapentin for off-label use[END_REF], ghost-written articles [START_REF] Steinman | Narrative review: the promotion of gabapentin: an analysis of internal industry documents[END_REF][START_REF] Gøtzsche | Ghost authorship in industry-initiated randomised trials[END_REF][START_REF] Grill | Kranke Geschäfte: wie die Pharmaindustrie uns manipuliert[END_REF][START_REF] Mundy | Dispensing with the truth[END_REF][START_REF] Angell | The truth about the drug companies: how they deceive us and what to do about it. Random House[END_REF][START_REF] Petersen | Biomedical conflicts of interest: a defence of the sequestration thesis-learning from the cases of Nancy Olivieri and David Healy[END_REF], intimidation of researchers [START_REF] Grill | Kranke Geschäfte: wie die Pharmaindustrie uns manipuliert[END_REF][START_REF] Mundy | Dispensing with the truth[END_REF][48][START_REF] Revill | Doctor accuses drug giant of 'unethical' secrecy[END_REF][START_REF] Revill | How the drugs giant and a lone academic went to war[END_REF][START_REF] Bosely | Junket time in Munich for the medical profession -and it´s all on the drug firms[END_REF][START_REF] Tanne | FDA places "black box" warning on antidiabetes drugs[END_REF][START_REF] Mello | Academic medical centers' standards for clinical-trial agreements with industry[END_REF][START_REF] Shuchman | Drug company threatens legal action over Canadian guidelines[END_REF][START_REF] Williams | Evening primrose oil for atopic dermatitis[END_REF][START_REF] Gornall | Industry attack on academics[END_REF] and misleading and false statements in research papers and marketing [START_REF] Steinman | Narrative review: the promotion of gabapentin: an analysis of internal industry documents[END_REF][START_REF] Grill | Kranke Geschäfte: wie die Pharmaindustrie uns manipuliert[END_REF][START_REF] Mundy | Dispensing with the truth[END_REF][START_REF] Angell | The truth about the drug companies: how they deceive us and what to do about it. Random House[END_REF][START_REF] Petersen | Biomedical conflicts of interest: a defence of the sequestration thesis-learning from the cases of Nancy Olivieri and David Healy[END_REF][START_REF] Braitwaite | Corporate crime in the pharmaceutical industry[END_REF][START_REF] Avorn | Powerful medicines: the benefits, risks, and costs of prescription drugs[END_REF][START_REF] Goozner | The $800 million pill: the truth behind the cost of new drugs[END_REF][60][START_REF] Graham | COX-2 inhibitors, other NSAIDs, and cardiovascular risk: the seduction of common sense[END_REF][START_REF] Avorn | Dangerous deception-hiding the evidence of adverse drug effects[END_REF][START_REF] Steinman | Characteristics and impact of drug detailing for gabapentin[END_REF][START_REF] Ziegler | The accuracy of drug information from pharmaceutical sales representatives[END_REF][START_REF] Perry | Neurontin: clinical pharmacologic opinion of Dr[END_REF].

We suggest that studies like ours should be done on other drugs than rofecoxib, preferably with newly marketed drugs associated with high expectations.

Conclusions

The basic principle of balanced reporting of benefits and harms seems to have been seriously distorted in abstracts on rofecoxib, although the harms were equally predictable as the benefits from the mechanism of action of the drug. Before the withdrawal of rofecoxib, abstracts mostly reported on gastrointestinal bleeding and were in favour of rofecoxib. The harms came in focus too late, when the drug had already been withdrawn, and when tens of thousands of patients had been harmed unnecessarily [3,[START_REF] Topol | Failing the public health-rofecoxib, Merck, and the FDA[END_REF][START_REF] Graham | Risk of acute myocardial infarction and sudden cardiac death in patients treated with cyclo-oxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: nested case-control study[END_REF].

Funding None.

Fig. 2

 2 Fig. 1 Search for relevant abstracts

Fig. 4

 4 Fig. 4 Conditions for which the effect of rofecoxib was mentioned


Mª Concepción

MD Sánchez Martínez
email: msanchezma@sego.es

Amparo Ruiz Simón

Mª Concepción Sánchez Martínez

Breast cancer during pregnancy

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Breast cancer associated with pregnancy is defined as breast cancer diagnosed during pregnancy, breastfeeding or during the first year postpartum.

The incidence of breast cancer during pregnancy ranges between 0.2% and 3.8% of all malignant breast tumors, but if only women of childbearing age (<45 years) are considered this prevalence reaches 7-14% [START_REF] Ring | Breast cancer and pregnancy[END_REF][START_REF] Pavlidis | Coexistence of pregnancy and malignancy[END_REF]. Although breast cancer is an uncommon condition in pregnant women, it is the most common malignancy associated with pregnancy after cervical carcinoma, with an estimated incidence of 1/3000 to 3/10000 pregnancies [START_REF] Pavlidis | Coexistence of pregnancy and malignancy[END_REF][START_REF] Anderson | Mammary cancers and pregnancy[END_REF]. There has been an increase in the incidence of breast cancer in women of childbearing age due to the delay in initiating pregnancies with respect to previous decades. The current average age of the pregnant woman with breast cancer is 36 years.

Classically, the prognosis in pregnant women has been thought to be worse than in non-pregnant women. However, currently, and based on case-control studies, the prognosis of pregnant women does not seem to differ from that of non-pregnant patients of the same age and stage of disease. It is possible that the worse prognosis initially attributed to pregnant women was due to a more advanced stage at diagnosis or a less standardized therapy [START_REF] Loibl | Breast carcinoma during pregnancy. International recommendations from an expert meeting[END_REF].

Clinical experience of breast cancer in pregnant women is very limited due to its rarity, and therefore making therapeutic decisions can be complicated. Breast cancer in this group of women requires a change in the usual therapeutic approach, a modification of diagnostic methods, and often an adjustment of treatment sequences depending on gestational age [START_REF] Gentilini | Breast cancer diagnosed during pregnancy and lactation: biological features and treatment options[END_REF]. It requires a multidisciplinary expert team as well as psychological and family support.

Tumor characteristics of breast cancer in pregnant women

Biological characteristics of breast cancers in pregnant women are similar to those in young non-pregnant women. However, these characteristics, as well as the greater aggressiveness, are determined by age rather than by pregnancy itself. The most frequent histological type of breast tumor in pregnant women is invasive ductal carcinoma with a high histological grade. It has been shown that breast tumors diagnosed during pregnancy are of greater size and have increased axillary involvement, lymphovascular invasion is common, as well as a lower incidence of hormonal receptor expression. The percentage of tumors expressing human epidermal growth factor receptor 2 (HER2) is inconsistent across studies, but appears to be higher in pregnant women with breast cancer [START_REF] Middleton | Breast carcinoma in pregnant women: assessment of clinicopathologic and immunohistochemical features[END_REF][START_REF] Euler | Breast cancer in young women aged 35 or less[END_REF].

Diagnosis of breast cancer during pregnancy

A breast examination is recommended in all women at first pregnancy visit. Although over 80% of breast masses are benign in pregnancy, any breast node that persists beyond 2-4 weeks should be analyzed [START_REF] Woo | Breast cancer in pregnancy: a literature review[END_REF]. If breast masses or skin changes appear during pregnancy, pertinent studies should be performed to rule out malignancy.

Given the physiological changes of the mammary gland during pregnancy, the fear of using certain diagnostic procedures (mammography) may cause delays in diagnosis, which in many cases translates into a worse prognosis. If indicated, a mammogram can be performed using proper abdominal shielding, although it should be noted that the false negative rate is higher in pregnant women because of the physiological changes in the mammary gland. Breast ultrasound is important in differentiating solid and cystic lymph nodes and in assessing their morphology. Additionally, this method is very useful to evaluate breast masses in pregnant women in whom interpretation of mammography results is difficult due to the increased breast density. The routine use of magnetic resonance imaging is not recommended for the diagnosis of breast nodules during pregnancy because the effectiveness of this technique has not been established in pregnant women and there are no safety data regarding the use of gadolinium in this population. Although fine-needle aspiration can be used to diagnose a breast mass in a pregnant patient, a core or excisional biopsy is required for a definitive diagnosis of invasive cancer. The potential for the development of a subsequent mammillary fistula after core or excisional biopsy is overestimated, with very few reports in the literature [START_REF] Loibl | Breast carcinoma during pregnancy. International recommendations from an expert meeting[END_REF].

Once breast cancer is diagnosed, assessment of metastatic spread should only include those studies needed for an effective treatment planning. Computerized tomography, magnetic resonance imaging and bone scintigraphy should be avoided during the first trimester of pregnancy. If metastatic disease is suspected, these studies may be performed from the second trimester on. On the contrary, mammography, chest X ray (using proper shielding) and ultrasounds may be used whenever they are required.

Prenatal follow-up assessment

Monitoring of pregnancy must be strict in patients with breast cancer. The accurate dating of gestation is fundamental to know the probable date of birth and, based on that, to assess the therapeutic options. A high-resolution ultrasound must be performed to assess the morphology of the fetus prior to treatment. Amniocentesis should be offered if there is an obstetric indication.

If the patient is going to receive chemotherapy, fetal growth assessment should be carried out using serial ultrasounds every 3-4 weeks prior to each chemotherapy cycle, as well as a fetal biophysical profile from week 28 to term and an assessment of the amniotic fluid, because its volume can decrease reversibly with the use some drugs [START_REF]Protocolo de cáncer de mama y embarazo[END_REF].

An umbilical artery doppler study is also recommended to assess the status of the placenta and the fetal middle cerebral artery to rule out fetal anemia [START_REF]Protocolo de cáncer de mama y embarazo[END_REF]. In patients treated with potentially cardiotoxic drugs such as anthracyclines, it is advisable to carry out serial fetal echocardiography studies during treatment and in the neonatal period.

The expected date of delivery must be accurately known. It is recommended that gestation continues to week 37, but with advances in neonatal medicine today it is sometimes possible for babies to survive only 30 weeks of gestation. Delivery should be done vaginally because of its lower morbidity and mortality compared with caesarean section, particularly if chemotherapy is planned to be continued after delivery [START_REF] Loibl | Breast carcinoma during pregnancy. International recommendations from an expert meeting[END_REF].

Maternal myelosuppression secondary to chemotherapy should be avoided and controlled during labor and the perinatal period to minimize the risk of infection and bleeding. Furthermore, chemotherapy administered close to delivery may lead to accumulation of drugs on the fetus or newborn, thereby increasing exposure and toxicity. Therefore, chemotherapy should not be given after the 35 th week of gestation if delivery is to be spontaneous. Chemotherapy should be avoided during the last 2-3 weeks before delivery to minimize the risk of maternal and fetal neutropenia, although some authors recommend no less than 4 weeks [START_REF] Loibl | Breast carcinoma during pregnancy. International recommendations from an expert meeting[END_REF]. After delivery, chemotherapy should be delayed by one or two weeks and breastfeeding should be discontinued.

Although placental metastases are rare, the placenta should be sent for histological examination [START_REF] Loibl | Breast carcinoma during pregnancy. International recommendations from an expert meeting[END_REF].

Treatment of breast cancer during pregnancy

If the psychological impact following a diagnosis of breast cancer is high, this is even greater if the diagnosis is compounded by a pregnancy. A diagnosis of pregnancy can affect the treatment and therefore the prognosis of the disease. The therapeutic approach to breast cancer during pregnancy requires the participation of a multidisciplinary team, which will choose the most appropriate treatment according to tumor stage. Based on the treatment required, and after informing of the potential risks to the fetus, the patient and her family will decide whether to continue or terminate pregnancy. Until the decision is taken, the physician should provide all the necessary support to the patient.

For decades it was thought that pregnancy could worsen disease prognosis, and therefore in many cases women were advised to terminate pregnancy. Nevertheless, data from several retrospective studies have shown that the survival of pregnant patients with breast cancer is not increased by termination of the pregnancy, even if the cancer has spread [START_REF] Gwyn | Breast cancer during pregnancy[END_REF][START_REF] Trivers | Association between reproductive factors and breast cancer survival in younger women[END_REF][START_REF] Clark | Breast cancer and pregnancy: the ultimate challenge[END_REF]. It may be reasonable to discuss termination of pregnancy in women with advanced or metastatic disease, or in very early pregnancies because of the teratogenic risk of chemotherapy during the first trimester [START_REF] Clark | Breast cancer and pregnancy: the ultimate challenge[END_REF]. Breast cancer treatments include surgery, chemotherapy, radiotherapy and endocrine therapy.

Surgery in the pregnant woman with breast cancer

Surgery can be performed safely during all trimesters of pregnancy with minimal fetal risk. Mastectomy is a good choice when radiotherapy can not be delayed until after delivery. Breast-conserving surgery, when used, is more commonly performed at the end of the second trimester and during the third trimester, and radiotherapy is delivered postpartum [START_REF] Kuerer | Conservative surgery and chemotherapy for breast carcinoma during pregnancy[END_REF].

The role of sentinel node mapping is controversial in pregnant patients because of concerns about the possible teratogenic effects of blue dye and the reliability of lymphatic mapping in the pregnant breast. Although some have used radioactive colloid alone in this subgroup of patients, sensitivity of lymphatic mapping is reduced when a single modality is used [START_REF] Amersi | The benefits and limitations of sentinel lymph node biopsy[END_REF].

Cytotoxic therapy in the pregnant woman with breast cancer

The therapeutic approach may vary depending on the length of gestation. The effect of polychemotherapy during the first trimester of pregnancy involves a risk of malformations that can reach 25%, whereas when only one drug is used the probability is 17%. This incidence of teratogenesis is much higher than in the general population (3%) [START_REF] Wiebe | Pharmacology of antineoplastic agents in pregnancy[END_REF][START_REF] Doll | Antineoplastic agents and pregnancy[END_REF].

Chemotherapy can be administered from the second trimester onwards with some modifications to minimize harm to the fetus. There are enough safety data to suggest that there is no risk of fetal malformation when chemotherapy is administered during the second and third trimester [START_REF] Gwyn | Breast cancer during pregnancy[END_REF][START_REF] Keleher | Multidisciplinary management of breast cancer concurrent with pregnancy[END_REF].

Other toxicities to consider are increased frequency of miscarriage, possibility of low birth weight, and hematologic toxicity in the fetus [START_REF] Kerr | Neonatal effects of breast cancer chemotherapy administered during pregnancy[END_REF][START_REF] Giacalone | Chemotherapy for breast carcinoma during pregnancy: A French national survey[END_REF]. Changes that occur during pregnancy, such as the generation of a third space (fetal-placental amniotic fluid), increased volume distribution, and changes in the metabolism and elimination of drugs, may determine different toxicity patterns that may also indirectly affect the fetus. There is also a risk of long-term toxicities on chemotherapy, but information on this issue is scarce [START_REF] Aviles | Long-term follow-up of children born to mothers with acute leukemia during pregnancy[END_REF].

Clinical experience of pregnant women undergoing chemotherapy for breast cancer has resulted in allowing the administration anthracycline-based regimens from the second trimester onwards with some security [START_REF] Azim | Anthracyclines for gestational breast cancer: course and outcome of pregnancy[END_REF]. With other active drugs, such as taxanes, vinorelbine or trastuzumab the experience is more limited [START_REF] Mir | Emerging therapeutic options for breast cancer chemotherapy during pregnancy[END_REF]. Docetaxel, paclitaxel and vinorelbine have been used without serious toxicity to the fetus being detected [START_REF] Mir | Emerging therapeutic options for breast cancer chemotherapy during pregnancy[END_REF].

For the moment, targeted therapies such as trastuzumab seem less safe because they are associated with anhydramnios [START_REF] Sekar | Trastuzumab use for metastatic breast cancer in pregnancy[END_REF]. To date, there is no experience with lapatinib or with bevacizumab. Use of bisphosphonates should also be avoided due to safety issues.

In the future, the development of prospective databases will provide additional information, especially in regard to possible long-term sequelae secondary to antitumor therapies.

Radiotherapy and endocrine therapy in the pregnant woman with breast cancer

Radiotherapy and endocrine therapy are not indicated during pregnancy due to the high risk of teratogenicity, and should be delayed until after delivery [START_REF] Marty | Teratogenic and mutagenic risks of radiotherapy: when and how to prescribe contraception[END_REF][START_REF] Fenig | Pregnancy and radiation[END_REF][START_REF] Isaacs | Tamoxifen as systemic treatment of advanced breast cancer during pregnancy--case report and literature review[END_REF].

Risk of breast cancer during pregnancy in carriers of BRCA

In women with a family history of breast cancer without mutational study, pregnancy does not confer an increased risk of breast cancer. However, some studies have noted a significant increase in breast cancer incidence in the two years following pregnancy in BRCA2 mutation carriers [START_REF] Cullinane | Effect of pregnancy as a risk factor for breast cancer in BRCA1/BRCA2 mutation carriers[END_REF]. Because these data have not been confirmed in other studies [START_REF] Tryggvadottir | BRCA2 mutation carriers, reproductive factors and breast cancer risk[END_REF], currently a known carrier of a BRCA mutation can not be advised against becoming pregnant, but it is our responsibility to inform these women of the available results.

Conclusions



Breast cancer during pregnancy is occurring with increasing frequency because more women are delaying pregnancy into their thirtieth and fortieth decades.



The prognosis in pregnant women is classically thought to be worse than in nonpregnant woman, which is probably mainly due to variable factors such as delayed diagnosis, rather than the actual pregnancy itself. Data indicate that the prognosis of pregnant women does not appear to differ from that of non-pregnant patients of the same age and stage of disease.



Techniques to diagnose breast cancer in a pregnant woman are similar to those used in non-pregnant women (mammography, ultrasound, biopsy), with comparable efficiency. Studies to assess metastatic spread, such as computerized tomography or magnetic resonance imaging, should be limited to those strictly necessary, and recommended only in cases of suspected metastasis.



Monitoring of pregnancy must be strict in patients with breast cancer.



The probable date of birth needs to be known to assess therapeutic options.



The therapeutic approach to a pregnant woman with breast cancer requires the collaboration of a multidisciplinary team. The treatment of the cancer needs to be carefully planned, with consideration given to the potential risks to the developing fetus. Therapeutic abortion is rarely indicated except in women with advanced or metastatic disease or very early in pregnancy (first trimester), because of the potential teratogenic effects of chemotherapy.



Although breast-conserving surgery has evolved as the major treatment in breast cancer, in pregnant women is recommended only in the final of the second trimester and third trimester because the radiotherapy has to be delayed after delivery.



Radiotherapy and endocrine therapy should not be applied during pregnancy and should be delayed until after delivery.



Pregnant women with breast cancer can be treated with anthracycline-based chemotherapy during the second and third trimesters of pregnancy with minimal risk to the fetus. There is less experience with other chemotherapy agents such as taxanes and vinorelbine, but to date no malformations have been described secondary to their use. For now, the use of bisphosfonates or biological therapies (trastuzumab, bevacizumab, lapatinib) can not be recommended.

Acknowledgements

The authors acknowledge the support of Pfizer Spain, which facilitated the necessary meetings to evaluate and discuss all the data presented in this review, and Dr.

Fernando Sánchez Barbero from HealthCo SL (Madrid, Spain) for assistance in the preparation of this manuscript.