Antonio Clavenna
email: antonio.clavenna@marionegri.it

Marco Sequi

DIFFERENCES IN THE DRUG PRESCRIPTIONS TO CHILDREN BY ITALIAN PAEDIATRICIANS AND GENERAL PRACTITIONERS

Keywords: Child, Drug prescriptions, Family Practice, Pediatrics

Differences in the drug prescriptions to children by Italian paediatricians and general practitioners

Antonio Clavenna, Marco Sequi, Maurizio

INTRODUCTION

Drug utilization studies performed in the Italian paediatric population found a prescription profile that appears to be specific to Italy, especially concerning antibiotics. [1][2][3][4][5] For these drugs, both quantitative and qualitative differences with respect to other European countries were found. [6] The antibiotic prescription prevalence was 3-4 fold higher in Italy than in the Netherlands or the UK, with a wide use of second choice antibiotics such as cephalosporins. [6] Moreover, a wide, irrational use of anti-asthmatics, in particular inhaled steroids, was observed. [7] According to some international studies, drug prescriptions are influenced by the type of physician writing them. [8][9][10][11][START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In fact, differences in prescription profiles between family paediatricians and general practitioners were found, in particular concerning antibiotics. [9,10,[START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In Italy, children are assigned to a paediatrician until they are 6 years old; afterwards, the parents can choose to remain with that paediatrician until child is 14 years old or to register the child with a general practitioner. All adolescents over 14 years of age are assigned to a general practitioner. No studies were performed in Italy to compare the prescribing profile of the two kinds of physicians.

Thus, an analysis of drugs prescribed to children 6-13 years old was performed in order to compare general practitioners' with family paediatricians' prescribing profiles.

METHODS

The data source was a database routinely updated for administrative and reimbursement reasons.

The database stores all community (i.e. out of hospital) prescriptions issued to individuals living in the Lombardy Region that are reimbursed by the National Health Service (NHS).

Data were managed and analysed using an anonymous patient code, after obtaining each local health unit director's authorisation. In order to obtain a comparable sample, only physicians who were in charge of children representing all the ages in the 6-13 year old range were included. Moreover, physicians below the 5-centile of the distribution of physicians by number of treated children were excluded. Children receiving six or more prescriptions were also excluded, since the percentage of chronically treated patients may be different between physicians and may influence the prescribing profile.

A total of 548,922 children 6-13 years of age was, therefore, selected (81% of the Lombardy Region's population 6-13 year old): 402,053 children were cared for by 1020 family paediatricians (88% of the total number of family paediatricians) and 146,869 children cared for by 2824 general practitioners (42% of the total). Prescribed drugs were classified according to the International Anatomic-Therapeutical-Chemical Classification system (ATC).

Prevalence data by children's age and type of physician were calculated by dividing the number of drug users by the total number of residents in each age group.

Moreover, a Mantel-Haentzel χ 2 test was performed in order to compare the drug prescription prevalence in children cared for by family paediatricians versus children cared for by general practitioners.

The area under the prevalence-time curve (AUC) from time 6 to 13 years (data plotted at mid-time interval) were calculated according to the linear trapezoidal rule. The slopes of the prevalence vs time curve were estimated by log-linear least square fit of the 6 and 13 year age points.

Comparisons were made using the t test. The percentage of family paediatricians and general practitioners who prescribed each single drug was calculated and a percentage ≥ 75% was considered a high degree of concordance.

Statistical analysis was performed using SAS software, version 9.1.

A P value < 0.05 was considered statistically significant.

RESULTS

During 2005, 239,296 children (43.6% of the population) received at least one drug prescription (table 1). The prevalence was highest at 6 years of age (53.9%), and decreased to 35% in children 13 years old (Figure 1).

The prevalence was slightly higher in children treated by general practitioners (44.2 versus 43.4%;

χ 2
M-H = 845, p <0.001). The greatest difference was observed in 13 year old children, with a prevalence of 38.7 versus 31.8% in children cared for by general practitioners and family paediatricians, respectively. A difference was found when comparing the slopes of the prevalence versus age curve (p<0.001).

The AUC 6-14 general practitioners/paediatricians prevalence ratio was 1.1.

The chance of receiving a drug prescription, adjusted for age, gender, and LHU of residence, was slightly higher in children cared for by general practitioners (OR 1.16; 95%CI 1.14-1.17).

Moreover, the chance was slightly higher for children in charge to female compared to male physicians (OR 1.08; 95%CI 1.07-1.10).

On average, each treated child received 2 prescriptions, without differences between the two physician type groups, while the mean number of medication packages prescribed to children cared for by family paediatricians was slightly higher (2.9 versus 2.6).

Antibiotics, anti-asthmatics, and anti-histamines were the most prescribed therapeutic classes, and covered 92.5% of the packages. The prevalence of antibiotics was nearly the same in the two groups (37%), while the prevalence of anti-asthmatics was slightly higher in children treated by family paediatricians (10.6 vs 9.9%) and, on the contrary, the prevalence of anti-histamines was higher in the general practitioner treated group (4.2 vs 3.6%).

Taking into account the ten most prescribed classes, the prevalence of antinflammatory and antirheumatic drugs was 5-fold higher, and the prevalence of intestinal antidiarrheals/antinfectives was 3.5 fold higher, in children treated by general practitioners.

On the contrary, the prevalence rates of anti-parasitic drugs and antivirals were 1.4 fold higher in the paediatrician treated group.

An analysis of antibiotic prescriptions by class was also performed; the prevalence of penicillins was higher in children cared for by family paediatricians (64.5 vs 54.2%), while the prevalence of macrolides and cephalosporins was higher in children cared for by general practitioners (32.9 vs 28.3%, and 32.2 vs 26.6%, respectively).

Amoxicillin+clavulanic acid was the most frequently prescribed drug independently of the type of physician, followed by amoxicillin and beclometasone. Fourteen out of the 15 most frequently prescribed drugs were the same in both groups (table 2).

Budesonide was among the most prescribed drugs only in the family paediatrician treated group and oxatomide only in the general practitioner group.

A total of 542 drugs were prescribed, 78% of which were prescribed by both the family paediatricians and the general practitioners. 74 drugs (14%) were prescribed only by the family paediatricians and 42 (8%) only by the general practitioners. In particular, 7/74 and 4/42 were sexual hormones (mainly oral contraceptives). Furthermore, 48 out of 74 (64%) and 36 out of 42 (86%) drugs were prescribed by only one physician to one child.

In all, 96% of the drugs were prescribed by less than 25% of the physicians, with a higher percentage among general practitioners compared to family paediatricians (97% versus 92%,

F

DISCUSSION

The prevalence of drug prescriptions was slightly higher in children treated by general practitioners with an OR of 1.2. On the contrary, children cared for by paediatricians received a greater number of packages. Despite these differences, the prescription profiles of general practitioners and family paediatricians were similar.

Only a few differences were found concerning the most prescribed therapeutic classes, as expected since the two types of physicians face the same diseases. However, the differences were greater for 9 some classes with a low prevalence rate, e.g. the prevalence of antinflammatory drugs was 5-fold higher in children treated by general practitioners, even if the most frequently prescribed drugs were the same in both the groups (ketoprofen, nimesulide, acetilsalicilic acid).

A large number of drugs was prescribed, and most of them were prescribed by both family paediatricians and general practitioners. Only a few drugs were specific of a type of physician, and nearly all of them were prescribed by one physician to one child.

It is interesting to note that while 13 drugs were prescribed by 75% or more family paediatricians, only 4 drugs were prescribed by 75% or more general practitioners. A wide variability in the drugs was observed for both groups of physicians, even if a greater percentage of drugs prescribed by less than 25% of the physicians was found for general practitioners (97% versus 92%). A lower concordance was, therefore, observed between general practitioners, and very few drugs can be considered "essential" on the basis of their prescribing attitude.

It is likely that this finding is influenced by the fact that general practitioners are in charge of a lower number of children compared to family paediatricians and that each general practitioner has to therefore deal with a narrower spectrum of diseases and therapeutic needs.

A low level of agreement emerged also when looking at the pharmaceutical preparations of the 4 drugs with a high degree of concordance among prescribers: 11 preparations were prescribed by 75% or more family paediatricians and only 2 by 75% or more general practitioners. In the case of amoxicillin and clarithromycin there were no pharmaceutical preparations prescribed by more than 50% of the general practitioners. However, this could indicate that many general practitioners prefer to prescribe only one trade name, while family paediatricians more commonly prescribe different trade names of the same drug, as was the case for clarithromycin, for which 3 different trade name oral suspensions were prescribed by more than 75% family paediatricians.

General practitioner seems to have less of a tendency to prescribe generic drugs, at least taking into account amoxicillin prescriptions. In fact, nearly 6 out of 10 general practitioners never prescribed The prescribing profile observed in this setting did not differ from that of other Italian contexts, in particular concerning the antibiotic prescription profile, characterized by a wide use of second line agents (cephalosporins or macrolides), in which Italy differs from other European countries. [6] A more common inappropriate use of antibiotics was observed among general practitioners than family paediatricians in three international studies. [10,[START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In this study, no differences were observed regarding antibiotic prevalence rate; however, differences were found concerning antibiotic classes, with the family paediatricians prescribing penicillins more commonly and general practictioners prescribing macrolides and cephalosporins more frequently. This finding partly reflects the profile observed in the Italian adult population. [START_REF] Mazzaglia | Exploring patient-and doctor-related variables associated with antibiotic prescribing for respiratory infections in primary care[END_REF] Amoxi-clavulanic acid was the most prescribed antibiotic and it was prescribed by more than 96% of physicians, despite the fact that amoxicillin is considered the antibiotic of choice for the most common infections in the paediatric age group [START_REF]Diagnosis and management of childhood otitis media in primary care. A national clinical guideline[END_REF][START_REF]Diagnosis and management of acute otitis media[END_REF][START_REF]Management of sore throat and indication for tonsillectomy. A national clinical guideline[END_REF] and is the most prescribed antibiotic in other countries [6]. Moreover, nearly 1 out of 10 general practitioners never prescribed amoxicillin.

In Italy, amoxicillin+clavulanic acid was probably preferred due to its twice daily schedule regimen that is supposed to give a better compliance compared to the amoxicillin dosage schedule and to an unproved concern of beta-lactamase-producing bacteria. This study therefore seems to confirm that different attitudes towards antibiotic prescribing exist between family paediatricians and general practitioners.

However, taking into account other drug classes, family paediatricians and general practitioners seem to share the same inappropriate prescriptions. In this regard, it is interesting to note that the prevalence of nebulised beclometasone, commonly prescribed without evidence of efficacy for the symptomatic treatment of upper respiratory tract infections (e.g. common cold, pharyngitis), as 11 previously reported in a study performed with a group of Italian family paediatricians [START_REF] Pandolfini | Italian family paediatricians and off-label prescriptions: loyal to regulatory or guideline standards[END_REF], was the same in the two groups.

The main limit of this study is that only drugs reimbursed by the national health service were evaluated. It is therefore possible that there are some differences in the prescribing profile of drugs non reimbursed or over the counter (e.g. analgesics, antitussive medicines, and prokinetics).

Moreover, this study was performed in a very homogeneous regional setting and the findings may not perfectly reflect the national situation. However, the study sample represents 12% of the 6-13 year old Italian population and the profile of drug prescriptions is similar to that observed in other national settings.

The findings from this study seem to document that there are few differences in the prescribing pattern between general practitioners and family paediatricians. Differences exist in particular for older children and for some drug classes. The inappropriateness of drug prescription to children is mostly independent from the type of physician. This should be taken into account in planning and performing educational interventions with the aim to improve the rational use of drugs in children. The prevalence of the most prescribed therapeutic classes was similar in the two groups. Some differences were found concerning the antibiotic classes, with family paediatricians prescribing penicillins more frequently and general practioners prescribing macrolides and cephalosporins more frequently.

A total of 542 drugs were prescribed, 78% of which were prescribed by both the family paediatricians and the general practitioners. In all, only 4 drugs were prescribed by 75% or more physicians.

The chance risk of receiving a drug prescription, adjusted for age, gender, and local health unit of residence, was slightly higher in children cared for by general practitioners (OR 1.16; 95%CI 1.14-1.17)

Conclusions. Few differences were found between the prescribing patterns of family paediatricians and those of general practitioners. Differences exist in particular for older children and for some drug classes. Drug utilization studies performed in the Italian paediatric population found a prescription profile that appears to be specific to Italy, especially concerning antibiotics. [1][2][3][4][5] For these drugs, both quantitative and qualitative differences with respect to other European countries were found. [6] The antibiotic prescription prevalence was 3-4 fold higher in Italy than in the Netherlands or the UK, with a wide use of second choice antibiotics such as cephalosporins. [6] Moreover, a wide, irrational use of anti-asthmatics, in particular inhaled steroids, was observed. [7] According to some international studies, drug prescriptions are influenced by the type of physician writing them. [8][9][10][11][START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In fact, differences in prescription profiles between family paediatricians and general practitioners were found, in particular concerning antibiotics. [9,10,[START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In Italy, children are assigned to a paediatrician until they are 6 years old; afterwards, the parents can choose to remain with that paediatrician until child is 14 years old or to register the child with a general practitioner. All adolescents over 14 years of age are assigned to a general practitioner. No studies were performed in Italy to compare the prescribing profile of the two kinds of physicians.

Thus, an analysis of drugs prescribed to children 6-13 years old was performed in order to compare general practitioners' with family paediatricians' prescribing profiles.

METHODS

The data source was a database routinely updated for administrative and reimbursement reasons.

The database stores all community (i.e. out of hospital) prescriptions issued to individuals living in the Lombardy Region that are reimbursed by the National Health Service (NHS).

Data were managed and analysed using an anonymous patient code, after obtaining each local health unit director's authorisation. The area under the prevalence-time curve (AUC) from time 6 to 13 years (data plotted at mid-time interval) were calculated according to the linear trapezoidal rule. The slopes of the prevalence vs time curve were estimated by log-linear least square fit of the 6 and 13 year age points.

Comparisons were made using the t test. The percentage of family paediatricians and general practitioners who prescribed each single drug was calculated and a percentage ≥ 75% was considered a high degree of concordance.

Statistical analysis was performed using SAS software, version 9.1.

A P value < 0.05 was considered statistically significant.

RESULTS

During 2005, 239,296 children (43.6% of the population) received at least one drug prescription (table 1). The prevalence was highest at 6 years of age (53.9%), and decreased to 35% in children 13 years old (Figure 1).

The prevalence was slightly higher in children treated by general practitioners (44.2 versus 43.4%;

χ 2
M-H = 845, p <0.001). The greatest difference was observed in 13 year old children, with a prevalence of 38.7 versus 31.8% in children cared for by general practitioners and family paediatricians, respectively. A difference was found when comparing the slopes of the prevalence versus age curve (p<0.001).

The AUC 6-14 general practitioners/paediatricians prevalence ratio was 1.1.

The risk chance of receiving a drug prescription, adjusted for age, gender, and LHU of residence, was slightly higher in children cared for by general practitioners (OR 1.16; 95%CI 1.14-1.17).

Moreover, the risk chance was slightly higher for children in charge to female compared to male physicians (OR 1.08; 95%CI 1.07-1.10).

On average, each treated child received 2 prescriptions, without differences between the two physician type groups, while the mean number of medication packages prescribed to children cared for by family paediatricians was slightly higher (2.9 versus 2.6).

Antibiotics, anti-asthmatics, and anti-histamines were the most prescribed therapeutic classes, and covered 92.5% of the packages. The prevalence of antibiotics was nearly the same in the two groups (37%), while the prevalence of anti-asthmatics was slightly higher in children treated by family paediatricians (10.6 vs 9.9%) and, on the contrary, the prevalence of anti-histamines was higher in the general practitioner treated group (4.2 vs 3.6%).

Taking into account the ten most prescribed classes, the prevalence of antinflammatory and antirheumatic drugs was 5-fold higher, and the prevalence of intestinal antidiarrheals/antinfectives was 3.5 fold higher, in children treated by general practitioners.

On the contrary, the prevalence rates of anti-parasitic drugs and antivirals were 1.4 fold higher in the paediatrician treated group.

An analysis of antibiotic prescriptions by class was also performed; the prevalence of penicillins was higher in children cared for by family paediatricians (64.5 vs 54.2%), while the prevalence of macrolides and cephalosporins was higher in children cared for by general practitioners (32.9 vs 28.3%, and 32.2 vs 26.6%, respectively).

Amoxicillin+clavulanic acid was the most frequently prescribed drug independently of the type of physician, followed by amoxicillin and beclometasone. Fourteen out of the 15 most frequently prescribed drugs were the same in both groups (table 2).

Budesonide was among the most prescribed drugs only in the family paediatrician treated group and oxatomide only in the general practitioner group.

A total of 542 drugs were prescribed, 78% of which were prescribed by both the family paediatricians and the general practitioners. 74 drugs (14%) were prescribed only by the family paediatricians and 42 (8%) only by the general practitioners. In particular, 7/74 and 4/42 were sexual hormones (mainly oral contraceptives). Furthermore, 48 out of 74 (64%) and 36 out of 42 (86%) drugs were prescribed by only one physician to one child.

In all, 96% of the drugs were prescribed by less than 25% of the physicians, with a higher percentage among general practitioners compared to family paediatricians (97% versus 92%,

F

DISCUSSION

The prevalence of drug prescriptions was slightly higher in children treated by general practitioners with an OR of 1.2. On the contrary, children cared for by paediatricians received a greater number of packages. Despite these differences, the prescription profiles of general practitioners and family paediatricians were similar.

Only a few differences were found concerning the most prescribed therapeutic classes, as expected since the two types of physicians face the same diseases. However, the differences were greater for 9 some classes with a low prevalence rate, e.g. the prevalence of antinflammatory drugs was 5-fold higher in children treated by general practitioners, even if the most frequently prescribed drugs were the same in both the groups (ketoprofen, nimesulide, acetilsalicilic acid).

A large number of drugs was prescribed, and most of them were prescribed by both family paediatricians and general practitioners. Only a few drugs were specific of a type of physician, and nearly all of them were prescribed by one physician to one child.

It is interesting to note that while 13 drugs were prescribed by 75% or more family paediatricians, only 4 drugs were prescribed by 75% or more general practitioners. A wide variability in the drugs was observed for both groups of physicians, even if a greater percentage of drugs prescribed by less than 25% of the physicians was found for general practitioners (97% versus 92%). A lower concordance was, therefore, observed between general practitioners, and very few drugs can be considered "essential" on the basis of their prescribing attitude.

It is likely that this finding is influenced by the fact that general practitioners are in charge of a lower number of children compared to family paediatricians and that each general practitioner has to therefore deal with a narrower spectrum of diseases and therapeutic needs.

A low level of agreement emerged also when looking at the pharmaceutical preparations of the 4 drugs with a high degree of concordance among prescribers: 11 preparations were prescribed by 75% or more family paediatricians and only 2 by 75% or more general practitioners. In the case of amoxicillin and clarithromycin there were no pharmaceutical preparations prescribed by more than 50% of the general practitioners. However, this could indicate that many general practitioners prefer to prescribe only one trade name, while family paediatricians more commonly prescribe different trade names of the same drug, as was the case for clarithromycin, for which 3 different trade name oral suspensions were prescribed by more than 75% family paediatricians.

General practitioner seems to have less of a tendency to prescribe generic drugs, at least taking into account amoxicillin prescriptions. In fact, nearly 6 out of 10 general practitioners never prescribed The prescribing profile observed in this setting did not differ from that of other Italian contexts, in particular concerning the antibiotic prescription profile, characterized by a wide use of second line agents (cephalosporins or macrolides), in which Italy differs from other European countries. [6] A more common inappropriate use of antibiotics was observed among general practitioners than family paediatricians in three international studies. [10,[START_REF] Pradier | Factors related to the prescription of antibiotics for young children with viral pharyngitis by general practitioners and family paediatricians in southeastern France[END_REF][START_REF] Bocquet | Comparison of prescriptions by paediatricians and general practitioners: a population-based study in Franche-Comté from the database of Regional Health Insurance Fund[END_REF] In this study, no differences were observed regarding antibiotic prevalence rate; however, differences were found concerning antibiotic classes, with the family paediatricians prescribing penicillins more commonly and general practictioners prescribing macrolides and cephalosporins more frequently. This finding partly reflects the profile observed in the Italian adult population. [START_REF] Mazzaglia | Exploring patient-and doctor-related variables associated with antibiotic prescribing for respiratory infections in primary care[END_REF] Amoxi-clavulanic acid was the most prescribed antibiotic and it was prescribed by more than 96% of physicians, despite the fact that amoxicillin is considered the antibiotic of choice for the most common infections in the paediatric age group [START_REF]Diagnosis and management of childhood otitis media in primary care. A national clinical guideline[END_REF][START_REF]Diagnosis and management of acute otitis media[END_REF][START_REF]Management of sore throat and indication for tonsillectomy. A national clinical guideline[END_REF] and is the most prescribed antibiotic in other countries [6]. Moreover, nearly 1 out of 10 general practitioners never prescribed amoxicillin

In Italy, amoxicillin+clavulanic acid was probably preferred due to its twice daily schedule regimen that is supposed to give a better compliance compared to the amoxicillin dosage schedule and to an unproved concern of beta-lactamase-producing bacteria. Moreover, nearly 1 out of 10 general practitioners never prescribed amoxicillin, which is the first line antibiotic for the most common infections in children. [START_REF]Diagnosis and management of childhood otitis media in primary care. A national clinical guideline[END_REF][START_REF]Diagnosis and management of acute otitis media[END_REF][START_REF]Management of sore throat and indication for tonsillectomy. A national clinical guideline[END_REF] This study therefore seems to confirm that different attitudes towards antibiotic prescribing exist between family paediatricians and general practitioners.

However, taking into account other drug classes, family paediatricians and general practitioners seem to share the same inappropriate prescriptions. In this regard, it is interesting to note that the prevalence of nebulised beclometasone, commonly prescribed without evidence of efficacy for the previously reported in a study performed with a group of Italian family paediatricians [START_REF] Pandolfini | Italian family paediatricians and off-label prescriptions: loyal to regulatory or guideline standards[END_REF], was the same in the two groups.

The main limit of this study is that only drugs reimbursed by the national health service were evaluated. It is therefore possible that there are some differences in the prescribing profile of drugs non reimbursed or over the counter (e.g. analgesics, antitussive medicines, and prokinetics).

Moreover, this study was performed in a very homogeneous regional setting and the findings may not perfectly reflect the national situation. However, the study sample represents 12% of the 6-13 year old Italian population and the profile of drug prescriptions is similar to that observed in other national settings.

The findings from this study seem to document that there are few differences in the prescribing pattern between general practitioners and family paediatricians. Differences exist in particular for older children and for some drug classes. The inappropriateness of drug prescription to children is mostly independent from the type of physician. This should be taken into account in planning and performing educational interventions with the aim to improve the rational use of drugs in children.

General practitioner

Paediatricians

  all paediatric prescriptions reimbursed by the Health Service and dispensed by the retail pharmacies of all the 15 local health units (LHU) in the Lombardy Region, in northern Italy, between 1 January and 31 December 2005.

6 A

 6 logistic regression analysis was performed to evaluate the association between drug prescription, type and gender of physician, and the age, gender, and LHU of residence of the child.

  children cared for by general practitioners had a 50% lower chance of receiving generic amoxicillin compared to children cared for by family paediatricians.

5 The

 5 analysis involved all paediatric prescriptions reimbursed by the Health Service and dispensed by the retail pharmacies of all the 15 local health units (LHU) in the Lombardy Region, in northern Italy, between 1 January and 31 December 2005.In order to obtain a comparable sample, only physicians who were in charge of children representing all the ages in the 6-13 year old range were included. Moreover, physicians below the 5-centile of the distribution of physicians by number of treated children were excluded. Children receiving six or more prescriptions were also excluded, since the percentage of chronically treated patients may be different between physicians and may influence the prescribing profile.A total of 548,922 children 6-13 years of age was, therefore, selected (81% of the Lombardy Region's population 6-13 year old): 402,053 children were cared for by 1020 family paediatricians (88% of the total number of family paediatricians) and 146,869 children cared for by 2824 general practitioners (42% of the total). Prescribed drugs were classified according to the International Anatomic-Therapeutical-Chemical Classification system (ATC).Prevalence data by children's age and type of physician were calculated by dividing the number of drug users by the total number of residents in each age group.Moreover, a Mantel-Haentzel χ 2 test was performed in order to compare the drug prescription prevalence in children cared for by family paediatricians versus children cared for by general practitioners.

6 A

 6 logistic regression analysis was performed to evaluate the association between drug prescription, type and gender of physician, and the age, gender, and LHU of residence of the child.

  children cared for by general practitioners had a 50% lower chance of receiving generic amoxicillin compared to children cared for by family paediatricians.

  upper respiratory tract infections (e.g. common cold, pharyngitis), as

18 Figure 1 -

 181 Figure 1-Trend of prevalence by age and type of physician.

Table 1 -Characteristics of the population.

 1

	Such interventions should involve all the health care professionals and not only family
	paediatricians.

14. Mazzaglia G, Caputi AP, Rossi A, et al.

(2003)

. Exploring patient-and doctor-related variables associated with antibiotic prescribing for respiratory infections in primary care.

Eur J Clin Pharmacol. 59:651-7. 15. Scottish Intercollegiate Guidelines Network (SIGN). (2003) Diagnosis and management of childhood otitis media in primary care. A national clinical guideline. Scottish Intercollegiate Guidelines Network (SIGN). Edinburgh (Scotland) 16. American Academy of Pediatrics Subcommittee on Management of Acute Otitis Media. (2004) Diagnosis and management of acute otitis media. Pediatrics 113:1451-65 17. Scottish Intercollegiate Guidelines Network (SIGN). (1999) Management of sore throat and indication for tonsillectomy. A national clinical guideline. Scottish Intercollegiate Guidelines Network (SIGN). Edinburgh (Scotland).

Table 2 -The 15 most prescribed drugs in order of prevalence.

 2 % of packages covered by the 15 most prescribed drugs. The gray background highlights drugs prescribed by 75% or more physicians.

	Family paediatricians

*

Table 3 -Most prescribed pharmaceutical preparations of the four shared drugs.

 3

	Drug	Trade Name	Formulation	treated	FP (%)	GP (%)
				children (%)
	Co-amoxiclav (15)* Augmentin ®	400mg/5 ml + 57mg/5 dry powder	49.9	99.9	79.2
			for oral suspension
		Clavulin ®	400mg/5 ml + 57mg/5 dry powder	18.3	90.8	10.8
			for oral suspension
		Augmentin ®	875/125 mg tablets	10.9	89.6	15.3
		Augmentin ®	875/125 mg sachets	10.9	88.1	14.6
	Amoxicillin (42)	Zimox ®	5 g/100 ml powder for oral	24.1	91.3	49.9
			suspension
		Zimox ®	500 mg chewable tablets	12.2	71.1	31.5
		Amoxicillina ® 5 g/100 ml powder for oral	11.0	67.3	15.7
			suspension
		Zimox ®	1 g chewable tablets	10.7	65.4	31.6
		Velamox ®	1 g chewable tablets	8.7	45.6	35.5
	Beclometasone (13) Clenil ®	0.8/2 ml suspension for inhalation	76.5	99.8	91.3
		Prontinal	0.8/2 ml suspension for inhalation	22.8	83.4	40.0
	Clarithromycin (18) Klacid ®	125 mg/5ml Granules for Oral	31.7	95.7	46.7
			Suspension
		Macladin ®	125 mg/5ml Granules for Oral	20.7	86.9	37.2
			Suspension
		Veclam ®	125 mg/5ml Granules for Oral	16.4	80.0	25.5
			Suspension
		Klacid ®	250 mg tablets	8.1	58.5	27.1
		Macladin ®	250 mg tablets	4.6	35.2	20.2
	*( ) total number of preparations. Pharmaceutical preparations prescribed by 75% or more physicians are reported in bold. FP= family
	paediatricians; GP= general practitioners

18 Figure 1-Trend of prevalence by age and type of physician.

	DIFFERENCES IN THE DRUG PRESCRIPTIONS TO CHILDREN BY ITALIAN Abstract
	PAEDIATRICIANS AND GENERAL PRACTITIONERS. Purpose. To compare family paediatricians and general practitioners' habits on drug prescribing to
	children.
	Antonio Clavenna, Marco Sequi, and Maurizio Bonati. Methods. Prescriptions reimbursed by the Health Service and dispensed by the retail pharmacies of
	15 local health units in the Lombardy Region, Italy, during 2005 were analysed. Only family
	Laboratory for Mother and Child Health, Department of Public Health, Mario Negri Institute for paediatricians and general practitioners who were in charge of children of all the ages in the 6-13
	Pharmacological Research, Milan, Italy year old age range were included.
	F o r Correspondence to: Antonio Clavenna, Laboratory for Mother and Child Health, Department of Public Health, "Mario Negri" Institute for Pharmacological Research, via Giuseppe La Masa 19, Results. During 2005, 239,296 children (43.6% of the selected population) received at least one F drug prescription. o r The prevalence was higher in children treated by general practitioners (44.2 versus 43.4%), in
	P P particular in children 13 years old, with a prevalence of 38.7 versus 31.8% in children cared for by Milan 20156, Italy
	e phone: +39 02 39014 559, fax: +39 02 3550924, e-mail: antonio.clavenna@marionegri.it e general practitioners and family paediatricians, respectively.
		e r e r
		R R
		e e
	General practitioner	Paediatricians	v i e v i e
			w w
			1 2

  =11; p<0.001). In all, 13 drugs were prescribed by 75% or more family paediatricians and only 4 drugs were prescribed by 75% or more general practitioners (table2).

	respectively, χ 2 The drug with the highest degree of concordance was amoxicillin+clavulanic acid, prescribed by
	more than 96% of family paediatricians and general practitioners. The other common drugs were
	amoxicillin, beclometasone and clarithromycin. These four drugs covered 68% of the treated
	children and the 54% of the packages. A total of 91 pharmaceutical preparations (identified by trade
	name, strength and formulation) containing the four leading drugs was prescribed, 87 of which
	(96%) were shared by family paediatricians and general practitioners.
	Only two pharmaceutical preparations were prescribed by 75% or more family paediatricians and o r general practitioners: Augmentin ® (amoxicillin+clavulanic acid) 400/57 mg dry powder for oral
	P suspension and Clenil ® (beclometasone) 0.8mg/2 ml nebulised suspension. No pharmaceutical
	e preparation containing amoxicillin or clarithromycin was prescribed by more than 50% of the
	general practitioners. (table 3)	e r
	Among physicians prescribing amoxicillin, 91% of family paediatricians and 44% of the GPs R prescribed a generic specialty (χ 2 =504; p<0.001). The percentage of children receiving generic e amoxicillin was 41% in those cared for by family paediatricians and 22% in those cared for by v i e general practitioners (χ 2 =1525; p<0.001).
		w
		8

Table 1 -

 1 Such interventions should involve all the health care professionals and not only family paediatricians. Characteristics of the population.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	12

Table 3 -

 3 Most prescribed pharmaceutical preparations of the four shared drugs. total number of preparations. Pharmaceutical preparations prescribed by 75% or more physicians are reported in bold. FP= family paediatricians; GP= general practitioners

	Drug	Trade Name	Formulation	treated	FP (%)	GP (%)
				children (%)
	Co-amoxiclav (15)* Augmentin ®	400mg/5 ml + 57mg/5 dry powder	49.9	99.9	79.2
			for oral suspension
		Clavulin ®	400mg/5 ml + 57mg/5 dry powder	18.3	90.8	10.8
			for oral suspension
		Augmentin ®	875/125 mg tablets	10.9	89.6	15.3
		Augmentin ®	875/125 mg sachets	10.9	88.1	14.6
	Amoxicillin (42)	Zimox ®	5 g/100 ml powder for oral	24.1	91.3	49.9
			suspension
		Zimox ®	500 mg chewable tablets	12.2	71.1	31.5
		Amoxicillina ® 5 g/100 ml powder for oral	11.0	67.3	15.7
			suspension
		Zimox ®	1 g chewable tablets	10.7	65.4	31.6
		Velamox ®	1 g chewable tablets	8.7	45.6	35.5
	Beclometasone (13) Clenil ®	0.8/2 ml suspension for inhalation	76.5	99.8	91.3
		Prontinal	0.8/2 ml suspension for inhalation	22.8	83.4	40.0
	Clarithromycin (18) Klacid ®	125 mg/5ml Granules for Oral	31.7	95.7	46.7
			Suspension
		Macladin ®	125 mg/5ml Granules for Oral	20.7	86.9	37.2
			Suspension
		Veclam ®	125 mg/5ml Granules for Oral	16.4	80.0	25.5
			Suspension
		Klacid ®	250 mg tablets	8.1	58.5	27.1
		Macladin ®	250 mg tablets	4.6	35.2	20.2
	*( )

European Journal of Clinical Pharmacology

Acknowledgments. Antonio Clavenna holds an educational fellowship granted by Boehringer Ingelheim Italia.

The authors would like to acknowledge Dr Angela Bortolotti, Dr Luca Merlino, and Dr Ida Fortino, of the Regional Health Ministry -Lombardy Region, Milan, for providing data.

This study has been funded in part by the Lombardy Region Health Ministry (Progetto "Epidemiologia dei farmaci -EPIFARM") Acknowledgments. Antonio Clavenna holds an educational fellowship granted by Boehringer Ingelheim Italia.

The authors would like to acknowledge Dr Angela Bortolotti, Dr Luca Merlino, and Dr Ida Fortino, of the Regional Health Ministry -Lombardy Region, Milan, for providing data.

This study has been funded in part by the Lombardy Region Health Ministry (Progetto "Epidemiologia dei farmaci -EPIFARM")


P Karina

I Papakostas

M Kostopoulos

G Bobos

D Pentheroudakis

Pectasides

Aristotelis Bamias
email: abamias@med.uoa.gr

M Karina

P Papakostas

I Kostopoulos

M Bobos

G Vourli

E Samantas

Ch Christodoulou

G Pentheroudakis

D Pectasides

M A Dimopoulos

G Fountzilas

A randomized phase III study of adjuvant platinum/docetaxel chemotherapy with or without radiation therapy in patients with gastric cancer

Keywords: Adjuvant chemotherapy, Gastric cancer, Docetaxel, Radiotherapy, TMA, ERCC1, HER2, MAP-Tau

A randomized phase III study of adjuvant platinum/docetaxel chemotherapy with or without radiation therapy in patients with gastric cancer

Aristotelis Bamias, M.

Introduction

Gastric cancer is the fourth most common cancer and the second highest cause of cancer deaths worldwide [START_REF] Van Cutsem | The neo-adjuvant, surgical and adjuvant treatment of gastric adenocarcinoma. Current expert opinion derived from the Seventh World Congress on Gastrointestinal Cancer, Barcelona[END_REF]. Each year in Europe, an estimated 192,000 new cases of gastric cancer are diagnosed, with the disease accounting for about 158,000 deaths in 2000 [START_REF] Ferlayj | Cancer incidence, mortality and prevalence worldwide, version 1.0[END_REF]. The treatment of choice for patients diagnosed with gastric cancer is surgical resection of the primary tumor and regional lymph nodes [START_REF] Catalano | Gastric cancer[END_REF]. However, even in patients presenting with early stage disease, about 60% eventually have local relapse or distant metastases after curative resection [START_REF] Ajani | Evolving chemotherapy for advanced gastric cancer[END_REF]. Various adjunctive therapies (such as chemotherapy, radiation therapy [RT], or immunotherapy) have been studied in an attempt to improve survival in patients with resectable gastric cancer [START_REF] Macdonald | Clinical overview: adjuvant therapy of gastrointestinal cancer[END_REF][START_REF] Sastre | Chemotherapy for gastric cancer[END_REF][START_REF] Cunningham | ESMO Guidelines Task Force. ESMO Minimum Clinical Recommendations for diagnosis, treatment and follow-up of gastric cancer[END_REF]. The role of adjuvant therapies has only recently been clariWed, following the results of three well designed randomized studies with more than 500 patients included in each, all showing a survival beneWt [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF][START_REF] Cunningham | Participants Trial (2006) Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer[END_REF][10]. Nevertheless, diVerent strategies were used: chemoradiotherapy [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF], neoadjuvant plus adjuvant chemotherapy [START_REF] Cunningham | Participants Trial (2006) Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer[END_REF], and adjuvant chemotherapy [10]. Therefore, it is still unclear what the optimal adjuvant therapy is for resected gastric cancer.

Chemoradiotherapy has recently been associated with a signiWcant survival beneWt in gastric cancer [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF]. The chemotherapy regimen consisted of 5-Xuorouracil and leucovorin, a combination with minimal eYcacy in advanced disease. In addition, the quality of surgery in that study was questionable, with the majority of patients undergoing a D0 resection. These two points would therefore question the eYcacy of RT, if more eVective chemotherapy and/or more appropriate surgery had been applied. The dilemma becomes even more relevant when the toxicity associated with RT and the diYculties of monitoring the quality of RT are considered.

Several novel chemotherapeutic agents appear to have promising eYcacy in advanced disease. Docetaxel has shown considerable eYcacy as single-agent [START_REF] Sulkes | Docetaxel (Taxotere) in advanced gastric cancer: results of a phase II clinical trial. EORTC Early Clinical Trials Group[END_REF] and as part of a combination therapy [START_REF] Ajani | Evolving chemotherapy for advanced gastric cancer[END_REF][START_REF] Thuss-Patience | Docetaxel and continuous-infusion Xuorouracil versus epirubicin, cisplatin, and Xuorouracil for advanced gastric adenocarcinoma: a randomized phase II study[END_REF]. In particular, docetaxel and cisplatin have been reported to show at least additive eYcacy when used as combination therapy [START_REF] Roth | Swiss Group for Clinical Cancer Research (SAKK), The European Institute of Oncology (EIO) (2000) Docetaxel (Taxotere)-cisplatin (TC): an eVective drug combination in gastric carcinoma[END_REF], and their demonstrated eYcacy and tolerability make this regimen particularly applicable to the adjuvant setting [START_REF] Ridwelski | Combination chemotherapy with docetaxel and cisplatin for locally advanced and metastatic gastric cancer[END_REF].

Another approach to improve the eYcacy of adjuvant strategies is the detection of factors, which might aid the selection of patients with a better prognosis or an improved response to treatment. Biological markers (such as estrogen receptors, HER2 status, and KRAS mutations) are currently used in other neoplasms to select patients for speciWc therapies [START_REF] Smith | 2-year follow up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: a randomised controlled trial[END_REF][START_REF] Dowsett | Emerging biomarkers and new understanding of traditional markers in personalized therapy for breast cancer[END_REF][START_REF] De Castro-Carpeño | EGFR and colon cancer: a clinical view[END_REF]. Biological prognostic factors can be derived from genetic processes that represent crucial steps to carcinogenesis. Others can be predictive of response to therapy, as they are targets of chemotherapy or targeted therapies. Such markers are under study in gastric cancer but results are non-conclusive yet [START_REF] Scartozzi | Molecular biology of sporadic gastric cancer: prognostic indicators and novel therapeutic approaches[END_REF].

Based on the above data, this study aimed to compare the eYcacy of chemoradiotherapy, consisting of RT plus a modern chemotherapy regimen, with the same regimen without RT, as adjuvant treatment in patients in high-risk for relapse after curative resection of gastric cancer. In addition, we studied the prognostic and/or predictive value of molecular markers, namely epidermal growth factor receptor (EGFR), excision repair cross-complementing group-1 (ERCC1), HER2, Met proto-oncogene tyrosine kinase/hepatocyte growth factor receptor (MET/HGFR), MAP-Tau (microtubule-associated protein Tau), and PTEN/MMAC1 (phosphatase and tensin homolog, mutated in multiple advanced cancers 1).

Patients and methods

Patients

Patients with histologically conWrmed gastric adenocarcinoma (including adenocarcinoma of the gastroesophageal junction) were included in the study. Only surgeons performing a minimum of 20 operations for gastric cancer per year were accepted to participate. Patients were eligible for post-operative adjuvant therapy if: disease was absent from the peritoneal cavity and other distant organs, negative surgical margins were obtained, had serosal inWltration (pT3 based on American Joint Committee on Cancer criteria [START_REF] Greene | Stomach. In: AJCC cancer staging manual[END_REF]) or inWltrated lymph nodes; they had performance status 2 or lower according to the Eastern Cooperative Oncology Group criteria; they had no history of other malignancy except basal cell or squamous cell carcinoma of the skin; were at least 18 years of age; had no evidence of cardiac failure; had absolute neutrophil count >1,500 L ¡1 , platelet count >100,000 mL ¡1 , normal serum bilirubin, alanine transaminase and aspartate transaminase <2 times the upper limit of normal, and calculated creatinine clearance >60 mL min ¡1 ; and were of satisfactory nutritional status (weight increase following gastrectomy or minimum intake of 1,500 kcal day ¡1 ). The clinical protocol and collateral translational research studies were approved by the HeCOG Protocol Review Committee and by the Bioethics Committee of the Aristotle University of Thessaloniki School of Medicine. The study was registered at the Australian New Zealand Clinical Trials Registry (No. 308272). All patients gave their informed consent prior to study entry.

123

Treatment

Patients were randomized to one of the following regimens:

(1) Six cycles of docetaxel with cisplatin (group A) and ( 2) Six cycles of docetaxel with cisplatin and RT (group B). After the Wrst 45 patients (22 group A, 23 group B), the protocol was amended due to excessive nausea and vomiting and cisplatin was substituted by carboplatin.

The doses of the chemotherapeutic agents used were 75 mg m ¡2 docetaxel in 250 mL saline administered over a 1-h period; 75 mg m ¡2 cisplatin in 500 mL saline administered over a 1-h period or carboplatin to an area under the curve (AUC) of 5 in 500 mL saline or 5% dextrose administered over a 1-h period; treatment was administered every 3 weeks for six cycles. A minimum of 3 L of hydration was given with cisplatin administration. Before receiving the chemotherapy regimen, patients were given 5-HT 3 antagonists with 20 mg dexamethasone, 4 mg dimentindine maleate, and 50 mg ranitidine. On the day before and the 3 days after treatment, patients received 4 mg methyl prednisolone twice daily. Treatment was administered if ANC ¸1,500 and PLT ¸100,000, otherwise it was delayed for a week. Drug doses were reduced by 20% in case of grade 3 or 4 neutropenia or thrombocytopenia. G-CSF was used in case of repeated delays or neutropenic fever. Cisplatin was discontinued in case of nephrotoxicity, grade > 2. In case of stomatitis or diarrhea grade ¸2 treatment was delayed until toxicity was reduced to grade 1. Docetaxel was reduced by 20% in case of grade > 2 or repeated grade 2 hematological toxicity.

Radiation therapy (RT) was administered 3-4 weeks after the third chemotherapy cycle. RT was planned with dedicated computed tomography (CT) and a threedimensional planning system. It was delivered with linear accelerators with nominal energy of 6 and/or 18 MV, through parallel-opposed AP-PA Welds. RT consisted of fractionated external irradiation at a dose of 1.8 Gy per fraction given once daily 5 days per week (Monday through Friday) over a period of 5 weeks, for a total dose of 45 Gy. Target volume included the tumor bed, identiWed on the preoperative CT, the anastomoses, the stomach remnant, as well as the regional lymphatics. In cases of proximal T3 lesions, the medial left hemidiaphragm was also included in the radiation Welds. The regional lymph nodes irradiated were the perigastric, celiac, local paraortic, pancreaticoduodenal, portal hepatic, and splenic nodes. In patients with lesions of the antrum, the splenic nodes could be excluded. In patients with proximal lesions exclusion of the pancreaticoduodenal nodes was allowed. Doses were limited, so that less than 60% of the liver received more than 30 Gy and less than 30% of the cardiac volume received more than 40 Gy. An equivalent of at least three-fourths of one kidney was spared from doses higher than 20 Gy. The treatment protocol did not include a central quality-assurance review of the radiotherapy plan.

RT was delayed in case of grade 3 diarrhea or myelotoxicity and was resumed upon reduction to grade 1. Three further cycles of chemotherapy were administered 4-5 weeks after completion of RT. Prior to treatment, initiation, patients were assessed by abdominal computed tomography (CT), chest X-ray, complete blood count, biochemical examination, carcinoembryonic antigen, and carbohydrate antigen 19.9. These assessments were repeated 1 month after completion of therapy, every 6 months during the Wrst 5 years of the study, and yearly thereafter. Upper GI endoscopy was performed on an annual basis.

Tissue samples

Formalin-Wxed, paraYn-embedded tissue blocks from 69 gastrectomy specimens were collected. H&E sections were reviewed by a pathologist (MB), and the most representative tumor areas were marked for the construction of the tissue microarrays (TMAs). Nine specimens with tumor accounting for <5% of whole tissue area were not used for TMA construction, but whole tissue sections were used for immunohistochemistry.

TMA construction

Specimens were arrayed (5 cores per case, 0.6 mm in diameter) into two recipient paraYn blocks (Paraplast ® , McCormick, St. Louis, MO, USA) using a manual arrayer (Beecher Instruments, Sun Prairie, WI, USA). After construction, array blocks were placed in an incubator (60°C) for 15 min in order to assist Wlling in the cracks and defects at the core-wax interface that could cause the loss of cores on the slides.

Immunohistochemistry

Immunohistochemical labeling was performed according to standard protocols with slight modiWcations [START_REF] Williams | Tissue preparation for immunocytochemistry[END_REF], on serial 2.5-m thick sections from the original blocks or the two TMA blocks. All slides of the study were stained in one run for each antibody. All cases were stained also for vimentin (clone V9, Dako), Ki-67 (clone MIB1, Dako), and cytokeratin 8/18 (CAM5.2, clone 5D3, Novocastra), which were used as control stains for tissue immunoreactivity and Wxation, as well as for identiWcation of tumor cells (CAM5.2). Samples negative for the above antibodies were excluded from the study. The evaluation of all IHC sections was done simultaneously by two observers, (I. K. and M. B.), blinded as to the patients' clinical characteristics and survival data, according to previously described criteria [START_REF] Nakajima | The prognostic signiWcance of ampliWcation and overexpression of c-met and c-erb B-2 in human gastric carcinomas[END_REF][START_REF] Kwon | Prognostic value of expression of ERCC1, thymidylate synthase, and glutathione S-transferase P1 for 5-Xuorouracil/oxaliplatin chemotherapy in advanced gastric cancer[END_REF][START_REF] Mimori | Reduced tau expression in gastric cancer can identify candidates for successful Paclitaxel treatment[END_REF][START_REF] Scartozzi | Epidermal growth factor receptor (EGFR) status in primary colorectal tumors does not correlate with EGFR expression in related metastatic sites: implications for treatment with EGFR-targeted monoclonal antibodies[END_REF][START_REF] Perren | Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast[END_REF], with slight modiWcations for MET, ERCC1, and 123 MAP-Tau. BrieXy, for MET (clone 8F1, Novocastra), ERCC1 (clone 8F11, Thermo Fisher ScientiWc), and MAP-Tau (clone T1029, US Biological), a 4-tier range system was applied for intensity and staining pattern. Intensity was scored as follows: 0 = no staining, 1 = weakly positive, 2 = moderately positive, and 3 = strongly positive. The scoring of the staining pattern was based on the percentage of positive tumor cells: 0 = 0-5%, 1 = 6-25%, 2 = 26-50%, 3 = 51-100%). The localization of staining for each protein was also indicated, as cytoplasmic and cytoplasmic/membraneous for MET and nuclear, cytoplasmic and nuclear/ cytoplasmic for ERCC1 and MAP-Tau. In order to deWne the expression status of the above proteins, the total score was calculated as the sum of the intensity score and the staining pattern score. Cases with a total score of at least 2 were considered as positive (high-expressing tumors), whereas cases with a total score of 0-1 were considered as negative or low-expressing tumors. EGFR (clone 31G7, Zymed) intensity of reactivity was scored using a four-tier system [START_REF] Scartozzi | Epidermal growth factor receptor (EGFR) status in primary colorectal tumors does not correlate with EGFR expression in related metastatic sites: implications for treatment with EGFR-targeted monoclonal antibodies[END_REF]: 0 (negative), no staining or background staining; 1+ weak discontinuous membranous staining; 2+ moderate complete or incomplete membranous staining; 3+ strong and complete membranous staining. Cases were considered positive when more than 1% of tumor cells showed at minimum 1+ staining. HER2 (Dako) protein expression was scored 0-3+ according to the modiWed breast IHC Hercep Test protocol (Dako). PTEN (clone 6H2.1, Dako) protein expression (cytoplasmic, nuclear or both) was evaluated according to a previously established rank scale of 0-2 [START_REF] Perren | Immunohistochemical evidence of loss of PTEN expression in primary ductal adenocarcinomas of the breast[END_REF]. Vascular endothelial cells and/or Wbroblasts were used as control markers of staining intensity. Tumors with PTEN scores of 0 or 1 were considered to have PTEN loss.

Randomization and statistical analysis

Patients were centrally randomized and stratiWed according to study center, the depth of tumor inWltration and number of inWltrated nodes. The study was originally designed to identify a 20% increase in survival rate in the RT arm, assuming that the 5-year survival rate in arm A is 35%. Therefore, 200 patients were needed, in order to have a trial with 80% statistical power at a = 0.05 level of signiWcance. Taking into account a 3% withdrawal rate, 206 patients were needed for this trial. A 70 patients/year accrual rate was anticipated when the study was designed. This estimation was accurate for the Wrst year; however, accrual declined during the second and third year. After 3 years, only 147 patients were randomized due to the decrease of the accrual rate over time. Thus, the ScientiWc Committee of HeCOG decided to suspend the study.

Analyses were conducted on an intent-to-treat basis. Overall survival (OS) was measured from the date of randomization until death from any cause and surviving patients were censored at the date of last contact. Diseasefree survival (DFS) was measured from randomization until relapse. Death without veriWed disease progression was considered as an event in the DFS analysis. Time to event distributions were estimated using Kaplan-Meier curves and compared using the log-rank test. In order to identify the factors that had a signiWcant eVect on patients' OS and DFS, multivariate Cox regression analysis was performed. Variables included were age, number of involved nodes (0-7 vs. 8-15 vs>15), stage (T 1 /T 2 vs. T 3 /T 4 ), grade, histological subtype (intestinal vs. diVuse vs. mixed/unclassiWed), and randomization group. Statistical tests were two-sided and were performed to a signiWcance level of 0.05. Results of this study were presented according to reporting recommendations for tumor marker prognostic studies [START_REF] Mcshane | Reporting recommendations for tumor marker prognostic studies[END_REF].

Results

Patient characteristics

A total of 147 patients were registered in the study between April 2002 and April 2005. Four patients were considered ineligible and were therefore not included in the analysis: 1 patient had liver metastases at the time of surgery, 2 patients had second primaries (esophageal and head and neck) and 1 patient had pT2N0 disease. The study Xow is shown in Fig. 1. Selected baseline characteristics of the 143 eligible patients are summarized in Table 1. Seventy-one patients were randomized to receive adjuvant chemotherapy (arm A) and 72 were randomized to receive chemotherapy with RT (arm B). There were no signiWcant diVerences in major characteristics between the two treatment groups, with the exception of histological subtype (P = 0.007).

Treatment characteristics and toxicity

Treatment details and toxicity are shown in Table 2. Only treated patients with complete medical records were included in this analysis apart from the following cases: one patient (arm A) who did not receive protocol treatment and another patient (arm B) whose medical records were not available at the time of the analysis. Nine patients in group A (13%) did not complete 6 cycles of treatment: 1 patient discontinued due to toxicity, 4 discontinued following disease progression, 3 withdrew consent, while for one patient the reason for noncompletion was unknown. In group B, 22 patients (31%) did not complete six cycles of chemotherapy: 2 patients died, 5 discontinued due to toxicity, 3 experienced disease progression, 6 withdrew consent, 4 patients discontinued for other reasons, and 2 discontinued for unknown reasons. Discontinuation rate was signiWcantly higher for patients treated in arm B (13% vs. 31%, P = 0.014). Sixty patients (85%) from arm B received RT. Details of this treatment modality are shown in Table 3. DI and RDI for docetaxel and cisplatin were lower in arm B, as expected from the interruption of chemotherapy for the administration of RT.

Grade 3 or 4 toxicities observed in the two arms are shown in Table 3. The most common grade 3/4 toxicities for arms A and B (excluding alopecia) were non-febrile neutropenia (11% vs. 17%, respectively), febrile neutropenia (9% vs. 7%), and diarrhea (7% vs. 4%, respectively). These diVerences were not signiWcant. In addition, no sig-niWcant diVerences between group A and B were found in the requirements for: hospitalization (13 [19%] vs. 10 4a). After adjustment for randomization arm, the diVerence remained marginally signiWcant (Wald, P = 0.046). Similarly, median DFS for ERCC1 negative tumors was 11.8 months (95% CI 8.7-15), while the respective median for positive tumors was 33.1 months (95% CI 3.6-62.7; log-rank, P = 0.018, HR = 0.47, [95% CI: 0.25-0.89]; Figure 4b). After adjustment for randomization arm, the diVerence remained signiWcant (Wald, P = 0.016).

Discussion

We assessed the beneWt from the combination of RT with docetaxel and carboplatin/cisplatin chemotherapy compared to chemotherapy alone in patients with radically resected gastric cancer at high-risk for relapse. At a median follow-up of approximately 54 months, there were no diVerences between the two therapies in OS and DFS. Our entry criteria resulted in a cohort of high-risk patients, with most of them having stage III or IV disease. Contemporary surgical series form Europe and the USA estimate longterm DFS in similar populations below 40% [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF][START_REF] Cunningham | Participants Trial (2006) Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer[END_REF]. For this reason, we believe that our Wndings are relevant to the recent developments in adjuvant therapy in gastric cancer.

Recent randomized studies [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF][START_REF] Cunningham | Participants Trial (2006) Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer[END_REF][10] established adjuvant therapy as a standard approach, since all of them showed a survival beneWt over no adjuvant therapy. However, the optimal type of adjuvant therapy remains to be determined. Our Wndings indicate that RT may not add any beneWt to modern, eYcient chemotherapy combinations. This is of clinical relevance, considering the toxicity as well as problems of quality assurance associated with RT, particularly when used as adjuvant therapy in gastric cancer. Indeed, the administration of RT by many centers made monitoring of the quality of RT very diYcult. Every eVort should be made, however, for central quality-assurance review of the radiotherapy plan in clinical trials, similar to that adopted by the MacDonald study [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF]. Another explanation of the lack of a beneWt by the addition of RT may be the lower DI of chemotherapy in arm B because of the administration of RT between cycles 3 and 4. Thus, our results may reXect a lack of beneWt due to the interruption of the administration of the chemotherapy combination.

Other limitations of this study should also be considered in the interpretation of our results. This study was powered to demonstrate a 20% survival beneWt of adjuvant chemoradiotherapy over adjuvant chemotherapy alone. Since only 147 instead of the required 200 patients were randomized, it was underpowered to test the initial hypothesis. SpeciWcally, having observed 74 deaths (68% of the total events needed according to study design) the post hoc power was 66%. The possibility, therefore, that a beneWt by the addition of RT was not detected due to decreased power of the study should not be excluded.

Another assumption was that the chemotherapeutic combination would have had proven eYcacy in advanced disease. Although this is true for the combination of docetaxel/cisplatin, most patients received docetaxel/carboplatin, which is a less established regimen in advanced gastric cancer. The substitution was decided because nausea and vomiting were reported frequently for docetaxel/cisplatin. In the same context, regarding the choice of the chemotherapy regimen, other better-studied combinations, such as ECF, which has previously been used perioperatively [START_REF] Cunningham | Participants Trial (2006) Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer[END_REF], could have been used. In addition, despite having set criteria for the experience of surgeons, D1 and D2 resections were reported in only 34% of the eligible patients (50% among those with known type of resection). Nevertheless, the last two limitations should have, if anything, strengthened the contribution of RT. This was not, however, reXected in the results.

Although treatment was generally well tolerated, a sizeable subset of the patients did not complete protocol treatment. This subset was signiWcantly higher in the radiochemotherapy arm and consistent with the data reported by McDonald et al. [START_REF] Macdonald | Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction[END_REF]. In 6 cases, therapy was discontinued during RT, reXecting the signiWcant toxicity that this modality can be associated with.

In the absence of any signiWcant diVerences in survival between the randomization arms, we sought to determine whether molecular markers might identify groups of patients more likely to beneWt from the administration of adjuvant therapy. We have previously reported a study of molecular markers and their prognostic role in gastric cancer [START_REF] Skarlos | Expression of a molecular marker panel as a prognostic tool in gastric cancer patients treated postoperatively with docetaxel and irinotecan. A study of the Hellenic Cooperative Oncology Group[END_REF]. In this study, a panel of 5 biomarkers, Topo I, Topo I , VEGF, E-Cadherin, and thymidylate synthase (TS) had been evaluated in 46 patients treated postoperatively with irinotecan and docetaxel, with none of the biomarkers shown to be of prognostic value, probably due to the small number of patients.

In the current study, a diVerent panel of biomarkers was selected with a similar approach and rational. Hence, among a variety of potentially relevant molecules, ERCC1 and MAP-Tau were selected because they are involved in the process of 'recovery' from the platinum and taxane cytotoxic eVect, respectively [START_REF] Tanaka | Tau expression and eYcacy of paclitaxel treatment in metastatic breast cancer[END_REF][START_REF] Huang | ERCC1 polymorphism, expression and clinical outcome of oxaliplatin-based adjuvant chemotherapy in gastric cancer[END_REF][START_REF] Bellmunt | Gene expression of ERCC1 as a novel prognostic marker in advanced bladder cancer patients receiving cisplatin-based chemotherapy[END_REF]. Moreover, the expression of tyrosine kinase receptors (TKRs), such as HER2, EGFR and MET, has being thoroughly investigated [START_REF] Kopp | Impact of epidermal growth factor (EGF) radioreceptor analysis on long-term survival of gastric cancer patients[END_REF][START_REF] Garcia | Clinical signiWcance of the epidermal growth factor receptor and HER2 receptor in resectable gastric cancer[END_REF][START_REF] Lim | Using whole genome ampliWcation (WGA) of low-volume biopsies to assess the prognostic role of EGFR, KRAS, p53, and cMET mutations in advanced-stage non-small cell lung cancer (NSCLC)[END_REF] in solid tumors, but Wndings in gastric cancer are still controversial. Also, the role of the PTEN protein in gastric cancer is still unknown, whereas loss of heterozygosity of the relevant gene is found in many solid tumors.

The results of ERCC1 overexpression in our study appear to be inconsistent with the so far published data. Patients with positive ERCC1 (46 of 67, and 69%) had 123 signiWcantly longer OS and DFS compared to those who did not overexpress the protein. ERCC1 expression has previously been evaluated in metastatic gastric cancer by Kwon et al. [START_REF] Kwon | Prognostic value of expression of ERCC1, thymidylate synthase, and glutathione S-transferase P1 for 5-Xuorouracil/oxaliplatin chemotherapy in advanced gastric cancer[END_REF]. In that study, 64 patients with advanced gastric cancer were evaluated for ERCC1, among other markers. Seventy percent (70%) of the patients were ERCC1 positive and had a poor response to treatment. Two more studies conWrmed their Wndings [START_REF] Wei | ERCC1 mRNA levels and survival of advanced gastric cancer patients treated with a modiWed FOLFOX regimen[END_REF][START_REF] Matsubara | Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer[END_REF], but no published data in operable gastric cancer treated with docetaxel/cisplatin or carboplatin exist so far. In non-small cell lung (NSCLC) cancer, however, the role of ERCC1 [START_REF] Siddik | Cisplatin: mode of cytotoxic action and molecular basis of resistance[END_REF][START_REF] Reed | Platinum-DNA adduct, nucleotide excision repair and platinum based anti-cancer chemotherapy[END_REF] as a key protein in nucleotide repair of platinum-induced DNA intrastrand breaks has been conWrmed. Olaussen et al. [START_REF] Olaussen | DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy[END_REF] evaluated ERCC1 by immunohistochemistry (IHC) in 761 patients with operable NSCLC. ERCC1 negative patients had signiWcantly longer survival when treated with platinum-based chemotherapy. Nevertheless, expression of ERCC1 was associated with improved outcome in the whole population, indicating a prognostic role, independently of its predictive value. It is plausible that the prognostic role of ERCC1 expression prevailed in our study over its predictive value. Unfortunately, all of our patients received platinum-based chemotherapy and, therefore, we could not test this hypothesis in a cohort of non-treated patients. Nevertheless, our results appear to be contradictory and await validation in future studies with larger number of patients.

As for the Tau protein, it belongs to the microtubuleassociated proteins (MAP). The MAP group controls the assembly of and tubulin dimmers to form microtubules [START_REF] Nogales | Structure of the alpha beta tubulin dimer by electron crystallography[END_REF]. When bound to microtubules, MAP-Tau promotes their polymerization and stabilization [START_REF] Drechsel | Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau[END_REF]. Taxanes, on the other hand, although they have a similar way of interacting with tubulin [START_REF] Nogales | Structure of tubulin at 6.5 A and location of the taxol-binding site[END_REF], alter the tubulin dynamics in a way that eventually leads to cell cycle arrest and apoptosis [START_REF] Yvon | Taxol suppresses dynamics of individual microtubules in living human tumor cells[END_REF]. Wagner P et al. [START_REF] Wagner | Microtubule associated protein (MAP)-Tau: a novel mediator of paclitaxel sensitivity in vitro and in vivo[END_REF], published an analysis of patients with breast cancer who had pathologic complete response after neo-adjuvant chemotherapy with taxanes. Decreased MAP-Tau expression was associated with sensitivity to taxanes. These results have been validated in other studies, both in breast cancer [45] and NSCLC patients [START_REF] Rosell | Predicting response to paclitaxel/carboplatin-based therapy in non-small cell lung cancer[END_REF]. In gastric cancer, Mimori et al. [START_REF] Mimori | Reduced tau expression in gastric cancer can identify candidates for successful Paclitaxel treatment[END_REF] have published results from a study of MAP-Tau protein expression in 20 patients with metastatic gastric cancer. All MAP-Tau negative patients (6/20) responded to taxanes. Although there is little doubt of the emerging role of MAP-Tau as a predictive marker for taxane-based chemotherapy, no correlation with survival has, so far, been validated. In our study, 36 patients (54%) were MAP-Tau negative. There were no statistically signiWcant diVerences in OS or DFS, related with the protein expression. The predictive value of MAP-Tau could not be evaluated in our population, since both arms received docetaxel. Moreover, the small number of patients renders these results controversial.

Regarding HER2 protein expression, evaluation with IHC in gastric cancer is equally accurate as in breast cancer [START_REF] Takehana | Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, Xuorescence in situ hybridization and enzyme-linked immuno-sorbent assay[END_REF]. HER2 is overexpressed in 6-22% of patients with gastric cancer [START_REF] Jain | c-erbB-2 protooncogene expression and its relationship to survival in gastric carcinoma: an immunohistochemical study on archive material[END_REF][START_REF] Van Cutsem | EYcacy results from the ToGA trial: A phase III study of trastuzumab added to standard chemotherapy (CT) in Wrst-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC)[END_REF] and appears to result in favorable prognosis [START_REF] Jain | c-erbB-2 protooncogene expression and its relationship to survival in gastric carcinoma: an immunohistochemical study on archive material[END_REF], however, later publications did not conWrm this Wnding. On the contrary, poor prognosis, higher probability of lymph node involvement and increased number of postoperative relapses was reported in other studies [START_REF] Yonemura | Evaluation of immunoreactivity for erbB2 protein as a marker of poor short term prognosis in gastric cancer[END_REF][START_REF] Mizutani | Relationship of c-erbB-2 protein expression and gene ampliWcation to invasion and metastasis in human gastric cancer[END_REF]. In our study, 61 of 67 patients (91%) were HER2 negative, while 5 patients (7%) were positive. There was no correlation with OS or DFS.

123

The percentage of HER2 positive patients observed in our study (7%) is much lower than the 22% reported in the ToGA trial [START_REF] Van Cutsem | EYcacy results from the ToGA trial: A phase III study of trastuzumab added to standard chemotherapy (CT) in Wrst-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC)[END_REF], which included 3,800 patients centrally tested for HER2. The discrepancy might possibly be due to diVerences in the IHC evaluation criteria used (staining of 30% of cells was required in our study as opposed to 10% in the ToGA trial). Another possible explanation for the discordance might have been the observed high percentage of heterogeneous tumors in gastric cancer combined with the small number of tumors evaluated in our study.

In conclusion, our results indicate that the role of RT as an adjuvant treatment of gastric cancer may change with the evolution of modern chemotherapeutic combinations, which may prove to be more eVective than those previously used. The optimal adjuvant strategy and the incorporation of various modalities in such approaches should therefore be further investigated. Finally, more thorough evaluation of molecular markers may lead to the identiWcation of subsets of patients more likely to beneWt from diVerent strategies of adjuvant treatment.

Fig. 1

 1 Fig. 1 Flow of the study

Fig. 2

 2 Fig. 2 Kaplan-Meier curves for the OS (a) and DFS (b) of patients randomized to arm A (blue line) or arm B (red line)

Fig. 3

 3 Fig.[START_REF] Catalano | Gastric cancer[END_REF] Immunohistochemistry performed on tissue microarrays. a ERCC1 strong nuclear positivity; b ERCC1 staining in a small number of neoplastic cells similar to the staining intensity of stromal Wbroblasts (regarded as negative staining); c HER2 strong membraneous staining; d MAP-Tau intense cytoplasmic staining; original magniWcation £100; insets £200

Fig. 4

 4 Fig. 4 Kaplan-Meier curve of OS (a) and DFS (b) according to ERCC1 expression in 66 patients treated with adjuvant therapy. The red line corresponds to patients with positive ERCC1 expression and the blue line to patients with negative ERCC1 expression

Table 1

 1 Selective patient and tumor characteristics

	Age, years [median (range)]

Table 2

 2

	Treatment
	characteristics

Table 4

 4 EYcacy results and Multivariate Cox regression analysis

		Survival (months)	PFS (months)
	Group A	Median	95% CI	Median	95% CI
		55.3		38.1-72.5	37		13.6-60.3
		3 years		3 years
		61%			51%
	Group B	Median	95% CI	Median	95% CI
		44.5		28.6-60.5	33.1	18.4-47.8
		3 years		3 years
		57%			48%
		Multivariate analysis
		HR	95% CI.	P	HR	95% CI.	P
	Randomization group
	A	1	-	-	1	-	-
	B	1.20 0.75-1.91 0.448	1.04 0.66-1.63 0.879
	T
	T1 or T2 1	-	-	1	-	-
	T3 or T4 2.65 1.26-5.59 0.011	2.16 1.10-4.25 0.026
	Lymph nodes involved
	0-7	1	-	-	1	-	-
	8-15	2.92 1.66-5.15 <0.001 2.35 1.35-4.08 0.002
	>15	3.60 1.88-6.88 <0.001 4.07 2.17-7.66 <0.001
	Age	1.03 1.01-1.06 0.018	1.03 1.00-1.05 0.048

Table 5

 5 Immunohistochemical results in 67 tumor tissue samples available for protein analysis * For each marker staining could not be assessed in one sample

	Protein	Expression	Arm A	Arm B	Total
		Status N (%)	(N = 34)	(N = 32)	(N = 66)
	EGFR	Negative	22

Acknowledgments

The authors wish to thank Ms. Evita Fragou for monitoring the study, Ms. Maria Moschoni for coordinating the data management, Ms. Thalia Spinari for coordinating tumor tissue collection (all at the HeCOG Data OYce, Athens), and Sophia ChrisaW Ph.D. for excellent technical assistance. Supported in part by a HeCOG Research Grant: RD/2 ConXict of interest statement No author declared any conXict of interest.

Received allocated intervention: n=66 Received Docetaxel-Cisplatin/Carboplatin +RT: n=3 Did not receive protocol intervention or unknown intervention (Lost medical record): n=2 Docetaxel-Cisplatin/Carboplatin +RT Allocated to intervention: n=72 Received allocated intervention: n=68

123

[14%]), red blood cell transfusion (2 [3%] vs. 2 [3%]), platelet transfusion (2 [3%] vs. 1 [1%]), requirement for G-CSF (32 [46%] vs. 39 [59%]), or antibiotic treatment (8 [11%] vs. 12 [17%]).

EYcacy results

After a median follow-up of 53.7 months (range 0.1-77.8), 34 deaths and 37 relapses were observed in group A, while 40 deaths and 43 relapses were observed in group B. No diVerence in local recurrence rates was observed between the two arms (10% vs. 5%, P = 0.246). Median and 3-year OS and DFS for the two groups are shown in Table 4. No signiWcant diVerences between the two groups were found (Fig. 2).

In the multivariate analysis (Table 4), T stage and lymph node involvement were found to be independent prognostic factors for OS and lymph node involvement for DFS. After adjusting for the signiWcant factors, randomization group still did not have a signiWcant eVect on OS (HR = 1.20, 95% CI: 0.75-1.91, P = 0.448) or PFS (HR = 1.04, 95% CI: 0.66-1.63, P = 0.879).

Study of molecular markers

Tissue blocks for molecular marker studies were obtained from 67 patients (group A: 35, group B: 32). Staining could not be assessed for all of the examined markers in two cases. The results of immunostaining in the 67 patients with at least one evaluated marker are shown in Table 5, while ERCC1, HER2, and MAP-Tau staining patterns are shown in Fig. 3. There was no association of any marker with pT, N, diVerentiation, or TNM stage. In Table 6, distribution of protein expression according to histological subtype and tumor localization is presented. For gastric tumors, 5 out of