PhD Cécile Jeanpierre
email: cecile.jeanpierre@inserm.fr

Guillaume Macé

Mélanie Parisot

Vincent Morinière

Audrey Pawtowsky

Marion Benabou

Jelena Martinovic

Jeanne Amiel

Tania Attié-Bitach

Anne-Lise Delezoide

Philippe Loget

Patricia Blanchet

Dominique Gaillard

Marie Gonzales

Wassila Carpentier

Patrick Nitschke

Frédéric Tores

Laurence Heidet

Corinne Antignac

Rémi Salomon

Victor Dupouy

Jean Verdier

D Carles

A Clemenson

Chu St

Hôpital F Cornelis

Hôpital F Guimiot

Robert Debré

V Hennequin

Nancy Chu

S Khung

J-P Masutti

M-J Perez

RET and GDNF mutations are rare in fetuses with renal agenesis or other severe kidney development defects

Keywords: renal agenesis, renal hypodysplasia, variants, evolutionary conserved non-coding regions, CNVs

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

INTRODUCTION

Congenital abnormalities of the kidney and urinary tract (CAKUT) are frequently observed in children and represent a significant cause of morbidity, accounting for more than 40% of pediatric end-stage renal failure, and mortality. [START_REF] Sanna-Cherchi | Renal outcome in patients with congenital anomalies of the kidney and urinary tract[END_REF] Kidney development defects (KDD) include: (i) bilateral/unilateral renal agenesis (BRA/URA); (ii) renal hypodysplasia (RHD) characterized by a reduction in the number of nephrons leading to a small overall kidney size and frequent dysplasia with or without cysts; and (iii) multicystic dysplastic kidney (MCDK).

These various alterations can be observed together in a same patient or in different members of the same family, suggesting that they belong to a same continuum of phenotypes. Although most cases are sporadic and isolated, syndromic and familial cases suggest that genetic factors are involved. In particular, dominant mutations with variable penetrance have been found in several syndromic forms of KDD. The most frequently mutated genes are PAX2 in patients with renal-coloboma syndrome, EYA1 and SIX1 in patients with branchio-oto-renal (BOR) syndrome and HNF1B in patients with renal cysts and diabetes association. [START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF][START_REF] Kochhar | SIX1 mutation screening in 247 branchio-oto-renal syndrome families: a recurrent missense mutation associated with BOR[END_REF][START_REF] Orten | Branchio-oto-renal syndrome (BOR): novel mutations in the EYA1 gene, and a review of the mutational genetics of BOR[END_REF][START_REF] Ulinski | Renal phenotypes related to hepatocyte nuclear factor-1beta (TCF2) mutations in a pediatric cohort[END_REF][START_REF] Weber | Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study[END_REF] Analysis of genotype-phenotype correlations showed that the severity of the renal disease associated with these mutations is extremely variable. [START_REF] Heidet | Spectrum of HNF1B mutations in a large cohort of patients who harbor renal diseases[END_REF][START_REF] Krug | Mutation screening of the EYA1, SIX1 and SIX5 genes in a large cohort of patients harboring branchio-oto-renal syndrome calls into question the pathogenic role of SIX5 mutations[END_REF] Other less frequent syndromes including KDD are associated with mutations in developmental genes such as SALL1 (Townes-Brocks syndrome), WNT4 (Rokitanski syndrome), KAL1 (Kallman syndrome), FRAS1 and FREM1 (Fraser syndrome), GATA3 (RHD, hypoparathyroidism and sensorineural deafness) and GLI3 (Pallister-Hall syndrome). [START_REF] Alazami | FREM1 mutations cause bifid nose, renal agenesis, and anorectal malformations syndrome[END_REF][START_REF] Biason-Lauber | A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman[END_REF][START_REF] Botzenhart | Townes-Brocks syndrome: twenty novel SALL1 mutations in sporadic and familial cases and refinement of the SALL1 hot spot region[END_REF][START_REF] Cain | GLI3 repressor controls nephron number via regulation of Wnt11 and Ret in ureteric tip cells[END_REF][START_REF] Georgopoulos | Renal dysgenesis and KAL1 gene defects in patients with sporadic Kallmann syndrome[END_REF][START_REF] Pitera | Fras1, a basement membrane-associated protein mutated in Fraser syndrome, mediates both the initiation of the mammalian kidney and the integrity of renal glomeruli[END_REF][START_REF] Van Esch | GATA3 haplo-insufficiency causes human HDR syndrome[END_REF] Moreover, comparative genomic hybridization and familial studies have highlighted several chromosomal regions that could carry other genes involved in KDD. [START_REF] Sanna-Cherchi | Localization of a gene for nonsyndromic renal hypodysplasia to chromosome 1p32-33[END_REF][START_REF] Weber | Mapping candidate regions and genes for congenital anomalies of the kidneys and urinary tract (CAKUT) by array-based comparative genomic hybridization[END_REF].

Mammalian kidney development results from a reciprocal induction between the ureteric bud (UB) and the metanephric mesenchyme (MM). Signalling by GDNF secreted by the MM and the RET tyrosine kinase receptor and its co-receptor GFRα1 expressed on the UB plays a critical role for normal growth and branching of the UB. [START_REF] Chi | Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis[END_REF][START_REF] Meyer | Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney[END_REF] The RET gene encodes two major splicing isoforms, RET9 and RET51, that differ in their carboxy terminus. These two isoforms appear to have redundant roles for kidney development. [START_REF] De Graaff | Differential activities of the RET tyrosine kinase receptor isoforms during mammalian embryogenesis[END_REF][START_REF] Jain | Critical and distinct roles for key RET tyrosine docking sites in renal development[END_REF] The RET/GDNF signalling also plays a critical role during the development of the enteric nervous system. Homozygous knockout of Ret or Gdnf in mice leads to loss of enteric ganglia as well as severe kidney aplasia or hypodysplasia caused by a failure of UB outgrowth. [START_REF] Sanchez | Renal agenesis and the absence of enteric neurons in mice lacking GDNF[END_REF][START_REF] Schuchardt | Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret[END_REF] In humans, heterozygous loss-of-function mutations in RET resulting in haploinsufficiency are the most frequent alterations reported in patients with segmental intestinal aganglionosis known as Hirschsprung disease (HSCR). [START_REF] Angrist | Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease[END_REF][START_REF] Attie | Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease[END_REF] RET is also an oncogene involved, through activating mutations, in predisposition to multiple endocrine neoplasia type 2A (MEN2A) characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and/or parathyroid hyperplasia. [START_REF] Margraf | Multiple endocrine neoplasia type 2 RET protooncogene database: repository of MEN2-associated RET sequence variation and reference for genotype/phenotype correlations[END_REF] Several studies have reported the association of HSCR and MTC in patients with RET mutations affecting the cysteine-rich extracellular domain of the protein. Both activating and inactivating effects have been demonstrated for the C620R mutation (so called the Janus mutation) because, when expressed in kidney cells, it promotes cell proliferation and impairs migration and branching in three-dimensional culture in response to GDNF. [START_REF] Arighi | Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease[END_REF] Interestingly, association of HSCR with CAKUT has also been described and a RET mutation has been reported in few cases, [START_REF] Prato | Hirschsprung disease and congenital anomalies of the kidney and urinary tract (CAKUT): a novel syndromic association[END_REF] suggesting a common genetic basis for these two pathologies. Renal agenesis was also reported in a family with MTC and the Janus RET C620 mutation. [START_REF] Lore | Unilateral renal agenesis in a family with medullary thyroid carcinoma[END_REF] Recently, both activating and inactivating RET mutations have been reported in a small series of fetuses with renal agenesis. [START_REF] Skinner | Renal aplasia in humans is associated with RET mutations[END_REF] To better assess the role of the RET/GDNF signalling in KDD, we analysed coding and regulatory sequences of RET and GDNF in a series of 105 fetuses with severe bilateral defects.

PATIENTS AND METHODS

Patients

We studied a series of 105 fetuses with bilateral KDD contributing to anamnios or severe oligohydramnios and that had motivated termination of pregnancy. This included 65 cases with BRA, 24 cases with URA and an abnormal contralateral kidney (RHD in 8 cases, MCDK in 16 cases), 4 cases with bilateral RHD, 10 cases with bilateral MCDK, one case with RHD on one kidney and MCDK on the other and one case with URA and ureteral duplication. Kidney defects were defined by the fetopathologists of the Société Française de Foetopathologie upon histological examination. Five fetuses were from consanguineous families, 11 cases (including 4 sib-pairs) were from 7 non-consanguineous families in which the mother underwent several terminations of pregnancy for anamnios, and five other cases had relatives with renal abnormalities. Twenty-one fetuses had extra-renal abnormalities, namely uterine agenesis (6 cases), bifid uterus (1 case), epididymal hypoplasia or cysts (2 cases), or more complex syndromic associations (growth retardation, craniofacial dysmorphy, hand and limb anomalies, cardiopathy). No GLI3 mutation was identified in 3 fetuses with symptoms suggestive of Pallister-Hall syndrome. There was no indication of renal-coloboma or BOR syndromes in relatives of any of the studied fetuses. Moreover, we excluded the presence of HNF1B mutations / deletions in the fetuses with MCDK or RHD with cysts.

This study was fully approved by the Comité de Protection des Personnes pour la Recherche Biomédicale Ile de France 2 and informed consent was obtained from all parents.

DNA was extracted from frozen liver, lung, spleen or kidney samples. For three fetuses, RNA was also extracted from frozen kidney samples.

Sequencing of RET and GDNF coding and non-coding sequences

We screened the 20 RET exons encoding both protein isoforms (RET9: exons 1 to 19b, RET51: exons 1 to 19a, 20) and the three GDNF exons (non-coding exon 1 and coding exons 2 and 3). Evolutionary conserved non-coding regions (ECRs) located upstream of RET and GDNF and in RET intron 1 (http://www.dcode.org) were also analyzed. Their positions relative to exon 1 are given in Table 1. Finally, as variations in the 3'UTR could potentially disrupt genetic regulation by micro-RNAs, these regions were also analyzed. The 3'UTRs of RET9 and RET51 transcripts were identified from http://www.ensembl.org/. For analysis of the 3'UTR of GDNF, we focused on the first 1231 bp that were evolutionary conserved (Table 1

Analysis of RET cDNA

The effect of the c.1353 G>A variant (T451T) on splicing was analysed by RT-PCR, using primers located in exons 6 and 8. Quantitative RT-PCR was performed using the ABsolute Sybr green ROX mix (Thermo Scientific) and GAPDH was used as a control.

Analysis of copy number variations

For 41 samples, the quality and the available amount of DNA allowed us to perform genomic analysis on Illumina Infinium HumanOmni1 beadchips. Hybridizations were performed as recommended by the manufacturer. We used the GenomeStudio software for normalization and genotyping. For identification of copy number variations (CNVs), we used the GenomeStudio plug-in CNVpartition and the PennCNV algorithm with default parameters. [START_REF] Wang | PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data[END_REF]

RESULTS

Analysis of RET

Sequencing of the 20 exons of RET in 105 fetuses with severe bilateral KDD identified 7 previously unreported variations in the coding region, including one nonsense mutation, four missense and two neutral changes (Table 2). All variations were heterozygous and none of them was identified in 180 controls. The nonsense mutation resulted in a stop codon located 58 and 16 amino acids before the C-terminus of RET51 and RET9 isoforms, respectively.

PolyPhen-2 predicted variations D567N and V787I as probably damaging with scores of 0.984 and 0.979 respectively and L56M as possibly damaging with a score of 0.281. Although R57Q was predicted as benign by PolyPhen-2, it was qualified as possibly damaging with the previous version of PolyPhen (score=1.558) and it affects a very conserved aminoacid.

For fetuses with the R57Q, D567N and W1056X mutations and the P992P neutral variant, DNA from the parents was available. In all four cases, the variation was also present in the heterozygous state in the father, in whom presence of the two normal-sized kidneys was ascertained by renal echography. This demonstrates that none of these variants alone, even the nonsense mutation, is sufficient to explain the renal development defect. Several variations corresponding to known SNPs were also identified in the coding sequence as well as in flanking intronic sequences. We compared their frequencies in the KDD fetuses to controls (Table 3A). No significant difference was observed. Evolutionary sequence conservation has proven a valuable approach to identify genomic regions important for gene expression regulation. Using the ECR Browser (http://ecrbrowser.dcode.org), we identified 8 ECR regions that were at least 70% conserved until mouse (Table 1). Six ECRs were located upstream of the transcript region (ECR1, -1B, -1E, -2, -4 and -8,) and sequencing of these ECRs in the 105 samples led to the identification of 11 variants (Table 3A): five of them were referenced in the SNP database (http://www.ncbi.nlm.nih.gov/snp/)

and their frequencies were similar in cases and controls, whereas the 6 other were unreported variations that were heterozygous in 1% to 6% of the fetuses (allele frequencies between 0.005 and 0.03). We only identified the most frequent one, ECR2/504, in controls (Table 3A).

ECR6 and ECR7 were located in intron 1 and included 7 variants that were all referenced in the SNP database. One of these variants, ECR6/271, was significantly more frequent in the KDD fetuses than in controls (heterozygous in 14/96 fetuses vs 7/189 controls, P=0.002) (Table 3A). To confirm this result, we analysed a new series of 171 cases with bilateral KDD including 53 fetuses and 118 children. Based on analysis of all of the samples (variant heterozygous in a total of 24/267 fetuses/children) or fetuses only (variant heterozygous in a total of 17/149 fetuses), the variant frequency remains significantly higher in cases than in controls (P=0.04 or P=0.01, respectively). The presence of the variant in fetuses with BRA but also in fetuses and children with MCDK suggests that it could confer predisposition to the whole spectrum of KDD.

Sequencing of the 3'UTR of both RET9 and RET51 transcriptional isoforms revealed 8

referenced SNPs and 6 previously unreported rare variants (Table 3A). There was no significant difference in their frequencies in cases vs. controls.

Analysis of GDNF

Sequencing of GDNF coding and non-coding sequences identified 7 referenced SNPs and 6

unreported variants (Table 3B). Variant frequencies in the fetuses were not different from those in controls. Therefore, this study did not provide any indication of the involvement of this gene in the etiology of KDD.

CNV analysis at RET, GDNF and other genes linked to the RET/GDNF signalling

Finally, as CNVs could be responsible for an altered gene expression, we quantified the RET and GDNF loci in 41 of the fetuses, based on genomic data generated by DNA hybridization on Illumina HumanOmni1 chips. We extended this analysis to several other genes involved in the RET/GDNF pathway, namely the GDNF co-receptor encoding gene GFRA1, regulators of the expression of RET and/or GDNF (GDF11, PAX2, EYA1, SIX1, SIX2, SIX4, HOXA11, HOXD11, FOXC1, GATA3), repressors of the pathway (SPRY1, ROBO2, SLIT2), the activator SOX9 and target genes ETV4 and ETV5. [START_REF] Little | Kidney development: two tales of tubulogenesis[END_REF][START_REF] Lu | Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis[END_REF][START_REF] Reginensi | SOX9 controls epithelial branching by activating RET effector genes during kidney development[END_REF] For each gene, we analysed not only the gene itself but also surrounding sequences extending up to neighbouring genes on each side, in order to be sure to include regulatory sequences. In addition to two intronic CNVs, in SLIT2 and EYA1, each identified in 4 fetuses, we characterized a 1 kb heterozygous deletion spanning the GDNF non-coding exon 1 in one case. These three CNVs have been reported in controls (http://projects.tcag.ca/variation/), suggesting that they are polymorphic variations.

There was no CNV in any of the other tested genes.

DISCUSSION

The pattern of expression of RET and GDNF in fetal kidneys, the severe disruption of kidney development in Ret or Gdnf knockout mice, as well as large amounts of data generated from cellular and organ culture models, support the major role of the RET/GDNF signalling in control of branching morphogenesis during kidney development. [START_REF] Chi | Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis[END_REF][START_REF] Meyer | Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney[END_REF][START_REF] Jain | Critical and distinct roles for key RET tyrosine docking sites in renal development[END_REF][START_REF] Sanchez | Renal agenesis and the absence of enteric neurons in mice lacking GDNF[END_REF][START_REF] Schuchardt | Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret[END_REF][START_REF] Jain | Organotypic specificity of key RET adaptor-docking sites in the pathogenesis of neurocristopathies and renal malformations in mice[END_REF][START_REF] Jijiwa | A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia[END_REF][START_REF] Majumdar | Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during metanephric kidney development[END_REF][START_REF] Shakya | The role of GDNF/Ret signaling in ureteric bud cell fate and branching morphogenesis[END_REF][START_REF] Srinivas | Dominant effects of RET receptor misexpression and ligand-independent RET signaling on ureteric bud development[END_REF] Moreover, several RET mutations associated with MTC and/or HSCR have been shown to impair branching in a three-dimensional kidney cell culture model, [START_REF] Arighi | Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease[END_REF][START_REF] Degl'innocenti | Differential requirement of Tyr1062 multidocking site by RET isoforms to promote neural cell scattering and epithelial cell branching[END_REF] in agreement with a role of RET in kidney morphogenesis. It was thus expected that mutations in RET and GDNF could be responsible for kidney development defects in human. Accordingly, heterozygous RET mutations were previously reported in 30% of a small series of 29 fetuses with BRA or URA and one heterozygous GDNF mutation in a fetus with URA.

[29] However, our results, based on analysis of a larger series of 105 cases including 90 fetuses with either BRA or URA and contralateral RHD or MCDK do not confirm the high frequency of RET mutations in fetuses with renal agenesis or other severe abnormality of kidney development.

Discrepancy between the two studies could be explained by differences in the ethnic origin of the patients and/or by a bias due to the small number of patients in the study of Skinner et al..

We only report 7 potential mutations in the RET coding sequence (6.6%), and no mutation in the GDNF coding sequence. We did not analyse the sequence of the GDNF co-receptor encoding gene GFRA1 in this study because no mutation has ever been reported in this gene, neither in KDD nor in HSCR. [START_REF] Prato | Hirschsprung disease and congenital anomalies of the kidney and urinary tract (CAKUT): a novel syndromic association[END_REF][START_REF] Skinner | Renal aplasia in humans is associated with RET mutations[END_REF][START_REF] Myers | Investigation of germline GFR alpha-1 mutations in Hirschsprung disease[END_REF] As reported, [START_REF] Skinner | Renal aplasia in humans is associated with RET mutations[END_REF] the RET mutations that we identified in KDD fetuses were heterozygous, distributed all along the gene and most of them were missense variants. Only one nonsense mutation was identified and absence of tissue available for RNA and protein extraction precluded analysis of the impact of this mutation on the expression of the gene. As for HSCR, heterozygosity of the mutations suggests a mechanism based on haploinsufficiency, leading to a dysregulation of the RET signalling in kidney. However, one cannot exclude that some of the missense variants might be activating mutations with both gain-of-function and loss-of-function effects, as suggested for some of the mutations previously described. [START_REF] Skinner | Renal aplasia in humans is associated with RET mutations[END_REF] While Skinner et al. described the presence of two or three mutations in some fetuses, none of the fetuses in our series presented with several mutations. [START_REF] Skinner | Renal aplasia in humans is associated with RET mutations[END_REF] No mutation was common to both studies and only the L56M variant has previously been reported in a HSCR patient. [START_REF] Hofstra | RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems[END_REF] Absence of splicing defect in the kidney from the fetus with the T451T variant suggests that this neutral variant is a rare polymorphism rather than a causative mutation. Moreover, heterozygosity of the P992P variant in a fetus from a consanguineous family and his father suggests that the kidney development defect in this family is probably not linked to this RET variant. Finally, while Skinner et al. did not trace the inherited vs. de novo origin of the mutations in their fetuses, we demonstrate that the missense mutations R57Q and D567N as well as the W1056X nonsense mutation were inherited from healthy fathers. Although we cannot eliminate the hypothesis that somatic mosaicism could explain the absence of phenotype in the father, these results likely indicate that, even if these RET mutations impair kidney development, other genetic or epigenetic events affecting the RET/GDNF signalling must cooperate.

In addition to the 7 RET variations in the coding sequence, a total of 48 SNPs in coding and non-coding RET and GDNF sequences, as well as 21 new rare variants in non-coding sequences were identified in our series of fetuses (4 to 22 RET variants per fetus; 0 to 4 GDNF variant per fetus). This includes RET SNP variants A432A in exon 7 and G691S in exon 11, that have been reported as associated with CAKUT, [START_REF] Yang | RET Gly691Ser mutation is associated with primary vesicoureteral reflux in the French-Canadian population from Quebec[END_REF][START_REF] Zhang | A common RET variant is associated with reduced newborn kidney size and function[END_REF] and RET variants rs1864411, rs1864410, rs2435357 and rs2506004 in intron 1 that have been reported as part of a HSCR susceptibility haplotype. [START_REF] Burzynski | Identifying candidate Hirschsprung disease-associated RET variants[END_REF][START_REF] Pelet | Homozygosity for a frequent and weakly penetrant predisposing allele at the RET locus in sporadic Hirschsprung disease[END_REF] Based on the observation that allele frequencies in our cases were similar to controls, our data do not support a role in KDD for any of these variants. Four previously unreported variants in RET 5'-ECR regions were identified in a total of six patients and not in controls. However, the absence of clustering of these variants in one specific ECR is not in favour of a role in the dysregulation of RET expression. Moreover, discordant genotypes in the 3 pairs of fetuses belonging to families with recurrent KDD suggest that RET is not the culprit gene in these patients.

Interestingly, we identified one polymorphism in RET intron 1 (ECR6/271, rs2506012) with significant over-representation of the minor G allele in KDD fetuses. Analysis of transcription factor binding on the ECR6 sequence using the Genomatix (http://www.genomatix.de) and the Mapper (http://mapper.chip.org) softwares predicted creation of a site for the aryl hydrocarbon receptor nuclear translocator (ARNT) transcription factor by this variant. ARNT is a protein that is involved in translocation of the aryl hydrocarbon receptor (AHR) to the nucleus, following induction by ligands like dioxin and polycyclic aromatic hydrocarbons. Interestingly, ARNT and its receptor AHR are both expressed during kidney development (http://www.gudmap.org/) and activation of AHR has been reported to inhibit branching morphogenesis of metanephric kidneys. [START_REF] Falahatpisheh | Ligand-activated Ahr signaling leads to disruption of nephrogenesis and altered Wilms' tumor suppressor mRNA splicing[END_REF] Thus, this polymorphism could confer an increased susceptibility to environmental factors and could result in an altered regulation of RET. ARNT has also been identified as the beta subunit of the heterodimeric transcription factor HIF1 (hypoxia-inducible factor 1). As HIF1A is coexpressed with ARNT in cap mesenchyme and UB (http://www.gudmap.org/), an altered regulation of RET by HIF1 can also be proposed. While analysis of a second series of samples confirmed the difference in allele frequencies between KDD fetuses and controls, it resulted in a decrease in the level of significance (P=0.002 to P=0.01). Thus, analysis of a larger series of patients will be necessary to unambiguously draw conclusions. Moreover, due to the low frequency of this variant in the fetuses, its potential contribution to kidney development defect can only account for a small proportion of patients.

Quantification of RET transcript in KDD vs. control kidney samples revealed downexpression in the three KDD samples. There are several possible explanations for this result, including sample bias, cause or consequence of the altered differentiation process. One of the mechanisms involved in regulation of gene expression is through binding of micro-RNAs to 3'UTR sequences. Indeed, variations in 3'UTR and mutations in the micro-RNA encoding genes, resulting in an altered gene regulation, have been reported in several pathologies. [START_REF] Mencia | Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss[END_REF][START_REF] Simon | A mutation in the 3'-UTR of the HDAC6 gene abolishing the posttranscriptional regulation mediated by hsa-miR-433 is linked to a new form of dominant X-linked chondrodysplasia[END_REF][START_REF] Yang | Mechanistic insights into the link between a polymorphism of the 3'UTR of the SLC7A1 gene and hypertension[END_REF] In one case, the variation was a common polymorphism, significantly over-represented in patients vs. controls. [START_REF] Yang | Identification of a novel polymorphism in the 3'UTR of the L-arginine transporter gene SLC7A1: contribution to hypertension and endothelial dysfunction[END_REF] In the RET gene, a variant located in the 3'UTR has been reported to confer protection from HSCR. [START_REF] Griseri | A common variant located in the 3'UTR of the RET gene is associated with protection from Hirschsprung disease[END_REF] However, sequencing of RET and GDNF 3'UTRs in KDD fetuses did not allow us to identify new variants nor variants with a higher frequency in patients vs. controls. When the micro-RNAs that regulate RET expression during kidney development are identified, it will be worthwhile screening for mutations in these sequences.

Another genetic mechanism that could affect RET and/or GDNF expression and consequently kidney development is gene copy number variations. The only CNV of potential interest that we identified was a deletion spanning a CpG rich region and the non-coding exon 1 of GDNF. Although this CNV has been reported in normal controls (http://projects.tcag.ca/variation/), suggesting that it is a polymorphism, it could result in a decreased GDNF expression. However, it was present in only one fetus and thus cannot be considered as a significant event in KDD. Moreover, CNV analysis of 17 other genes encoding regulators or targets of the RET/GDNF pathway including the GDNF co-receptor encoding gene GFRA1, did not allow us to identify any CNV affecting these genes.

Altogether, this study clearly shows that RET and GDNF genomic alterations are not significantly associated with renal agenesis / hypodysplasia /multicystic dysplastic kidney in humans, suggesting that they play a minor role in KDD. These results are an important issue to be taken into account for genetic diagnosis of these defects. Interestingly, redundant receptor tyrosine kinase signalling, notably including FGF10/FGFR2, as well as a balance between positive and negative regulation of this signalling network, rather than RET/GDNF per se, was suggested to be the central pathway regulating branching for kidney development. [START_REF] Michos | Kidney development in the absence of Gdnf and Spry1 requires Fgf10[END_REF] Regulatory or target genes common to these redundant signalling events could be worthwhile testing as new candidate genes to explain kidney development defects.

  ). PCR and sequencing primers are available upon request.Fisher exact tests were used to compare frequencies of the variants in patients and controls.As 80% of the fetuses were of European origin, frequencies in controls were mostly evaluated by analysis of 189 unrelated Caucasians. Algerian and Turkish controls (34 and 33 cases, respectively) were also tested for polymorphisms present in patients originating from these countries. Moreover, we considered frequencies generated from the HapMap-CEU (120

	GDNF-ECR3 chr5:37850279-	3'-UTR	1231	82% (M)	opossum
		37851510
	Positions on chromosomes are given according to the NCBI36/hg18 built ;
	Table1 Evolutionary conserved sequences in 5' and 3' of RET and GDNF
	Evolutionary	Position on	Position	Size	Homology 1 More distant
	Conserved	chromosome	related to the	(bp)		species with
	Region		first exon			>70%
						homology
	RET-ECR1E	chr10:42794983-	5' (-97kb)	385	78% (O)	chicken
		42795367
	RET-ECR1B chr10:42803235-	5' (-89 kb)	464	78% (M)	frog
		42803698
	RET-ECR1	chr10:42880879-	5' (-11.6kb)	610	77% (M)	chicken
		42881488
	RET-ECR4	chr10:42883631-	5' (-8.9kb)	275	74% (M)	mouse
		42883905
	RET-ECR8	chr10:42883965-	5' (-8.5kb)	500	77% (M)	mouse
		42884464
	RET-ECR2	chr10:42886856-	5' (-5.7kb)	640	79% (M)	opossum
		42887495
	RET-ECR6	chr10:42895408-	intron 1	334	76% (M)	mouse/rat
		42895741
	RET-ECR7	chr10:42902036-	intron 1	315	72% (M)	opossum
		42902350
	GDNF-ECR1 chr5:37876398-	5' (-5.5kb)	469	81% (O)	opossum
		37876866
	GDNF-ECR2 chr5:37877173-	5' (-6.2kb)	349	75% (O)	opossum
		37877521

1 % of homology of the selected region with the mouse (M) or the opossum (O) sequence Statistical analyses chromosomes), Pilot.1.CEU (72 chromosomes) and AGI_ASAP (74 chromosomes) populations (http://www.ncbi.nlm.nih.gov/projects/SNP/). For statistical study of the RET variant rs2506012, we analysed a second series of 171 patients, including 53 fetuses and 118 living children, with the same spectrum of bilateral KDD as the first series.

Table 2

 2 Variants identified in the RET coding sequenceWe questioned whether the neutral variations c.1353 G>A (T451T) located in exon 7 and c.2976 G>A (P992P) in exon 18 could have an effect on splicing, using the mutation interpretation software Alamut (http://www.interactive-biosoftware.com/) and the Exonic Splicing Enhancer analysis software ESE finder (http://rulai.cshl.edu/tools/ESE/). While we did not identify any effect of the c.2976 G>A variant, both methods identified creation of sites for splicing factors SF2, SC25 and SPp40 by the c.1353 G>A variant (scores = 4.5, 2.5 and 4.5, respectively), suggesting that this variant might alter splicing of exon 7.[START_REF] Cartegni | Listening to silence and understanding nonsense: exonic mutations that affect splicing[END_REF] To test this hypothesis, we amplified a cDNA fragment spanning RET exons 6 to 8 from kidney samples obtained for fetus H637 with the c.1353 G>A exon 7 variant, two other fetuses with KDD and no RET mutation, and three control fetuses without renal pathologies. We did not identify any abnormal-sized band associated with the variant (data not shown). To analyse if the variant could decrease the amount of RET transcript, we performed quantitative RT-PCR using the same exon 6 and exon 8 primers. In the three KDD kidney samples, the level of RET

	Nucleotide	Variant	Consequence Exon Phenotype	Features of the variants
	Change 1
	c.166	p.L56M	Missense	2	BRA and	Possibly damaging 2
	C>A				uterine agenesis
	c.170	p.R57Q	Missense	2	MCDK	Benign 2
	G>A					Present in healthy father
	c.1353	p.T451T	Neutral	7	URA/MCDK	Potential effect on
	G>A					splicing 3
	c.1699	p.D567N	Missense	9	BRA	Probably damaging 2
	G>A					Present in healthy father
	c.2359	p.V787I	Missense	13	URA/RHD	Probably damaging 2
	G>A
	c.2976	p.P992P	Neutral	18	BRA	Present in healthy father
	G>A
	c.3167	p.W1056X Nonsense	19	BRA	Present in healthy father
	G>A
	1 c. positions are given according to the coding sequence of NM_020975
	2 identified with PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/);
	3 identified with Alamut (http://www.interactive-biosoftware.com/) and
	ESE finder (http://rulai.cshl.edu/tools/ESE/):

BRA : bilateral renal agenesis ; URA : unilateral renal agenesis ; RHD : renal hypodysplasia. MCDK: multicystic dysplastic kidney expression was 10 times lower than in the normal fetal kidney samples. However, RET expression in H637 kidney sample with the c.1353 G>A variant was not different from RET expression in the two other KDD kidney samples without RET mutation (data not shown). Altogether, these results did not allow us to validate any effect of the c.1353 G>A variant on RET expression. Whether the low level of RET expression in KDD kidneys is a cause or a consequence of the altered kidney development remains an open question.

Table 3A

 3A Polymorphic variants in the RET sequence

	3'UTR	RET9 2	c.3219+15 c.3219+128 c.3219+166 c.3219+731	C>T C>T A>T C>T		0.17 (0.15) 4 0.005 (-) 0.20 (0.22) 4 0.15 (0.15) 4	rs2075912 rs2075913 rs2565200
			c.3345+29	A>C		0.005 (-)
			c.3345+95	C>T		0.17 (0.22) 4	rs17028
			c.3345+388	G>A		0.23 (0.13) 4	rs3026782
	3'UTR	RET51 2	c.3345+576 c.3345+600 c.3345+1046 c.3345+1116	G>A T>A G>C T>C		0.005 (0.003) 0.15 (0.19) 0.01 (0.003) 0.20 (0.24)	rs2742240 rs2435355
			c.3345+1506	A>G		0.17 (0.19)	rs2742241
			c.3345+1582	G>A		0.005 (-)
			c.3345+1590	G>A		0.04 (0.03)
	1 positions are given according to the ECR sequence (see Table 1) ;
	2 c. positions are given according to NM_020975, except for RET9 3'UTR (NM_020630) ;
	3 frequency of the homozygous variant ;
	4 frequency in controls according to Pilot-CEU ;
	5 p = 0.002 ;
	(-) absent in controls (370 to 444 chromosomes) ;
	(nd) not determined
			Position	Variation in	Variation	Frequency of	SNP
				non coding	in coding	the minor	referenced
				sequence	sequence	allele in KDD	in NCBI
						fetuses
						(in controls)
			ECR1E/83	C>A		0.01 (-)	rs73262104
	5' evolutionary conserved	ECRs 1	ECR1E/176 ECR1B/36 ECR1B/136 ECR1B/262 ECR1B/293 ECR8/48 ECR2/63 ECR2/189 ECR2/230	C>A G>A C>T C>T G>A C>T T>C C>G delTCAC		0.01 (-) 0.23 (0.23) 0.01 (-) 0.01 (-) 0.005 (0.007) 0.29 (0.24) 0.005 (-) 0.47 (0.51) 0.005 (-)	rs1547930 rs12572318 rs7910199 rs2505992
			ECR2/504	C>T		0.03 (0.02)
	Intron 1 ECRs 1		ECR6/44 ECR6/198 ECR6/213 ECR6/224 ECR6/271 ECR7/27 ECR7/244	G>A C>T C>T G>T C>G C>T C>A		0.09 (0.10) 0.36 (0.37) 0.33 (0.28) 0.20 (0.23) 0.07 (0.02) 5 0.21 (0.19) 4 0.21 (0.18) 4	rs1897002 rs1864411 rs10900298 rs1864410 rs2506012 rs2435357 rs2506004
			c.73+53	G>A		0.15 (0.10) 3	rs12267460
			c.73+104	T>C		0.005 (nd)
			c.73+171/172	GC>AA		0.30 (0.35)	rs34327391
			c.73+173	insGGGCGGC		0.15 (0.10) 3
	Exonic and flanking intronic sequences 2		exon2 c.337+9 exon3 exon3 c.626-72/71 c.1264-5 exon7 c.1648+84 c.1648+88 exon11 c.2284+47 c.2285-102 c.2285-85 exon13 exon14	G>A delCC C>T A>G insC C>T C>T G>A	A45A V125V N199N A432A G691S L769L S836S	0.23 (0.29) 0.24 (0.28) 0.01 (nd) 0.005 (nd) 0.33 (0.34) 0.02 (0.009) 0.31 (0.29) 0.19 (0.22) 0.19 (0.22) 0.22 (0.22) 0.28 (0.28) 0.01 (nd) 0.005 (nd) 0.20 (0.22) 0.03 (0.04)	rs1800858 rs2435351 rs1800859 rs55810667 rs35906041 rs9282835 rs1800860 rs3026750 rs34827976 rs1799939 rs760466 rs3026766 rs1800861 rs1800862
			c.2608-24	G>A		0.20 (0.21)	rs2472737
			exon15		S904S	0.23 (0.26)	rs1800863
			c.2801+54	A>T		0.005 (nd)	rs3026772
			c.2801+72	G>A		0.005 (nd)	rs3026773
			exon18		R982C	0.05 (0.03)	rs17158558

Table 3B

 3B Polymorphic variants in the GDNF sequence

		Position	Variation in	Variation	Frequency of	SNP
			non coding	in coding	the minor	referenced
			sequence	sequence	allele in KDD	in NCBI
					fetuses
					(controls)
		ECR1/405	G>A		0.07 (0.11) 3	rs2975100
	5' 1	ECR1/2	C>A		0.07 (0.22) 4	rs2075680
		c.1-229	insGCC		0.01 (-)
	Exons 2	c.150 c.351+17 Exon3	T>C C>T	R143R	0.24 (0.33) 1 0.005 (-) 0.02 (0.03) 5	rs2973033 rs36010631
		ECR3/57	G>T		0.005 (nd)
		ECR3/93	T>A		0.005 (nd)	rs45535335
	3'UTR 1	ECR3/363 ECR3/450 ECR3/529	A>T G>A C>T		0.005 (nd) 0.02 (0.02) 0.01 (nd)	rs45611430 rs58787312
		ECR3/795	G>A		0.007 (-)
		ECR3/988	T>C		0.05 (0.03)

positions are given according to the ECR sequence (see Table1) ;

c. positions are given according to NM_000514 ;

frequency in controls according to Pilot-CEU ;

frequency in controls according to JBCI-allele ;

frequency in controls according to AGI_ASAP population : (-) absent in controls (296 to 452 chromosomes) ; (nd) not determined

"The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JMG and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licence-forms)."

ACKNOWLEDGMENTS

We thank Dr C Baumann, C. Bernabe-Dupont, B Gilbert, L. Olivier-Faivre and F. Prieur for providing some fetal and/or parental DNAs and Sophie Saunier for helpful discussion.

FOOTNOTES

Funding This work was supported by Institut National de la Santé et de la Recherche Médicale, Agence Nationale de la Recherche (ANR 06-MPAR-034-01), GIS-Institut des Maladies Rares (AAE07007KSA) and Programme Hospitalier de la Recherche Clinique Assistance Publique (AOM07129).

Competing interest None

Patients consent obtained

Ethics approval This study was conducted with the approval of the the Comité de Protection des Personnes pour la Recherche Biomédicale Ile de France 2


M.D Darius Soonawala
email: d.soonawala@lumc.nl

Rutger A Middelburg

Matthias Egger

Jan P Vandenbroucke

Olaf M Dekkers

Keywords: non-inferiority, meta-analysis, systematic review

Referee comments in black, authors' response in blue, changes in red and italic.

General response to the referees' comments:

We think that we may not have explained our hypothesis and the focus of our study well enough. The question that we address in this study is whether a verdict of 'proven' noninferiority for experimental treatment somehow hides a consistently lesser effect than standard treatment. We have revised the introduction as follows, to emphasize the focus of our paper:

There is concern that non-inferiority trials might be deliberately designed to conceal that a new treatment is somewhat less effective than a standard treatment. [START_REF] Gotzsche | Lessons from and cautions about noninferiority and equivalence randomized trials[END_REF][START_REF] Garattini | Non-inferiority trials are unethical because they disregard patients' interests[END_REF] Systematic use of too large non-inferiority margins or systematic biases of design, conduct or reporting of non-inferiority trials may skew results in favour of new treatments. [START_REF] Smith | Medical journals are an extension of the marketing arm of pharmaceutical companies[END_REF][START_REF] Lexchin | Pharmaceutical industry sponsorship and research outcome and quality: systematic review[END_REF][START_REF] Chan | Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles[END_REF][START_REF] Turner | Selective publication of antidepressant trials and its influence on apparent efficacy[END_REF][START_REF] Mathieu | Comparison of registered and published primary outcomes in randomized controlled trials[END_REF][START_REF] Leucht | Pretrial medication bias in randomized antipsychotic drug trials[END_REF] In this meta-analysis we examined one type of systematic bias. If trialists systematically compare slightly less effective new treatments to standard treatments, the combined results from a meta-analysis of many trials in which experimental treatments gain a verdict of non-inferiority, would be expected to favour the standard treatment. To test this hypothesis we performed a meta-analysis of non-inferiority trials published in clinical journals and assessed the average effect of experimental treatments compared with standard treatments. Importantly, the combined estimate from the meta-analysis will not be influenced by the choice of the non-inferiority margins.

Referee: 1 Comments to the Author

To the Author.

This paper represents a considerable effort in establishing the value of non-inferiority trials.

The paper is well written, concise and quite critical in establishing strengths and weaknesses of the study. However to recognize the limitations is not a reason to allow a conclusion. The strong message derived from this paper is in fact not reliable because, as the AA report them selves, the recognition of bias does not rule out that the experimental treatments are inferior to the standard interventions. The study is based on 30 percent of the non inferiority trials only. There has been no attempts to find unpublished trials. No evaluation has been made on the quality of the recruited trials.

There are two points that should be included in the paper 1. a critical analysis of the delta utilized

We acknowledge that this is an important issue, but please note that the focus of our paper is on the combined results from the meta-analysis. The combined estimate from the meta-analysis will not be influenced by the choice of the noninferiority margins. An assessment of the appropriateness of non-inferiority margins in relation to the outcome examined was outside the scope of this study. This would have required an in-depth analysis of each trial with subject matter knowledge on each topic. We have revised the introduction to emphasize the focus of our paper. Please see our 'general response to the referees' comments'.

2. an analysis of the standard therapy; it should be established if the standard represents the best available therapy. It is in fact obvious that if poor standards are selected everything may become non inferior.

The reviewer is right in pointing out that this is an important issue in noninferiority and in superiority trials in which an active comparator treatment is used, but please note that the focus of our paper is on the combined results from the meta-analysis. An assessment of the appropriateness and efficaciousness of the standard treatments would have required an in-depth analysis of each trial with subject matter knowledge on each topic. This was outside the scope of this study. As suggested by the reviewer we have added an analysis to assess how standard treatments performed in view of the expected efficacy as stated by trialists.

Methods:

We compared the observed incidence of the primary endpoint in the group that received standard treatment with the expected incidence, as specified by the trialists. The result was expressed as a ratio. If needed, the inverse of this ratio was calculated, so that ratios below 1 consistently indicated that the standard treatment performed better than was expected at the design stage of the trial, and ratios above 1 indicated that the standard treatment performed worse than was expected. This ratio could not be calculated for studies that did not report separately the expected, or the observed incidence of the primary endpoint, or studies reporting continuous endpoints.

Results:

The ratio of the observed and expected incidence of the primary endpoint in the group that received standard treatment could be calculated for 112 comparisons. Fifty-three percent of comparisons (n=59) had a ratio below 1, indicating that the standard treatment performed better than was expected at the design stage of the trial and 46% (n=51) above 1, indicating that the standard treatment performed worse than was expected. Two ratios were exactly 1. The mean ratio was 0.941 (95% CI 0.859 to 1.030), meaning that on average the chosen standard treatments performed slightly better than was estimated at the design stage of the trials. Stratified by source of funding, this ratio was 0.974 (95% CI 0.865 -1.097) for 55 studies funded by industry and 0.906 (95% CI 0.760 to 1.080) for 31 studies funded by public sources. These two estimates did not differ significantly (p=0.5 from t-test for equality of means).

Discussion:

The combined estimate will be influenced by the efficaciousness of standard treatment. If the standard treatment is not effective, the experimental treatment is in fact tested against 'placebo' in a non-inferiority design. Although we did not assess whether the chosen standard treatment represented the best available comparator, we did assess how standard treatments performed in view of what Furthermore efficacy is only one part of the problem; the possibility that a treatment could be no less effective but more toxic should be excluded before any conclusion is reached.

We agree. There is a trade-off between efficacy and toxicity. In our metaanalysis, six studies deemed the experimental treatment to be clinically inferior based on a secondary 'toxicity end-point'. Systematic assessment of toxicity in a meta-analysis would require that we judge the trade-off between the benefit and the drawback for each individual experimental treatment and come to a combined endpoint. Such an analysis would be quite subjective and was beyond the scope of our study. We have added a paragraph in the discussion addressing the issue of toxicity. p13:

Of the 175 comparisons in this meta-analysis, we considered the experimental treatment to be non-inferior in 130 (74%) and superior in 15 (9%). Although in 6 of these 145 comparisons the authors deemed the experimental treatment to be clinically inferior based on a secondary end-point, the majority of published noninferiority trials can be used to support the registration of a new treatment. The added value and safety of these treatments may not always be self-evident and may not always be demonstrated in the trial. The follow-up time and the sample size of trials is limited, making it improbable that rarer side effects or long term side effects are detected.

From a statistical standpoint, analysis has been conducted according to standard methodology. However, the relevance of the primary analysis is questionable, since a result close to unity should be expected, while focus on differences in precision of the estimates might be of more interest.

We think that we may not have explained our hypothesis and the focus of our study well enough. The question that we address in this study is whether a verdict of 'proven' non-inferiority for experimental treatment somehow hides a consistently lesser effect than standard treatment. We have revised the introduction as follows, to emphasize the focus of our paper: See our 'general response to the referees' comments'.

Besides a critical analysis of delta utilized, the evaluation of the differences between observed and hypothesized point estimates would be welcome too. The papers aims at providing evidence on the current debate about the pros and cons of non inferiority trials which, in Authors words: " can be deliberately designed to conceal that a new treatment is less effective than a standard one" To do this Authors performed a metanalysis of all non inferiority RCTs published between 1991 and February 2009 searching the 115 journals included into PubMed selection of core clinical journals. Authors found that the average relative risks comparing experimental with standard treatment was close to 1 thus concluding that non inferiority trials are not systematically less effective than standard treatments. Authors acknowledge that their study has several important limitations, and that further studies are warranted before a firm conclusions about the benefit risk profile of this sort of trials can be reached. Overall, this reviewer has reservations about: • the process through which Authors reached their conclusion -see point 2) below; • the presentation of the results -see point 1) and 3) below • the failure to analyze in greater depth some of their results -see point 4 below.

Specific Comments 1. Abstract The abstract is unbalanced, and somewhat falsely reassuring. It in fact emphasizes that "….non inferiority trials published in core clinical journals are not systematically less effective than the standard treatment". The thorough examination of the limitations of the review, which is in the discussion, does not allow to come safely to this conclusion.

We have modified the conclusion of the abstract as follows:

In this meta-analysis of non-inferiority trials the average relative risk comparing experimental with standard treatments was close to 1. The experimental treatments that gain a verdict of non-inferiority in published trials do not appear to be systematically less effective than the standard treatments. Importantly, publication bias and bias in the design and reporting of the studies cannot be ruled out and may have skewed the study results in favour of the experimental treatments. Further studies are required to examine the importance of such bias.

2.

Methods Besides the limitations of their search strategy that the Authors discuss on page 11 (Discussion section lines 19-39), it should be noted that there are also other reasons why the search strategy can be challenged as of its ability to identify all relevant studies. Authors in fact do not discuss the consequences of their choice to base their search on the 3 main key words "noninferiority", "noninferiority", "equivalence". It is, I believe, possible that poorer quality studies are also those poorly indexed, and this potential loss would disproportionately affect studies with more methodological problems. Specifically, a "negative study" (i.e a study that found no statistically significant difference between new and standard treatments) may not be labelled as "non inferiority" and be used in practice to claim demonstration of "non inferiority study". I recognize that this is an

F o r R e v i e w O n l y
issue that does not have an easy solution but given that cannot be ruled out should have inspired greater caution to the Authors. Obviously, this would have required a completely different sampling strategy (ie. full identification of all studies conducted in a specific disease/condition and then classification -according to the directions and statistical significance of the results). It would at least be legitimate to mention this issue in the discussion at least to hypothesize what would have happened to this metanalysis had these studies been systematically searched and included?.

Thank you for this comment. To pick up your challenge we did a quick full text search of a limited number of journals that were included in our meta-analysis We did indeed find a number of additional non-inferiority trials that were not mentioned as such in title or abstract. Interestingly, just under half of these 'missed trials' were designed as two-step trials, meaning that the methods section stated that a test for superiority would be performed in case of a verdict of non-inferiority. In two thirds of these trials the point estimate favoured experimental treatment and in half of the trials the authors' deemed experimental treatment to be statistically superior. This 'quick and dirty' sensitivity analysis suggests that if anything, adding the trials found in full text would have skewed the results of our meta-analysis in favour of experimental treatment. This would not invalidate or change our main conclusion which focussed on the question whether a verdict of 'proven' non-inferiority somehow hides a consistently lesser effect than standard treatment. Thank you for pointing this out. The trials with a 'two-step analysis' that are the cause of this finding (they are the ones that lead to the favouring of the experimental treatment) may become the focus of our next effort, as they seem special! However, they were not the focus of this report. In the discussion we address this issue as follows (p11):

Our study has several strengths and limitations. We aimed to include all the noninferiority trials published in these journals, irrespective of the type of endpoints or measures of effect. We restricted the search to the group of core clinical journals, as defined in PubMed, which is the same group of journals as in the Abridged Index Medicus (AIM). This selection covers a wide range of journals from many clinical specialties. Our results may therefore be representative for non-inferiority trials published in other journals. However, external validity may be limited to higher quality journals. If non AIM journals are of lower quality, the characteristics of non-inferiority trials published in those journals might be different. Furthermore, our search would have missed trials that do not mention the non-inferiority design in the abstract, the title or as a key word. The characteristics of such trials might also differ.

3.

Results section page 8 and 9. This metanalysis somewhat refers to a non real situation. "Non inferiority trials" -if used for registration purposes -are, in real life, presented to regulatory bodies in isolation (at most an handful of them). In my view it does not make much sense to look at their overall significance and direction without taking this into account. At the bottom of page 8 Authors state that: "…….in 74% comparisons experimental treatment was non inferior ". This means that, in most cases, these trials can be used to support the registration of a new intervention no matter how good they were designed. Authors acknowledge this on page 12 but then they do not draw the consequences of it. If one wants address and discuss what happens in reality, namely the way drugs are licensed this can (and should) be the main finding of the study. To me that result is much more important than saying that "…of the 130 comparisons judged to be non-inferior 58% favoured the experimental treatment and 42% the standard …" (see page 8 line 45-8). The latter looks to me an elegant statistical results but of very limited (and as said, somewhat misleading) practical significance.

We agree that it is important for readers to realize that the majority of published non-inferiority trials can be used to support the registration of a new intervention. However, this was not the main focus of this meta-analysis (see our 'general response to the referees' comments'). In the discussion we now do draw the conclusion from the fact that "…….in 74% comparisons experimental treatment was non inferior " (p13).

Of the 175 comparisons in this meta-analysis, we considered the experimental treatment to be non-inferior in 130 (74%) and superior in 15 (9%). Although in 6 of these 145 comparisons the authors deemed the experimental treatment to be clinically inferior based on a secondary end-point, the majority of published noninferiority trials can be used to support the registration of a new treatment.

4.

Influence of source of funding This should have been explored more thoroughly. On page 9 (lines 43-49) Authors state that "the main results and the results from the stratifies analysis where similar with the two statistical model were similar except for a difference in those stratified by funding sources". Readers would legitimately like to know more on that as one of the main concern about the (mis)use of inferiority trials is exactly the possible inappropriate use in commercially sponsored studies. Findings reported in table 2 should, in other words be better discusses as the category of industry funded trials is the one whose upper 95% CI is 1.00) Thus more information should be provided and discussed.

We now discuss this finding on p13:

The finding that studies sponsored by industry were more likely to have results favouring sponsored treatments is in line with other reports. 15,25 26 Systematic bias has been suggested as a possible explanation. Our finding could also be due to the play of chance.

5.

Representativeness of the sample Authors state in the discussion section that their sample is representative (page 11 lines 24-39). This reviewer is surprised by the distribution of the "fields" of the "non inferiority trials" shown in Table 1. In particular the proportion of oncology trials seem disproportionately low and the fact that 1out of 5 come from a lumped category "others" is rather uninformative.

The Abridged Index Medicus does not include some important journals on oncology such as the Journal of Clinical Oncology and journal of the national cancer institute. Furthermore, in a systematic review of 327 publications Lange and Freitag found only a slightly larger proportion of oncology trial (8.7%) (Lange S, Freitag G. Choice of delta: requirements and reality--results of a systematic review. Biom J 2005;47:12-27). The most common 'fields in medicine' in the category 'other' were: general medicine (3%), pulmonary medicine (2%%), gastroenterology (2%), anaesthesiology (1%), intensive care medicine (1%) and neurology (1%). We have added this in the Results section on p 8:

Other fields were obstetrics and gynaecology (7%), oncology (6%), rheumatology (6%), surgery (5%), psychiatry (3%), general medicine (3%), pulmonary medicine (2%), gastroenterology (2%), anaesthesiology (1%), intensive care medicine (1%) and neurology (1%). ABSTRACT Background There is concern that non-inferiority trials might be deliberately designed to conceal that a new treatment is less effective than a standard treatment. To test this hypothesis we performed a meta-analysis of non-inferiority trials to assess the average effect of experimental treatments compared with standard treatments.

Methods One hundred and seventy non-inferiority treatment trials published in 121 core clinical journals were included. Trials were identified in a search of PubMed (1991 to February 20, 2009). Combined relative risk (RR) from meta-analysis comparing experimental with standard treatments was the main outcome measure.

Results

The 170 trials contributed a total of 175 independent comparisons of experimental with standard treatments. The combined RR for all 175 comparisons was 0.994 (95% CI 0.978 to 1.010) using a random effects model and 1.002 (95% CI 0.996 to 1.008) using a fixed-effects model. Of the 175 comparisons, experimental treatment was considered to be non-inferior in 130 (74%). The combined relative risk for these 130 comparisons was 0.995 (95% CI 0.983 to 1.006) and the point estimate favoured the experimental treatment in 58% (n=76) and standard treatment in 42% (n=54). The median non-inferiority margin (RR) prespecified by trialists was 1.31 (interquartile range [IQR] 1.18 to 1.59).

Conclusion

In this meta-analysis of non-inferiority trials the average relative risk comparing experimental with standard treatments was close to 1. The experimental treatments that gain a verdict of non-inferiority in published trials do not appear to be systematically less effective than the standard treatments. Importantly, publication bias and bias in the design and reporting of the studies cannot be ruled out and may have skewed the study results in favour of the experimental treatments.

INTRODUCTION

Non-inferiority trials are increasingly published in the medical literature, increasingly used in drug licensing, and have at the same time come under increased scrutiny and criticism, up to the allegation that they are unethical. [START_REF] Ware | Equivalence trials[END_REF][START_REF] Siegel | Equivalence and noninferiority trials[END_REF][START_REF] Djulbegovic | Scientific and ethical issues in equivalence trials[END_REF][START_REF] Agostino | Non-inferiority trials: design concepts and issues -the encounters of academic consultants in statistics[END_REF][START_REF] Hung | Some fundamental issues with noninferiority testing in active controlled trials[END_REF][START_REF] Kaul | Trials and tribulations of non-inferiority: the ximelagatran experience[END_REF][START_REF] Lange | Choice of delta: requirements and reality--results of a systematic review[END_REF][START_REF] Henanff | Quality of reporting of noninferiority and equivalence randomized trials[END_REF][START_REF] Piaggio | Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement[END_REF][START_REF] Gotzsche | Lessons from and cautions about noninferiority and equivalence randomized trials[END_REF][START_REF] Fueglistaler | Non-inferiority trials in surgical oncology[END_REF][START_REF] Garattini | Non-inferiority trials are unethical because they disregard patients' interests[END_REF][START_REF] Kaul | Good enough: a primer on the analysis and interpretation of noninferiority trials[END_REF] A verdict of "non-inferiority" leaves readers with the impression that a new experimental treatment is as good as an established standard treatment and that the two can be used interchangeably. However, in such trials, noninferiority is statistically accepted whenever an experimental treatment is unlikely to be worse than an established treatment by more than a pre-specified amount, the so called non-inferiority margin. If a relatively wide margin is chosen, new treatments that are actually less beneficial might wrongly be considered as equally effective. This may lead to acceptance and use of new therapies that are actually less effective in a clinically relevant way. [START_REF] Gotzsche | Lessons from and cautions about noninferiority and equivalence randomized trials[END_REF][START_REF] Garattini | Non-inferiority trials are unethical because they disregard patients' interests[END_REF] There is concern that non-inferiority trials might be deliberately designed to conceal that a new treatment is somewhat less effective than a standard treatment. [START_REF] Gotzsche | Lessons from and cautions about noninferiority and equivalence randomized trials[END_REF][START_REF] Garattini | Non-inferiority trials are unethical because they disregard patients' interests[END_REF] Systematic use of too large non-inferiority margins or systematic biases of design, conduct or reporting of noninferiority trials may skew results in favour of new treatments. [START_REF] Smith | Medical journals are an extension of the marketing arm of pharmaceutical companies[END_REF][START_REF] Lexchin | Pharmaceutical industry sponsorship and research outcome and quality: systematic review[END_REF][START_REF] Chan | Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles[END_REF][START_REF] Turner | Selective publication of antidepressant trials and its influence on apparent efficacy[END_REF][START_REF] Mathieu | Comparison of registered and published primary outcomes in randomized controlled trials[END_REF][START_REF] Leucht | Pretrial medication bias in randomized antipsychotic drug trials[END_REF] In this meta-analysis we examined one type of systematic bias. If trialists systematically compare slightly less effective new treatments to standard treatments, the combined results from a meta-analysis of many trials in which experimental treatments gain a verdict of non-inferiority, would be expected to favour the standard treatment. To test this hypothesis we performed a metaanalysis of non-inferiority trials published in clinical journals and assessed the average effect of experimental treatments compared with standard treatments. Importantly, the combined estimate from the meta-analysis will not be influenced by the choice of the non-inferiority margins.

METHODS

Eligibility and search strategy

We searched for non-inferiority trials on February 20 th 2009 using PubMed (National Library of Medicine) using the text words "noninferiority" or "non inferiority" or "equivalence" combined with the text words "clinical trial" or "trial" or "trials" or "study" or "studies", limiting the search to publications from 1991 onwards. The initial search was restricted to six general medicine journals (Annals of Internal Medicine, BMJ, JAMA, Lancet, New England Journal of Medicine and PLoS Med). In a second step the search was extended to include the other 115 journals included in PubMed's selection of core clinical journals (see http://www.nlm.nih.gov/bsd/aim.html for a list of these journals). Although equivalence trials (trials that specify both a lower and an upper equivalence margin) [START_REF] Dunnett | An alternative to the use of two-sided tests in clinical trials[END_REF][START_REF]Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). E9: Statistical principles for clinical trials[END_REF] were not eligible for inclusion, we included the term equivalence in our search strategy in order not to miss noninferiority trials that had been described as equivalence trials.

Study selection

All two-arm parallel group non-inferiority trials of an experimental treatment compared with standard therapy were included, independent of the intervention examined in the trial.

Articles that were published electronically ahead of print were also considered.

Data extraction

The following information was extracted independently by two investigators (DS, RM): year of publication, journal, subject area (cardiovascular medicine, infectious diseases, obstetrics and gynaecology, rheumatology, surgery or other), primary endpoint, non-inferiority margin for primary endpoint, expected incidence of the primary endpoint in the standard arm, and the point estimate for the comparison of experimental with standard treatment. The primary endpoint was classified into three categories: (i) mortality alone or as part of a combined endpoint, (ii) clinical disease, (iii) surrogate endpoint (imaging or laboratory test). Disagreements were resolved in consultation with a third investigator (OD). If trials presented more than one primary endpoint, the endpoint for which the sample size had been calculated was used. If it was unclear for which endpoint sample size calculations were done, or if no such calculations were reported, one of the primary endpoints was randomly selected. In trials that included several non-inferiority comparisons using the same standard treatment, for example when testing two dosages of an experimental therapy, one comparison was selected at random and included in the analysis. If a study reported both intention-to-treat-and per-protocol analyses, the result used by the author to determine whether the intervention was non-inferior was extracted. If this was not clear, the perprotocol results were used. The funding source was independently classified by two investigators (DS, OD) as industry, public or mixed funding. The provision of study drugs by industry was considered as a source of industry funding.

Data synthesis and analysis

The confidence intervals and non-inferiority margins reported by the investigators were used to classify the results as superior, non-inferior, inconclusive or inferior according to the definitions given in the extension of the Consolidated Standards of Reporting Trials (CONSORT) statement to non-inferiority trials. [START_REF] Piaggio | Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement[END_REF] Superiority was assumed if the experimental treatment was significantly (p<0.05) more efficacious than the standard treatment. Noninferiority was assumed if the 95% confidence interval did not include the non-inferiority margin. Results were classified as inconclusive if the 95% confidence interval included the non-inferiority margin. Treatments were assumed to be inferior if the entire 95% confidence interval was significantly worse than the non-inferiority margin.

Results of comparisons were expressed as ratio measures, which we call relative risks throughout this paper. If the trial reported risk ratios or hazard ratios or odds ratios from statistical models these were used in the analyses. For trials that reported risk differences, we calculated the risk ratio. For studies reporting continuous endpoints (for example blood pressure) results were converted to odds ratios using the method described by Hasselblad and Hedges, [START_REF] Hasselblad | Meta-analysis of screening and diagnostic tests[END_REF] and the odds ratio was then used in further calculations. This method assumes logistic distributions with equal variances in the two treatment groups. Under this assumption the natural logarithm of the odds ratio equals a constant multiplied by the standardized difference between means. If needed, the inverse of the relative risk was calculated, so that ratios above 1 consistently favoured the standard treatment and ratios below 1 favoured the experimental treatment. The relative risks from individual studies were combined using random-effects models. In addition to combining relative risks for all studies, we performed stratified meta-analyses according to whether results were interpreted as inferior, non-inferior or superior, by type of effect measure, by type of endpoint, by source of funding, by journal and according to whether the judgment of the result was based on an intention-to-treat analysis or not. In a random-effects meta-regression model we analyzed the influence of the source of funding.

Pre-specified non-inferiority margins were also expressed as relative risks. Margins that were reported as a difference in incidence were converted to a relative risk by dividing the expected incidence of the primary endpoint in the standard treatment arm plus (for morbidity or mortality) or minus (for beneficial endpoints) the pre-specified margin by the expected incidence of the primary endpoint in the standard treatment arm. For example, if the expected mortality rate in the standard treatment arm was 10% and the pre-specified margin was set at 2%, the margin converted to a relative risk of 1.2 (10 plus 2 divided by 10).

Margins could not be expressed as a relative risk for studies that did not report the expected incidence, or studies reporting continuous endpoints. We examined the median and the distribution of non-inferiority margins and examined whether margins differed across the subgroups of trials mentioned above. We compared the observed incidence of the primary endpoint in the group that received standard treatment with the expected incidence, as specified by the trialists. The result was expressed as a ratio. If needed, the inverse of this ratio was calculated, so that ratios below 1 consistently indicated that the standard treatment performed better than was expected at the design stage of the trial, and ratios above 1 indicated that the standard treatment performed worse than was expected. This ratio could not be calculated for studies that did not report separately the expected, or the observed incidence of the primary endpoint, or studies reporting continuous endpoints.

RESULTS

Literature search and study characteristics

We identified 532 potentially eligible articles, and excluded 362 studies for the reasons shown in Figure 1. A total of 170 studies which were published in 43 different journals were included (see Appendix Table 1, 2 and 3 for bibliographic details). Five articles reported the results for two separate comparisons. In total, 175 comparisons were therefore included in the analyses. The oldest non-inferiority study in our selection dates from 1993. [START_REF] Carlier | A randomised controlled trial to test equivalence between retinyl palmitate and beta carotene for vitamin A deficiency[END_REF] Seventyeight percent of included studies date from 2004 onwards reflecting an increase in noninferiority trials in the last five years.

The characteristics of the 170 non-inferiority trials are described in Table 1. Of the general medical journals, the New England Journal of Medicine published the largest number of noninferiority trials; our search found no non-inferiority trials in PLoS Medicine. Most trials were from cardiovascular medicine (n=47, 28%) or infectious disease (n=43, 25%). Other fields were obstetrics and gynaecology (7%), oncology (6%), rheumatology (6%), surgery (5%), psychiatry (3%), general medicine (3%), pulmonary medicine (2%), gastroenterology (2%), anaesthesiology (1%), intensive care medicine (1%) and neurology (1%). The majority reported risk differences (n=106, 61%); 36 studies reported continuous endpoints, which were converted to odds ratios for the present analysis. Figure 2 shows the type of endpoint, number of participants, point estimate, confidence interval and pre-specified margin for 33 comparisons that were reported on the relative risk scale.

For 130 comparisons (74%) we considered the experimental treatment to be non-inferior according to the published criteria. [START_REF] Piaggio | Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement[END_REF] Of note, in 6 of these 130 comparisons the authors deemed the experimental treatment to be clinically inferior based on a secondary end-point.

For 27 comparisons (15%) results were inconclusive, for 15 comparisons (9%) superior and for 3 comparisons (2%) inferior. In 20 instances our assessment differed from the authors' verdict: in 9 instances we judged the result to be superior where the authors' verdict was non-inferior, in 6 instances to be inconclusive as opposed to inferior and once to be noninferior instead of inferior. The authors' verdict was more favourable to the experimental treatment in 4 comparisons, each time judging the result to be non-inferior instead of inconclusive.

Meta-analysis

The funnel plot showed a symmetrical distribution of results around relative risk 1 (Figure 3). Forty-seven percent of comparisons (n=82) had a point-estimate above 1 (favouring standard treatment) and 53% (n=93) below 1 (favouring experimental treatment). Of the 130 comparisons judged to be non-inferior, the point estimate favoured experimental treatment in 58% (n=76) and standard treatment in 42% (n=54). The combined relative risk for all 175 comparisons was 0.994 (95% CI 0.978 to 1.010) using a random effects model and 1.002 (95% CI 0.966 to 1.008) using a fixed-effects model. The combined relative risk for comparisons judged to be non-inferior was 0.995 (95% CI 0.983 to 1.006). Table 2 shows stratified random-effects meta-analyses according to trial result, measure of effect, type of endpoint, source of funding, by two journal strata, and according to whether the judgment of the result was based on an intention-to-treat analysis or not. Using a random effects model, the combined estimate for trials funded by industry was 0.978 (95% CI 0.956 to 1.000). The combined result for trials funded by public sources was 1.008 (95% CI 0.980 to 1.038). These two estimates did not differ significantly (p=0.15 from random-effects metaregression). All meta-analyses were also performed using a fixed-effects model and are presented in Appendix Table 4. The main result and the results from the stratified analyses were similar for the random-and fixed-effects meta-analyses except for a difference in the result stratified by funding source. The ratio of the observed and expected incidence of the primary endpoint in the group that received standard treatment could be calculated for 112 comparisons. Fifty-three percent of comparisons (n=59) had a ratio below 1, indicating that the standard treatment performed better than was expected at the design stage of the trial and 46% (n=51) above 1, indicating that the standard treatment performed worse than was expected. Two ratios were exactly 1.

Non-inferiority margins

The mean ratio was 0.941 (95% CI 0.859 to 1.030), meaning that on average the chosen standard treatments performed slightly better than was estimated at the design stage of the trials. Stratified by source of funding, this ratio was 0.974 (95% CI 0.865 -1.097) for 55 studies funded by industry and 0.906 (95% CI 0.760 to 1.080) for 31 studies funded by public sources. These two estimates did not differ significantly (p=0.5 from t-test for equality of means).

DISCUSSION

In this meta-analysis of trials using a non-inferiority design, experimental treatments were regarded as non-inferior to standard treatments in the majority of studies. The combined relative risk for these studies comparing experimental with standard treatments was close to 1. For non-inferiority trials published in core clinical journals, this finding contradicts the hypothesis that new treatments that gain a verdict of non-inferiority are systematically less effective than standard treatments.

Our study has several strengths and limitations. We aimed to include all the non-inferiority trials published in these journals, irrespective of the type of endpoints or measures of effect.

We restricted the search to the group of core clinical journals, as defined in PubMed, which is the same group of journals as in the Abridged Index Medicus (AIM). This selection covers a wide range of journals from many clinical specialties. Our results may therefore be representative for non-inferiority trials published in other journals. However, external validity may be limited to higher quality journals. If non AIM journals are of lower quality, the characteristics of non-inferiority trials published in those journals might be different. Furthermore, our search would have missed trials that do not mention the non-inferiority design in the abstract, the title or as a key word. The characteristics of such trials might also differ.

We examined two aspects of non-inferiority trials: firstly we combined the results of a large number of non-inferiority trials in a meta-analysis, secondly we examined the non-inferiority margins chosen by the investigators. Importantly, the combined estimate from the metaanalysis will not be influenced by the choice of the non-inferiority margin. The combined estimate will be influenced by the efficaciousness of standard treatment. If the standard treatment is not effective, the experimental treatment is in fact tested against 'placebo' in a non-inferiority design. Although we did not assess whether the chosen standard treatment represented the best available comparator, we did assess how standard treatments performed in view of what trialists had expected. On average the standard treatments performed slightly better than was estimated at the design stage of the trials. Our study did not address other important issues pertaining to non-inferiority trials. For example, we did not assess whether a non-inferiority trial was the appropriate design to use (or whether a superiority design would have been more appropriate) or whether the choice of the noninferiority margin that was used for the power calculation and statistical testing made clinical sense. The non-inferiority margin is often criticized as being arbitrary, unacceptably wide or even fraudulent. 8 10 The selection of the non-inferiority margin should be based on a combination of statistical reasoning and clinical judgment. [START_REF] Piaggio | Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement[END_REF][START_REF]Committee for Medicinal Products for Human Use (CHMP) guideline on the choice of the non-inferiority margin[END_REF] Others have reviewed the rationale for the size of the margins in non-inferiority trials. [START_REF] Lange | Choice of delta: requirements and reality--results of a systematic review[END_REF][START_REF] Henanff | Quality of reporting of noninferiority and equivalence randomized trials[END_REF] They found that the majority of trials did not justify the choice of the margin and that less than 20% reported a clinical consideration. An in-depth analysis of each trial with subject matter knowledge on each topic would have been required to judge whether the choice of the margin was adequate. This was beyond the aim of the present analysis. Does our meta-analysis rebuke some of the criticism aimed at non-inferiority trials? We found that the combined relative risk for all studies was close to 1. This contradicts the hypothesis that in non-inferiority trials the experimental treatment is generally less effective than the standard treatment. We believe that this is an important, re-assuring finding, considering the criticism that has been levelled at non-inferiority trials. [START_REF] Ware | Equivalence trials[END_REF][START_REF] Siegel | Equivalence and noninferiority trials[END_REF][START_REF] Djulbegovic | Scientific and ethical issues in equivalence trials[END_REF][START_REF] Agostino | Non-inferiority trials: design concepts and issues -the encounters of academic consultants in statistics[END_REF][START_REF] Hung | Some fundamental issues with noninferiority testing in active controlled trials[END_REF][START_REF] Kaul | Trials and tribulations of non-inferiority: the ximelagatran experience[END_REF][START_REF] Lange | Choice of delta: requirements and reality--results of a systematic review[END_REF][START_REF] Henanff | Quality of reporting of noninferiority and equivalence randomized trials[END_REF][START_REF] Piaggio | Reporting of noninferiority and equivalence randomized trials: an extension of the CONSORT statement[END_REF][START_REF] Gotzsche | Lessons from and cautions about noninferiority and equivalence randomized trials[END_REF][START_REF] Fueglistaler | Non-inferiority trials in surgical oncology[END_REF][START_REF] Garattini | Non-inferiority trials are unethical because they disregard patients' interests[END_REF] Several issues should nevertheless be considered when interpreting this result. Firstly, current standards for drug approval stipulate that a new treatment should be better than placebo and (at least) non-inferior to the established options. This means that demonstrating non-inferiority can legally suffice for the licensing of a new drug. The underlying assumption is often that a "non-inferior" treatment has added value regarding other properties, such as ease of use, lower costs, or fewer adverse effects, which might offset a small loss in efficacy. Sometimes such superior properties, such as costs, are self-evident and do not have to be demonstrated in a trial. Claiming that an agent has less adverse effects should however be based on evidence. A separate analysis of the adverse event data, analyses of combined endpoints or a meta-analysis of several trials might be appropriate and informative to demonstrate superiority in this respect. Of the 175 comparisons in this meta-analysis, we considered the experimental treatment to be non-inferior in 130 (74%) and superior in 15 (9%). Although in 6 of these 145 comparisons the authors deemed the experimental treatment to be clinically inferior based on a secondary end-point, the majority of published non-inferiority trials can be used to support the registration of a new treatment. The added value and safety of these treatments may not always be self-evident and may not always be demonstrated in the trial. The follow-up time and the sample size of trials is limited, making it improbable that rarer side effects or long term side effects are detected. The finding that studies sponsored by industry were more likely to have results favouring sponsored treatments is in line with other reports. 15,25 26 Systematic bias has been suggested as a possible explanation. Our finding could also be due to the play of chance.

Secondly, for superiority trials it has repeatedly been described how the outcome may be skewed in favour of the experimental treatment by making convenient choices when designing the study or reporting or publishing the result. This may involve the choice of (the dosage of) the comparator drug, the choice of patients, endpoints or of the type of analysis. 27 19 It may also involve selective reporting of data or changing the prespecified endpoint after a study is completed. [START_REF] Chan | Empirical evidence for selective reporting of outcomes in randomized trials: comparison of protocols to published articles[END_REF] It is plausible that such mechanisms affect the results of non-inferiority trials. In other words, biased choices in study design and bias due to selective reporting of outcomes may make it more likely that an experimental treatment is considered non-inferior after completion of the trial. We did not have access to the study protocols of the included articles and relied on what was reported as the primary endpoint. Also, we restricted our search to studies that have been published. Unpublished trials are more likely to favour standard treatment. [START_REF] Turner | Selective publication of antidepressant trials and its influence on apparent efficacy[END_REF] Therefore if publication bias would be an issue, our results might be skewed in favour of experimental treatments. All these potential sources of bias would remain unnoticed in our meta-analysis. Although the funnel plot showed a symmetrical distribution of results around relative risk 1, this does not rule out biases. This leaves the possibility that our finding of an overall relative risk close to unity is skewed in favour of experimental treatment.

Thirdly, the statistical verdict of non-inferiority permits licensing of a drug even if the trial result shows that it is somewhat less effective than standard. Therefore some treatments that are approved based on non-inferiority testing may be less effective compared to the standard therapy with respect to the primary endpoint. A cascade of non-inferiority trials is possible, in which each next experimental treatment is slightly less effective than the previously established "standard". After several generations of non-inferiority trials ineffective interventions could be licensed, leading to deteriorating patient care. [START_REF] Agostino | Non-inferiority trials: design concepts and issues -the encounters of academic consultants in statistics[END_REF][START_REF] Fueglistaler | Non-inferiority trials in surgical oncology[END_REF] This outcome has been called 'bio-creep'. [START_REF] Fleming | Current issues in non-inferiority trials[END_REF] Our results are relevant in this context. Our study showed that of the 130 comparisons judged to be non-inferior, the point estimate favoured the standard treatment in 42% of trials. Biocreep could occur if two or three trials in succession belong to this 42% category and if each next trial adopts the previous demonstrated non-inferior treatment as the new active control treatment. Importantly, our study provided no empirical evidence for or against the existence of biocreep.

In conclusion, the number of non-inferiority trials published in clinical journals has greatly increased. We found that the experimental treatments that gain a verdict of non-inferiority in trials published in core clinical journals are not systematically less effective than the standard treatments. Biases in design, reporting and publication cannot be ruled out and may have skewed the study results in favour of experimental treatments. Continued vigilance is required to assure that non-inferiority trials are used appropriately.

KEY MESSAGES

• There is concern that non-inferiority trials might be deliberately designed to conceal that a new treatment is less effective than a standard treatment. There is little empirical evidence at present to support this notion, however.

• The combined relative risk from 170 randomized trials using a non-inferiority design and published in core clinical journals in recent years was close to 1, neither favouring the experimental, nor the standard treatment.

• In the majority of trials, the new treatments were considered to be non-inferior. For these trials the combined relative risk was also close to 1.

• The experimental treatments that gain a verdict of non-inferiority do therefore not appear to be systematically less effective than the standard treatments.

• The evidence from published non-inferiority trials might still be distorted by publication bias, or by a biased choice of standard treatments. Further studies are required to clarify the risk of bias in non-inferiority trials.

  Efficacy of experimental treatments compared with standard treatments in non-inferiority trials: A metaanalysis of randomized controlled trials. -10-0935 Efficacy of experimental treatments compared with standard treatments in non-inferiority trials: A meta-analysis of randomized controlled trials.

  had expected. On average the standard treatments performed slightly better than was estimated at the design stage of the trials.

  Efficacy of experimental treatments compared with standard treatments in non-inferiority trials:A meta-analysis of randomized controlled trials.

  Further studies are required to examine the importance of such bias. Keywords non-inferiority, meta-analysis, systematic review 2

  Statistical analyses were done in Comprehensive Meta-Analysis (version 2.0, Biostat, Englewood, New Jersey, USA) and Stata (version 10.0, Stata Corporation, College Station, Texas, USA).

  The margin was expressed as a relative risk for 33 comparisons and could be converted from a risk difference to a relative risk for 91 comparisons. The median prespecified noninferiority margin was 1.31 (IQR 1.18 to 1.59). Stratified according to trial result, the median margin was 1.42 (range 1.21 to 4.75) for 3 comparisons judged to be inferior, 1.33 (IQR 1.14 to 1.51) for 23 comparisons judged to be inconclusive, 1.31 (IQR 1.19 to 1.59) for 92 comparisons judged to be non-inferior and 1.45 (IQR 1.20 to 1.75) for 6 comparisons judged to be superior. Stratified by type of endpoint the median margin was 1.34 (IQR 1.19 to 1.50) for comparisons that had mortality as (part of a combined) endpoint, 1.38 (IQR 1.20 to 1.70) for comparisons in which clinical disease was the endpoint and 1.23 (IQR 1.15 to 1.35) for comparisons reported as a surrogate endpoint. Stratified according to source of funding the median margin was 1.30 (IQR 1.17 to 1.50) for 62 trials funded by industry, 1.31 (IQR 1.12 to 1.63) for 33 trials funded by public sources and 1.35 (IQR 1.23 to 1.55) for 19 trials with mixed funding.

Figure 1 .Figure 2 .

 12 Figure 1. Summary of the search strategy and study selection.

Figure 3 .

 3 Figure 3. Funnel plot of the standard error by the log relative risk for 175 comparisons.

Endpoint type Participants (n) Relative risk (95% CI)

						Non-inferiority
						margin
	Olshansky 29	mortality	988	0.67	(0.43 -1.03)	1.22
	Bousser 30	clinical disease	4576	0.71	(0.39 -1.30)	1.50
	Agnelli 31	clinical disease	2048	0.74	(0.50 -1.09)	1.70
	Fleshman 32	clinical disease	863	0.84	(0.62 -1.13)	1.23
	Tebbe 33	mortality	3089	0.85	(0.64 -1.12)	1.50
	Surgical therapy 34	clinical disease	863	0.86	(0.63 -1.17)	1.23
	Schellhammer 35	mortality	813	0.87	(0.74 -1.03)	1.25
						1.25
						1.23
						1.14
						1.23
						1.32
						1.35
						1.09
						1.23
						1.17
						2.00
						1.12
						1.18
						1.08
						1.15
						2.00
						1.20
						1.67
						1.25
						1.20
						1.50
						1.47
						3.00
						1.35
						1.43
						2.00
						2.00

Table 1 .

 1 Characteristics of the 170 non-inferiority trials included in the meta-analysis.

	Study characteristics	n	%
	Journal
	New England Journal of Medicine	33	(19%)
	Lancet	28	(16%)
	Circulation	11	(6%)
	Journal of the American Medical Association	10	(6%)
	British Medical Journal	8	(5%)
	Obstetrics and Gynaecology	8	(5%)
	Paediatrics	8	(5%)
	Other (from 36 different journals)	64	(38%)
	Field in medicine
	Cardiovascular medicine	47	(28%)
	Infectious disease	43	(25%)
	Obstetrics and Gynaecology	12	(7%)
	Oncology	10	(6%)
	Rheumatology	10	(6%)
	Surgery	9	(5%)
	Psychiatry	5	(3%)
	Other	34	(20%)
	Type of comparison
	Drug	133	(78%)
	Procedure	23	(14%)
	Device	14	(8%)
	Source of funding
	Industry	94	(55%)
	Public or charity	46	(27%)
	Mixed	19	(11%)
	Not reported	11	(7%)
	Median number of participants per comparison (range)*	467 (40 to 20332)
	Effect measure*
	Risk difference	106	(61%)
	Ratio measure	33	(19%)
	Hazard Ratio	20
	Risk Ratio	7
	Odds Ratio	6
	Continuous	36	(21%)
	Main analysis used to judge the result*
	Intention-to-treat analysis	95	(54%)
	Modified intention-to-treat-or per protocol analysis	80	(46%)
			18

* characteristics for 175 comparisons from the 170 trials.

Table 2 .

 2 Random-effects meta-analyses of 175 comparisons of experimental and standard treatments from 170 non-inferiority trials. All meta-analyses in this table were performed using a random effects model. All meta-analyses were also performed using a fixed effects model and are presented in Appendix Table4. The main result and the results from the stratified analyses were similar for the random-and fixed-effects meta-analyses except for a difference in the result stratified by funding source.

	Number of	Relative risk (95% CI)*
	comparisons

*

Acknowledgement

We acknowledge the contribution of Theo Stijnen, PhD, Professor of Medical Statistics, Department of Medical Statistics and Bioinformatics, Leiden University Medical Centre for critical discussion of the analyses and of J.W. Schoones, MA, Walaeus Library, Leiden University Medical Centre for assistance with the search strategy.

Permission has been granted by Professor T. Stijnen and drs. J.W. Schoones for mentioning their name in acknowledgement. Neither was compensated for their contribution.

Funding

There was no dedicated funding for this project.

Conflict of interest statement

We declare we have no conflict of interest.

Author's contribution and individual conflict of interest statement

D. Soonawala: "I declare that I participated in the preparation of the protocol, data extraction, data analysis and in writing the manuscript and that I have seen and approved the final version. I have no conflicts of interest. As the corresponding author I had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the analysis and will act as guarantor for the paper" This material has not been published previously in a substantively similar form. The manuscript has been carefully edited by M. Egger who has an excellent mastery of the English language.