Echanisms of inactivation for the same pathway. Indeed, the loss of CDKN2A also leads to the deletion of p16INk4A, which encodes a cyclin kinase inhibitor controlling RB protein activity through CDK4 in G1/S. Furthermore, p14ARF may have several TP53-independent activities [22] and TP53 mutations may have gain-of-function effects [23]. Thus, the various events (CDKN2A loss and TP53 mutation) may contribute in an additional manner to the transformed phenotype. Meta-analyses are often performed on clinical data, but more rarely on biological data. Meta-analysis is much more powerful than individual analysis but has several drawbacks: 1) Different studies use different methodologies. Indeed, in the studies analysed here, different methods 
were used to assess the frequency of both FGFR3 (snapshot or sequencing) and TP53 (direct sequencing or FASAY) mutations. Furthermore, different exons were explored in different studies: exons 7, 10, 15 or 
7, 10, 13, 15 for FGFR3 and exons 5 to 8, 4 to 9, 4 to 11 or 2 to 11 for TP53. Differences due to the choice of exons studied should 15481974 be negligible; mutations of exon 15 of FGFR3 account for only 3 of all identified mutations when exons 7, 10, and 15 are considered, and mutations in exons 2, 3, 4, 9, 10 and 11 of TP53 mutations account for only 6.3 of all identified mutations when exons 2 to 11 of this gene are considered. FASAY and direct sequencing are very different in nature (FASAY being a functional assay and more BIBS39 biological activity sensitive), but they give very similar results [24]. Most of the tumours exploredTable 4. TP53 BIBS39 site mutation rates in FGFR3-wild-type and FGFR3-mutated tumours, according to a combination of stage and grade.TP53 mutation ratepTaG1G2 (n = 242) FGFR3 mutant FGFR3 wild-type 4.7 (8/170) 4.2 (3/72) pTaG3 (n = 42) 5.9 (1/17) 20 (5/25) 0.37 pT1G2 (n = 65) 16.7 (7/42) 21.7 (5/23) 0.74 pT1G3 (n = 260) 37 (20/54) 48.5 (100/206) 0.17 pT2-4 (n = 195) 43.5 (10/23) 51.2 (88/172) 0.P-value Fisher’s exact test 0.99 doi:10.1371/journal.pone.0048993.tFGFR3 and TP53 Mutations in Bladder Cancerby the FASAY method in our analysis were pTaG1-G3 or pT1G2 tumours. A comparison of the FASAY data with direct sequencing data for the same tumours showed the frequencies of TP53 mutation obtained with these two techniques to be very similar. 2) This meta-analysis may also have been biased by the lack of review of stage and grade determination. The stages and grades assigned to tumours may therefore differ between the studies included in the meta-analysis. This could be problematic for pT1 tumours, because some pTa tumours may have been overstaged and classified as pT1 tumours [25]. This meta-analysis provides a good example of the effect of confounding factors that are not taken into account. Such factors may not only modify the magnitude of any association, but also create spurious associations. Larger sample sizes and more detailed data are therefore required for a valid statistical analysis. In such conditions, meta-analysis is an effective approach, making it possible to draw reliable conclusions, because it builds on the strengths of several studies that may not be sufficiently powerful individually to generate conclusive results. However, metaanalyses can only take into account the confounding factors that were actually measured. Other unknown variables may affect the relationship between the pathway and disease severity and remain undetected. All the publications retained for this analysis had a high qual.Echanisms of inactivation for the same pathway. Indeed, the loss of CDKN2A also leads to the deletion of p16INk4A, which encodes a cyclin kinase inhibitor controlling RB protein activity through CDK4 in G1/S. Furthermore, p14ARF may have several TP53-independent activities [22] and TP53 mutations may have gain-of-function effects [23]. Thus, the various events (CDKN2A loss and TP53 mutation) may contribute in an additional manner to the transformed phenotype. Meta-analyses are often performed on clinical data, but more rarely on biological data. Meta-analysis is much more powerful than individual analysis but has several drawbacks: 1) Different studies use different methodologies. Indeed, in the studies analysed here, different methods were used to assess the frequency of both FGFR3 (snapshot or sequencing) and TP53 (direct sequencing or FASAY) mutations. Furthermore, different exons were explored in different studies: exons 7, 10, 15 or 7, 10, 13, 15 for FGFR3 and exons 5 to 8, 4 to 9, 4 to 11 or 2 to 11 for TP53. Differences due to the choice of exons studied should 15481974 be negligible; mutations of exon 15 of FGFR3 account for only 3 of all identified mutations when exons 7, 10, and 15 are considered, and mutations in exons 2, 3, 4, 9, 10 and 11 of TP53 mutations account for only 6.3 of all identified mutations when exons 2 to 11 of this gene are considered. FASAY and direct sequencing are very different in nature (FASAY being a functional assay and more sensitive), but they give very similar results [24]. Most of the tumours exploredTable 4. TP53 mutation rates in FGFR3-wild-type and FGFR3-mutated tumours, according to a combination of stage and grade.TP53 mutation ratepTaG1G2 (n = 242) FGFR3 mutant FGFR3 wild-type 4.7 (8/170) 4.2 (3/72) pTaG3 (n = 42) 5.9 (1/17) 20 (5/25) 0.37 pT1G2 (n = 65) 16.7 (7/42) 21.7 (5/23) 0.74 pT1G3 (n = 260) 37 (20/54) 48.5 (100/206) 0.17 pT2-4 (n = 195) 43.5 (10/23) 51.2 (88/172) 0.P-value Fisher’s exact test 0.99 doi:10.1371/journal.pone.0048993.tFGFR3 and TP53 Mutations in Bladder Cancerby the FASAY method in our analysis were pTaG1-G3 or pT1G2 tumours. A comparison of the FASAY data with direct sequencing data for the same tumours showed the frequencies of TP53 mutation obtained with these two techniques to be very similar. 2) This meta-analysis may also have been biased by the lack of review of stage and grade determination. The stages and grades assigned to tumours may therefore differ between the studies included in the meta-analysis. This could be problematic for pT1 tumours, because some pTa tumours may have been overstaged and classified as pT1 tumours [25]. This meta-analysis provides a good example of the effect of confounding factors that are not taken into account. Such factors may not only modify the magnitude of any association, but also create spurious associations. Larger sample sizes and more detailed data are therefore required for a valid statistical analysis. In such conditions, meta-analysis is an effective approach, making it possible to draw reliable conclusions, because it builds on the strengths of several studies that may not be sufficiently powerful individually to generate conclusive results. However, metaanalyses can only take into account the confounding factors that were actually measured. Other unknown variables may affect the relationship between the pathway and disease severity and remain undetected. All the publications retained for this analysis had a high qual.
