The recent reports have led to the suggestion that urothelial carcinoma develop through at least two molecular pathways, one related to FGFR3 and one related to TP53 [23
]. The aim of such a pathway-driven approach was to identify strong and comprehensible gene signatures associated with carcinogenesis of bladder cancer [24
]. In previous findings, FGFR3 mutations in nonmuscle invasive bladder carcinoma were reported to be associated with low recurrence rate [22
]. Further studies revealed contradictory findings. In a large prospective study [25
], FGFR3 mutations were associated with increased recurrence rate only in TaG1 tumors. Bladder cancer development is a complex multistep process and the pathway from normal to malignant urothelium is not clearly understood. Molecular alterations in tumors are not isolated and the study of single determinants does not reflect accurately the cascade of molecular aberrations.
We previously found that 9p22 LOH was able to discriminate potentially progressing tumors from non-progressing ones, in patients presenting with primary or recurrent NMI-BC, and that LOH at 9p22 had an independent prognostic value for progression to muscle invasive disease. Observation of LOH at a specific chromosomal marker in cells from the tumor suggests the presence of a closely linked tumor suppressor gene, the loss of which is involved in pathogenesis of the tumor. Specific locus at 9p21-22, referred to as CDKN2A, encodes two proteins, p16INK4A and p14ARF, which are frequently inactivated in bladder cancers [14
]. The protein p16 plays an important role in cell cycle, and blocks the cell division at G1/S checkpoint [27
]. The prognostic value of loss of p16 was studied in the literature [18
]. Moreover, association between 9p22 LOH and loss of p16 function have been previously demonstrated by immunohistochemical analysis in NMI-BC [14
]. These findings have led us to address the question whether the combined FGFR3/9p22 status might be predictive for clinical outcome in NMI-BC.
As previously described, we found FGFR3 mutations in 50% of all tumors [28
]. We also noted in line with published findings that the mutation frequency of FGFR3 was significantly associated with tumor stage and grade [25
]. FGFR3 mutations characterize low grade and low stage NMI-BC with favourable histological features. The 9p22 LOH was more frequently detected in high grade and high stage tumors, but difference was not so markedly detected than FGFR3 mutational status.
Losses at 9p22 were similarly distributed in groups with or without FGFR3 mutations [28
]. Thus, a correlation of FGFR3 mutations and alterations at 9p22 does not exist in NMI-BC. We could not conclude on the fact that losses on 9p22 occur earlier than FGFR3 mutations. However, the presence of allelic losses at 9p22 might modulate the mechanisms of FGFR3 mutations on the disease recurrence and progression.
Our observations suggest that allelic losses at 9p22 locus or FGFR3 mutations are not significantly associated with changes leading to tumor recurrence and global progression when these molecular alterations are taking into account separately, except for deletion of genes at 9p22 that may be more likely to lead to muscle invasion.
In spite of limitations due to the low number of cases, we think our findings are of great value and may participate in the understanding of the conflicting results reported in the literature concerning the prognostic value of the FGFR3 mutational status. In the present series, we did not found significant association with isolated FGFR3 status and propensity for recurrence or progression. Nevertheless, our findings emphasized that the clinical relevance of the FGFR3 mutational status depended on the allelic status at 9p22. In tumors showing allelic losses at 9p22 locus, the presence of FGFR3 mutation (in 50% of these cases) characterized low malignant tumors with favourable clinical outcome that are at low risk of recurrence and progression. By contrast, combination of 9p22 LOH and absence of FGFR3 mutation was associated with tumors at high malignant potential with early recurrence, progression and muscle invasion. Interestingly, the risk of recurrence was slightly higher in tumors with FGFR3 mutation when no allelic loss was found at 9p22 (recurrence-free survival rate at 2 years: 61.6% versus 71.1%). Nevertheless, difference did not reach significance. As tumors without 9p22 LOH represents 75% of the NMI-BC of our study, it is not surprising that large prospective studies report a higher recurrence rate in NMI-BC with FGFR3 mutations [25
Recently, Sylvester and co-workers retrospectively analyzed 2596 EORTC bladder tumor patients and proposed a scoring system predicting the risk of recurrence and of progression of NMI-BC [2
]. We decided to integrate this tool in the multivariate analysis in order to reduce the number of integrated variables and, thus, to keep the statistical relevance for the Cox model. In our multivariate analysis, the combined FGFR3/9p22 status was associated with an increased risk of recurrence, progression and muscle-invasion independent of other prognostic parameters such as age and Bladder Calculator. The tumors displaying wtFGFR3 and 9p22 LOH had a risk of recurrence and progression increased by 5.5 and 8-fold, respectively. In spite of the weak rate of events, strong significant differences were detected that bore witness of strong value of such a marker combination. Another interest of our methodology was to evaluate any patient with NMI-BC at any time of the follow-up, reflecting every day clinical practice.
However, the short follow-up of our cohort constituted an important limitation to end in such observations. Our follow-up data were not able to evaluate the correlation between the combined FGFR3/9p22 status and progression, in subgroups of patients stratified on stage and grade, due to the small number of progression observed. Given the mid-term follow-up of our cohort, we cannot conclude whether the combined mutational status impact the patient survival. Longer follow-up and larger cohort remain warranted.
The findings of this study support the notion that only a combination of molecular markers may characterize a subgroup of bladder cancer with poor prognosis. Our prognostic evaluation of the combined FGFR3/9p22 status seems to be interesting to better identify patients at high risk of poorer clinical outcome. The strengths of our study were the prospective design, the relative high number of cases, and the homogeneity of our cohort, consisting in NMI-BC. One concern was the possible presence of concomitant CIS in our tumor cohort. We systematically performed random quadrant biopsies in all cases and found 7 CIS cases. However, the possibility of undetected concomitant CIS cannot be entirely ruled out because resection was performed under white-light cystoscopy. Such a consideration is important as CIS plays an important role in tumor progression and may affect the genetic profile. Nevertheless, no significant association between CIS and combined FGFR3/9p22 status was found.